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A multidisciplinary approach to tackle the problem
of the zoonotic parasite Anisakis in fish
Miguel Bao Domínguez
BSc Marine Science – University of Vigo, Spain
Doctorate itinerary of the Master in Aquaculture – University of Vigo, Spain
A thesis presented for the degree of Doctor of Philosophy in Systems Biology at the
University of Aberdeen
April 2017
2
Declaration
I declare that this thesis has been composed by me and has not been accepted in any
previous application for a degree. All quotations have been distinguished by quotation
marks and the sources of information specifically acknowledged.
Chapter 2 has been published jointly as:
Bao, M., Garci, M. E., Antonio, J. M., Pascual, S., 2013. First report of Anisakis simplex
(Nematoda, Anisakidae) in the sea lamprey (Petromyzon marinus). Food control, 33(1),
81-86. doi: 10.1016/j.foodcont.2013.02.009.
Contribution
I have done all the work, with the exception of the following contributions: J. M. A.
contributed to perform sea lamprey necropsies, and to the enzymatic peptic digestion of
the viscera and muscle tissues. M. E. G. contributed during sea lamprey field sampling
and photography. S. P. contributed to the molecular analysis which was carried out by
staff member (Mariana Noelia Cueto Rivas) of the “Instituto de Investigaciones Marinas
– Consejo Superior de Investigaciones Científicas” (IIM-CSIC) (Vigo, Spain), performed
the statistic tests and supervised the final content of the work.
Chapter 3 has been published jointly as:
Bao, M., Mota, M., Nachón, D. J., Antunes, C., Cobo, F., Garci, M. E., Pierce, G. J.,
Pascual, S., 2015. Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and
twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers:
zoonotic and ecological implications. Parasitology Research, 114(6), 2143-2154. doi:
10.1007/s00436-015-4403-5.
Contribution
I have done all the work, with the exception of the following contributions: M. M., D. J.
N., C. A., F. C., and M. E. G contributed collecting the fish and parasite data. S. P.
contributed to the molecular analysis which was carried out by staff member (Mariana
Noelia Cueto Rivas) of the IIM-CSIC. G. P. designed the statistical analysis. All co-
authors contributed to the final content of the work.
Chapter 4 has been published jointly as:
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Bao, M., Strachan, N. J. C., Hastie, L. C., MacKenzie, K., Seton, H. C., Pierce, G. J.,
2017. Employing visual inspection and Magnetic Resonance Imaging to investigate
Anisakis simplex s.l. infection in herring viscera. Food Control, 75, 40-47. doi:
10.1016/j.foodcont.2016.12.030.
Contribution
I designed the study and have done all the work, with the exception of the following
contributions: H. S. developed the MRI protocol. L. H. contributed to Anisakis inspection
in herring. K. M. performed the morphological identification of ascaridoid samples. H. S.
contributed to the Magnetic Resonance Imaging (MRI) investigations, analysis of results
and writing of the paper. N. S. and G. P. contributed to the ideas behind the study and
reviewed the paper. All co-authors contributed to the final content of the work.
Chapter 5 has been produced jointly and is currently under review as:
Bao, M., Pierce, G. J., Strachan, N. J. C., Martínez, C., Fernández, R., Theodossiou, I.
(under review). Consumers’ attitudes and willingness to pay for Anisakis-free fish.
Fisheries Research.
Contribution
I have done all the work, with the exception of the following contributions: G. J. P., N. S.
C. M., R. F. and I. T. contributed to questionnaire design and distribution. G. J. P., N. S.
and I. T. helped analysing results and supervised the final content of the work.
Chapter 6 has been published jointly as:
Bao, M., Pierce, G. J., Pascual, S., González-Muñoz, M., Mattiucci, S., Mladineo, I.,
Cipriani, P., Bušelić, I., Strachan, N. J. C., 2017. Assessing the risk of an emerging
zoonosis of worldwide concern: anisakiasis. Scientific reports, 7, 43699. doi:
10.1038/srep43699.
Contribution
I have done all the work, with the exception of the following contributions: N. S.
contributed to developing the quantitative risk assessment model. I. M., I. B., S. M. and
P. C. provided data of Anisakis spp. infection levels in anchovy from the Mediterranean
Sea. S. P. provided data of Anisakis spp. infection levels in anchovy from North-East
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Atlantic waters. N.S. and G.P. supervised the work. All co-authors contributed to the final
content of the work.
Miguel Bao Domínguez
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Acknowledgements
Non foi sen tempo!, which in my homeland (Galicia) could be translated as: about time!
But, first, I would like to thank many people that helped me to successfully conclude this
PhD project.
The work presented in this thesis started five years ago (2012) at “Instituto de
Investigaciones Marinas – Consejo Superior de Investigaciones Científicas (IIM-CSIC)”.
I am sincerely thankful to my Spanish supervisor (Dr. Santiago Pascual) and to many
work colleagues at IIM-CSIC who helped me, directly or indirectly, while I was studying
the epidemiology of Anisakis in sea lamprey and shads of Western Iberian Peninsula
rivers. Santi, I will always thank you for giving me the opportunity to face this challenge,
the facilities and resources of the ECOBIOMAR Group to develop my research, and for
the enlightening supervision of my work. José, Garci, Mariana and María Teresa thank
you very much for your invaluable help during the field work, sampling and lab work,
and for the splendid time I had with you all. Many thanks to all my PhD colleagues,
Álvaro, Jorge, Marcos, María Gregori, María Llarena, Lorena, and to Helena for your
help, encouragement and for the nice chats/time talking about science and common live.
You are great scientists and inspiring persons. Thanks a lot to Prof. Ángel Guerra and Dr.
Ángel F. González for your warm welcome to ECOBIOMAR, for your help, for the very
helpful conversations/advices, and for the good times at the office and during the coffee
time. I also want to extend my thanks to ALL the colleagues of the IIM-CSIC for your
help any time I needed it. It was superb being part of your family during those years.
I would like to acknowledge the University of Aberdeen for not only funding me, but also
providing me with an excellent environment of work/education and continuous
opportunities of training. I also acknowledge the EU FP7 PARASITE project for the
funding, and for the opportunity to meet and learn from outstanding experts in the field
of Anisakis and associated diseases.
My profound gratitude to my Aberdeen-based supervisors Prof. Norval Strachan, Prof.
Graham Pierce and Prof. Ioannis Theodossiou. Firstly, Graham, I am eternally thankful
for the opportunity you gave me to come to Aberdeen. Norval and Graham my deepest
gratitude for your time, patience, encouragement, guidance, teaching, constant support
and inspiring supervision of my thesis. Ioannis thank you very much for the teaching and
supervision of the economic part (chapter five) of the thesis. I sincerely thank Dr. Lee
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Hastie for his help during fish sampling at the Oceanlab, for your help/company at the
office, and for many other collaborations we carried out together and really nice moments
(for instance, many tasks within the EU FP7 PARASITE project, “sea lice” and
“freshwater pearl mussel” projects, chapter four, etc.). Thanks a lot to Dr. Ken MacKenzie
for reviewing chapter one, for your collaboration in chapter four, for providing me the
“anisakid” bibliography I wasn’t able to find anywhere else, for your collaboration/help
in many other projects (“macroparasites of shads” paper, etc.), and for the really nice
moments (for instance, good times at the EU FP7 PARASITE project meeting at
Hamburg and in the 9th International Symposium on Fish Parasites in Valencia, etc.). I
wish you all the best.
I also wish to re-express my sincere gratitude to all the co-authors and collaborators of
the chapters that compose this thesis, which contribution was much appreciated and has
been properly detailed above. M. E. Garci, J. M. Antonio, M. Mota, D. J. Nachón, C.
Antunes, F. Cobo, L. Hastie, K. MacKenzie, H. Seton, C. Martínez, R. Fernández, M.
González-Muñoz, S. Mattiucci, I. Mladineo, P. Cipriani and I. Bušelić, a million thanks
to you all!
Thanks a lot to the lunch/badminton club friends (Eakapon, Ahmed, Douglas, Ottavia,
Yeh-Fang, Ayham, Abdo and Manu) for the good times and exciting conversations we
had together. You made my life in the grey Aberdeen much more enjoyable. My best
wishes for you all in your future in which I hope we will meet again. Also, many thanks
to my office colleagues (Niall, Ewan, Alan, Josh) for your help with English grammar
and nice conversations at work.
Thanks to my lifetime friends from Vigo (some still living there and other living abroad).
Thank you for being there every time I come back home and for the daily funny chats we
have thanks to the social media.
This thesis is dedicated to my family, and so I wish to express my endless gratitude to
them. Especially dedicated to Mum and Dad, thanks for your never-ending love,
understanding, support and confidence in me. Thanks to my little brother for your
understanding when I left home, for your support, and for being at home with Mum and
Dad. Also, thanks to the rest of my family and to my family members in law for your
understanding, support and permanent caring. Last, but certainly not least, no
acknowledgement would be complete without giving thanks to my future wife, Belén.
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For moving with me since the first day to Aberdeen and for being brave throughout these
years, for listening to me in the hardest times, for your understanding, constant
encouragement and endless trust, for your love and support, and for keeping me attached
to the real world. You all were my major source of inspiration. This thesis is also yours.
Thanks to ALL of you.
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Table of Contents Abstract .......................................................................................................................... 14
Chapter 1 - General introduction and thesis outline ................................................. 17
1.1 Anisakid nematodes and their significance as aetiological agents of a fish-borne
parasitic zoonosis (anisakidosis) ................................................................................. 17
1.2 The genus Anisakis ........................................................................................... 17
1.3 Anisakis spp. life cycle ecology ....................................................................... 19
1.4 Can Anisakis spp. pose a risk in freshwater environments? ............................. 20
1.5 Anisakis spp. in fish hosts ................................................................................ 21
1.6 Detection methods of Anisakis spp. larvae in fish ........................................... 22
1.7 Consumer perception of risk due to the presence of Anisakis spp. in fish ....... 23
1.8 Anisakiasis and its incidence in the human population ................................... 24
1.9 Objectives and thesis outline ............................................................................ 25
1.10 References .................................................................................................... 27
Chapter 2 - First report of Anisakis simplex (Nematoda, Anisakidae) in the sea
lamprey (Petromyzon marinus) .................................................................................... 33
2.1 Abstract ............................................................................................................ 33
2.2 Introduction ...................................................................................................... 34
2.3 Materials and methods ..................................................................................... 36
2.3.1 Sampling ................................................................................................... 36
2.3.2 Necropsy ................................................................................................... 36
2.3.3 Artificial peptic digestion ......................................................................... 36
2.3.4 Molecular analysis .................................................................................... 37
2.3.5 Quantitative descriptors ............................................................................ 37
2.4 Results .............................................................................................................. 37
2.5 Discussion ........................................................................................................ 43
2.6 Acknowledgements .......................................................................................... 46
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2.7 References ........................................................................................................ 47
Chapter 3 - Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and twaite
shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers:
zoonotic and ecological implications ........................................................................... 50
3.1 Abstract ............................................................................................................ 50
3.2 Introduction ...................................................................................................... 51
3.3 Materials and methods ..................................................................................... 53
3.3.1 Sampling ................................................................................................... 53
3.3.2 Necropsy and visual inspection ................................................................ 53
3.3.3 Artificial enzymatic digestion ................................................................... 56
3.3.4 Molecular analysis .................................................................................... 56
3.3.5 Quantitative descriptors and statistical analysis ....................................... 56
3.4 Results .............................................................................................................. 58
3.4.1 Parasite inspection .................................................................................... 58
3.4.2 Genetic identification ................................................................................ 59
3.4.3 Infection data ............................................................................................ 60
3.4.4 Statistical modelling ................................................................................. 60
3.5 Discussion ........................................................................................................ 65
3.5.1 Where are shad infected by Anisakis nematodes? .................................... 65
3.5.2 How do shad acquire Anisakis parasites? ................................................. 67
3.5.3 Statistical analysis ..................................................................................... 68
3.5.4 Risk assessment ........................................................................................ 69
3.6 Acknowledgements .......................................................................................... 70
3.7 References ........................................................................................................ 72
Chapter 4 - Employing visual inspection and Magnetic Resonance Imaging to
investigate Anisakis simplex s.l. infection in herring viscera..................................... 78
4.1 Abstract ............................................................................................................ 78
4.2 Introduction ...................................................................................................... 80
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4.3 Materials and methods ..................................................................................... 81
4.3.1 Ascaridoid nematode sampling in herring viscera .................................... 81
4.3.2 MRI protocol ............................................................................................. 81
4.4 Results .............................................................................................................. 83
4.4.1 Anisakis simplex s.l. infection in herring viscera ...................................... 83
4.4.2 MRI results ............................................................................................... 85
4.5 Discussion ........................................................................................................ 89
4.5.1 Anisakis simplex s.l. habitat in the visceral cavity of herring ................... 89
4.5.2 Parasite detection with MRI ..................................................................... 91
4.6 Acknowledgements .......................................................................................... 92
4.7 Authors’ contributions ..................................................................................... 92
4.8 References ........................................................................................................ 94
Chapter 5 - Consumers’ attitudes and willingness to pay for Anisakis-free fish .... 98
5.1 Abstract ............................................................................................................ 98
5.2 Introduction .................................................................................................... 100
5.3 Materials and methods ................................................................................... 102
5.3.1 Survey and sample selection ................................................................... 102
5.3.2 Questionnaire design ............................................................................... 102
5.3.3 Willingness to pay for Anisakis-free fish ................................................ 103
5.3.4 Data processing ....................................................................................... 104
5.3.5 Data analysis: descriptive statistics ......................................................... 104
5.3.6 Statistical analysis: generalised additive modelling ............................... 105
5.4 Results ............................................................................................................ 105
5.4.1 Data analysis: descriptive statistics ......................................................... 105
5.4.2 Data analysis: generalised additive models for WTP ............................. 124
5.5 Discussion ...................................................................................................... 127
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5.5.1 Consumer awareness of the presence of Anisakis in fish and raw fish
consumption behaviour ......................................................................................... 127
5.5.2 Purchase and consumption behaviour of consumers in relation to the
presence of Anisakis in fish .................................................................................. 127
5.5.3 WTP for Anisakis-free fish ..................................................................... 128
5.5.4 WTP protesters ....................................................................................... 130
5.5.5 Study limitations ..................................................................................... 131
5.6 Conclusions .................................................................................................... 131
5.7 Acknowledgements ........................................................................................ 131
5.8 Authors’ contributions ................................................................................... 132
5.9 References ...................................................................................................... 133
Chapter 6 - Assessing the risk of an emerging zoonosis of worldwide concern:
anisakiasis .................................................................................................................... 138
6.1 Abstract .......................................................................................................... 138
6.2 Introduction .................................................................................................... 139
6.3 QRA Section .................................................................................................. 142
6.3.1 Materials and methods ............................................................................ 142
6.3.2 Risk assessment ...................................................................................... 152
6.3.3 Results ..................................................................................................... 164
6.4 General discussion ......................................................................................... 168
6.5.1 Estimating the number of anisakiasis cases in Spain .............................. 168
6.5.2 Comparison of the two Risk Characterization methods and potential biases
169
6.5.3 Anchovy is the main fish species causing anisakiasis ............................ 171
6.5.4 Home-prepared raw and marinated anchovies are the main fish specialties
causing anisakiasis ................................................................................................ 171
6.5.5 Viability of Anisakis spp. in untreated anchovy meals and their inactivation
in the human stomach ........................................................................................... 172
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6.5.6 Implementation and validity of the exponential dose response model for
Anisakis spp. ......................................................................................................... 172
6.5.7 Pathogenicity of Anisakis spp. species and susceptibility of humans ..... 173
6.5.8 Sensitization and allergy to Anisakis spp. ............................................... 173
6.5.9 Post-mortem migration of Anisakis spp. from fish viscera to the muscle
174
6.5.10 Risk mitigation strategies ........................................................................ 175
6.5.11 Extending the QRA method to other countries and other parasites ........ 175
6.5 Conclusions .................................................................................................... 176
6.6 Acknowledgements ........................................................................................ 177
6.7 Authors’ contributions ................................................................................... 177
6.8 References ...................................................................................................... 179
Chapter 7 - General discussion, future work and conclusions ............................... 184
7.1 Overview ........................................................................................................ 184
7.1.1 Anisakis spp. in anadromous fish – potential health risk for humans and
wildlife 184
7.1.2 Anisakis spp. in freshwater ecosystems – ecological implications ......... 185
7.1.3 Anatomical location of Anisakis spp. in anadromous fish – epidemiological
insights 186
7.1.4 Anatomical location of Anisakis spp. in clupeids – epidemiological insights
187
7.1.5 Magnetic Resonance Imaging – potential new technology for parasite
investigation and detection ................................................................................... 188
7.1.6 The problem of Anisakis spp. in fishery products – socioeconomic,
legislative and parasite control issues ................................................................... 189
7.1.7 Assessing the risk of anisakiasis and its incidence in population – public
health insights ....................................................................................................... 193
7.2 Future work .................................................................................................... 195
7.2.1 QRA for allergy to Anisakis spp. ............................................................ 195
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7.2.2 QRA for human/wildlife disease from Anisakis spp. in anadromous fish
196
7.2.3 Assessing the risk of anisakiasis in other fish preparations/species and
countries ................................................................................................................ 197
7.2.4 Categorizing the health risk posed by parasitized fishery products ........ 198
7.2.5 Extending the contingent valuation study to other countries, and cost-
benefit analysis for Anisakis-free fishery products ............................................... 199
7.3 Conclusions .................................................................................................... 199
7.4 References ...................................................................................................... 201
List of publications ...................................................................................................... 209
Overview of completed training activities ................................................................ 212
Abstract (Spanish) ...................................................................................................... 214
Abstract (Galician) ..................................................................................................... 218
Appendices ................................................................................................................... 222
Supplementary material of Chapter 4 ....................................................................... 222
Supplementary material of Chapter 5 ....................................................................... 224
Supplementary material of Chapter 6 ....................................................................... 237
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Abstract
Parasitic nematodes of the genus Anisakis have complex life cycles in the marine
environment, involving a wide variety of aquatic organisms from all major seas
worldwide. During their life cycle, marine mammals (in particular cetaceans) serve as
definitive or final hosts, small crustaceans serve as intermediate hosts, and fish and
cephalopods serve as intermediate or paratenic (transport) hosts. These parasites are
mainly known to be causative agents of an emerging food-borne zoonotic disease called
anisakiasis, as well as allergic reactions in sensitized individuals. Humans may become
infected and develop disease by consumption of raw or lightly cooked fishery products
parasitized with living Anisakis spp. larvae. Anisakis spp. can also cause economic losses
to the fishing industry and consumer distrust in fishery products. For all these reasons,
Anisakis spp. have attracted increasing interest and concern within academia, the fishing
and food industries, health professionals, food safety authorities and consumers.
The present thesis mainly focuses on providing new insights, understandings and methods
in relation to the epidemiology and life cycle ecology of Anisakis spp. in fish hosts (three
anadromous (sea lamprey, allis shad and twaite shad) and one marine (Atlantic herring)
fish species), and the socioeconomic and public health importance of their presence in
fishery products, using different approaches.
The epidemiology of Anisakis spp. was studied in anadromous fish captured in Western
Iberian rivers. The occurrence of A. simplex (s.s.) in sea lamprey, and mixed infections of
A. simplex (s.s.) and A. pegreffii in allis and twaite shads was reported for the first time.
Results highlighted the role of anadromous fish as transport hosts of Anisakis spp. from
marine to freshwater environment, and the zoonotic relevance of the findings due to the
gastroallergic threats posed by these parasites.
Infection levels and sites of infection of Anisakis spp. in the visceral cavity of Atlantic
herring from the North Sea were investigated, by shifting the focus towards marine fish.
This study was part of a large EU FP7 project (PARASITE project (GA no. 312068))
aiming to provide epidemiological data on zoonotic parasites (i.e. Anisakis spp.) in many
commercially important fish species from European waters. Visual inspection showed
that Anisakis spp. larvae tend to accumulate in the viscera at the posterior end of the
terminal blind sac of the stomach, around the ductus pneumaticus, in Atlantic herring and
in allis and twaite shads (family Clupeidae). This is probably caused by the “Y” shape of
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the stomach of these clupeids, which carries the food and Anisakis spp. larvae to the
terminal blind sac during digestion, from where they emerge into the viscera. In maturing
fish, gonads would act as physical barriers/traps for larvae, therefore, preventing their
migration to the muscle or other organs and favouring the formation of Anisakis spp.
accumulations in this region. Magnetic Resonance Imaging (MRI) was investigated as a
potential methodology for the detection of Anisakis spp. larvae in fish for the first time.
Results demonstrated the capacity of MRI to detect Anisakis spp. larvae in the viscera of
whole herring (previously identified by visual inspection as detailed above) in situ, in a
3D environment, and in a non-invasive and non-destructive way. Results also showed the
ability of MRI to detect Anisakis spp. and Pseudoterranova sp. larvae in fish muscle.
For the first time, a survey-based contingent valuation study was performed to elicit the
maximum willingness to pay (WTP) of Spanish fish consumers for a fish product “free”
of Anisakis spp. larvae. The survey also investigated consumer attitudes regarding the
presence of these parasites in fishery products, especially focused on those species
frequently consumed by respondents of the questionnaire. Results indicated that a
majority of consumers were willing to pay for the product, showing a modal WTP of 10%
extra over a typical fish price at market (or 6.60€/kg of fish, considering 6€/kg the
common fish price). Results revealed that over a quarter of consumers had avoided to
consume fish (mainly European hake), and that a majority of them would avoid fish
consumption in the future, due to the presence of Anisakis spp. Overall, the study provided
further evidence to conclude that the presence of Anisakis spp. in fishery products is an
important health and aesthetic issue for Spanish fish consumers.
Finally, a quantitative risk assessment approach was performed to assess the risk of
anisakiasis caused by consumption of non-previously frozen home-made raw and
marinated anchovy meals, and to then estimate the burden of disease in Spain. On this
occasion, the study was focussed on raw/marinated anchovies because they were
identified as the main vehicle for anisakiasis in the literature. In addition, the frequency
of consumption of untreated (non-previously frozen) raw and marinated home-made
anchovy meals was estimated using respondents’ answers from the questionnaire
mentioned above. A dose-response relationship for Anisakis spp. was developed for the
first time and the probability of anisakiasis was calculated to be 9.56×10-5 per meal. The
total annual number of anisakiasis cases requiring medical attention was estimated to be
between 7,700 and 8,320. Results suggest that anisakiasis is a highly underestimated and
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underdiagnosed zoonosis in Spain, probably due to misdiagnosis, not being diagnosed or
not reported. The study is of interest for fishing and food industries, health professionals,
academy and consumers, and can be used to inform policy (e.g. by food safety authorities)
and to reduce the incidence of disease as follows: firstly, by highlighting the need of
adequate cold storage of anchovies and early evisceration, in order to prevent post-
mortem migration of Anisakis spp. larvae from anchovy viscera to flesh which may
increase the risk of disease, as well as parasite monitoring to control the parasite along
the fish value chain. Secondly, by education campaigns to encourage freezing of fish
meant to be consumed as raw or lightly cooked, with a particular target on those
individuals who apparently know about prevention methods of anisakiasis but appeared
to refuse freezing fish prior to raw consumption.
In conclusion, this thesis contributes to a better understanding of the life cycle ecology
and epidemiology of Anisakis spp. in anadromous and marine fish hosts, attitudes of
consumers regarding parasitized fishery products and how much they would value such
product “free” of parasites, as well as to understand how particular habits of fish
consumption may result in zoonotic disease (i.e. anisakiasis), and the burden of disease
for the whole population. This thesis provides further evidence of the socioeconomic,
legislative and public health issues caused by the presence of Anisakis spp. in fishery
products and may be used to improve their quality and safety, and to reduce the incidence
of disease in the human population. Future studies are recommended to assess the
potential health risk posed by the presence of Anisakis spp. allergens in fishery products.
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Chapter 1 - General introduction and thesis outline
1.1 Anisakid nematodes and their significance as aetiological agents of a fish-borne
parasitic zoonosis (anisakidosis)
Some members of the family Anisakidae (phylum Nematoda, superfamily Ascaridoidea),
particularly species belonging to the genera Anisakis, Pseudoterranova and
Contracaecum, are parasitic nematode species of a wide range of aquatic organisms and
have indirect life cycles in the aquatic environment (mainly marine) (Mattiucci and
Nascetti, 2008). Adult anisakids parasitize the alimentary tract of marine mammals
(cetaceans (Anisakis spp.), pinnipeds (Pseudoterranova spp. and Contracaecum spp.) and
fish-eating birds (Contracaecum spp.)) which serve as definitive or final hosts (Mattiucci
and Nascetti, 2008). Fish and cephalopods serve as intermediate or paratenic (transport)
hosts, and small crustaceans serve as intermediate hosts of anisakid larvae (Mattiucci and
Nascetti, 2008). Humans may become accidental hosts through consumption of raw or
lightly cooked parasitized fish products (Audicana and Kennedy, 2008). These anisakids
(especially Anisakis spp.) are of considerable medical and economic concern worldwide
since they are responsible for 1) a fish-borne zoonosis known as anisakidosis (anisakiasis
when it refers only to Anisakis spp. as causative agent), and 2) economic losses to the
fishing industry due to negative effects on the marketability of fishery products (Audicana
and Kennedy, 2008; Buchmann and Mehrdana, 2016; Chai et al., 2005; D’amico et al.,
2014; EFSA-BIOHAZ, 2010; Llarena-Reino et al., 2015; Mattiucci et al., 2017, 2013b;
Mattiucci and D’Amelio, 2014; McClelland, 2002; Shamsi, 2016).
The following sections will focus on the genus Anisakis, the zoonotic parasite central to
the work presented in this thesis.
1.2 The genus Anisakis
To date, nine species of Anisakis have been confirmed using genetic and/or biochemical
methods, i.e. A. simplex sensu stricto, A. pegreffii, A. berlandi, A. ziphidarum, A. nascettii,
A. paggiae, A. physeteris, A. brevispiculata and A. typica (Mattiucci et al., 2017, 2014;
Mattiucci and D’Amelio, 2014). Development of genetic tools during the last thirty years
has allowed the identification of the nine Anisakis species (larval and adult stages) using
multilocus allozyme electrophoresis (MAE), and DNA based methods, such as
polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of
18
rDNA and DNA sequence analysis of nuclear (entire internal transcribed spacer (ITS-1,
5.8S, ITS-2) region of the rDNA and partial sequence of the elongation factor 1 alpha1
nuclear DNA gene (EF1 α-1 nDNA)) and two mitochondrial genes (mtDNA cox2 and
rrnS) (Cavallero et al., 2011; D’Amelio et al., 2000; Mattiucci et al., 2017, 2016, 2014,
2009, 2005, 2002, 2001, 1997; Mattiucci and D’Amelio, 2014; Mattiucci and Nascetti,
2008, 2006; Nascetti et al., 1986; Paggi et al., 1998; Valentini et al., 2006; Zhu et al.,
2000).
Morphological identification of Anisakis spp. to the species level is difficult and
sometimes impossible. The morphological characters of diagnostic value are very few
(i.e. features of the excretory system, alimentary canal, position of the vulva and length
of the spicules, and allometric characters such as tail length/total body length ratio, etc.),
are applicable to adult specimens only, and are often only relevant to males (Mattiucci et
al., 2014; Mattiucci and D’Amelio, 2014; Mattiucci and Nascetti, 2008). Indeed, the
situation is even more complicated for larval stages parasitizing fish (those infective for
humans), where morphological identification can only be achieved at the genus level, and
this is mainly based on the morphology and length of the ventriculus, and the
presence/absence of a caudal spine (mucron) (Berland, 1961; Mattiucci and D’Amelio,
2014; Mattiucci and Nascetti, 2008). According to Berland (1961), morphological
features of larvae divide the genus into two main morphotypes, i.e. Anisakis type I and
Anisakis type II.
Phylogenetic inference based on combined nuclear and mitochondrial DNA sequence
datasets of Anisakis specimens belonging to all species described so far supported the
existence of four distinct clades: the first clade comprises the A. simplex complex or A.
simplex sensu lato (A. simplex s.s., A. pegreffii and A. berlandi) and a second clade is
formed by A. ziphidarum and A. nascettii; the third clade comprises A. physeteris, A.
brevispiculata, and A. paggiae; and finally A. typica forms a separate phylogenetic
lineage, thus comprising the forth clade within the group (Cavallero et al., 2011; Mattiucci
et al., 2017, 2014; Mattiucci and D’Amelio, 2014). In addition, it appears that using
morphological and molecular methods those Anisakis species belonging to the first and
second clades have Type I larvae (sensu Berland, 1961) and species of the third clade
have Type II larvae (sensu Berland, 1961) (Mattiucci et al., 2017, 2014, 2005; Mattiucci
and D’Amelio, 2014). Thus, accurate identification of the larval stages of Anisakis spp.
is very important to understand their epidemiological significance, since they are the
19
causative agents of human anisakiasis, but this can only be achieved by genetic/molecular
methods (Mattiucci et al., 2017, 2005; Mattiucci and D’Amelio, 2014; Mattiucci and
Nascetti, 2008).
1.3 Anisakis spp. life cycle ecology
Anisakis spp. have a heteroxenous life cycle comprising five stages and four moults
(Højgaard, 1998; Klimpel et al., 2004; Klimpel and Palm, 2011; Ugland et al., 2004).
Cetaceans are the final or definitive hosts for Anisakis spp., and Anisakis spp. adults can
be often found free in the lumen of the stomach or sometimes forming clusters of
roundworms embedded in the mucosa and submucosa (Mattiucci and D’Amelio, 2014;
Young, 1972). Anisakis spp. adults reproduce and eggs are released into the marine
environment with the host’s faeces (Young, 1972). Apparently, the hatching of eggs
occurs in the water, and it is the third larval stage (L3) which emerges from the egg, after
two moults. The L3 larva is still surrounded by the cuticle of the second stage larva, which
appears to increase its buoyancy in the pelagic environment (Højgaard, 1999; Køie et al.,
1995). The free-swimming ensheathed larva may be ingested by small crustaceans
(mainly euphausiaceans and copepods), which become the intermediate hosts (Gregori et
al., 2015; Klimpel et al., 2004; Køie, 2001; Sluiters, 1974; Smith, 1983). Infected
crustaceans can then be eaten by the final host (e.g. baleen whales), where the infective
L3 grows and moults to reach the fourth larval stage and then reaches adulthood, thereby
closing the life cycle (Klimpel and Palm, 2011; Smith, 1983).
In the marine food chain, infected crustaceans may be eaten by small fish and cephalopods
which become second intermediate or paratenic hosts, and these may be predated upon
and so transfer the larvae to larger piscivorous fish which also serve as paratenic hosts
(Klimpel et al., 2004; Smith, 1983). The ability of the larvae to reinfect fish through the
food chain together with their long life expectancy in fish hosts (they may be able to
survive for at least two years in Atlantic cod (Hemmingsen et al., 1993) or three years in
Atlantic herring (Smith, 1984a)) may enable accumulations of hundreds to thousands of
Anisakis spp. larvae in large and old predatory fish feeding extensively on infected
intermediate and paratenic crustacean/fish hosts (Berland, 2006; EFSA-BIOHAZ, 2010;
Hemmingsen et al., 2000; Klimpel et al., 2004; Klimpel and Palm, 2011; Mladineo and
Poljak, 2014). Consequently, a positive relationship between fish size or age and overall
Anisakis spp. infection levels (i.e. prevalence and abundance) may be expected, and has
been reported in several fish species, including Atlantic herring, Atlantic cod, European
20
hake, European anchovy, blue whiting, Atlantic mackerel and grey gurnard (Hemmingsen
et al., 2000; Levsen and Karl, 2014; Levsen and Lunestad, 2010; Madrid et al., 2016,
2012; Mladineo and Poljak, 2014). However, there are some exceptions to this pattern,
which is not completely understood and not always observed (e.g. beaked redfish, blue
whiting), probably due to different feeding ecology, differences in migratory routes or
increased immunity against larvae in larger host fish (Berland, 2006; Gómez-Mateos et
al., 2016; Klapper et al., 2015; Priebe et al., 1991).
Finally, large numbers of fish/cephalopod species have been identified as
intermediate/paratenic hosts for Anisakis spp. (Mattiucci and D’Amelio, 2014). Indeed,
the European Food Safety Authority (EFSA) Panel on Biological Hazards (BIOHAZ)
delivered a scientific opinion on food safety related to parasites in fishery products, and
concluded that “For wild-catch fish, no sea fishing grounds can be considered free of A.
simplex” (EFSA-BIOHAZ, 2010). The use of fish/cephalopod intermediate/paratenic
hosts greatly facilitates the dispersion of Anisakis spp. larvae in the ocean, and their
successful transmission to the final host (e.g. toothed whales), so completing the life cycle
(Abollo et al., 2001; Klimpel et al., 2004; Klimpel and Palm, 2011; Smith, 1983). In the
life cycle of Anisakis spp. humans may accidentally become infected (i.e. become
accidental hosts) by ingestion of parasitized raw or lightly cooked fishery products, but
the parasite cannot reach adulthood and eventually dies (Audicana and Kennedy, 2008;
EFSA-BIOHAZ, 2010; Mattiucci and D’Amelio, 2014).
1.4 Can Anisakis spp. pose a risk in freshwater environments?
In 2010, the EFSA-BIOHAZ concluded with the scientific opinion that “All wild caught
seawater and freshwater fish must be considered at risk of containing any viable parasites
of human health concern if these products are to be eaten raw or almost raw” (EFSA-
BIOHAZ, 2010). However, after a careful study of the document, it may be said that
EFSA had not specifically identified Anisakis spp. as a health hazard when fishery
products originate from freshwater ecosystems. It seems that EFSA was actually referring
to other zoonotic parasites, such as the trematodes Clonorchis spp. and Opisthorchis spp,
as causative agents of zoonoses from freshwater origin. Likewise, the Spanish Agency
for Consumer Affairs, Food Safety and Nutrition (AECOSAN) stated that fish from
continental waters (e.g. rivers, lakes, ponds) do not cause anisakiasis, even if they are
consumed raw (AECOSAN, n.d.). However, anadromous fish (e.g. Atlantic salmon
(Salmo salar)) can acquire Anisakis spp. during the marine phase of their life cycle, and
21
transport them to freshwater ecosystems during their upstream reproductive migration
(Noguera et al., 2009). Thus, in the present thesis, the epidemiology of Anisakis spp. in
anadromous fish captured in Western Iberian Peninsula rivers was investigated to assess
if this parasite may pose a health risk for humans and wildlife who consume parasitized
anadromous fish (chapters 2 and 3).
1.5 Anisakis spp. in fish hosts
Fish hosts may become infected through ingestion of Anisakis spp. larvae from infected
crustacean intermediate hosts or from smaller infected fish. The Anisakis spp. larvae will
then emerge from the fish stomach and pass to the visceral body cavity, where it can be
found free in the cavity and/or, frequently encapsulated (coiled in a spiral in a capsule of
host origin) on the surface of several organs and tissues (e.g. liver, pyloric caeca, gonads,
etc.) (Figure 1.1) or embedded in the fish musculature (Figure 1.2) (Abollo et al., 2001;
Karl, 2008; Smith and Wootten, 1978). However, the site of Anisakis spp. larvae
encapsulation and the relative frequency of occurrence of the Anisakis spp. larvae in each
organ are highly variable between fish individuals and species (Smith and Wootten,
1978). For instance, Anisakis spp. tends to encapsulate in the liver and mesenteries of
Atlantic cod (Gadus morhua) in higher numbers than in muscle (Smith and Wootten,
1978; Strømnes and Andersen, 2003; Young, 1972), whereas the larvae are more frequent
in the muscle of Alaska salmon (Oncorhynchus spp.) than in viscera (Karl et al., 2011).
Figure 1.1. Anisakis spp. in situ in the body cavity of allis shad (Alosa alosa).
22
Figure 1.2. Visual evidence of the presence of Anisakis spp. larvae in allis shad flesh.
In addition, post-mortem migration of Anisakis spp. from fish viscera to muscle has been
reported to occur in a number of species (Cipriani et al., 2016; Karl, 2008; Karl et al.,
2002; Smith, 1984b). However, the underlying processes determining the anatomical
location of Anisakis spp. within fish hosts and in vivo and post-mortem migration
behaviour are still largely unknown (EFSA-BIOHAZ, 2010; Strømnes and Andersen,
1998). The anatomical location of Anisakis spp. in Atlantic herring (Clupea harengus)
has been investigated in the present thesis (chapter 4).
1.6 Detection methods of Anisakis spp. larvae in fish
The visceral cavity and muscle of fish can be inspected for the presence of Anisakis spp.
larvae by a number of methodologies that can be used individually or in combination,
such as visual inspection, slicing, candling, pressing, artificial enzymatic digestion and
UV illumination (EFSA-BIOHAZ, 2010). The numbers and anatomical location of the
parasites can be recorded, despite the methods differing in their speed and accuracy. In
fish processing plants, the fast non-destructive candling method (i.e. brief visual
inspection of fish fillets on a light table) is commonly applied to detect and remove visible
nematodes from fish fillets. However, the method is not suitable for accurate estimation
of the total number of Anisakis spp. larvae present in the muscle (EFSA-BIOHAZ, 2010;
Karl and Leinemann, 1993; Levsen et al., 2005). For scientific research purposes, time-
consuming and expensive invasive artificial enzymatic digestion (Figure 1.3), and
combined pressing and UV illumination methods are frequently applied for quantitative
determination of Anisakis spp. larvae in fish viscera and muscle, achieving high levels of
accuracy (Karl and Leinemann, 1993; Levsen et al., 2005; Llarena-Reino et al., 2013b).
23
Figure 1.3. A. Artificial enzymatic digestion of fish muscle in a magnetic stirrer
multiplate. B. Anisakis spp. larva sieved from digestion contents.
The procedures applied by the industry cannot therefore completely guarantee the
absence of Anisakis spp. larvae in fishery products reaching the market (Levsen and
Lunestad, 2010; Llarena-Reino et al., 2012). The development of new technologies for
nematode detection in fish and fishery products is therefore of great interest for the fishing
industry, consumers and academia. The potential use of Magnetic Resonance Imaging as
a non-invasive and non-destructive parasite detection tool in whole fish specimens and
fish muscle samples is studied and the results presented and discussed (chapter 4).
1.7 Consumer perception of risk due to the presence of Anisakis spp. in fish
The occurrence of Anisakis spp. larvae and its allergens in fish poses a risk for human
health and also affects the aesthetic quality of the fishery product. The visual impact of
the parasite in the viscera and muscle of fish can cause distrust and commercial rejection
of fishery products by consumers, resulting in negative effects on marketing and
economic losses to the fishing industry (Abollo et al., 2001; D’amico et al., 2014; Karl,
2008; Levsen et al., 2005; Llarena-Reino et al., 2015, 2013a). The coverage of the
“Anisakis problem” by the media may also generate social alarm, thus, causing more
distrust in consumers and monetary losses to the industry (Abollo et al., 2001; Karl, 2008;
Mattiucci and D’Amelio, 2014). Moreover, inspection procedures to control and remove
visible parasites carried out by the industry introduce additional costs to the commercial
processing (Abollo et al., 2001; Llarena-Reino et al., 2015). Despite previous evidence,
little is known about the attitudes of fish consumers in relation to the presence of Anisakis
24
spp. in fish, and how much they would value the removal of Anisakis spp. from fishery
products. In the present thesis, the willingness to pay for Anisakis-free fish of Spanish
fish consumers and their behaviour regarding the presence of Anisakis spp. in fish have
been investigated, and results are presented and discussed (chapter 5).
1.8 Anisakiasis and its incidence in the human population
Human anisakiasis is a fish-borne zoonotic disease caused exclusively by members of the
genus Anisakis (Audicana and Kennedy, 2008). To date, the only Anisakis species
genetically identified as aetiological agents of human anisakiasis belong to A. simplex s.s.
and A. pegreffii (Mattiucci and D’Amelio, 2014). The transmission of Anisakis spp. to
humans is typically associated with the consumption of parasitized raw or undercooked
fish meals, such as traditional anchovies in vinegar consumed in Spain (Figure 1.4)
(Audicana and Kennedy, 2008). Anisakis spp. infection can cause mild to severe
gastrointestinal symptoms (e.g. epigastric pain, vomiting, diarrhoea, etc.), which may be
associated with hypersensitivity symptoms (e.g. urticaria, angioedema and anaphylaxis)
(Audicana and Kennedy, 2008; Daschner et al., 2000). Anisakis spp. is therefore the
causative agent of human anisakiasis, of which there are gastric, intestinal, ectopic and
allergic forms (Audicana and Kennedy, 2008). In addition, allergy to Anisakis spp. can
also occur in sensitized individuals by exposure to Anisakis spp. allergens from
contaminated fish products (Audicana and Kennedy, 2008; EFSA-BIOHAZ, 2010).
Figure 1.4. Home-prepared Spanish “boquerones en vinagre” (anchovies in vinegar)
ready to eat.
25
Since anisakiasis was first described in The Netherlands in the nineteen-sixties by Van
Thiel, the number of cases reported worldwide has increased, with Japan numbering
approximately 2,000 cases diagnosed annually (EFSA-BIOHAZ, 2010). In Europe, the
majority of the cases have been described in Spain and Italy (Alonso-Gómez et al., 2004;
Del Rey Moreno et al., 2013; EFSA-BIOHAZ, 2010; Mattiucci et al., 2013a; Pampiglione
et al., 2002). However, the actual burden of disease (i.e. total number of annual anisakiasis
cases, incidence of anisakiasis) in European countries is largely unknown. A quantitative
risk assessment has been performed to assess the risk of anisakiasis caused by raw and
marinated anchovy meals in Spain and estimate the number of Spanish cases (chapter 6).
1.9 Objectives and thesis outline
The work presented in this multidisciplinary thesis has focused on providing new insights
and methodologies and has the following objectives/aims:
1) To investigate the epidemiology of Anisakis spp. in anadromous fish species captured
in rivers of Western Iberian Peninsula.
2) To investigate the anatomical location of Anisakis spp. in Atlantic herring, and to
investigate whether Magnetic Resonance Imaging can be used as a new technology to
detect Anisakis spp. in fish samples.
3) To determine the monetary value of a fish product “free” of Anisakis spp. for Spanish
fish consumers.
4) To assess the risk for humans posed by the presence of Anisakis spp. in fish and the
burden of anisakiasis in the whole Spanish population.
The present thesis is structured into seven chapters. The five main chapters (2 to 6) are
all based on manuscripts (published or under review) and, in consequence, there is a
certain amount of repetition of background material across these chapters.
Chapter 1 provides a general introduction to the current knowledge about the family
Anisakidae and especially about the genus Anisakis and its life cycle. It provides an
explanation of the importance of investigating the presence of the parasite in anadromous
fish captured in rivers. It provides an overview of the current knowledge about the
anatomical location of the parasite within fish hosts, and about the parasite detection
methods currently used, and why it is important to investigate these aspects. Finally, it
explains the significance of Anisakis spp. from economic and social, and health points of
26
view. Chapters 2 and 3 investigate the epidemiology of Anisakis spp. in sea lamprey
(Petromyzon marinus) and in shads (A. alosa and A. fallax) captured in rivers of Western
Iberian Peninsula to assess the zoonotic and ecological implications of the findings.
Chapter 4 focuses on the use of visual inspection of Anisakis spp. in Atlantic herring to
investigate their anatomical location in this fish host, and to determine if Magnetic
Resonance Imaging can be used as a new technology to investigate and detect Anisakis
spp. in fish samples. Chapter 5 uses the survey-based contingent valuation method to
determine the willingness to pay of Spanish fish consumers for a fish product “free” of
Anisakis spp., and to investigate the knowledge and behaviour of consumers regarding
the presence of the parasite in fish and its associated diseases (i.e. anisakiasis). Chapter
6 describes the results of a quantitative risk assessment study for the risk of human
anisakiasis caused by ingestion of parasitized raw and marinated anchovies in Spain.
Finally, chapter 7 includes the final discussion, future perspectives and concluding
remarks based on the results produced in this thesis.
27
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30
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100, 199–214. doi:10.1645/12-120.1
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Testini, M., Villan, M., 2002. Human anisakiasis in Italy: a report of eleven new
cases. Pathol. Res. Pract. 198, 429–434.
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4
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Parasitology 16, 93–163. doi:10.1016/S0065-308X(08)60573-4
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Ascaridoidea, Anisakidae) third-stage larvae in paratenic fish hosts. Parasitol. Res.
89, 335–341. doi:10.1007/s00436-002-0756-7
Strømnes, E., Andersen, K., 1998. Distribution of whaleworm (Anisakis simplex,
Nematoda, Ascaridoidea) L3 larvae in three species of marine fish; saithe
(Pollachius virens (L.)), cod (Gadus morhua L.) and redfish (Sebastes marinus (L.))
from Norwegian waters. Parasitol. Res. 84, 281–285. doi:10.1007/s004360050396
Ugland, K.I., Strømnes, E., Berland, B., Aspholm, P.E., 2004. Growth, fecundity and sex
ratio of adult whaleworm ( Anisakis simplex ; Nematoda, Ascaridoidea, Anisakidae)
in three whale species from the North-East Atlantic. Parasitol. Res. 92, 484–489.
doi:10.1007/s00436-003-1065-5
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Colom-Llavina, M.M., Nascetti, G., 2006. Genetic relationships among Anisakis
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among some ascaridoid nematodes based on ribosomal DNA sequence data.
Parasitol. Res. 86, 738–744. doi:10.1007/PL00008561
33
Chapter 2 - First report of Anisakis simplex (Nematoda, Anisakidae) in
the sea lamprey (Petromyzon marinus)
2.1 Abstract
A total of 64 sea lampreys Petromyzon marinus caught in Galician rivers in 2012 (NW
Spain), was dissected and examined to evaluate the presence of marine parasitic
Anisakidae nematodes following microscopic and PCR analysis. The results revealed that
sea lampreys are infected by third-larval stage of Anisakis simplex. Internal organs,
especially the gonads, and flesh, hosted from 1 to 10 Anisakis larvae with prevalence
values reaching up to 60%. This is the first time that this infection has been described in
the sea lamprey. A plausible hypothesis is that sea lamprey might have acquired larvae
once feeding on flesh or the products of tissue cytolysis from infected teleosts (e.g.
Atlantic salmon). In Galician (NW Spain) waters, the sea lamprey could act as a transport
host for A. simplex from marine to freshwater ecosystems. This finding has
epidemiological relevance considering the zoonotic and gastro-allergic condition of this
parasite.
Keywords: Petromyzon marinus, Sea lamprey, Anisakis simplex, Galicia (NW Spain).
34
2.2 Introduction
The sea lamprey (Petromyzon marinus L. 1758) is an aquatic, jawless vertebrate
belonging to the Order Petromyzontiformes (Renaud, 2011). Although sea lampreys are
anadromous, migrating to the sea during their parasitic phase, some populations are
permanent freshwater residents in North American Great Lakes (Renaud, 2011; Wilkie et
al., 2004). Their life cycle includes microphagic filter-feeding larvae (ammocoete) in a
freshwater habitat. After four to six years, the ammocoetes suffer a radical metamorphosis
into post-metamorphic juvenile, which migrate downstream to the sea (Cobo et al., 2010;
Lança et al., 2011; Renaud, 2011). In Galician waters, the downstream migration of
juveniles occurs in the river from October to May. Although most sea lampreys begin the
parasitic stage of their life cycle in the estuary, a significant number of individuals have
already fed in freshwater during their migration (Cobo and Silva, 2012; Hardisty and
Potter, 1971). The main hosts for post-metamorphic juveniles identified in Galician rivers
correspond to the anadromous fishes Atlantic salmon (Salmo salar), Twaite shad (Alosa
fallax), Sea trout (Salmo trutta), the brackish fish Golden grey mullet (Liza aurata) and
the freshwater fish Brown trout (S. trutta) (Cobo & Silva 2012; pers. com.). After that,
adults begin their marine trophic phase when they parasitize several species of marine
mammals, sharks and teleosts (Farmer, 1980; Nichols and Tscherter, 2011; Wilkie et al.,
2004). They feed by sucking mainly blood, and much lesser extent of other body fluids
and products of tissue cytolysis (Farmer, 1980; Renaud et al., 2009), from such hosts,
attaching to them by the toothed circular sucking mouth (Igoe et al., 2004). It is
considered that this marine trophic phase lasts one to two years (Cobo and Silva, 2012;
Renaud, 2011). However, this marine life history is not well known, especially the
dynamics of lamprey/host relationships (Farmer, 1980; Halliday, 1991; Hardisty and
Potter, 1971; Lança et al., 2011; Wilkie et al., 2004). Even though homing behaviour has
not been demonstrated (Renaud, 2011), anadromous adult sea lampreys return to the river
attracted by pheromones (bile acids) released into the water by ammocoetes (Mateus et
al., 2012; Renaud, 2011). The upstream migration begins in the river between December
to June (Cobo and Silva, 2012) when they stop feeding, reproduce and die (Beamish,
1980; Hardisty and Potter, 1971).
Anisakis spp. (Rudolphi, 1809) is a worldwide marine parasitic nematode that completes
its cycle in cetaceans and sometimes pinnipeds, which act as final hosts (Abollo et al.,
1998; Klimpel et al., 2004; Smith and Wootten, 1978; Young, 1972). Cetaceans acquire
35
the infective larvae (stage L3) by feeding on marine zooplankton or on teleosts or
cephalopods that act as intermediate or paratenic hosts (Klimpel et al., 2004; Marcogliese,
1995). Humans could become an accidental host if they consume raw, marinated or
undercooked fish that contains at least one viable L3 larva, and which may then develop
into a severe pathology (anisakiasis and/or allergy) (EFSA, 2010). In general, anisakid
larvae may be responsible for four forms of illness in consumers: gastric, intestinal, and
ectopic anisakidosis and allergic forms. Additionally Anisakis simplex is now known to
be associated with occupational seafood allergy (Audicana and Kennedy, 2008). Human
anisakiasis has become a public health problem, whose incidence is increasing in Spain
in recent years. This increase could be due to a higher infection of captured fishes,
improvements in the diagnosis of disease and the incorporation of foreign dining habits
(Japanese sushi and sashimi, South American ceviche, etc.) into Spanish culture, and
other typical national dishes (pickled anchovies, etc.) (Ministerio de Sanidad y Consumo,
2006). In recent years some regulations and scientific opinions have been developed for
improving detection and prevention of this parasite and associated illnesses. Freezing or
heat treatments remain the most effective processes guaranteeing the killing of parasitic
larvae, under well-defined conditions. Nevertheless, because A. simplex allergens are
highly resistant to heat and freezing to date there is no preventive treatment to protect the
consumer against allergic hazards (EFSA, 2010).
It is notable that sea lamprey supports a seasonal and artisanal freshwater fishery of great
importance to local economies in Galicia (NW Spain) and Portugal where it is considered
a delicacy (Andrade et al., 2007; Cobo et al., 2010; Mateus et al., 2012). Specifically, sea
lamprey fisheries in Galician waters are only allowed in Ulla river, Tea (tributary of
Minho river) and in the International Section of the Minho river (TIM) (BOP, 2012; DOG,
2012). In the lower part of TIM hundreds of Spanish boats use gillnets (“lampreeiras”) to
catch between 22,000 and 34,000 sea lampreys per year (Comandancia Naval de Miño,
pers. com.). Moreover in the Ulla river approximately 15 boats use sea lamprey traps
(“butrones”) and declare from 1,000 to 5,000 catches per year (Carril Fishermen
Association, pers. com.). The socioeconomic importance of sea lampreys is well reflected
by their high commercial value, usually about 20-50 € per animal (Mateus et al. 2012,
pers. com.).
36
Bearing in mind that A. simplex is considered an emergent biological hazard in fishery
products the epidemiological information of this hazard in the sea lamprey and its role as
transport host in the marine-freshwater mainstream are herein analysed.
2.3 Materials and methods
2.3.1 Sampling
Sampling was carried out in two batches during 2012: the first batch corresponded to 50
sea lampreys caught by fishermen during their upstream spawning migration (between
February to April) in the Ulla river (Galicia, NW Spain). Sea lampreys were transported
alive to the laboratory, a journey lasting from 1 h to 1.5 h. Some specimens were
necropsied immediately while others were kept in tanks with freshwater and abundant
aeration. The second batch comprised 14 post-spawning sea lampreys caught in June from
the Ulla and Tea rivers. Due to the poor condition of post-spawning specimens they were
frozen to further analysis.
2.3.2 Necropsy
Data on total weight, total length (from the distal part of the oral disc to the end of the
caudal fin) and sex were recorded for each specimen. Following that, a longitudinal
section was performed from the anus to the last pair of branchiopores to expose the
internal organs. Macroscopic observation for anisakid larvae was made without removing
the internal organs from the visceral cavity. Then, each internal organ was re-inspected
under stereomicroscope for Anisakis spp. larvae. Finally, the head and branchial region
of the lamprey were separated from the rest of the body, which was frozen for artificial
muscle digestion to be carried out later. The branchial region was dissected and the gill
pouches were extracted and observed. The large gonads, both male and female, were
visually inspected under magnifying glass and transilluminator and further subjected to
an artificial peptic digestion. All Anisakis spp. found were removed and placed in
individual eppendorfs with ethanol 70% for molecular diagnosis.
2.3.3 Artificial peptic digestion
The artificial digestion of sea lampreys (both flesh and gonads) was carried out on the
basis of an optimized artificial digestion protocol (Llarena-Reino et al., 2013). The flesh
was digested at 37-40 °C for 5-6 h (30 min for gonads) in an ACM-11806 Magnetic Stirrer
Multiplate, using a weight/volume pepsin ratio of 1:20, understanding that ratio as 1 g of
flesh for 20 ml of a 0.5% pepsin solution in HCl 0.063 M (pH 1.5). The digestion solution
37
was decanted through a sieve and the remains of digestion and nematodes were inspected
under stereomicroscope. All Anisakis spp. found were placed in individual Eppendorf
with ethanol 70% for further molecular diagnosis.
2.3.4 Molecular analysis
Genomic DNA from each Anisakis spp. was isolated using MACHEREY-NAGEL
NucleoSpin® Tissue kit following manufacturer-recommended protocols. The entire ITS
(ITS1, 5.8S rDNA gene and ITS2) was amplified using the forward primer NC5 (5’-GTA
GGT GAA CCT GCG GAA GGA TCA TT-3´) and the reverse primer NC2 (5´-TTA
GTT TCT TTT CCT CCG CT-3´). PCR reactions were carried out in a total volume of
25 µl containing 100 ng of genomic DNA, 10 µM of each primer, 2.5 µl of 10x buffer,
0.5 µl of dNTPs and 5 U/µl of Taq DNA polymerase (From Thermus Aquaticus BM,
recombinant, Roche). PCR cycling parameters included denaturation at 94 ºC for 2 min,
followed by 35 cycles of 94 ºC for 30 s, annealing at 55º C for 30 s, and extension at 72
ºC for 1min. 15s, and a final extension at 72 ºC for 7 min. PCR products were purified
using ExoSAP-IT© following manufacturer recommended protocols, with some
modifications. We added 4 µl of reactive ExoSAP-IT© and incubated the mix for 15 min.
at 37 ºC. For inactivating the reagents added we incubated the mix 20 min at 80 °C.
Samples with DNA concentration in clean reaction of 20 ng/µl were sequenced by
SECUGEN® (Madrid). All sequences were subjected to a homology search through Basic
Local Alignment Search Tool (BLAST) searches in the National Center for
Biotechnology Information (NCBI) database.
2.3.5 Quantitative descriptors
The quantitative descriptors of parasite populations found in sea lampreys, such as
prevalence, mean abundance and mean intensity were calculated as described in Bush et
al. 1997. Statistical inference of relationships between parasite counts and host data was
achieved by Statistica v6.0.
2.4 Results
The biometric parameters of the sea lampreys are shown in Table 2.1. In fresh animals
from the first sampling batch, the Anisakis spp. larvae found inside the visceral cavity of
sea lampreys were alive. In fact, some moved spontaneously, while others reacted to the
manipulation with forceps. All larvae were found stretched or rolled on the internal
organs, almost exclusively in the gonads, or free in the visceral cavity (Fig. 2.1.A). In the
38
gut no larva was observed. Some larvae were found in the kidney and a single larva
appeared anchored on a gill pouch and another one in the liver of a sea lamprey (Fig.
2.2.A). Further observations under the stereomicroscope showed nematodes
morphologically similar to Anisakis spp. According to the ITS amplified regions of 750
bp and their sequence homology searches (Blast values of 100%) the nematode species
infecting the viscera, gonad and flesh of sea lampreys analyzed belong to A. simplex.
Nematode sequences deposited in the Gen Bank (accession No. KC663441-KC663498)
correspond to larvae collected from both batches.
Table 2.1. Total length and standard deviation (TL ± SD, cm), total weight and standard
deviation (TW ± SD, g) of 50 sea lampreys caught from batch 1 in Ulla river at
Pontecesures (NW Spain) (42º42´56´´N; 8º40´03´´W ED50), and 14 post-spawning sea
lampreys in batch 2 from the Tea river (42º11´12´´N; 8º30´40´´W) and Ulla river at
Ximonde (42º44´45´´N; 8º27´51´´W). Total Prevalence (P); prevalence on visceral cavity
(Pv); prevalence on flesh (Pf); mean abundance, intensity and standard deviation in
visceral cavity (mAv ± SD, mIv ± SD); total abundance range (A) and abundance range
on flesh (Af) of A. simplex larvae.
Batch 1
TL ± SD TW ± SD P Pv Pf mAv ±
SD
range
mIv ±
SD
range
A Af
74.59 ±
4.18
751.00 ±
109.23
60%
(30/50)
44%
(22/50)
38%
(19/50)
1.06 ±
1.64
[0-6]
2.41 ±
1.71
[1-6]
[0-9] [0-5]
Batch 2
TL ± SD TW ± SD P Pv Pf mAv ±
SD
range
mIv ±
SD
range
A
Af
67.04 ±
5.69
774.07 ±
223.59
43%
(6/14)
29%
(4/14)
36%
(5/14)
0.29 ±
0.47
[0-1]
1 ± 0
[1]
[0-10]
[0-9]
In defrosted post-spawning animals from the second sampling batch, the internal organs
were generally in poor condition (Fig. 2.1.B). The female gonads were degenerated with
few or no eggs, while the male gonads were degenerated too. A total of 4 A. simplex
larvae were found in the rests of the peptic digestion of the internal organs, while a total
of 17 A. simplex larvae were found from the digested flesh (Fig. 2.2.B).
39
Figure 2.1.A: appearance of a larval Anisakis sp. on a female gonad of sea lamprey; B:
degenerated female gonad of a post-spawning sea lamprey.
Figure 2.2. Batch 1 A: percentage and location of the A. simplex larvae found in the sea
lampreys; batch 2. B: percentage and location of the A. simplex larvae found in the post-
spawning sea lampreys.
40
The quantitative descriptors of A. simplex populations found in sea lampreys are shown
in Table 2.1. In the first sampling batch, a total of 8 sea lampreys were infected on muscle
but no infection was recorded in internal organs, whereas the internal organs of 11 sea
lampreys were parasitized with no muscle infection. Furthermore, 11 sea lampreys had
A. simplex larvae both on internal organs and in muscle. In post-spawning sea lampreys
of the second sampling batch, the infection was higher in flesh than in the visceral cavity,
unlike the first sampling batch. The number of Anisakis larvae was statistically
independent of the batch and sex of sea lampreys (Table 2.2). Moreover, the relationships
between sea lamprey size/weight vs. total parasite numbers (Fig. 2.3) were not
statistically significant (Table 2.2), although the number of A. simplex larvae in the flesh
of sea lamprey was closely related to the number of parasites found in the viscera (F=
10.1260; R2= 0.1265; p= 0.002).
Table 2.2. Statistical significance (p-values) of the relationships between parasite counts
and host data. Data for batch and sex correspond to Mann-Whitney U-tests. Data for TL
and TW correspond to ANOVA analysis of log-transformed data.
Batch Sex TL TW
Total No. 0.3122 0.2218 0.6427 0.4940
Viscera 0.1616 0.3680 0.1122 0.4994
Flesh 0.9400 0.6517 0.4217 0.5913
41
Batch 1
42
Batch 2
Figure 2.3. Relationships between size/weight vs. total parasite numbers in sea lampreys from batches 1 and 2.
43
2.5 Discussion
The high prevalence and remarkable mean abundance observed in this study show that A.
simplex has the status of component parasite of sea lamprey in freshwater ecosystems
from Galician rivers (Bush et al., 1990). The infection in the flesh is quite high, counting
up to 9 larvae in a single sea lamprey. Migration of the larvae from the visceral cavity to
the flesh is coincident with the finding of a statistically significant relation between the
number of A. simplex on the internal organs and those in the flesh.
As some studies have demonstrated, sea lamprey feeds mainly on blood (Farmer, 1980;
Renaud et al., 2009). However it is unlikely that A. simplex larvae could be transferred to
lampreys if their diet is restricted to blood and tissue fluids withdrawn from superficial
wounds on their hosts. Ingestion of parasites already present in host fish appears to be the
only route for infection of lampreys if direct feeding on invertebrates is discounted.
Accordingly with Lethbridge et al. (1983), there is scientific evidence that in some
circumstances lampreys actively consume the flesh of their host. In fact, there are
previous records of Anisakis spp. infection in river lamprey Lampetra fluviatilis (Sobecka
et al., 2009), and in Arctic lamprey Lethenteron camtschaticum (Appy and Anderson,
1981), which also belongs to Petromyzontidae family. However, in these cases, the river
and Arctic lampreys feed mainly on flesh (Renaud et al., 2009). On the other hand, sea
lamprey also parasitizes a wide variety of teleost fish species infected with anisakids,
especially the Atlantic salmon S. salar, when they congregate in inshore waters prior to
their upstream migration (Hardisty and Potter, 1971; Kelly and King, 2001). In addition,
sea lamprey selects the largest hosts most frequently and it shows specificity for site of
attachment; scars are most frequent in the lower half of the middle third of the body
(Beamish, 1980; Farmer, 1980; Hardisty and Potter, 1971). The Red Vent Syndrome in
Atlantic salmon is associated with A. simplex (Noguera et al., 2009) and it is usual that
sea lamprey parasitizes Atlantic salmon, particularly on the ventral regions (Fig. 2.4A)
(Beamish 1980; pers. com.).
The life cycle of A. simplex in the marine ecosystem of Galicia is well established
(Pascual and Abollo, 2005). Based on the above mentioned citation we herein propose a
modification in the life cycle of A. simplex in Galician waters which includes Agnatha as
a potential paratenic host (Fig. 2.5). A plausible hypothesis for the role of P. marinus in
the parasite life cycle is that sea lamprey might have acquired larvae when it was
parasitizing and feeding on flesh and the products of tissue cytolysis from infected teleosts
44
(for potential sea lamprey hosts in the study sampling area, infected with A. simplex, see
Abollo et al. (2001)). Furthermore, A. simplex could be acquired from infected teleosts in
freshwater (e.g. sea trout in Fig. 2.4B) by post-metamorphic juveniles or in the estuary or
open ocean by adult sea lampreys, because there are enough reports of sea lamprey attacks
in all of these areas.
Fig. 2.4. A: Atlantic salmon caught in the river Lérez (Galicia) showing a wound caused
by sea lamprey in the ventral region, a possible route of Anisakis simplex transmission;
B: post-metamorphic juvenile of sea lamprey anchored to a sea trout in the Tea river
(42°11’12’’N; 8°30’40’’W).
45
Figure 2.5. Modified life cycle of Anisakis simplex in Galician waters.
46
We have to bear in mind that the sea lamprey is a commercially important species in
Galicia and Portugal. Although sea lamprey is not consumed raw and the epidemiological
relevance of anisakiasis in the region is low (Ubeira et al., 2000), the remarkable
prevalence and abundance of A. simplex in the flesh of the sea lampreys observed in this
study suggest a potential human risk for allergic reactions due to thermostable allergens
which should be evaluated even if no live larvae reach the consumer (EFSA, 2010).
Monitoring of parasite contaminants introduced from the marine environment to the
freshwater ecosystem is thus desirable to manage this emergent public health risk.
2.6 Acknowledgements
The authors thank the staff members of the ECOBIOMAR group at IIM-CSIC for
technical support. We thank sincerely the personnel of the “Service Nature Conservation
of the Xunta de Galicia”, with special mention to Pablo Caballero, Daniel Costal and José
Antonio Garea for their important collaboration and supply of some post-spawning
individuals. We are also grateful to Cofradía de Pescadores de Carril and the
Comandancia Naval del Miño for providing sea lamprey fishing data.
47
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Northwest Atlantic. Can. J. Fish. Aquat. Sci. 48, 832–842.
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& Potter, I.C. (Ed.), The Biology of Lampreys. Academic Press Inc., London, pp.
127–206.
Igoe, F., Quigley, D.T.G., Marnell, F., Meskell, E., O’Connor, W., Byrne, C., 2004. The
sea lamprey Petromyzon marinus (L.), river lamprey Lampetra fluviatilis (L.) and
brook lamprey Lampetra planeri (Bloch) in Ireland: General biology, ecology,
distribution and status with recommendations for conservation. Biol. Environ. Proc.
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species, Lampetra fluviatilis (L.), Lampetra planeri (Bloch) and Petromyzon
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Almeida, P.R., 2011. Can muscle fatty acid signature be used to distinguish diets
during the marine trophic phase of sea lamprey (Petromyzon marinus, L.)? Comp.
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helminths in a southern hemisphere lamprey (Geotria australis Gray), with a
discussion of the significance of feeding mechanisms in lampreys in relation to the
acquisition of parasites. Acta Zool. 64, 79–83. doi:10.1111/j.1463-
6395.1983.tb00785.x
Llarena-Reino, M., Piñeiro, C., Antonio, J., Outeriño, L., Vello, C., González, Á.F.,
Pascual, S., 2013. Optimization of the pepsin digestion method for anisakids
inspection in the fishing industry. Vet. Parasitol. 191, 276–283.
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to fish. Rev. Fish Biol. Fish. 5, 336–371. doi:10.1007/BF00043006
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Lampreys of the Iberian Peninsula: distribution, population status and conservation.
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sobre prevención de la parasitosis por anisakis en productos de la pesca
suministrados por establecimientos que sirven comida a los consumidores finales o
a colectividades. Ministerio de Sanidad y Consumo. Gobierno de España, España.
Nichols, O.C., Tscherter, U.T., 2011. Feeding of sea lampreys Petromyzon marinus on
minke whales Balaenoptera acutorostrata in the St Lawrence Estuary, Canada. J.
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Bricknell, I., Wallace, S., Cook, P., 2009. Red vent syndrome in wild Atlantic
salmon Salmo salar in Scotland is associated with Anisakis simplex sensu stricto
(Nematoda: Anisakidae). Dis. Aquat. Organ. 87, 199–215. doi:10.3354/dao02141
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pollution. Trends Parasitol. 21, 204–206. doi:10.1016/j.pt.2005.03.005
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Lamprey Species Known To Date, in: FAO Species Catalogue for Fishery Purposes
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characteristics of the dentition, buccal glands and velar tentacles of the adults of the
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species of Poland. Oceanol. Hydrobiol. Stud. 38, 129–137. doi:10.2478/v10009-
009-0015-7
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2000. Anisaquiosis y alergia. Un estudio seroepidemiológico en la Comunidad
Autónoma Gallega., Documentos Técnicos de Salud Pública, Serie C, no 8.
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Xunta de Galicia. Spain., Santiago de Compostela.
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50
Chapter 3 - Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758),
and twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian
Peninsula Rivers: zoonotic and ecological implications
3.1 Abstract
Spawning individuals of allis shad, Alosa alosa (Linnaeus, 1758), and twaite shad, Alosa
fallax (Lacépède, 1803), were sampled from three rivers on the Atlantic coast of the
Iberian Peninsula (Ulla, Minho, Mondego) during 2008 to 2013 to assess the presence of
the zoonotic marine parasite Anisakis spp. larvae. The results revealed that both shad
species were infected by third-larval stage Anisakis simplex s.s. and Anisakis pegreffii.
The latter is reported in mixed infections in both shad species of Western Iberian
Peninsula for the first time. In A. alosa, the prevalence of Anisakis infection can reach
100%, while in A. fallax, prevalence was up to 83%. Infected individuals of the former
species also often contain much higher number of parasites in theirs internal organs and
flesh: from 1 to 1,138 Anisakis spp. larvae as compared to 1 to 121 larvae, respectively.
In general, numbers of A. pegreffii were higher than those of A. simplex s.s. Our results
suggest that in the marine environment of the Western Iberian Peninsula, both
anadromous shad species act as paratenic hosts for A. simplex s.s. and A. pegreffii, thus
widening the distribution of the infective nematode larvae from the marine to the
freshwater ecosystem. This finding is of great epidemiological relevance for wildlife
managers and consumers, considering the zoonotic and gastroallergic threats posed of
these parasites.
Keywords: Alosa, Anisakis, Iberian Peninsula, Anadromous, Freshwater, Gastroallergic.
51
3.2 Introduction
Allis shad, Alosa alosa (Linnaeus, 1758), and twaite shad, Alosa fallax (Lacépède, 1803),
are anadromous members of the family Clupeidae. In their marine phase, they live mainly
in coastal waters and have a pelagic lifestyle; they migrate to rivers for spawning
(Aprahamian et al., 2003; Baglinière et al., 2003). Historically, their distributions along
the eastern Atlantic coast extended from Iceland and Norway in the north to Morocco in
the south (Aprahamian et al., 2003). In addition, A. fallax is found throughout the
Mediterranean Sea although rare in the Marmara and Black Sea, whilst A. alosa formerly
occurred in the western Mediterranean (Ceyhan et al., 2012; Faria et al., 2012).
Both species are protected and fishing regulated by Spanish (BOP, 2013; DOG, 2012)
and Portuguese (Ministério da Agricultura do Desenvolvimento Rural e das Pescas, 2000;
Ministério da Agricultura Pescas e Alimentação, 1987; Ministério do Ambiente, 1999;
Ministério do Planeamento e da Administração do Território, 1989; Ministério dos
Negócios Estrangeiros, 2008; Presidencia da República, 1959) legislation. In addition, A.
alosa is classified as “endangered” and A. fallax as “vulnerable” in the red Book of the
Portuguese Vertebrates (Cabral et al., 2006) and both species are considered as vulnerable
by some authors in Spain (Doadrio et al., 2011). In Galicia (NW of Spain), A. alosa is
classified as vulnerable in the Galician List of Threatened Species (DOG, 2007), and both
shad species are considered as endangered in Galician rivers by some authors (Solórzano,
2004).
Currently, the international section of River Minho (ISRM), along the border between
Spain and northern Portugal, holds what seems to be the only stable A. alosa stock in this
region (Mota et al., 2015). It suffered a dramatic drop in annual catches, by about 90%,
after the first half of the twentieth century, but numbers subsequently stabilized (Mota et
al., 2015; Mota and Antunes, 2011). Alosa fallax occurs in the river Ulla (Cobo et al.,
2010; Nachón et al., 2013; Silva et al., 2013) and also inhabits the river Minho (EHEC.
INRA - UPPA ECOBIOP. CIIMAR, 2012). Migration of A. alosa occurs mainly from
March to June in the river Minho (Mota et al., 2015; Mota and Antunes, 2011). The
upstream reproductive migration of A. fallax seems to take place between March and July
in the river Ulla (Nachón et al., 2013) and between April and June in the river Minho (M.
Mota unpublished data).
52
These anadromous species, especially A. alosa, have considerable ecological,
sociocultural and economic importance in Galicia, in the vicinity of the river Minho, and,
especially, in Portugal (Pereira et al., 2013), as for the whole of their distribution range
(Baglinière, 2000). Commercial fishing is permitted in the lower ISRM (BOP, 2013).
Spanish vessels caught around 2,000 A. alosa per year with trammel nets in 2010/11 and
2011/12 (data provided by Comandancia Naval de Tui). Portuguese catches are similar
(Mota and Antunes, 2011). The socioeconomic importance of A. alosa is reflected in their
high commercial value, which can range between 10 and 16 €/kg, depending on the year.
Spanish A. fallax catches were less than 200 fish per year in 2010/11 and 2011/12 (data
provided by Comandancia Naval de Tui). Sport fishing is permitted in the river Minho
for both species (BOP, 2013) and also for A. fallax in the river Ulla (DOG, 2012).
Nematodes of the genus Anisakis are marine parasites belonging to the family Anisakidae,
for which cetaceans serve as the main final hosts. It is considered that euphausiaceans and
copepods act as intermediate hosts and the anisakids also use a huge variety of fish and
cephalopods as paratenic or transport hosts (Mattiucci and Nascetti, 2008). Humans may
become an accidental host when they eat at least one live larva of Anisakis spp. from raw
or undercooked seafood products. This ingestion can cause clinical pathology, namely
anisakiasis or gastroallergic problems associated to thermostable allergens from third-
stage larvae. The anisakids with most zoonotic relevance are Anisakis simplex s.s.
(Rudolphi, 1809) and Anisakis pegreffii (Campana-Rouget and Biocca, 1955), which both
belong to A. simplex complex and which are the etiological agents responsible for an
increasing number of clinical cases worldwide (Arizono et al., 2012; Juric et al., 2013;
Mattiucci et al., 2013). These two Anisakis species seem to have different host, life cycle
and distribution preferences within European waters, although they are known to co-
infect fish hosts along the Spanish and Portuguese Atlantic coast and the Alboran Sea
(Mattiucci and Nascetti, 2008, 2006).
In Galicia, A. alosa and A. fallax are usually prepared fried in thin slices or baked. In
Portugal, several recipes exist, especially for A. alosa, including its roe, which is
considered to be a delicacy, but in all cases, the fish is well-cooked (Pereira et al. 2013,
C. Antunes and M. Mota, personal observation). However, consumers may still
experience a reaction to thermostable parasite allergens, if present.
53
The present paper provides an overview of the epidemiology of Anisakis spp. in both shad
species in this area, with the aim of identifying the different species present and obtaining
quantitative descriptors of parasite populations. We highlight the zoonotic risk for
wildlife and consumers and discuss the ecological implications of our findings on these
two vulnerable shad species.
3.3 Materials and methods
3.3.1 Sampling
Several sample batches of shad (Table 3.1) were caught by experimental or professional
fishing (trammel net) or by sport fishing in Minho, Ulla and Mondego rivers (Fig. 3.1),
covering March to August from 2008 to 2013 to include the whole migration season (for
more detailed descriptions of the sampling sites, see Mota et al. (2015), Mota and Antunes
(2011) and Nachón et al. (2013)).
3.3.2 Necropsy and visual inspection
Data on total weight (TW), total length (TL) and sex were recorded for each specimen. A
longitudinal section was performed from the cloaca to the operculum and then upwards
to expose internal organs. Internal organs were removed and macroscopic observation
was carried out to detect free Anisakis larvae around the peritoneum in the empty visceral
cavity. Internal organs were then inspected for presence of Anisakis larvae or frozen for
subsequent visual inspection. Most stomachs were separated from viscera and visually
inspected, in order to identify the preferences of nematodes for tissue location. Next, some
samples of the visceral cavity (without stomach) and fish flesh were subjected to further
enzymatic digestion, in order to confirm the visual inspection. The branchial region of
most shad belonging to batches 2, 6 and 7 (Table 3.1) was dissected, and gill arches were
extracted and examined for Anisakis larvae.
All nematodes found by macroscopic observation or enzymatic digestion were separated
and conserved in ethanol 70%. Then, every anisakid larva from each tube was
individually examined and identified at genus level under a stereomicroscope. Only
anisakid nematodes belonging to the genus Anisakis were included in the present study.
Finally, a random selection of parasite samples from individual shad and organs was
stored for molecular identification of Anisakis species.
54
Table 3.1. Sampling batches.
Species Batch n River Date Fishing method Fish organs inspected Method of inspection
Aa 1 160 Minho March to August, 2009
to 2011
Trammel net Visceral cavity and stomach Visual
Aa 2 9 Minho 6 May 2013 Trammel net Gill, visceral cavity, stomach
and flesh
Visual and artificial digestion
(visceral cavity, stomach and
flesh)
Aa 3 9 Mondego 14 May 2012 (five fish)
and 15 May 2013 (four
fish)
Trammel net Visceral cavity and stomach Visual
Af 4 148 Ulla March to July, 2011 and
2012.
Trammel net Visceral cavity and flesh
(only 18 fish)
Visual and artificial digestion (of
flesh)
Af 5 6 Ulla 1 May to 8 June 2008 Sport fishing Visceral cavity, stomach and
flesh
Visual and artificial digestion
Af 6 27 Minho 29 April 2011 Trammel net Visceral cavity, stomach and
flesh (only seven fish)
Visual and artificial digestion (of
flesh)
Af 7 42 Minho 14 May 2012 Trammel net Gills, visceral cavity,
stomach and flesh
Visual and artificial digestion
Method of inspection: adult specimens were inspected for Anisakis nematodes following the visual scheme established by the European Regulation
EU 853.
Aa Alosa alosa, Af Alosa fallax, n number of individuals.
55
Figure 3.1. Map of the study area showing the location of the three rivers.
56
3.3.3 Artificial enzymatic digestion
The artificial digestion of the flesh and visceral cavity of shad was carried out on the basis
of an optimized artificial digestion protocol (Llarena-Reino et al., 2013b). The flesh was
digested at 37-40 °C during approximately 3-4 h (3 h for visceral cavity material) in an
ACM-11806 Magnetic Stirrer Multiplate, using a weight/volume pepsin ratio of 1:20,
understanding that ratio as 20 ml of a 0.5% pepsin solution in HCl 0.063 M (pH 1.5) for
1 g of flesh. The digestion solution was decanted through a sieve, and the residues of
digestion and nematodes were inspected under a stereomicroscope. All Anisakis spp.
found were placed in individual tubes with 70% ethanol.
3.3.4 Molecular analysis
Genomic DNA from 72 Anisakis spp. larvae was individually isolated using a
MACHEREY-NAGEL NucleoSpin® Tissue kit following manufacturer-recommended
protocols. The entire internal transcribed spacer (ITS) (ITS1, 5.8S rDNA gene and ITS2)
was amplified using the forward primer NC5 (5´- GTA GGT GAA CCT GCG GAA GGA
TCA TT-3´) and reverse primer NC2 (5´- TTA GTT TCT TTT CCT CCG CT-3´). PCR
reactions were carried out in a total volume of 25 µl containing 100 ng of genomic DNA,
10 µM of each primer, 2.5 µl of 10× buffer, 0.5 µl of dNTPs and 5 U/µl of Taq DNA
polymerase (from Thermus Aquaticus BM, recombinant, Roche). PCR cycling
parameters included denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for
30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min 15 s and a final
extension at 72 °C for 7 min. PCR products were purified using illustra ExoStar 1-Step
following manufacturer-recommended protocols, with some modifications. We added 4
µl of reactive illustra ExoStar 1-Step and incubated the mix for 15 min at 37 °C. For
inactivation of the reactive agent added, we incubated the mix 20 min at 80 °C. Samples
with DNA concentration in clean reaction of 20 ng/µl were sequenced by SecuGen®
(Madrid). All sequences were subjected to a homology search through Basic Local
Alignment Search Tool (BLAST) searches in the National Center for Biotechnology
Information (NCBI) database.
3.3.5 Quantitative descriptors and statistical analysis
Quantitative descriptors of parasite populations found in shad, such as prevalence, mean
abundance and mean intensity, were calculated as described by Bush et al. (1997).
57
Factors affecting the parasite burden of both shad species were investigated using a
generalized additive modelling (GAM) framework as implemented in Brodgar 2.7.4
(http://www.brodgar.com/). The response variable was the number of Anisakis spp. larvae
found in the visceral cavity (including stomach) of fish, using visual methods. The
explanatory variables considered for the model selection process were as follows: TL,
TW, sex, condition factor (K [K = 100 × (TW/TL3)]), river, river section, year, day of the
year (expressed as a fraction of 365 days) and observer. All data series were explored for
outliers, collinearity, heterogeneity of variance and interactions between variables and to
visualize the relationships between response and explanatory variables, following the
protocol proposed by Zuur et al. (2010).
The sampling date was expressed as a fraction of the calendar year (yearfrac). Moreover,
yearfrac was correlated with K; hence in order to remove the season (yearfrac) effect from
the latter variable, it was “de-seasonalized” by regressing against yearfrac (treating
yearfrac as a smoother [k=4]). Thus, in the models, K is substituted by resulting residuals,
becoming “res K”. Note that K is derived from TL and TW and therefore was not included
in the same models as TW. The variables river section and observer could not be included
in the same model, since, for some samples, the two variables are confounded.
For the A. alosa dataset, Anisakis numbers approximated to a Gaussian distribution after
cubic-root transformation and we therefore used a Gaussian GAM with identity link
function. For the A. fallax, the data were strongly skewed with an excess of zeroes, but a
quasi-Poisson GAM (with log link) provided a satisfactory solution. In both cases,
forward selection was applied to identify the best models. For Gaussian GAMs, the
optimum model was the one with the lowest value for the Akaike information criterion
(AIC, Akaike 1974) provided that deviance explained was reasonably high and individual
explanatory variables had significant effects. For the quasi-Poisson models, the AIC is
not available and selection was based on the latter two criteria. Smoothers obtained by
cross-validation for effects of TL, yearfrac and res K on Anisakis abundance in A. fallax
were unrealistically complex, and models were refitted after setting a maximum k value
of 4. Final models were checked for robustness to addition of further explanatory
variables as well as for problems such as influential data points or trends in residuals.
58
3.4 Results
3.4.1 Parasite inspection
The visceral cavity of A. alosa specimens was frequently clearly infected, as seen by
visual inspection. The larvae were found rolled or free on the exterior surface of the
internal organs, especially the pyloric caeca, connective tissue fat and gonads. Moreover,
they were also found in the surface of intestine, liver and spleen. In addition, visual
inspection of the flesh revealed the presence of marks probably caused by Anisakis larvae.
Accumulation of Anisakis larvae was usually observed at the posterior end of the terminal
blind sac of the stomach. When the accumulation was clearly evident, the stomach wall
appeared broken, presumably due to the parasites’ migration from the stomach to the
visceral cavity (Fig. 3.2). No Anisakis was observed in the gills.
Figure 3.2. Accumulation of anisakid larvae at the posterior end of the terminal blind sac
of an A. alosa stomach from the river Minho.
On the contrary, based on visual inspection, the visceral cavity of A. fallax generally
seemed to be lightly infected. Nevertheless, occasionally, several internal organs
appeared clearly infected (Fig. 3.3.). No Anisakis larvae were detected visually in the
flesh or gills.
Visual inspection indicated a prevalence of Anisakis of 100% for A. alosa, but only 35%
for A. fallax.
59
Figure 3.3. Several Anisakis spp. larvae accumulated outside the posterior end of the
terminal blind sac of the stomach of A. fallax fished in the river Minho.
3.4.2 Genetic identification
All isolated anisakid larvae were initially examined under the stereomicroscope enabling
identification to the genus level. Furthermore, several Anisakis spp. larvae from every
batch were subjected to molecular diagnosis. According to the ITS amplified regions of
750 bp and searches for sequence homology (Blast values of 100%), the nematode species
identified from both shad belong to A. simplex s.s. and A. pegreffii. Parasite sequences
were deposited in GenBank (Accession numbers KP857639-KP857649). Based on the
molecular work, A. pegreffii is more numerous than A. simplex s.s. in the samples,
although there was some variation between rivers (Table 3.2).
Table 3.2. Percentages (%) of Anisakis larvae (n=72) molecularly identified by genetic
markers as A. alosa and A. fallax caught in different rivers.
Sampling River n Anisakis larvae A. pegreffii (%) A. simplex s.s. (%)
Alosa alosa
Minho 33 57.58 42.42
Mondego 13 69.23 30.77
Alosa fallax
Ulla 14 64.29 35.71
Minho 12 66.67 33.33
Both species of Anisakis have been diagnosed in the flesh of A. alosa from the river
Minho. Four larvae were tested genetically, three of which were A. simplex s.s. and one
60
A. pegreffii. The single larva found in the flesh of A. fallax from the river Ulla was
genetically identified as A. simplex s.s.
3.4.3 Infection data
The quantitative descriptors of Anisakis spp. larvae of both shad species from all sampling
batches are shown in Table 3.3. Alosa alosa showed higher values than A. fallax for every
quantitative descriptor of Anisakis infection. Thus, following visual and enzymatic
digestive detection methods (i.e. in batches 2 and 7), A. alosa from the river Minho
showed total mean abundance and intensity parameters hundreds of times higher than A.
fallax from the same river, although it should be noted that the species were sampled in
different years (i.e. 2013 and 2012, respectively). In relation to infection in the flesh, A.
alosa was clearly infected with moderate values of Anisakis abundance up to a maximum
of 13 larvae per fish. On the other hand, Anisakis infection of A. fallax flesh was almost
absent, with only a single larva found in one fish of 73 inspected following enzymatic
digestive methods. Notwithstanding the foregoing, the vast majority of larvae were
located in the visceral cavity (including stomach) for both shad species. In addition, A.
alosa usually showed an aggregation of Anisakis larvae in the visceral cavity, located at
the posterior end of the terminal blind sac of the stomach (Fig. 3.2). Specimens from the
river Minho (batch 2) showed an overall mean of 313.83 larvae per aggregation (range 8
to 884 larvae).
3.4.4 Statistical modelling
Both final models were satisfactory in terms of an absence of highly influential data points
and of trends in residuals. Results from the GAMs indicated that the numbers of parasites
in A. alosa were significantly related to TL (p<0.0001), yearfrac (p<0.0001), residual K
(p<0.0006), year (p<0.0001) and river (p=0.001). The years with the lowest numbers of
parasites were 2010 and 2011, whilst 2012 and 2013 had highest values. Moreover,
samples from the river Mondego showed fewer parasites than those from the river Minho.
The model explained 71.5% of deviance. Smoothers presented in Fig. 3.4 suggest that the
number of parasites in the visceral cavity of A. alosa increased with TL and residual K.
A significant effect was also shown for yearfrac, with a decreasing number of parasites
until April, followed by a rise until the end of May.
61
Table 3.3. Quantitative descriptors of Anisakis spp. larvae
Alosa alosa
Batch 1 (n= 160). Visual methodologies.
Organ n TL ± SD TW ± SD P (%) mA ± SD [range] mI ± SD [range]
Stomach 51 63.32 ± 4.34 2,428 ± 656.02 76.57 97.88 ± 135.85 [0-509] 128.00 ± 142.58 [1-509]
Viscera 51 63.48 ± 4.68 2,378 ± 743.92 96.08 180.73 ± 161.53 [0-796] 188.10 ± 160.51 [10-796]
Whole viscera 144 64.40 ± 3.89 2,351 ± 721.15 95.83 143.81 ± 166.92 [0-877] 150.06 ± 167.74 [1-877]
Batch 2 (n=9). Visual plus enzymatic digestive methodologies.
Organ n TL ± SD TW ± SD P (%) mA ± SD [range] mI ± SD [range]
Stomach 9 63.93 ± 5.92 2,711.11 ± 727.92 66.66 5.56 ± 9.25 [0-29] 8.33 ± 10.44 [1-29]
Viscera 100 611.67 ± 344.03 [108-1,137] 611.67 ± 344.03 [108-1,137]
Whole viscera 100 617.22 ± 348.24 [108-1,137] 617.22 ± 348.24 [108-1,137]
Flesh 88.88 3.56 ± 4.10 [0-13] 4.00 ± 4.14 [1-13]
Total 100 620.78 ± 348.82 [108-1,138] 620.78 ± 348.82 [108-1,138]
Batch 3 (n=9). Visual methodologies.
Organ n TL ± SD TW ± SD P (%) mA ± SD [range] mI ± SD [range]
Stomach 9 55.33 ± 2.87 1,731.11 ± 221.06 22.22 0.22 ± 0.44 [0-1] 1 ± 0 [1]
Viscera 100 215.00 ± 209.19 [4-588] 215.00 ± 209.19 [4-588]
Whole viscera 100 215.22 ± 209.23 [4-588] 215.22 ± 209.23 [4-588]
Alosa fallax
Batch 4 (n=148). Visual methodologies. In addition, the flesh of 18 fish was subjected to enzymatic digestion.
Organ n TL ± SD TW ± SD P (%) mA ± SD [range] mI ± SD [range]
Viscera 148 45.20 ± 5.24 830.59 ± 330.37 3.38 1.61 ± 9.89 [0-89] 47.80 ± 28.86 [14-89]
62
Abundance range and intensity range are presented between brackets.
P Prevalence, mA ± SD mean abundance and standard deviation, mI ± SD mean intensity and standard deviation, n number of fish sampled, TL ±
SD (cm) total length and standard deviation, TW ± SD (g) total weight and standard deviation.
a Zero larvae in stomach.
Flesh 18 42.06 ± 4.12 508.14 ± 147.74 0 0 0
Batch 5 (n=6). Visual plus enzymatic digestive methodologies.
Organ n TL ± SD TW ± SD P (%) mA ± SD [range] mI ± SD [range]
Stomach 6 39.55 ± 5.39 643.43 ± 275.10 0 0 0
Viscera = Total 83.33 44.17 ± 51.17 [0-121] 53.00± 51.85 [1-121]
Flesh 16.66 0.17 ± 0.41 [0-1] 1 ± 0
Batch 6 (n=27). Visual methodologies. In addition, the viscera and flesh of 7 fish were subjected to visual and enzymatic digestive methodologies.
Organ n TL ± SD TW ± SD P (%) mA ± SD [range] mI ± SD [range]
Stomach 26 38.15 ± 3.43 497.12 ± 181.74 0 0 0
Viscera 27 38.16 ± 3.37 497.04 ± 178.21 22.22 1.26 ± 3.36 [0-13] 5.67 ± 5.35 [1-13]
Whole visceraa 7 37.29 ± 1.67 441.43 ± 48.21 14.29 0.57 ± 1.51 [0-4] 4 ± 0
Flesh 0 0 0
Batch 7 (n=42). Visual plus enzymatic digestive methodologies.
Organ n TL ± SD TW ± SD P (%) mA ± SD [range] mI ± SD [range]
Stomach 40 36.73 ± 2.65 494.40 ± 93.36 10 0.15 ± 0.53 [0-3] 1.50 ± 1.00 [1—3]
Viscera 60 5.55 ± 10.60 [0-47] 9.25 ± 12.44 [1-47]
Whole viscera = Total 42 36.70 ± 2.58 493.45 ± 91.21 64.29 5.52 ± 10.39 [0-47] 8.59± 11.96 [1-47]
Flesh 0 0 0
63
A
64
Figure 3.4. Left to right are relationships between number of Anisakis spp. larvae found in the visceral (including stomach) of shad, using visual
methods, and explanatory variables as visualized by fitting GAMs. Smoothers for the effect of total length (cm), fraction of the calendar year
(yearfrac) and residuals of condition factor (res K) of A. alosa (A) and A. fallax (B).
B
65
The numbers of parasites in A. fallax were significantly related to TL (p<0.0001), yearfrac
(p=0.0003), residual K (p<0.0005), sex (p<0.0007) and river (p<0.0001). The samples
with the fewest parasites were those from the river Ulla, whilst males showed more
parasites than females. The model explained 35.8% of deviance. As for the A. alosa
model, the smoothers presented in Fig. 3.4 suggest that the number of parasites in the
visceral cavity of A. fallax increased with TL and residual K. Again, the number of
parasites decreased from the end of March until the end of April followed by a rise until
the middle of July.
3.5 Discussion
The marine parasitic nematode A. simplex is well reported in various Alosa spp. (Hogans
et al., 1993; Landry et al., 1992; Shields et al., 2002). However, epidemiological studies
of parasites in European shad remain scarce. Knezevic et al. (1978) and Quignard and
Douchement (1991) reported Anisakis sp. larvae from A. fallax nilotica and A. fallax,
respectively. Moravec (2001) reported A. simplex in a specimen of A. alosa from the river
Elbe. Rokicki et al. (2009) reviewed the presence of A. simplex in A. fallax from Baltic
Sea. Recently, Mota et al. (2015) presented the first report of A. pegreffii in A. alosa from
the river Minho.
3.5.1 Where are shad infected by Anisakis nematodes?
Several previous studies have shown the usefulness of anisakid nematodes as biological
tags for fish stock characterization in European waters (Kuhn et al., 2011; MacKenzie,
2002; Mattiucci et al., 2008). In relation to this, the presence of a mixed infection of A.
pegreffii and A. simplex s.s. in A. alosa and A. fallax of the western Iberian Peninsula is
in agreement with previous epidemiological information for other fish species studied in
western Iberian marine waters. Along the eastern coast of the Atlantic Ocean, the
distribution of A. simplex s.s. seems to have a southern limit around the Strait of Gibraltar.
Anisakis pegreffii is the main species of Anisakis in the Mediterranean, and it is also
widely distributed along East Atlantic Ocean down to the Antarctic Peninsula (Kuhn et
al., 2011; Mattiucci and Nascetti, 2008). The west Iberian Peninsula coast represents an
oceanic area where several fish species have been found with such mixed infections, as
were two toothed whale species belonging to the family Delphinidae, short-beaked
common dolphin, Delphinus delphis (Linnaeus, 1758), and long-finned pilot whale,
Globicephala melas (Traill, 1809), in NW Iberian Peninsula waters (Abollo et al., 2003,
66
2001; Hermida et al., 2012; Mattiucci et al., 2014, 2007, 2004; Mattiucci and Nascetti,
2008).
Within this area of sympatry, analysis of mixed infections has revealed different relative
proportions of Anisakis species depending on the geographical distribution of the host
fish species (Hermida et al., 2012; Mattiucci et al., 2014, 2007, 2004). The fact that shad
from Galician and Portuguese rivers had a higher proportion of A. pegreffii than A.
simplex s.s. might suggest three different hypotheses: Firstly, previous parasitological
studies carried out along the western Iberia coast have showed an increasing relative
proportion of A. pegreffii (and the opposite for A. simplex s.s.) from North (Galicia) to
South (coasts of South Portugal) in horse mackerel, Trachurus trachurus (Linnaeus,
1758) (Mattiucci et al., 2008). Abollo et al. (2003) found the highest prevalence of A.
simplex s.s. in the north of the Iberian Peninsula, decreasing towards the south, and the
opposite tendency for A. pegreffii, which had the highest prevalence in the Alboran Sea
and the lowest in the Cantabrian Sea. Other studies have shown a higher relative
abundance of A. pegreffii in blackspot seabream, Pagellus bogaraveo (Brünnich, 1768),
from Portuguese waters of the Iberian Coast (Hermida et al., 2012). Hybrids of A. simplex
s.s. and A. pegreffii can be found in some fish species in this area, and occasionally, other
Anisakis spp. are found (Abollo et al., 2003; Hermida et al., 2012; Marques et al., 2006;
Sequeira et al., 2010). Generally, studies carried out in Galician waters have shown mixed
infections with a higher proportion of A. simplex s.s. in blue whiting, Micromessistius
poutassou (Risso, 1827), European hake, Merluccius merluccius (Linnaeus, 1758) and T.
trachurus (Abollo et al., 2003; Mattiucci et al., 2008, 2004). In addition, single infections
of A. simplex s.s. were confirmed genetically in four cephalopod and seven fish species,
whilst mixed infections were confirmed in seven fish species (Abollo et al., 2001).
Interestingly, sea lamprey, Petromyzon marinus (Linnaeus, 1758), from the river Ulla and
river Tea (tributary of the river Minho) showed a high prevalence of A. simplex s.s. (Bao
et al., 2013). In addition, a single larva of A. pegreffii was found in one P. marinus from
the river Ulla (M. Bao unpublished data). Hence, we suggest that both shad species
migrate southward temporarily to feeding grounds off central or southern Portugal, thus
acquiring a relatively high proportion of A. pegreffii.
A second possibility is that there is immigration of shad from Mediterranean or NW
African stocks. This explanation seems fairly unlikely due to previously suggested
homing behaviour (Alexandrino, 1996; Sabatié et al., 2000), the existence of three
67
different halogroups throughout the Atlantic basin and the geographic distribution of
genetic diversity within both shad species, which suggests existence of a strong but
permeable barrier between Atlantic and Mediterranean populations (Faria et al., 2012;
Jolly et al., 2012). Moreover, both Moroccan shad populations are considered almost
extinct (Sabatié and Baglinière, 2001).
Thirdly, it is possible that the predominance of A. pegreffii is extending northwards. In
relation to this, a recent parasitological study carried out on M. poutassou from ICES
fishing area Div. VIIIc found a slightly higher proportion of A. pegreffii (six larvae of A.
pegreffii and four larvae of A. simplex s.s.) (Llarena-Reino et al., 2013a). However, to
date, as far as we known, other paratenic fish species from coastal waters of NW Iberian
Peninsula do not show a higher proportion of A. pegreffii. Further studies will be needed
to determine which, if any, of these explanations is correct.
3.5.2 How do shad acquire Anisakis parasites?
Alosa alosa is mainly a zooplanktophagous fish, the preferred prey of which is mainly
Mysidacea, Euphausiacea (e.g. Nyctiphanes couchii (Bell, 1853)) and copepods, and fish
are secondary prey (Aprahamian et al., 2003; Mota et al., 2015; Taverny and Elie, 2001a).
In contrast, A. fallax is essentially ichthyophagous (Assis et al., 1992; Taverny and Elie,
2001a) and zooplankton (such as N. couchii) constitutes secondary prey (Taverny and
Elie, 2001a). The euphausiid N. couchii has been recently diagnosed as the intermediate
host of both A. pegreffii and A. simplex s.s. in Galician waters (Gregori et al., 2015). Thus,
both shad species might gain their mixed infection of both Anisakis species by feeding on
infected zooplankton (such as N. couchii) or other transport hosts during the marine
trophic phase. Accumulation of anisakids by continuous reinfection through the diet is
well reported in several fish species (Mladineo and Poljak, 2014 and references therein).
Moreover, positive correlations of fish length and age with the number of larvae
accumulated have been found in several fish species (Levsen and Lunestad, 2010;
Mladineo and Poljak, 2014; Strømnes and Andersen, 2003). Bearing in mind the latter
findings and the fact that Anisakis infection numbers vary with fish species, fishing area
and season (Mladineo et al., 2012 and references therein), it is possible that A. alosa
present higher numbers of larvae than A. fallax because the former feeds intensively on
zooplankton while the latter feeds mainly on small pelagic fish, such as sand smelt,
Atherina boyeri (Risso, 1810) (Nachón et al., 2013), which supposedly have a low
Anisakis burden.
68
From an ecological point of view, it was suggested that transport hosts of A. simplex s.s.
are mainly benthic or demersal, whilst those of A. pegreffii are mainly pelagic. Hosts with
mixed infections, like shad, are mesopelagic or benthopelagic (Mattiucci et al., 1997).
Subsequently, it has been suggested that these parasites use pelagic and demersal food
chains to complete their life cycles (Mattiucci et al., 2014; Mattiucci and Nascetti, 2008).
Both shad species seem to use pelagic and neritic environments and also have schooling
behaviour, although A. fallax seems to have a distribution pattern more dependent on
estuarine environment, especially in younger individuals (Taverny and Elie, 2001b). The
ranges of depth distribution of both shad species, recorded by observers on board the
commercial fleet fishing over the continental shelf (generally >100 m depth) in the NW
Iberian Peninsula waters, seem to be in accordance with these results; nevertheless, both
species usually appear in the oceanic zone and in the epipelagic environments. Bearing
in mind that the observer data does not cover the coastal zone, it can be said that A. alosa
occurs between 9 and 311 m (mean depth 174 m) while A. fallax occurs between 18 and
390 m (mean depth 148 m) (data provided by Vigo IEO).
In the Sado estuary (Portugal), bottlenose dolphin, Tursiops truncatus (Montagu, 1821),
predates A. fallax (Aprahamian et al., 2003 and references therein). Furthermore, Black
Sea T. truncatus predates Alosa sp. (Gladilina and Gol’din, 2014). In fact, A. alosa and
other members of the subfamily Alosinae have been shown to respond to ultrasonic clicks
from delphinids, which is consistent with a prey-predator relationship among these
species (Wilson et al., 2011). Hence, shad are a suitable transport host for A. simplex s.s.
and A. pegreffii in order to reach a suitable final host in marine or brackish environment
of the Iberian coast.
3.5.3 Statistical analysis
In both shad species, numbers of parasites were positively related to (the partial effects
of) fish length and (seasonally adjusted) condition factor. In addition, numbers of
parasites fell to a minimum in April and then increased again to around the end of May
(A. alosa) or middle of July (A. fallax). There were also significant differences between
rivers (both species), years (A. alosa only) and sexes (A. fallax only).
In part, these results are expected: Levsen and Lunestad (2010) found a highly significant
effect of fish host size on total Anisakis larval abundance in another clupeid, Atlantic
herring, Clupea harengus (Linnaeus, 1758), from Norwegian waters. Furthermore,
69
Anisakis larvae accumulate with the increasing fish age and length in other fish species
(Mladineo and Poljak, 2014; Strømnes and Andersen, 2003).
The seasonally adjusted condition factor also showed a positive correlation with parasite
abundance. Despite the fact that parasites may be detrimental to their host (Rokicki et al.,
2009), this positive relationship could arise simply because good condition and high
parasite burden both reflect a high feeding rate (Mladineo and Poljak, 2014).
The seasonal pattern in infestation is less easily explained. The spawning migration into
freshwater has been reported to have effects on parasites of shad (Aprahamian et al.,
2003), and bearing in mind that the Anisakis larvae could not be acquired in such
concentrations by shad in freshwater habitats, a progressive decrease in parasite burden
over time spent in freshwater is plausible. Whilst A. fallax may feed during the spawning
migration (Nachón et al., 2013), A. alosa does not feed while migrating (Mota et al.,
2015), and in any case, Anisakis spp. are marine parasites. Thus, the rise in average
parasite burden from April to around the end of May (A. alosa) or middle of July (A.
fallax) possibly indicates the later arrival of fish with higher parasite burdens. However,
it is not obvious why this should occur and further research will be needed to confirm this
trend and investigate the causes.
3.5.4 Risk assessment
The role of anadromous shad populations in the life cycle of Anisakis spp. from western
Iberian Peninsula waters has important public health implications. Anisakis simplex s.s.
and A. pegreffii are the main zoonotic nematodes so far recognized as causing human
anisakiasis and gastroallergic reactions (Arizono et al., 2012; Juric et al., 2013; Mattiucci
et al., 2013). The European Food Safety Agency (EFSA) published a scientific opinion
on risk assessment of parasites in fishery products (EFSA, 2010). There, it was recognized
that all wild-caught marine and freshwater fish must be considered at risk of containing
viable parasites of human health concern if these products are eaten raw or almost raw.
Shad products are consumed fresh locally in the Iberian Peninsula and also in France (Elie
et al., 2000) and are likely to be reasonably safe, due to Portuguese and Galician cultural
traditions of eating heavily cooked food. Nevertheless, a potential health human risk
exists since allergic reactions could occur due to thermostable allergens even if no live
larvae reach the consumer (Arcos et al., 2014; Baird et al., 2014; Sharp and Lopata, 2014).
The recognition of several thermostable Anisakis antigens provoking allergic reactions,
70
which can be highly aggressive and generate severe clinical manifestations, suggests that
surveillance and epidemiological awareness should be encouraged. Apart from a few EU
fish production value chains, sufficient monitoring data are not available. Therefore, it is
not possible to identify which fish species and fishing grounds present a health hazard
with respect to the presence of allergenic parasites. Indeed, apart from recent findings in
P. marinus (Bao et al., 2013), no data are available to confirm that no viable parasites or
their allergens are present in fishery products derived from anadromous fish species
caught in freshwater ecosystems of the NW Iberian Peninsula.
The role of shad species as transport hosts for parasites from the marine to the freshwater
ecosystem is also very noticeable, and the transport in the opposite direction may also
occur. In this regard, Bao et al. (2013) suggested the possibility that post-metamorphic
juvenile of P. marinus might act as paratenic host of Anisakis spp., transporting them
from freshwater to seawater in NW Iberian peninsula waters. In addition, the
haematophagous feeding of post-metamorphic P. marinus on both A. fallax (Silva et al.,
2013) and A. alosa (Silva et al., 2014) has been documented in this region. Furthermore,
it was demonstrated that European otter, Lutra lutra (Linnaeus, 1758), predates on shad
species (Aprahamian et al., 2003) and they were also reported as an accidental host of
Anisakis spp. in one Spanish river (Torres et al., 2004), so wildlife risks for terrestrial
mammals should be considered. Likewise, Shields et al. (2002) reported infection by A.
simplex in the American shad, Alosa sapidissima (Wilson, 1811), in two Oregon rivers.
On this occasion, the authors suggested that this parasite-host relationship has led to an
ecological expansion of Anisakis spp. into rivers and may present an emerging health risk
for wildlife and human consumers.
Overall, the results highlight the fact that anadromous fish species may be a significant
source of gastroallergens and represent an ecological transport mechanism that transfers
the parasite risk from the sea to the freshwater ecosystem.
3.6 Acknowledgements
The authors would like to thank M. N. Cueto and J.M. Antonio (ECOBIOMAR) for their
excellent technical support and also Rodrigo López for making the map of the study area.
We also thank the personal of the Vigo IEO, for providing information about shad
captures at sea collected on the basis of national program (AMDES) included in the
European Data Collection Framework (DCF) project. We are also grateful to
71
Comandancia Naval de Tui for providing fishing data. M. Bao is supported by a PhD
grant from the University of Aberdeen and also by financial support of the contract from
the EU Project PARASITE (grant number 312068). This study was partially supported
by a PhD grant from the Portuguese Foundation for Science and Technology (FCT)
(SFRH/BD/44892/2008) and partially supported by the European Regional Development
Fund (ERDF) through the COMPETE-Operational Competitiveness Programme and
national funds through Foundation for Science and Technology (FCT), under the project
PEst-C/MAR/LA0015/2013. The authors thank the staff of the Station of Hydrobiology
of the USC “Encoro do Con” due their participation in the surveys. This work has been
partially supported by the project 10PXIB2111059PR of the Xunta de Galicia and the
project MIGRANET of the Interreg IV B SUDOE (South-West Europe) Territorial
Cooperation Programme (SOE2/P2/E288). D.J. Nachón is supported by a PhD grant from
the Xunta de Galicia (PRE/2011/198).
72
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Chapter 4 - Employing visual inspection and Magnetic Resonance
Imaging to investigate Anisakis simplex s.l. infection in herring viscera
4.1 Abstract
Anisakis spp. and Pseudoterranova spp. are anisakid nematodes with complex life cycles
that utilise crustacean, fish and, finally, marine mammal hosts. Many species of marine
fish are used by these larval anisakids as paratenic or intermediate hosts, and these are
capable of infecting human consumers, causing public health, economic and social
concerns worldwide. They are responsible for a fish-borne zoonosis called anisakidosis
and associated allergic problems in humans, monetary loss to the fishing industry and the
avoidance of fish products by consumers. An understanding of the parasite/fish
relationship and the development of improved methods and tools for parasite detection
are important to provide better insights to their biology, as well as technology to control
the quality and safety of fishery products. In the present study, Anisakis simplex s.l.
infection levels and location within the viscera of Atlantic herring (Clupea harengus)
were visually investigated to provide a better understanding of settlement behaviour
within the host fish, and to test if Magnetic Resonance Imaging (MRI) is a useful
technology to detect anisakids in the viscera of whole herring. MRI potential to detect
anisakids in fish muscle was also tested. Visual inspection determined an ascaridoid
prevalence of 76% (> 90% A. simplex s.l. (likely A. simplex s.s.) in herring viscera, with
69% of the parasites located outside the hind stomach and intestine areas. MRI
demonstrated a capacity to detect A. simplex s.l. accumulations in the viscera of whole
herring, A. simplex s.l. larvae and their movements within fish muscle, and
Pseudoterranova sp. larva in fish muscle. Visual inspection of herring showed that A.
simplex s.l. larvae frequently accumulate in the posterior end of the terminal blind sac of
the stomach, around the ductus pneumaticus, as had previously been reported in herring
and other clupeids. It has been hypothesized that the food digestion process, visceral
organ topography and physicochemical conditions of fish tissues may play an important
role in determining the encapsulation site and in vivo migration behaviours of A. simplex
s.l. larvae within fish hosts. MRI showed potential to investigate the occurrence of
anisakids and possibly other macroparasites of fish, and to detect anisakids in fishery
products in situ, in a 3D environment and in a non-invasive and non-destructive way.
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Further investigation is required to determine if MRI can be used as a routine anisakid
inspection tool by the fishing industry.
Keywords: Anisakis, Pseudoterranova, Magnetic Resonance Imaging, parasite
detection, herring, fish.
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4.2 Introduction
The anisakid nematodes Anisakis spp., Pseudoterranova spp. and Contracaecum spp.
display complex life cycles in aquatic ecosystems which involve crustaceans as
intermediate hosts, fish and cephalopods as paratenic or intermediate hosts, and fish-
eating birds and marine mammals as definitive hosts (Mattiucci and Nascetti, 2008). They
are the etiological agents of a fish-borne parasitic zoonosis in humans (anisakidosis)
caused by the ingestion of raw or inadequately cooked fishery products infected with
larval anisakids (Audicana and Kennedy, 2008; Mattiucci and D’Amelio, 2014) (the
taxonomic details of the ascaridoid nematodes mentioned in this study are presented in
supplementary material). In addition, several studies have demonstrated that the genus
Anisakis can cause clinical allergic responses (Audicana and Kennedy, 2008; Carballeda-
Sangiao et al., 2016; Daschner et al., 2000; Nieuwenhuizen, 2016). These anisakids are
of importance not only for public health, but also for economic considerations and social
concerns, as they can reduce the marketability of fishery products and provoke consumer
aversion to fish (D’amico et al., 2014; EFSA-BIOHAZ, 2010; Llarena-Reino et al., 2015;
Mattiucci and D’Amelio, 2014). Thus, the development of accurate and fast detection
methods for anisakids in fishery products is of great importance for the fishing industry
and inspection services in order to ensure food safety and quality. However, routine use
of such devices is restricted because of technical limitations.
A number of methods have been developed and tested to detect whole infective third-
stage larvae of anisakids in fish, including visual inspection and candling (EFSA-
BIOHAZ, 2010), artificial peptic digestion (Llarena-Reino et al., 2013), UV-press (Karl
and Leinemann, 1993), and other imaging technologies (e.g. X-rays, electromagnetism
and spectroscopy) (McClelland, 2002; Sivertsen et al., 2012). Non-destructive visual and
candling methods are less effective for detecting anisakid larvae embedded deep within
fish tissue than destructive methods such as peptic digestion and UV-press (EFSA-
BIOHAZ, 2010; Karl and Leinemann, 1993; Levsen et al., 2005). Molecular,
immunological and proteomic methods have also been developed to detect genomic DNA
(Herrero et al., 2011), allergens (Rodríguez-Mahillo et al., 2010) and thermostable
proteins (Carrera et al., 2016) of anisakids in fish samples. To the best of our knowledge,
no method has been developed to detect Anisakis spp. larvae within unprocessed whole
fish.
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Magnetic Resonance Imaging (MRI) is widely used in clinical diagnosis and research
(Cheng et al., 2013). MRI can also be used for studying the physicochemical properties
and anatomical structure of fish (Cheng et al., 2013; Mathiassen et al., 2011; Patel et al.,
2015) including for example belly bursting in herring (Veliyulin et al., 2007), fat
measurement in rainbow trout cutlets (Collewet et al., 2013), and distribution of adipose
tissues in live fish (Wu et al., 2015). However, it has not yet been used to detect fish
parasites.
The aim of this study was to determine Anisakis spp. infection levels and the site of
infection (i.e. habitat or tissue) within the visceral cavity of Atlantic herring (Clupea
harengus) by visual inspection, to determine if MRI can be used to detect Anisakis spp.
accumulations in the viscera of whole herring and to test the potential of MRI to detect
anisakids (i.e. Anisakis and Pseudoterranova) in fish muscle.
4.3 Materials and methods
4.3.1 Ascaridoid nematode sampling in herring viscera
The visceral cavities of 209 thawed herring caught from the North Sea (ICES fishing area
IVa), during the summers of 2013 (n= 59) and 2014 (n= 150), were visually inspected for
ascaridoid larvae (Anisakis spp. and Hysterothylacium spp.) using the press and candling
method. Briefly, the internal organs were removed from fish specimens, placed into a
transparent polythene bag, flattened by a hydraulic press to approximately 1-2 mm
thickness, and inspected for ascaridoid larvae on a candling table in a dark room.
Ascaridoid location on the internal organs was reported by inspecting the following
habitats: pyloric caeca, fore intestine, hind intestine, fore stomach, hind stomach, swim
bladder, liver and gonad. Ascaridoid infection parameters, including prevalence, mean
abundance and mean intensity were calculated following Bush et al. (1997). A number of
ascaridoids (n= 20) were morphologically identified to the genus level using a
stereomicroscope following Berland (1989).
4.3.2 MRI protocol
Three independent experiments were performed to detect anisakids in fish samples at
room temperature using a 4.7 T horizontal field preclinical MRI scanner (Magnex, UK
and MR Solutions, UK), as detailed below.
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4.3.2.1 Detection of Anisakis spp. accumulation in the visceral cavity of whole herring
A whole herring was randomly selected from the sample, thawed in the fridge, wrapped
in a polythene bag and scanned with MRI the following day. MRI was carried out in the
4.7 preclinical system using a transmit-receive quadrature birdcage RF coil of in-house
design with 72 mm internal diameter. Five MRI measurements comprising 40 sagittal (n=
2), 40 axial (n=1) and 40 coronal (n=2) MRI slices of the fish viscera were acquired using
Multi-slice Spin-Echo (MSSE). The MSSE protocol used imaging bandwidth 25 KHz,
echo time (TE) = 25 ms, recovery time (TR) = 3000 ms, matrix size 256 x 256 pixels,
field of view (FOV) 75 mm giving an in-plane resolution of 293 µm, slice thickness 0.5
mm and a single average. The total acquisition time of each MRI scan was approximately
13 minutes. MRI images were displayed and analysed using Image J software
(https://imagej.nih.gov/ij/).
4.3.2.2 Detection of Anisakis spp. larvae in fish muscle
To test the capability of MRI to detect Anisakis spp. larvae in thick white fish muscle,
fresh Anisakis spp. parasites (n=5) were extracted from the visceral cavity of two whole
herring bought locally (Aberdeen, Scotland). A piece of fish muscle approximately 8 cm
long x 4 cm wide x 3 cm high was cut from a fish fillet (pollack, Pollachius pollachius)
and the parasites were introduced into the centre of the muscle with forceps. The sample
was wrapped in polythene before being placed in the MRI system for scanning. This
experiment acquired the MRI signal with a receive-only surface coil of 35 mm diameter
to improve the signal to noise ratio (SNR) compared to the first experiment. MSSE-MRI
was again used, this time to acquire 20 sagittal slices of the pollack muscle and the total
acquisition time was approximately 13 minutes. Then, a time series of five slices at four
time points, 13 minutes apart, was carried out to determine if the parasites were moving.
4.3.2.3 Detection of Pseudoterranova sp. in fish muscle
The muscle of a monkfish (Lophius piscatorius) caught in the North Sea in the summer
of 2015 was found to be infected with a single Pseudoterranova sp. larva, commonly
named as sealworm or codworm, during a routine fish parasite survey. A piece of
monkfish muscle approximately 3.5 cm long x 3.5 cm wide x 1.5 cm high containing the
codworm was cut, wrapped in polythene then scanned using three MSSE-MRI
measurements of 15 axial slices, using the 35 mm diameter receive-only surface coil. This
acquisition used a 35 mm FOV giving an in-plane resolution of 137 µm and was repeated
for repetition times (TR) of 1000 ms, 3000 ms and 6000 ms to find the value for best
83
contrast between muscle and codworm. The longest MSSE acquisition with TR = 6000
ms lasted 25 minutes and 40 seconds. The sample was also investigated using three
dimensional gradient echo (3DGE) with TE/TR of 15 ms/50 ms, matrix size 256 x 256 x
128 pixels, FOV 40 mm giving an isotropic resolution of 156 µm, a single average and
acquisition time 27 minutes. The resulting 3D data set was rendered and displayed using
the volume viewer and 3D viewer plugins in ImageJ.
4.4 Results
4.4.1 Anisakis simplex s.l. infection in herring viscera
Ascaridoid infection descriptors as well as the biometric parameters obtained from the
herring are shown in Table 4.1. Visual inspection showed that the visceral cavity of
herring was frequently infected by ascaridoids (prevalence of 76%), mainly Anisakis spp.
(>90%) and less frequently Hysterothylacium spp. larvae. Morphological identification
of ascaridoid subsamples (n=20) confirmed Anisakis spp. infection, which certainly
belongs to the species complex A. simplex s.l., and is likely to be A. simplex s.s., since it
appears to be the only Anisakis species present in Atlantic herring from NE Atlantic
waters (Cross et al., 2007; Levsen and Lunestad, 2010; Mattiucci and Nascetti, 2006;
Tolonen and Karlsbakk, 2003). No suspected Hysterothylacium spp. larvae were
collected for further morphological or genetic identification. Based on a previous study,
these larvae would likely be Hysterothylacium aduncum (Tolonen and Karlsbakk, 2003).
Table 4.1. Quantitative descriptors of ascaridoid nematode populations found in the
visceral cavity of herring (n= 209).
TL ± SD (range) TW ± SD (range) Ascaridoid nematodes (mainly A. simplex s.l. and
infrequently Hysterothylacium spp.)
P (%) mA ± SD (range) mI ± SD (range)
254 ± 37 (149-348) 174 ± 36 (40 – 435) 76 9.4 ± 16.2 (0 - 90) 12.4 ± 16.4 (1 - 90)
TL and TW, total length (mm) and total weight (g); P, mA and mI, prevalence, mean
abundance and mean intensity; SD, standard deviation; range, minimum and maximum
value.
Anisakis simplex s.l. larvae were observed on the surface of internal organs, especially on
the lower portion of the visceral cavity (i.e. hind stomach and hind intestine) (69%) (Table
4.2). Anisakis simplex s.l. infection on the pyloric caeca was also frequent (21%), whilst
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infection on fore-stomach and intestine, liver, gonad and swim bladder was rare (<5% for
each habitat) (Table 4.2).
Table 4.2. Frequency, percentage and location (habitat or tissue) of A. simplex s.l. on the
visceral cavity of sampled herring (n= 209).
Habitat in viscera Frequency (percentage)
Pyloric caeca 411 (21 %)
Fore intestine 60 (3 %)
Hind intestine 292 (15 %)
Fore stomach 32 (2 %)
Hind stomach 1060 (54 %)
Swim bladder 11 (1%)
Liver 46 (2%)
Gonad 46 (2%)
Total internal organs 1958 (100%)
Accumulations of A. simplex s.l. larvae (i.e. defined here as aggregations of 5 or more
larvae) were observed frequently (32%, 51 out of 158 infected herrings) at the posterior
end of the terminal blind sac of the stomach (i.e. hind stomach), where the ductus
pneumaticus opens (Figure 4.1).
Figure 4.1. Anisakis simplex s.l. larvae accumulation in situ in the visceral cavity of
whole Atlantic herring. 1- pyloric caeca, 2- stomach, 3- gonad, 4- Anisakis accumulation,
5- swim bladder, 6- intestine.
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4.4.2 MRI results
4.4.2.1 Detection of A. simplex s.l. accumulation in the visceral cavity of whole herring
Figure 4.2A shows the ventral area of a whole herring before it was scanned using MRI.
Figures 4.2B and 4.2C show example sagittal slices which demonstrate the potential of
the MSSE-MRI method to identify internal organs and tissues including pyloric caeca,
stomach, swim bladder, muscle, spine and insertion of the dorsal fin. MRI also detected
A. simplex s.l. larvae accumulations in situ, within the visceral cavity of whole herring.
MRI results were validated by subsequent visual inspection of the dissected herring
(Figure 4.2D). A total of 53, 8 and 3 A. simplex s.l. larvae were observed in the hind
stomach, hind intestine and gonad, respectively, during visual inspection. This
experiment shows that the existing MSSE-MRI protocol can detect A. simplex s.l.
accumulations in herring viscera.
4.4.2.2 Detection of A. simplex s.l. in fish muscle
Figure 4.3 and the supplementary material show that MRI detected the introduced A.
simplex s.l. larvae in the fish muscle, and was able to detect A. simplex s.l. that had
penetrated into the muscle from the site of introduction. The black spaces seen inside the
muscle are air cavities created during the introduction of the larvae into the fish muscle
using forceps. Time series images from this study which are available in the
supplementary material also demonstrated the potential of MRI to detect larvae
movements within the muscle.
4.4.2.3 Detection of Pseudoterranova sp. in fish muscle
Figure 4.4 demonstrates the feasibility of using MRI to detect codworm infection in fish
muscle. The 3DGE method and the MSSE method detected the Pseudoterranova sp. larva
(Figure 4.4B and 4.4C, respectively) but MSSE-MRI gave a clearer image once it was
rendered using the volume viewer in Image J (Figure 4.4C).
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Figure 4.2A. Photograph of the whole herring before scanning. B and C. Sagittal MRI
scan of the herring showing an A. simplex s.l. larvae accumulation and internal organs. D.
Photograph of the dissected herring confirming the A. simplex s.l. larvae accumulation in
situ detected by the MRI scan. Dashed lines- area of scanning, dashed circles- habitat of
A. simplex s.l. larvae accumulation, 1- pyloric caeca, 2- stomach, 3- gonad, 4- swim
bladder, 5- Anisakis accumulation, 6- muscle, 7- spine, 8- dorsal fin.
87
Figure 4.3. Two magnetic resonance images A and B were taken approximately 13
minutes apart and show an A. simplex s.l. larva moving inside the fish muscle (white
arrow).
88
Figure 4.4A. Pseudoterranova sp. larva infecting monkfish muscle. B. 3D MRI scan
showing the Pseudoterranova sp. larva processed using ImageJ 3D viewer and C. ImageJ
volume viewer.
89
4.5 Discussion
4.5.1 Anisakis simplex s.l. habitat in the visceral cavity of herring
The site of Anisakis spp. infection (i.e. habitat) and their in vivo migration behaviour
within the fish host have been discussed previously (Berland, 2006; Mladineo and Poljak,
2014; Sluiters, 1974; Smith, 1984; Smith and Hemmingsen, 2003; Strømnes, 2014;
Strømnes and Andersen, 2003, 1998; Young, 1972). It was suggested that the visceral
organ topography (i.e. spatial arrangements and contiguity of organs within the body
cavity) may influence which tissues are occupied by Anisakis spp. in fish (Smith, 1984;
Smith and Hemmingsen, 2003). Berland (2006) suggested that, in gadoids, Anisakis spp.
emerge from the stomach and encounter the fish liver, in which they become
encapsulated. However, during periods of starvation the liver almost completely
disappears and in vivo migration of Anisakis spp. from the stomach to the muscle, which
is in direct contact with the stomach in such conditions, may occur (Berland, 2006).
Mladineo and Poljak (2014) suggested that the point of the gastrointestinal tract where
the Anisakis spp. emerge and the organ located near to that point determine the settlement
area in the fish host. However, Strømnes and Andersen (1998) suggested that other factors
such as biochemical mechanisms (i.e. availability of nutrients in fish), in addition to
random migration of Anisakis spp. larvae emerging from the gut, may influence their in
vivo migratory behaviour and encapsulation site. These authors also found that the growth
of the larvae was significantly correlated with the fat content of the tissue in which the
larvae were encapsulated (Strømnes and Andersen, 2003). Recently, Strømnes (2014)
showed that Anisakis spp. migratory behaviour was influenced by lipid concentration in
in vitro studies. Strømnes (2014) also suggested that in the fish host, when Anisakis spp.
larvae emerge from the stomach, they randomly radiate to the body cavity (in accordance
with Young’s (1972) hypothesis), even though the migratory distance would tend to be
reduced at higher lipid concentration levels. This implies an increased probability of
Anisakis spp. larvae accumulating in high-fat host tissues.
In this study, A. simplex s.l. habitat in the visceral cavity of Atlantic herring was
investigated by visual methods, with the hind stomach being the highest area of larval
accumulation (Figure 1 and Table 2). This has been observed previously by Tolonen and
Karslbakk (2003) and by Sluiters (1974), who reported that penetration of worms was
only observed in the caudal part of the caecal stomach and that most larvae were
encapsulated adjacent to the ductus pneumaticus within the visceral cavity. Moreover,
90
this habitat of A. simplex s.l. was also seen in anadromous allis shad (Alosa alosa) and
twaite shad (Alosa fallax), which are also members of the family Clupeidae (Bao et al.,
2015). Thus, it appears that A. simplex s.l. tend to form accumulations in this visceral
cavity habitat in these clupeids, probably caused by the “Y” shape of their stomach, which
carries the food and anisakids to the caecal stomach during digestion, where they emerge.
It appears that fish gonads act as physical barriers/traps for A. simplex s.l. larvae and so
obstruct in vivo migration to the muscle during maturation (Figure 1). After spawning,
(1) the gonads reduce dramatically in size, (2) physicochemical conditions (e.g.
mesenteric fat) in the visceral cavity change (McPherson et al., 2011), and (3) the distance
from the hind stomach to the muscle becomes narrow, probably facilitating A. simplex s.l.
in vivo migration to muscle.
Accumulations of A. simplex s.l. also occur in the pyloric caeca, probably due to the
worms emerging from inside the pyloric region of the stomach or intestine to the caeca
following the digestion process, or migrating from the hind stomach habitat to the caeca
and/or vice versa. Interestingly, A. simplex s.l. infection in the liver was reported as
accidental in herring (Table 2), and not important in shads (Bao et al., 2015), whereas the
liver is usually found to be heavily infected in fish of the order Gadiformes (e.g. European
hake (Merluccius merluccius), blue whiting (Micromesistius poutassou) and Atlantic cod
(Gadus morhua) (Llarena-Reino et al., 2015; Mladineo and Poljak, 2014; Smith and
Wootten, 1978). In gadoids, the liver is situated contiguous to the stomach (Smith and
Hemmingsen, 2003) and it is the most important organ for fat storage (Eliassen and Vahl,
1982). However, in herring it is not in contact with the hind stomach and is not important
as a fat store (Lovern and Wood, 1937).
It can therefore be hypothesised that (1) it is the food digestion process that probably
determines the site from which Anisakis spp. emerge from the digestive tract, (2) the
visceral organ topography and the fat content of fish organs, which all are associated with
the life cycle (e.g. age, size, diet, reproduction, season, etc.), will influence Anisakis spp.
in vivo migration behaviour and their preference for habitat of encapsulation within fish
hosts. Other intrinsic factors among different Anisakis spp. may be involved in
determining Anisakis spp. behaviour within fish hosts. For instance, A. simplex s.s. has
been reported in European hake and chub mackerel (Scomber japonicus) muscle at higher
infection intensities than those seen for A. pegreffii (Cipriani et al., 2015; Suzuki et al.,
2010). Further investigation is required to improve understanding of Anisakis spp.
91
behaviour within fish hosts (i.e. in vivo migration and encapsulation habitat) and here
MRI investigation can help.
4.5.2 Parasite detection with MRI
Currently, the most accurate and commonly used anisakid detection methods in fish
musculature are candling and slicing, enzymatic digestion and UV-press which are
invasive and destructive (EFSA-BIOHAZ, 2010; Karl and Leinemann, 1993; Levsen et
al., 2005; Llarena-Reino et al., 2013). Smith and Hemmingsen (2003) reported that during
common parasite fish sampling, internal organs cannot be inspected for anisakids as a
three-dimensional environment because the visceral cavity is opened and manipulated
and the organs are therefore no longer in their original position. It is important to identify
which organs may be occupied or traversed by the anisakids migrating to the muscle, and
Smith and Hemmingsen (2003) suggested that MRI may provide accurate imaging of the
visceral organ topography of inspected fish. Further, Cheng et al. (2013) suggested that
MRI has great potential for use in fish quality classification, fish freshness discrimination
and parasite detection.
To the best of our knowledge this is the first time that MRI has been utilised to investigate
anisakid parasites in fish. This study demonstrated the potential of MRI to detect A.
simplex s.l. larvae accumulations in the visceral cavity of whole herring which were
previously observed by visual inspection, to detect A. simplex s.l. larvae in fish muscle
and follow their movements, and to detect Pseudoterranova sp. larvae infecting fish
muscle. It is likely that MRI investigation of fresh fish would improve the accuracy of
parasite imaging, as post-mortem degradation of viscera may occur, spoiling the original
anatomical structure of the fish (Veliyulin et al., 2007).
This study demonstrated that MRI has the potential for non-invasive and non-destructive
detection of anisakids in whole fish viscera and fish muscle in situ in a 3D environment.
This is of interest from both academic (i.e. to investigate anisakids behaviour within the
fish host) and applied (i.e. MRI can be used to detect anisakids) points of view. Wu et al.
(2015) used MRI to investigate the distribution of adipose tissues in intact and live fish.
MRI could also be explored as a potential method to investigate in vivo and post-mortem
Anisakis spp. migration behaviour in live and dead fish. It has also potential applicability
in fishery surveys and in screening of food products for anisakids, for example by
identifying common areas of anisakid infection in fish samples, thereby helping to ensure
92
quality and safety. Since MRI is non-destructive and non-invasive the process of scanning
itself will not affect the safety or quality of the product (Cheng et al., 2013). It is also
likely that MRI can be used to investigate other fish macroparasites, for instance,
didymozoid trematodes and cestodes (Gymnorhynchus spp., Molicola spp.) which affect
the quality and safety of fishery products (Llarena-Reino et al., 2015).
MRI has a number of challenges that need to be addressed before it can be applied in the
industry for routine detection and monitoring (online or off-line) of anisakids parasites in
fishery products in an automatic, cost-efficient and fast way. These challenges include:
expensive hardware with high maintenance costs (MRI); current speed to acquire pictures
is slow (several minutes per fish) and industry would likely require at least 1 fish per
second; its operators require specialised training (it is challenging to process and study
the spectral image for parasite detection) (Cheng et al., 2013; Mathiassen et al., 2011;
Patel et al., 2015; Veliyulin et al., 2007); MRI cannot be applied in frozen fishery products
(almost no signal from ice, water must be liquid). Finally, for research purposes, imaging
of live or anaesthetized fish is challenging because MRI is highly motion-sensitive. To
conclude, further investigations are required to determine if MRI can count total anisakid
larvae numbers, and if solutions can be found for the above challenges before MRI can
be used for routine parasite inspection by the fishery industry.
4.6 Acknowledgements
We thank Jamie Dawson and William Page for their work detecting anisakids in herring
samples. M. Bao is supported by a PhD grant from the University of Aberdeen and also
by financial support of the contract from the EU Project PARASITE (grant number
312068). This work received funding from the MASTS pooling initiative (The Marine
Alliance for Science and Technology for Scotland) and their support is gratefully
acknowledged. MASTS is funded by the Scottish Funding Council (grant reference
HR09011) and contributing institutions.
4.7 Authors’ contributions
MB designed the overall study and HS developed the MRI protocol. MB and LH
performed Anisakis inspection in herring. KM performed morphological identification of
anisakid samples. MB and HS performed the MRI investigations, analysed the results and
drafted the paper. NS, GP contributed to the ideas behind the study and the writing of the
93
paper. All authors critically reviewed the paper and agreed the final content of the version
to be published.
94
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in cod and marine mammals in British home waters. J. Appl. Ecol. 9, 459–485.
98
Chapter 5 - Consumers’ attitudes and willingness to pay for Anisakis-
free fish
5.1 Abstract
The presence of parasitic nematodes of the genus Anisakis and/or their proteins in seafood
poses a risk to human health through a fish-borne zoonosis, namely anisakiasis that can
cause gastrointestinal disease, and allergy. The presence of Anisakis may also dissuade
consumers from purchasing fishery products, resulting in economic losses to the fishing
industry. This is the first time a survey-based contingent valuation study has been
performed to investigate consumers’ willingness to pay for Anisakis-free fish, and to
analyse consumers’ responses to the presence of Anisakis in fishery products. Consumers
indicated a willingness to pay 10% above the usual fish price at market (6.60€/kg
compared with 6€/kg). The majority of consumers (77%) were willing to pay extra for an
Anisakis-free product. Past reluctance to purchase/consume fish due to the presence of
Anisakis was reported by >25% of consumers, with hake being the most frequently
rejected species. Almost one third of consumers (29%) indicated that they would stop
consuming/purchasing fish in the future due to the presence of Anisakis, whilst a similar
proportion of consumers (31%) would cease consumption only if there were a high chance
of Anisakis presence in fish. In initial statistical models with single explanatory variables,
consumers’ willingness to pay was significantly related to their gender, stated past and
future avoidance of fish consumption/purchase due to the presence of Anisakis, stated
past avoidance of cod, hake and mackerel, stated consumption of sardines, and to their
perception of the degree of risk of future development of anisakiasis and/or allergy to
Anisakis. The final (best fitting) multifactorial model included a number of the previous
explanatory variables (gender, stated future avoidance of fish and stated past avoidance
of cod) but also included significant effects of stated consumption of raw or lightly
cooked hake, stated consumption of raw fresh fish at home and allergy status of
consumers. These results revealed two main types of reaction to the presence of Anisakis
in fish: the avoidance of eating parasitized fish, and a willingness to pay above market
price to avoid adverse effects on health and food quality. Overall, the results suggest that
the presence of Anisakis in fish is an important health and aesthetic issue for consumers,
and this is of relevance for the fishing and food industries as well as for food safety
authorities. Improvements in parasite inspections and development of technologies to
99
prevent Anisakis infection in fishery products would likely both improve the economic
sustainability of the industry and benefit public health.
Keywords: Anisakis, willingness to pay, contingent valuation, anisakiasis, fish parasite,
fishing industry.
100
5.2 Introduction
Fish-borne zoonotic parasites are of high public health and socio-economic concern (Chai
et al., 2005; Dorny et al., 2009; EFSA-BIOHAZ, 2010). Aquatic helminths (e.g. cestodes,
trematodes and nematodes) are the etiological agents of a number of fish-borne zoonoses
(e.g. diphyllobothriasis, trematodiasis, anisakiasis) (Chai et al., 2005). In particular,
anisakiasis is a zoonosis caused by gastrointestinal (rarely ectopic) parasitism by marine
parasites of the genus Anisakis (Nematoda: Anisakidae) that typically use fish and
cephalopods as intermediate or transport hosts within their life cycle (EFSA-BIOHAZ,
2010; Klimpel et al., 2004; Mattiucci and D’Amelio, 2014). They can cause human
anisakiasis which may be associated with allergic symptoms following the consumption
of raw or lightly cooked fishery products containing live Anisakis. Allergy to Anisakis
can also occur in sensitized individuals, resulting from consumption of fishery products
contaminated with Anisakis allergens (Audicana and Kennedy, 2008; Carballeda-Sangiao
et al., 2016; EFSA-BIOHAZ, 2010; Mattiucci and D’Amelio, 2014).
Approximately 20,000 anisakiasis cases were reported worldwide prior to 2010, of which
over 90% were from Japan, where it is estimated that around 2,000 cases are diagnosed
annually (EFSA-BIOHAZ, 2010). Recently, the annual number of anisakiasis cases in
Spain was estimated to be around 8,000 cases (Bao et al., 2017). There has been a global
increase in the number of epidemiological studies describing Anisakis infection levels in
different commercial fish species (Bao et al., 2013, 2015, Cipriani et al., 2015, 2016;
Gómez-Mateos et al., 2016; Levsen and Karl, 2014; Madrid et al., 2016), as well as the
number of studies reporting new human anisakiasis cases (Amir et al., 2016; Carrascosa
et al., 2015; Del Rey Moreno et al., 2013; Mattiucci et al., 2013; Mladineo et al., 2016;
Muwanwella et al., 2016; Shih-Wei et al., 2015; Shimamura et al., 2016; Sohn et al.,
2015).
Presence of anisakids may reduce the marketability and commercial value of fishery
products due to food safety and quality implications, reducing consumer confidence and
thus provoking economic losses to the fishing industry (Abollo et al., 2001; D’amico et
al., 2014; Llarena-Reino et al., 2015; Mattiucci and D’Amelio, 2014; McClelland, 2002).
For example, in 1987, fish sales dropped 80% and many fishery employees lost their jobs
in Germany after a television broadcast which showed anisakids (Anisakis sp.) crawling
out of fish fillets, leading to a loss of consumer trust (Karl, 2008). It has been estimated
that economic losses due to anisakids in fish flesh among fish processors have reached
101
several millions of dollars (Bonnell (1994) cited in Llarena-Reino et al., 2015). In
addition, inspection procedures to control and remove visible nematodes also introduce
additional costs to the commercial processing (Abollo et al., 2001; Hemmingsen et al.,
1993; Llarena-Reino et al., 2015; McClelland, 2002). The detection and removal of
Pseudoterranova decipiens (Nematoda: Anisakidae) from the flesh of demersal fish
(especially Atlantic cod (Gadus morhua)) has been estimated to cost Atlantic coast
Canadian fish processors a total of $26.6 to $50 million per year, due to downgrading and
discarding of products (McClelland (2002) and references therein). Social concerns
caused by the negative perception of these parasites by consumers have also arisen in a
number of Southern European countries (e.g. Italy and Spain) in the last 20 years
(D’amico et al., 2014; Llarena-Reino et al., 2015 and references therein). In Spain, fishery
operators reported concern about the possible rejection of fishery products caused by
anisakids and other fish parasites and their negative effects on consumer confidence and
business profits (Llarena-Reino et al., 2015).
Recently, experts from FAO/WHO (Food and Agriculture Organization of United
Nations and World Health Organization) ranked anisakids 4th out of 24 food-borne
zoonotic parasites in terms of relevance to international trade (FAO/WHO, 2014). In the
European Union, the Rapid Alert System for Food and Feed (RASFF) reported, between
2010 and November 2016, a total of 289 RASFF notifications of parasitic infestation in
fish and fishery products, of which 234 (81%) were suspected to be Anisakis; 50 (21%)
of them were notified by Spain and 46 (20%) were notified by other countries due to
Anisakis infestation in fishery products from Spain (RASFF, n.d.).
Contingent valuation (CV) is a survey-based methodology for placing monetary values
on non-market resources (Carson, 2000). It measures the willingness to pay (WTP) as
reflected in the stated preferences of survey respondents regarding the use of or the benefit
from a product, service or public good not transacted in the markets (Carson, 2000, 2012).
Several CV studies have been performed worldwide to estimate WTP for a reduction in
the likelihood and severity of fatal and chronic diseases (Basu, 2013; Brandt et al., 2012;
Hadisoemarto and Castro, 2013; Milligan et al., 2010; Udezi et al., 2010; Yasunaga et al.,
2006) as well as to guarantee food safety (Sundström and Andersson, 2009; Wang et al.,
2009; Wang and Huo, 2016). For instance, Basu (2013) has used CV to estimate the WTP
of U.S. adults aged 50 or above for a prescription drug to prevent Alzheimer´s disease,
and Sundström and Andersson (2009) have used CV to estimate Swedish consumers’
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WTP for reducing the risk of infection by Salmonella bacteria in chicken fillets. To the
best of our knowledge, no CV study has been performed to estimate the WTP of
consumers for preventing the presence of anisakid nematodes (or any other fish parasite)
in fishery products.
The goal of this CV study is to provide an understanding of how fish consumers may
value the eradication of Anisakis in fishery products and to determine if WTP varies in
predictable ways with individuals’ perceptions of risks, fish consumption habits and other
socio-demographic characteristics. Consumers’ attitudes regarding the presence of
Anisakis in fishery products as well as their knowledge about anisakiasis and prevention
methods will also be investigated.
5.3 Materials and methods
5.3.1 Survey and sample selection
A questionnaire survey was developed to enable determination of WTP for Anisakis-free
fish by CV. A pilot version of the questionnaire was conducted with 18 staff and students
from the University of Aberdeen. Subsequently, an online version of the questionnaire
was produced and distributed among the partners of the EU project PARASITE, who are
experts on Anisakis and associated human diseases, for discussion and revision.
The online questionnaire (see supplementary material) was tested by project partners,
then finalised and disseminated via the Internet to the general Spanish population. It was
publicised on the website of the EU project PARASITE, on social media, press and radio,
by e-mails sent to professional and personal contacts and by “word of mouth”.
5.3.2 Questionnaire design
The questionnaire comprised a total of 44 questions (i.e. mainly closed questions and few
open-ended questions). In a covering letter the respondents were informed that: 1) the
questionnaire was part of the EU project PARASITE; 2) their responses would be treated
as confidential and anonymous; and 3) only people aged 18 or over should answer the
questionnaire. The questionnaire was organised in sections to gather information about:
1) socio-demographic information about the respondent (sex, age, nationality, education,
job status, occupation, income, etc.); 2) allergy and health status; 3) fish consumption
behaviour (frequency of consumption of fish, fish species consumed, preparation of
fishery products eaten (e.g. fresh, raw), etc.); 4) knowledge about Anisakis (awareness of
Anisakis and how to avoid Anisakis infection); 5) attitudes to the presence of Anisakis in
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fish (e.g. past avoidance of purchase or consumption of fish); 6) WTP for Anisakis-free
fish; 7) attitudes to hypothetical future scenarios regarding the presence of Anisakis in
fish (i.e. future avoidance of purchasing or consuming parasitized fish) and 8) perception
of risk (perceived likelihood of suffering anisakiasis or related allergy in the future).
Immediately before the WTP question (question 23), respondents were informed about:
1) the occurrence of Anisakis in many fish species; 2) how they may accidentally infect
humans; 3) the probability and severity of Anisakis related diseases and 4) how Anisakis
infection can be prevented. This information sheet ended with a compulsory question
(question 22). Respondents had to confirm or deny that they had read the information
provided before they completed the questionnaire. The aim was to provide enough
information to respondents, to allow them to make an informed decision of their WTP for
Anisakis-free fish (Carson, 2000).
5.3.3 Willingness to pay for Anisakis-free fish
The following hypothetical scenario was used to determine the WTP for Anisakis-free
fish (question 23):
‘Suppose that a technology or treatment were discovered that guaranteed that every
Anisakis (and related allergens) would be completely removed from fish, so the treatment
was 100% effective. Suppose further that there are no side-effects on fish quality. Then,
imagine that your favourite fish (e.g. cod) costs 6€ per kilo at the supermarket. What is
the maximum price you would be willing and able to pay for the same fish product free
of Anisakis and their allergens?’.
The method used in this CV survey was based on the “payment cards” approach, in which
respondents select their maximum WTP from a range of fixed amounts (Klose, 1999).
The responses were specified as follows: “I would not buy this product, 0% extra or 6
€/Kg (I will buy it at the stated price (6 €)), 10% extra or 6.60€/Kg, 20% extra or 7.20
€/Kg… to 100% extra or 12 €/Kg or more”.
Those respondents (hereinafter “protesters”) who reported a zero WTP (i.e. those
respondents who responded “I would not buy this product” or “0% extra”) were given the
opportunity by means of a contingency multiple response question (question 23A) to state
their reasons for refusing to buy or not paying extra for the Anisakis-free fish product.
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5.3.4 Data processing
The questionnaire was made available during January-October in 2015 and, in total 729
responses were received. Two responses were largely incomplete and were excluded from
further analysis. In addition, respondents who did not consume fish (question 14, n=2) or
who did not answer the WTP question (question 23, n=80) were excluded. The sample
available for the descriptive analysis thus included 645 responses (see section 5.3.5).
In addition, data from those protesters who were not interested in the Anisakis-free fish
product (n=38), i.e. those who responded in the WTP question (question 23) that they
‘would not buy this product”, were not used for statistical modelling (see section 5.3.6)
because these respondents stated non-interest in the hypothetical product and their
inclusion could have led to unpredictable biases in the results (Sundström and Andersson,
2009).
5.3.5 Data analysis: descriptive statistics
Thirty-three questions were included in the descriptive analysis. A number of questions
(n=11; 18C1-18C10, 18C12) were not analysed here because they formed part of a
parallel risk assessment study (Bao et al., 2017).
In order to analyse fish consumption behaviour of respondents and to estimate the rates
of consumption of raw or lightly cooked fish meals (section 5.4.1.3), it was assumed that
a consumer ate 1, 12 or 52 meals per year where their answers referred to annually,
monthly or weekly fish consumption (questions 16_1 to 16_14; 18A_1 and 18A_2). It
was also assumed that respondents who did not answer questions 16 and 18A consumed
0 fish meals per year. Prior to answering question 18 (i.e. Do you eat raw or lightly cooked
fish?) respondents were informed that raw or lightly cooked fish preparations included
fish that was undercooked, in vinegar or lemon, marinated, smoked, salted, dried, or
served as sushi, sashimi or ceviche. It was assumed that a respondent knew prevention
methods against Anisakis when they answered “Yes” to both “cooking” and “freezing”
options (question 20A). This was based on the view that raw, lightly cooked, marinated
and cold-smoked fish preparations are not sufficient to kill all Anisakis that may be
present in a fish product, while freezing or heat treatments are considered the most
effective methods to guarantee the death of the parasite (EFSA-BIOHAZ, 2010).
105
5.3.6 Statistical analysis: generalised additive modelling
The formulation of the main response variable (WTP) as a percentage value, potentially
transformable to fit a normal distribution, lent itself to the use of ordinary linear regression
or Gaussian generalised additive modelling (GAM). The response variable was recoded
from percentages to a scale of 0 to 10 and transformed (log X+1) to achieve approximate
normality.
Explanatory variables (n=67) were of two general types, categorical and ordinal. Where
the latter had sufficient unique values they could be treated as continuous variables, thus
allowing non-linear effects to be investigated, otherwise they were treated as categorical.
Since there was no expectation that all effects would be linear, the final methodology
adopted for the initial analysis was GAM. Data exploration was performed to detect
outliers, heterogeneity of variance, collinearity, interactions between variables, and
exploration of the relationships between response and explanatory variables, following
the protocol provided by Zuur et al. (2010), in Brodgar 2.7.4 (http://www.brodgar.com/).
The model was fitted by forwards selection, at each round adding the variable which had
the most significant effect and which most reduced the AIC (and normally also led to the
biggest increase in % deviance explained). For further details about the response and
explanatory variables included in the model, see supplementary material.
5.4 Results
5.4.1 Data analysis: descriptive statistics
Unless otherwise stated, all percentages in this section refer to the sample available for
the descriptive analysis (n= 645 respondents).
5.4.1.1 Socio-demographic characteristics
Table 5.1 shows the socio-demographic characteristics of respondents (n= 645) a majority
of whom were female (61%). The modal age class of respondents was between 30 and 49
years old (57%), while 17% of respondents were between 18 and 29 years old and 23%
were between 50 and 64 years old. Almost all respondents were born in Spain and a
majority (63%) were living in Galicia (NW of Spain). Just over half of respondents were
married or cohabiting (54%). Most respondents lived in households with more than 1
adult (87%), and a majority in households with no children (60%). Most had university
education (77%), and were employed or self-employed (75%) with 26% working in
106
science and development. Approximately half of respondents had an annual household
income between 12,001€ and 36,000€ (44%).
Table 5.1. Socio-demographic characteristics of respondents (n= 645), obtained from the
online questionnaire (see supplementary material) disseminated via the Internet to the
Spanish population.
Variable definition Respondents’ choices or
recodifications from questionnaire
Frequency of
responses (%)
Question
number
Gender
Female
Male
Not available
391 (61%)
195 (30%)
59 (9%)
1
Age
18-29 years old
30-49 years old
50-64 years old
65 years and over
Not available
108 (17%)
369 (57%)
148 (23%)
16 (2%)
4 (1%)
2
Nationality
Spanish
Other
628 (97%)
17 (3%)
3
Area of residence
Centre of Spain
Galicia (Community NW Spain)
Cantabrian Sea region
Mediterranean Sea region
Andalucía (Community South of Spain)
Canary Islands
Not available
102 (16%)
407 (63%)
55 (9%)
42 (7%)
18 (3%)
15 (2%)
6 (1%)
4
Marital status
Married or cohabiting
Single
Not available
351 (54%)
247 (38%)
47 (7%)
5
Adults in household 1 adult in household 77 (12%) 6
107
2, 4, 5, 6, 21 or 30 adults in household
Not available
564 (87%)
4 (1%)
Children in household 0 kid in household
1, 2, 3 and 4 kids in household
Not available
389 (60%)
241 (37%)
15 (2%)
7
Education
Primary education
Secondary education
University degree
Not available
15 (2%)
120 (19%)
497 (77%)
13 (2%)
8
Employment status
Employed
Self-employed
Unemployed
Retired
Inactive (e.g. students, domestic activity,
child rearing)
Not available
415 (64%)
71 (11%)
61 (9%)
33 (5%)
54 (8%)
11 (2%)
9
Occupation
Fishing sector
Processing fish sector
Science and development
Human health activities
Education and training
Energy & Water supplies
Extraction of minerals, etc.
Metal goods, engineering, etc.
Other manufacturing industries
Construction
Distribution, hotels, etc.
17 (3%)
11 (2%)
168 (26%)
43 (7%)
75 (12%)
14 (2%)
6 (1%)
26 (4%)
6 (1%)
8 (1%)
34 (5%)
10
108
5.4.1.2 Self-reported health and allergy status
A high proportion (73%) of respondents ranked their health status as good (Table 5.2).
Approximately a third (31%) stated that they had some type of allergy. A contingency
multiple response question (question 13A) was asked to identify the type of allergy.
Overall, 15% (n=96) had allergy to house mites, 0% (n=1) to fish, 1% (n=8) to shrimp,
1% (n=9) to Anisakis, 1% (n=6) to other fish-related food, 4% (n=28) to other food, and
17% (n=110) had other types of allergy.
Table 5.2. Health and allergy status of respondents (n= 645).
Transport & communication
Banking, finance, etc.
Other
Not available
21 (3%)
37 (6%)
165 (26%)
14 (2%)
Income 0€ - 12,000€
12,001€ - 24,000€
24,001€ - 36,000€
36,001€ - 48,000€
48,001€ - 60,000€
60,001€ - 100,000€
100,001€ and above
Not available
65 (10%)
141 (22%)
144 (22%)
99 (15%)
71 (11%)
44 (7%)
8 (1%)
73 (11%)
11
Variable definition Respondents’ choices from
questionnaire
Frequency of
responses (%)
Question
number
Health status Excellent
Good
Fair
Poor
108 (17%)
474 (73%)
57 (9%)
6 (1%)
12
Allergy Yes 199 (31%) 13
109
No
Not available
435 (67%)
11 (2%)
Allergy type (contingency
multi-response question)
13A
Allergy to fish Yes
No
Not available
1 (0%)
633 (98%)
11 (2%)
13A_1
Allergy to shrimp Yes
No
Not available
8 (1%)
626 (97%)
11 (2%)
13A_2
Allergy to other seafood Yes
No
Not available
6 (1%)
628 (97%)
11 (2%)
13A_3
Allergy to Anisakis Yes
No
Not available
9 (1%)
625 (97%)
11 (2%)
13A_4
Allergy to other food Yes
No
Not available
28 (4%)
606 (94%)
11 (2%)
13A_5
Allergy to house dust mites Yes
No
Not available
96 (15%)
538 (83%)
11 (2%)
13A_6
Other type of allergy Yes
No
Not available
110 (17%)
524 (81%)
11 (2%)
13A_7
110
5.4.1.3 Fish consumption behaviour
A majority of respondents (54%) consumed fish 2 or 3 times per week, and fish was most
frequently consumed fresh (84%) (Table 5.3). Respondents rarely ate fish only at
restaurants (2%) and usually ate fish at home (49%) or in both places (48%) (Table 5.3).
The main fish species consumed was hake (Table 5.4). A substantial minority of
respondents consumed raw or lightly cooked fish (40%) (Table 5.3). Overall, a third of
respondents consumed raw or lightly cooked fish at home (33%) and the same proportion
(33%) consumed raw or lightly cooked fish at restaurants (Table 5.3). A low proportion
of consumers (10%) ate raw or lightly cooked fresh fish at home (Table 5.3). Salmon or
trout (35%) followed by tuna (23%) and anchovy (22%) were the main fish species
consumed raw by respondents (Table 5.3). On average, 10 raw or lightly cooked fish
meals were consumed per year, of which 64% were eaten at the respondents’ home (Table
5.5).
Table 5.3. Fish consumption behaviour of respondents (n= 645).
Variable definition Respondents’ choices from
questionnaire
Frequency of
responses (%)
Question
number
Frequency of fish
consumption
1 per month
1 per week
2-3 times per week
4 times or more per week
Not available
28 (4%)
220 (34%)
346 (54%)
45 (7%)
6 (1%)
14
Which kind of fishery
product?
15
Fresh fish Yes
No
539 (84%)
106 (16%)
15_1
Frozen fish Yes
No
339 (53%)
306 (47%)
15_2
Surimi products Yes
No
134 (21%)
511 (79%)
15_3
Other (e.g. canned) Yes 252 (39%) 15_4
111
No 393 (61%)
Which fish species? 16
Frequency of hake
consumption
No hake consumption
Yearly
Monthly
Weekly
Not available
33 (5%)
51 (8%)
315 (49%)
245 (38%)
1 (0%)
16_1
Frequency of anchovy
consumption
No anchovy consumption
Yearly
Monthly
Weekly
Not available
212 (33%)
168 (26%)
219 (34%)
45 (7%)
1 (0%)
16_2
Frequency of monkfish
consumption
No monkfish consumption
Yearly
Monthly
Weekly
Not available
167 (26%)
190 (29%)
240 (37%)
47 (7%)
1 (0%)
16_3
Frequency of megrim
consumption
No megrim consumption
Yearly
Monthly
Weekly
Not available
133 (21%)
97 (15%)
269 (42%)
145 (22%)
1 (0%)
16_4
Frequency of blue whiting
consumption
No blue whiting consumption
Yearly
Monthly
Weekly
Not available
379 (59%)
131 (20%)
112 (17%)
22 (3%)
1 (0%)
16_5
112
Frequency of horse
mackerel consumption
No horse mackerel consumption
Yearly
Monthly
Weekly
Not available
191 (30%)
161 (25%)
226 (35%)
66 (10%)
1 (0%)
16_6
Frequency of cod
consumption
No cod consumption
Yearly
Monthly
Weekly
Not available
123 (19%)
134 (21%)
314 (49%)
73 (11%)
1 (0%)
16_7
Frequency of pollack
consumption
No pollack consumption
Yearly
Monthly
Weekly
Not available
392 (61%)
157 (24%)
85 (13%)
10 (2%)
1 (0%)
16_8
Frequency of mackerel
consumption
No mackerel consumption
Yearly
Monthly
Weekly
Not available
298 (46%)
163 (25%)
141 (22%)
42 (7%)
1 (0%)
16_9
Frequency of sardine
consumption
No sardine consumption
Yearly
Monthly
Weekly
Not available
152 (24%)
230 (36%)
215 (33%)
47 (7%)
1 (0%)
16_10
Frequency of tuna
consumption
No tuna consumption
Yearly
85 (13%)
76 (12%)
16_11
113
Monthly
Weekly
Not available
234 (36%)
249 (39%)
1 (0%)
Frequency of salmon or
trout consumption
No salmon or trout consumption
Yearly
Monthly
Weekly
Not available
152 (24%)
99 (15%)
252 (39%)
141 (22%)
1 (0%)
16_12
Frequency of John dory
consumption
No John dory consumption
Yearly
Monthly
Weekly
Not available
411 (64%)
155 (24%)
56 (9%)
22 (3%)
1 (0%)
16_13
Frequency of consumption
of other fish species
No other fish consumption
Yearly
Monthly
Weekly
Not available
373 (58%)
84 (13%)
118 (18%)
70 (11%)
1 (0%)
16_14
Where do you eat fish? Home
Other (restaurant)
Both
Not available
316 (49%)
14 (2%)
309 (48%)
6 (1%)
17
Raw fish consumption Yes
No
Not available
261 (40%)
380 (59%)
4 (1%)
18
Where and how often?
(contingency multi-
response question)
18A1
114
Frequency of raw fish
consumption at home
No raw consumption at home
Yearly
Monthly
Weekly
Not available
424 (66%)
45 (7%)
120 (19%)
51 (8%)
5 (1%)
18A1_1
Frequency of raw fish
consumption at restaurant
No raw consumption at restaurant
Yearly
Monthly
Weekly
Not available
428 (66%)
83 (13%)
112 (17%)
17 (3%)
5 (1%)
18A1_2
Consumption raw fish at
home
(contingency multi-
response question)
18A2
Consumption already
prepared fish at home
Yes
No
Not available
188 (29%)
450 (70%)
7 (1%)
18A2_1
Consumption raw fresh
fish at home
Yes
No
Not available
67 (10%)
571 (89%)
7 (1%)
18A2_2
Consumption raw frozen
fish at home
Yes
No
Not available
70 (11%)
568 (88%)
7 (1%)
18A2_3
Which fish species to
consume raw?
(contingency multi-
response question)
18B
Raw anchovy consumption Yes
No
144 (22%)
497 (77%)
18B_1
115
Not available 4 (1%)
Raw sardine consumption Yes
No
Not available
16 (2%)
625 (97%)
4 (1%)
18B_2
Raw hake consumption Yes
No
Not available
22 (3%)
619 (96%)
4 (1%)
18B_3
Raw mackerel
consumption
Yes
No
Not available
16 (2%)
625 (87%)
4 (1%)
18B_4
Raw salmon or trout
consumption
Yes
No
Not available
226 (35%)
415 (64%)
4 (1%)
18B_5
Raw tuna consumption Yes
No
Not available
149 (23%)
492 (76%)
4 (1%)
18B_6
Raw monkfish
consumption
Yes
No
Not available
8 (1%)
633 (98%)
4 (1%)
18B_7
Raw cod consumption Yes
No
Not available
33 (5%)
608 (94%)
4 (1%)
18B_8
Raw megrim consumption Yes
No
Not available
1 (0%)
640 (99%)
4 (1%)
18B_9
Raw squid consumption Yes
No
9 (1%)
632 (98%)
18B_10
116
Table 5.4. Estimated fish consumption rate per year of different fish species by
respondents (n= 645).
Not available 4 (1%)
Raw other fish
consumption
Yes
No
Not available
28 (4%)
613 (95%)
4 (1%)
18B_11
Fish species Meals consumed per year
by all respondents
Meals per respondent
and year
Percentage of the total
meals per fish species
Hake 16,571 26 17%
Tuna 15,832 25 16%
Megrim 10,865 17 11%
Salmon or trout 10,455 16 11%
Cod 7,698 12 8%
Horse mackerel 6,305 10 6%
Monkfish 5,514 9 6%
Sardine 5,254 8 5%
Anchovy 5,136 8 5%
Mackerel 4,039 6 4%
Blue whiting 2,619 4 3%
John dory 1,971 3 2%
Pollack 1,697 3 2%
Other 5,140 8 5%
Total meals 99,096 154 100%
117
Table 5.5. Estimated consumption rate (per year) of raw or lightly cooked fish by
respondents, categorised according to location of meal (n= 645).
5.4.1.4 Awareness and knowledge of prevention methods against Anisakis or “worms in
fish”
Most respondents (95%) had previously heard about Anisakis or “cod worms” or “fish
worms” (Table 5.6). Most respondents (82%) reported knowledge of prevention methods,
and subsequently answered a contingency question (question 20A) designed to identify
those who genuinely knew about prevention methods (Table 5.6). More than three
quarters of respondents (78%) were aware that freezing is recommended to prevent
adverse effects for the consumer from Anisakis-infected fish, while 52% were aware that
cooking is recommended. However only 35% correctly identified both prevention
methods against Anisakis (i.e. cooking and freezing). Two respondents (0%) (2 out of 528
who reported knowledge of prevention methods) did not provide any correct answer.
Table 5.6. Awareness of Anisakis or “worms in fish” and knowledge of prevention
methods against them of respondents (n= 645).
Place of
consumption
Raw or lightly cooked
meals consumed per year
by consumers surveyed
Raw or lightly cooked
meals per respondent
and year
Percentage of the total
raw or lightly cooked
meals per place of
consumption
Home 4,137 6 64%
Other places
(e.g. restaurant)
2,311 4 36%
Total raw meals 6,448 10 100%
Variable definition Respondents’ choices from
questionnaire
Frequency of
responses (%)
Question
number
Awareness of Anisakis Yes
No
Not available
612 (95%)
27 (4%)
6 (1%)
19
Knowledge of Anisakis
prevention methods
Yes 528 (82%) 20
118
5.4.1.5 Past behaviour of respondents regarding the presence of Anisakis in fish
More than a quarter (27%) of respondents reported that they had on at least one occasion
stopped consuming or purchasing fish due to the presence of Anisakis (Table 5.7). Again,
in order to determine which fish species were avoided, a contingency multiple response
question (question 21A) was asked, with hake (19%) being the fish species most
frequently avoided by the respondents (Table 5.7).
No
Not available
104 (16%)
13 (2%)
Prevention methods
(contingency multi-
response question)
20A
Raw or lightly cooked fish Yes
No
Not available
2 (0%)
629 (98%)
14 (2%)
20A_1
Gutting the fish Yes
No
Not available
94 (15%)
537 (83%)
14 (2%)
20A_2
Cooking the fish Yes
No
Not available
333 (52%)
298 (46%)
14 (2%)
20A_3
Marinating or smoking
fish
Yes
No
Not available
9 (1%)
622 (96%)
14 (2%)
20A_4
Freezing the fish Yes
No
Not available
505 (78%)
126 (20%)
14 (2%)
20A_5
119
Table 5.7. Previous avoidance of fish due to the presence of Anisakis of respondents (n=
645).
Variable definition Respondents’ choices from
questionnaire
Frequency of
responses (%)
Question
number
Historical fish avoidance
due to Anisakis
Yes
No
Not available
176 (27%)
410 (64%)
59 (9%)
21
Main fish species avoided
(contingency multi-
response question)
21A
Historical hake avoidance Yes
No
Not available
122 (19%)
464 (72%)
59 (9%)
21A_1
Historical cod avoidance Yes
No
Not available
18 (3%)
568 (88%)
59 (9%)
21A_2
Historical Atlantic pomfret
avoidance
Yes
No
Not available
49 (8%)
537 (83%)
59 (9%)
21A_3
Historical sardine
avoidance
Yes
No
Not available
4 (1%)
582 (90%)
59 (9%)
21A_4
Historical salmon or trout
avoidance
Yes
No
Not available
3 (0%)
583 (90%)
59 (9%)
21A_5
Historical monkfish
avoidance
Yes
No
Not available
9 (1%)
577 (89%)
59 (9%)
21A_6
120
5.4.1.6 Willingness to pay for Anisakis-free fish
The majority of respondents (94%) confirmed having read the information sheet about
Anisakis and their related human diseases prior to responding to the WTP question (Table
5.8). The modal WTP selected by 26% of respondents was 10% extra or 6.60 €/Kg (Figure
5.1). Around three quarters (77%) of respondents were willing to pay extra for Anisakis-
free product. The distribution of the WTP variable is skewed to the right (Figure 5.1),
with a median WTP of 20% extra and an interquartile range of 10% to 30% extra.
Among protesters (n=146), a majority (67%) considered that there should not be an
additional fee for safe food, 22% reported “other reasons” for being unwilling to pay
extra, 14% considered that the risk of negative consequences of Anisakis was too small
to warrant any extra price for the product, and 7% considered that the proposed scenario
was unrealistic. An open-ended question was available for those protesters who stated
“other reasons” to explain their view. They stated two main views: 1) they would freeze
Historical tuna avoidance Yes
No
Not available
4 (1%)
582 (90%)
59 (9%)
21A_7
Historical blue whiting
avoidance
Yes
No
Not available
32 (5%)
554 (86%)
59 (9%)
21A_8
Historical megrim
avoidance
Yes
No
Not available
11 (2%)
575 (89%)
59 (9%)
21A_9
Historical mackerel
avoidance
Yes
No
Not available
6 (1%)
580 (90%)
59 (9%)
21A_10
Historical anchovy
avoidance
Yes
No
Not available
29 (4%)
557 (86%)
59 (9%)
21A_11
121
or cook fish instead of buying Anisakis-free fish (13 out of 32 protesters) or 2) they
distrusted the technology/treatments to produce Anisakis-free fish (8 out of 32 protesters).
Table 5.8. Willingness to pay for Anisakis-free fish by respondents (n= 645).
Variable definition Respondents’ choices from
questionnaire
Frequency of
responses (%)
Question
number
Have you read the
information sheet?
Yes
No
605 (94%)
40 (6%)
22
Willingness to pay for
Anisakis free-fish
I would not buy this product
0% extra: 6€ €/Kg
10% extra: 6.60 €/Kg
20% extra: 7.20 €/Kg
30% extra: 7.80 €/Kg
40% extra: 8.40 €/Kg
50% extra: 9 €/Kg
60% extra: 9.60 €/Kg
70% extra: 10.20 €/Kg
80% extra: 10.80 €/Kg
90% extra: 11.40 €/Kg
100% extra: 12 €/Kg or more
38 (6%)
108 (17%)
170 (26%)
141 (22%)
66 (10%)
21 (3%)
56 (9%)
5 (1%)
9 (1%)
2 (0%)
3 (0%)
26 (4%)
23
Protesters (No interest in
product or not WTP extra)
(contingency multi-
response question)
23A
The risk is too small Yes
No
Willing to pay for the product
20 (3%)
126 (20%)
499 (77%)
23A_1
The scenario in unrealistic Yes
No
10 (2%)
136 (21%)
23A_2
122
Figure 5.1. Distribution of the willingness to pay, by Spanish fish consumers, for fish
free of Anisakis and their related allergens.
5.4.1.7 Future behaviour of respondents regarding the presence of Anisakis in fish
Over a quarter (29%) of respondents would always stop consuming or purchasing fish
due to the presence of Anisakis or worms (Table 5.9). A similar proportion (31%) would
cease buying or eating fish if there was a high chance of worms being present, whilst
more than a third (38%) would not avoid fish purchasing or consumption, because they
consider that cooking or freezing would kill any worm present (Table 5.9). A contingency
single response question (question 24A) was subsequently asked to those respondents
(n=386) who stated that they would cease consuming or purchasing fish due to Anisakis
to elicit their reasons. Health risk (45%) or both health risk and aesthetic issues (i.e.
Willing to pay for the product 499 (77%)
Not paying extra for safe
food
Yes
No
Willing to pay for the product
98 (15%)
48 (7%)
499 (77%)
23A_3
Other
(open-ended question)
Yes
No
Willing to pay for the product
32 (5%)
114 (18%)
499 (77%)
23A_4
123
fishery products with Anisakis are not appetising for respondents) (48%) were indicated
as their main reasons.
Table 5.9. Stated preference by respondents (n= 645) regarding future consumption of
fish, considering the presence of Anisakis.
5.4.1.8 Risk of development of anisakiasis or allergy to Anisakis
The majority of respondents (86%) perceived that the risk of suffering disease in the
future was small or moderate (Table 5.10). A low proportion (5%) stated their chances of
suffering disease as high or absolute (Table 5.10).
Table 5.10. Perceived risk of development of anisakiasis or allergy to Anisakis by
respondents (n= 645).
Variable definition Respondents’ choices from
questionnaire
Frequency of
responses (%)
Question
number
Future fish avoidance due
to Anisakis
Yes, always
Yes, but only if high chance of worms
No, cooking or freezing kill worms
Not available
187 (29%)
199 (31%)
244 (38%)
15 (2%)
24
Reasons future avoidance
due to Anisakis
(contingency single-
response question)
Health risk
Unappetising
Both
Other
No, cooking or freezing kill worms
Not available
174 (27%)
22 (3%)
186 (29%)
4 (1%)
244 (38%)
15 (2%)
24A
Variable definition Respondents’ choices from
questionnaire
Frequency of
responses (%)
Question
number
Self-perceived risk of
future development of
anisakiasis or allergy
No chance
Slight chance
Moderate chance
44 (7%)
384 (60%)
167 (26%)
25
124
5.4.2 Data analysis: generalised additive models for WTP
The results from the first round of GAMs (models with single explanatory variables)
indicated that the WTP for Anisakis-free fish was significantly associated (p<0.05) with
eight explanatory variables, of which the most highly significant was the stated future
avoidance by respondents of eating or purchasing fish due to presence of Anisakis
(p=0.0018) (Table 5.11). The respondents who indicated that they would cease eating or
purchasing fish in the future due to presence of Anisakis were willing to pay more than
those who did not say they would cease eating fish. Respondents who perceived a higher
risk of suffering adverse effects of ingesting Anisakis or their allergens (i.e. anisakiasis or
allergy to Anisakis) in the future were willing to pay more. Respondents who had avoided
eating cod, hake, mackerel or fish in general in the past were willing to pay more than
those who had not avoided eating these species. Respondents who did not consume
sardines were willing to pay more than those who consumed this fish species. Women
were willing to pay more than men. No significant relationship (p≥ 0.05) was observed
for age, nationality, area of residence, marital status, number of household members,
education, job status, occupation, income, health and allergy status, frequency, kind and
place of fish consumption, raw or lightly cooked fish consumption, or awareness and
prevention knowledge of Anisakis.
Table 5.11. Effects of individual explanatory variables on WTP (the coefficient indicates
the direction of the effect). Showing only variables with significant effects in models with
single explanatory variables (p≤ 0.05).
High chance
Absolutely chance
Not available
22 (3%)
11 (2%)
17 (3%)
Variable definition Type Effect
(coefficient)
p AIC N Question
number
Avoidance (future)
(1- Yes, always; 2- Yes,
but only if high chance
of worms; 3- No,
Categorical Negative
(-0.07170
-0.09690)
0.00184 167.66 598 24
125
The final best model, included three of the variables mentioned above (i.e. gender,
avoidance of cod consumption in the past, avoidance of fish consumption/purchase in the
cooking or freezing kill
worms)
Avoidance cod (past)
(1- Yes, 2- No)
Categorical Negative
(-0.20555)
0.00218 159.26 552 21A_2
Avoidance hake (past)
(1- Yes, 2- No)
Categorical Negative
(-0.08681)
0.00328 160.02 552 21A_1
Gender
(1- Female, 2- Male)
Categorical F > M
(-0.06071)
0.0161 160.58 550 1
Anisakiasis/Allergy
risk
(0- no chance, 1- slight
chance, 2- moderate
chance, 3- high
chance, 4- absolute
chance)
Continuous
(k=4)
Positive 0.0192 177.21 591 25
Avoidance (past)
(1- Yes, 2- No)
Categorical Negative
(-0.05820)
0.0259 163.71 552 21
Avoidance mackerel
(past)
(1- Yes, 2- No)
Categorical Negative
(-0.2529)
0.028 163.85 552 21A_10
Sardine consumption
(0- No sardine
consumption, 1-
Respondents who
answered: Yearly,
Monthly, and Weekly)
Categorical Negative
(-0.05685)
0.0327 173.27 606 16_10
Income Continuous
(k=4)
0.0543 155.64 540 11
126
future) but also included associations with (a) whether respondents ate raw or lightly
cooked fresh fish at home, and, raw or lightly cooked hake, and (b) the allergy status of
the respondent (Table 5.12). Respondents who indicated that they did not have allergy
were willing to pay more than those who had allergy. Respondents who ate raw or lightly
cooked hake were willing to pay more than those who did not consume this fish
preparation. Respondents who ate raw or lightly cooked fresh fish at home were willing
to pay less than those who did not.
Table 5.12. Summary of best model for WTP identified by forwards selection. The table
gives the coefficient, indicating the direction of the effect, and the probability for each
significant effect, plus the sample size (N), % of deviance explained (%DE) and the AIC
value.
Variable definition Type Coefficient p Question
number
Gender (1-Female, 2-Male) Categorical -0.06640 0.01266 1
Allergy (1-Yes, 2-No) Categorical 0.05514 0.04044 13
Consumption raw fresh fish at home
(0- Yes, 1-No)
Categorical 0.10225 0.01832 18A2_2
Raw hake consumption (1- Yes, 2- No) Categorical -0.19819 0.00662 18B_3
Avoidance cod (past)
(1- Yes, 2- No)
Categorical -0.20685 0.00258 21A_2
Avoidance (future)
(1- Yes, always; 2- Yes, but only if high
chance of worms; 3- No, cooking or
freezing kill worms)
Categorical -0.07770
-0.07889
0.01513
0.01078
24
N 484
%DE 7.4%
AIC 128.98
127
5.5 Discussion
This is the first time a specific survey has been performed to analyse perceptions and
attitudes of fish consumers regarding the presence of the parasitic nematode Anisakis in
fishery products as well as their WTP to pay for Anisakis-free fish.
5.5.1 Consumer awareness of the presence of Anisakis in fish and raw fish
consumption behaviour
The results indicated that most respondents had heard about Anisakis or worms in fish
before completing the questionnaire, even though fewer of them were aware of prevention
methods. The consumption of raw or lightly cooked fish was relatively common (40%),
and additionally, a low proportion (10%) of respondents utilised fresh (not previously
frozen) fish when preparing raw or lightly cooked fish at home. Thus, both the lack of
knowledge about Anisakis prevention methods as well as some potentially risky eating
behaviours (i.e. consumption of raw fish that was not previously frozen) by a number of
individuals, suggest that there would be a significant risk of anisakiasis and allergic
episodes for some Spanish fish consumers, which is supported by a large number of
publications (Amo Peláez et al., 2008; Audicana and Kennedy, 2008; Caballero et al.,
2012; Carballeda-Sangiao et al., 2016; Carrascosa et al., 2015; Daschner et al., 2005,
2000; Del Rey Moreno et al., 2013; Jurado-Palomo et al., 2010; Puente et al., 2008). Bao
et al. (2017) recently assessed the risk of anisakiasis for Spanish fish consumers due to
ingestion of non-previously frozen raw and marinated anchovies, using quantitative risk
assessment, estimating a total annual number of anisakiasis cases between 7,700 and
8,320 and concluding that anisakiasis is a highly underreported and underdiagnosed
disease in Spain.
5.5.2 Purchase and consumption behaviour of consumers in relation to the presence
of Anisakis in fish
Results showed that more than one quarter of consumers who responded to the survey
had ceased, on at least one occasion, purchasing or consuming fish due to the presence of
Anisakis. Amongst respondents, hake was both the most frequently consumed species
(Table 5.4) and the most frequently avoided fish species (Table 5.7). European hake can
be infected with hundreds of Anisakis (Llarena-Reino et al., 2013; Tejada et al., 2014)
meaning that the presence of these parasites is likely to be observed by consumers. Thus,
it appears that the stated past avoidance of hake by consumers is the consequence of its
high level of Anisakis infection. In fact, it has been reported that hake was the species
128
associated with the highest number of complaints by consumers to fish sellers in Galicia
about the presence of anisakids (Llarena-Reino et al., 2015). Interestingly, Atlantic
pomfret (Brama brama) was the second most frequently rejected fish species in the
present study (Table 5.7) as well as being the second most frequent cause of complaints
by consumers to fish sellers in Galicia (Llarena-Reino et al., 2015). To the best of our
knowledge, there is no evidence of Anisakis infection in Atlantic pomfret in the Northeast
Atlantic Ocean, but the presence of large cestodes (Gymnorhynchus spp.) in pomfret
muscle is well known and is targeted during veterinary inspections (Llarena-Reino et al.,
2015). Therefore, it is possible that there is a misunderstanding among consumers (and
fish sellers) about what it is an Anisakis and how to distinguish it from other fish parasites.
It also appears that some consumers would avoid fish and/or make complaints if their fish
were contaminated with any kind of visible parasites.
Nearly two thirds of respondents (60%) would cease buying or eating fish, to a greater or
lesser extent, due to the presence of Anisakis. Clearly, such behaviour could result in
economic losses to the fishing industry, as was previously observed in Germany during
the “nematode crisis” in 1987 (Karl, 2008). Health risk and aesthetic aspects (i.e. aversion
to fish due to the presence of Anisakis), were stated by respondents as the main reasons
for not buying fish. D’amico et al. (2014) reviewed the evolution of Anisakis risk
management in Europe, in particular Italy, and concluded that the presence of anisakids
in fishery products represents a public health issue and causes loss of product quality,
making it repugnant to the consumer.
5.5.3 WTP for Anisakis-free fish
A clear majority of respondents (77%) were willing to pay more for fish free from
Anisakis and its allergens. The modal additional amount was 10% (or 6.60€/Kg rather
than 6.00€/Kg) for the product. These findings were similar to those reported for a study
by Wang et al. (2009), in which the majority of respondents (79%) were willing to pay a
premium at retail for safer fish after implementation of a traceability system in China. In
the Chinese study, on average, respondents were willing to pay 6% extra for the safe fish
product over the price of non-traced products of uncertain safety. It can be suggested then
that, despite the fact that most consumers are willing to pay for safer fish, some of them
would pay a relatively low extra price.
129
Results of the GAM analysis indicated that several categories of respondents were willing
to pay more for fish free from Anisakis; those who had previously avoided or would in
future avoid fish consumption or purchase due to the presence of Anisakis, respondents
who ate raw or lightly cooked hake, and those who perceived themselves to be more at
risk from adverse effects of Anisakis. Women were also willing to pay more than men.
This may suggest that these individuals are more concerned about negative effects (health
and/or aesthetic issues) of Anisakis. Those respondents who had allergies were willing to
pay less than those who did not have allergies, which may suggest that the latter would
be willing to pay to maintain their non-allergic status, whilst those who already had
allergy would be less concerned.
Respondents who ate raw fresh fish at home were less willing to pay than those who did
not report this consumption behaviour. The former were more likely to be exposed to
viable Anisakis and consequently to disease. It could be suggested that they do not care
about Anisakis and their related diseases, however further investigation will be needed.
Respondents who did not consume sardines were willing to pay more than those who
consumed this fish species, however we do not have a plausible explanation for this
relationship, and again further research is required. To sum up, the GAM modelling
identified greater WTP for Anisakis-free fish among female consumers, consumers who
already ceased and would cease to eat or purchase fish due to Anisakis presence, people
with no allergies and people who perceived themselves to have higher risk of suffering
disease in future.
Overall, consumers revealed two general kind of attitudes to the presence of Anisakis in
fish: 1) WTP for Anisakis-free fish to avoid adverse effects (i.e. considering health risks
and aesthetic aspects) and 2) avoidance of eating or purchasing parasitized fish. Thus,
consumer WTP clearly depends on the perception of the risk associated with Anisakis in
fish, and this finding is consistent with other WTP studies. Sundström and Andersson
(2009) found a positive relationship between WTP to reduce the risk of Salmonella in
chicken fillets and concern of consumers about food-related diseases. In addition, Basu
(2013) reported that, on average, a higher WTP for a drug that prevents Alzheimer´s
disease was observed in respondents with higher perceived risks of developing the disease
in the future.
130
The findings suggest that the presence of anisakids in fish presents two main issues for
the Spanish fishing industry. First is the loss of income due to the attitudes of consumers
towards parasitized fishery products. The second is that food business operators (FBOs)
are required to satisfy EU legislation and ensure the protection of consumers with regard
to the food safety of fishery products (EC, 2004). In particular, EC Regulation 853/2004
states that “food business operators must not place fishery products that are obviously
contaminated with parasites on the market for human consumption” (EC, 2004). In
relation to this, respondents indicated that hake was the most frequently avoided fish
species due to the presence of Anisakis. Although it is not currently possible to completely
eliminate Anisakis from fish destined for human consumption, the problem needs to be
addressed by the fishing industry and official inspection services. For instance, the
improvement of parasite detection monitoring, and/or the development of new technology
that can help guarantee fish free of Anisakis should be pursued.
The idea that their food might contain Anisakis, or worms, tends to evoke a strong reaction
in consumers. Indeed, a likely outcome of increased awareness of anisakids is a reduction
in consumption of parasitized fish, as was evident from some responses to the
questionnaire (and the aforementioned German example). Ultimately, public perception
of risk has components that could be described as irrational in that they are not entirely
predictable from the probability of a hazardous event occurring. The perceived
seriousness of a risk depends, for example, on moral and social values (Lee, 1981). There
is, thus, no substitute for empirical data on perceived risk as a basis for a risk management
strategy. Equally, it is important to consider that the framing of a questionnaire can
influence the responses and great care was therefore taken in framing the questions here.
5.5.4 WTP protesters
It is common in contingent valuation studies to have a proportion of respondents
providing “protest” statements. For example disagreeing with the payment vehicle and
scenario, raising ethical concerns or believing that the product should be provided by
alternative means (Sundström and Andersson, 2009). Sundström and Andersson (2009)
reported that 43% of Swedish protesters (i.e. those who were unwilling to pay for safer
chicken) considered that the risk of suffering salmonellosis by consumption of chicken
fillets was too small to motivate any price differences, 34% considered the scenario to be
unrealistic, 11% provided the view that chicken fillets should be safe and 11% had other
motivations. In the present study, 14% of protesters viewed the risk of adverse effects
131
from Anisakis in fish as too small to pay any premium, 7% considered that the scenario
was unrealistic, 67% considered that one should not have to pay any premium for having
safe food, and 22% gave other reasons (mainly, 1) freezing and cooking is safe for
Anisakis and 2) lack of confidence in the technology to obtain the Anisakis-free product).
5.5.5 Study limitations
This questionnaire survey was performed with the aim of being representative of Spanish
fish consumers. However, 63% of the respondents were from Galicia, 77% had a
university degree and 26% had a job in science or development sector, characteristics
which are not representative for the whole of Spain (Spanish Statistical Office). GAM
results suggest that these biases are not important since area of residence, education level
and occupation were not related with WTP. However, additional data from other regions
and sectors may increase the robustness of the results.
Finally, we should highlight that respondents were self-selecting and those with
knowledge of or interest in the subject may well have been more likely to respond. This
should not be taken to indicate that there is less public concern about Anisakis than
suggested by our study, and certainly not that public concern could become an issue in
the future.
5.6 Conclusions
Overall, the results suggest that the zoonotic fish parasite Anisakis is of concern for a
majority of Spanish fish consumers due to food safety and quality issues. The results
suggest two main types of responses of consumers with regard to fishery products
parasitized by Anisakis; 1) to cease consuming parasitized fish, 2) a willingness to pay
extra price for Anisakis-free fish. In relation to this, the results suggest that a majority of
consumers would be willing to pay extra for such product, indicating a modal willingness
to pay of 10% above the usual fish price at market (6,60€/Kg compared with 6€/Kg).
Finally, these findings provide further evidence about the social issues and subsequent
potential economic losses, in addition to the legislative and public health implications,
caused by the presence of Anisakis in fishery products, and this is of special interest for
the fishing and food industries, food safety authorities and official inspection services.
5.7 Acknowledgements
We thank our partners of the PARASITE project (EU FP7 PARASITE project (GA no.
312068)), for their help during the creation of the questionnaire. We thank Pablo from
132
CETMAR for his help during electronic implementation of questionnaire. Miguel Bao is
supported by a PhD grant from the University of Aberdeen and also by financial support
of the contract from the EU Project PARASITE (grant number 312068). Ethical approval
from the University of Aberdeen for the dissemination of the questionnaire was obtained.
5.8 Authors’ contributions
MB produced the questionnaire, analysed the results and drafted the paper. GJP, NS, IT,
CM and RF contributed to questionnaire creation and divulgation. GJP, NS and IT helped
analysing results and drafting the paper. All authors critically reviewed the paper and
agreed the final content of the version to be published.
133
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Chapter 6 - Assessing the risk of an emerging zoonosis of worldwide
concern: anisakiasis
6.1 Abstract
Anisakiasis is an emerging zoonosis caused by the fish parasitic nematode Anisakis. Spain
appears to have the highest reported incidence in Europe and marinated anchovies are
recognised as the main food vehicle. Using data on fishery landings, fish infection rates
and consumption habits of the Spanish population from questionnaires, we developed a
quantitative risk assessment (QRA) model for the anchovy value chain. Spaniards were
estimated to consume on average 0.66 Anisakis per untreated (non-frozen) raw or
marinated anchovy meal. A dose-response relationship was generated and the probability
of anisakiasis was calculated to be 9.56×10-5 per meal, and the number of annual
anisakiasis cases requiring medical attention was predicted between 7,700 and 8,320.
Monte Carlo simulations estimated post-mortem migration of Anisakis from viscera to
flesh increases the disease burden by >1000% whilst an education campaign to freeze
anchovy before consumption may reduce cases by 80%. However, most of the
questionnaire respondents who ate untreated meals knew how to prevent Anisakis
infection. The QRA suggests that previously reported figures of 500 anisakiasis per year
in Europe is a considerable underestimate. The QRA tool can be used by policy makers
and informs industry, health professionals and consumers about this underdiagnosed
zoonosis.
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6.2 Introduction
Most emerging infectious diseases are of zoonotic origin and consequently involve spill-
over from animal to human populations (Taylor et al., 2001; Wolfe et al., 2007). A number
of factors can contribute to the emergence of an infectious disease, such as ecological
changes (including those due to economic development and agricultural land use or
changes in marine activity), human demographics and behaviour, international travel and
commerce, technology and industry, microbial adaptation/change, and breakdown in
public health measures (Morse, 1995).
Anisakis spp. are nematode parasites found in a wide range of marine organisms. Their
life cycle ecology involves cetaceans as final hosts, zooplankton as intermediate hosts
and fish and cephalopods as intermediate or paratenic hosts (i.e. transport host in which
survival but no larval development occurs, even though this phase may be crucial for
successful transfer of the parasite to the definitive host and completion of its life cycle
(EFSA-BIOHAZ, 2010)) (EFSA-BIOHAZ, 2010; Mattiucci and D’Amelio, 2014).
Within the Anisakis spp. life cycle, humans may become accidental hosts in which the
parasite can survive for a short period of time but cannot reproduce (Audicana and
Kennedy, 2008; Pravettoni et al., 2012). Humans become infected by eating raw or
undercooked fish that contain viable Anisakis spp. third stage larvae (Audicana and
Kennedy, 2008). The ingestion of live Anisakis spp. larvae may cause human disease (i.e.
anisakiasis). The severity of anisakiasis varies from mild to severe and can have gastric,
intestinal, ectopic and allergic forms (Audicana and Kennedy, 2008; Baird et al., 2014;
EFSA-BIOHAZ, 2010; Pravettoni et al., 2012). The disease is thought to be frequently
misdiagnosed and underdiagnosed since symptoms of anisakiasis are usually not specific
(Carrascosa et al., 2015; EFSA-BIOHAZ, 2010; Kim et al., 2013; Toro et al., 2004), with
rare outbreaks (Cabrera, 2010) and person to person transmission non-feasible.
The genus Anisakis comprises nine species of which two (A. simplex s.s. and A. pegreffii)
have been confirmed as zoonotic pathogens (Mattiucci and D’Amelio, 2014). Anisakiasis
is an emerging human health problem and is also of economic concern because of the
potential negative effects on consumer confidence and the marketability problems
associated with infested fishery products (Baird et al., 2014; D’amico et al., 2014; EFSA-
BIOHAZ, 2010; Llarena-Reino et al., 2015; Mattiucci and D’Amelio, 2014). Recent
increases in medical case reports of anisakiasis have been observed in a number of
countries around the world, and may be due to improved public health diagnoses (i.e.
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improved techniques and expertise), and/or behaviour change through increasing global
demand for seafood and a growing preference for raw or lightly cooked food, especially
in many Western countries (EFSA-BIOHAZ, 2010; Llarena-Reino et al., 2015). The
European Food Safety Authority (EFSA) Panel on Biological Hazards (BIOHAZ)
reported approximately 20,000 anisakiasis cases worldwide prior to 2010, with >90%
from Japan (EFSA-BIOHAZ, 2010). In Europe, Spain is considered to have the highest
incidence of anisakiasis, predominantly through consumption of the traditional marinated
dish “anchovies in vinegar” (EFSA-BIOHAZ, 2010). However the actual anisakiasis
burden in the human population is unknown because of the scarcity of epidemiological
data (EFSA-BIOHAZ, 2010). There is a need to determine and understand the burden of
disease associated with this zoonotic nematode in the human population and to identify
measures to reduce its incidence.
European anchovy (Engraulis encrasicolus), which is the anchovy species usually
consumed in Spain, is a small pelagic (i.e. living in the water column) marine fish which
tends to aggregate in large shoals, especially near the coast (Whitehead et al., 1988). Its
distribution in the Eastern North and Central Atlantic Ocean extends from the North Sea
to South Africa and it is also found throughout the Mediterranean and in the Black and
Azov Seas (Whitehead et al., 1988). This species is of high economic interest in Spain
with total capture production of 36,148 t reported in 2013 (Food and Agriculture
Organization of the United Nations (FAO)) (FAO, 2016). Imports of fresh or refrigerated
anchovy into Spain derive mainly from Italy, France and Morocco (totalling 8,757 t. in
2013) (Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA, pers.
comm.)). There are also exports from Spain, mainly to Italy and Morocco (2,949 t in 2013)
(MAGRAMA, pers. comm.). Spanish production, imports and exports can vary
considerably year on year (MAGRAMA, 2013). Anisakis simplex s.l. (probably belonging
to A. simplex s.s. and A. pegreffii, since mixed infection of both Anisakis species are
commonly found in fish species inhabiting the Iberian Atlantic coast (Mattiucci and
D’Amelio, 2014)) and A. pegreffii have been reported in European anchovy from the Gulf
of Cádiz and Strait of Gibraltar (Iberian Atlantic coast) (Rello et al., 2009), and from
Mediterranean Sea (Cipriani et al., 2016; Šimat et al., 2015), respectively, and their
prevalence and intensity of infection in this fish may vary depending on the fishing area
and season (Cipriani et al., 2016; Rello et al., 2009; Šimat et al., 2015).
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Quantitative risk assessment (QRA) is a science-based methodology that estimates the
probability and severity of an adverse event (Cassin et al., 1998), e.g. health risk to
individuals or populations due to exposure of zoonotic parasites through ingestion of
contaminated fish meals. Used alongside Monte Carlo simulation methods, QRA
estimates the human health risk simulating the uncertainty (lack of knowledge) and
variability of the associated model parameters (Vose, 2000). The process involves four
main stages (Codex Alimentarius Commision, 1998; European Commision, 1997):
(1) Hazard identification: identifies the pathogen (e.g. Anisakis spp.) of concern,
determines whether it is actually a hazard, and identifies the vehicle of
transmission (e.g. raw and marinated anchovies).
(2) Exposure assessment: determines the number of Anisakis spp. ingested per meal
(i.e. the dose).
(3) Hazard characterization: gives a quantitative or qualitative assessment of the
adverse effects of the pathogen on humans; more specifically a dose-response
model can be implemented, which mathematically models the response (i.e. the
impact and its variability) following exposure to different doses.
(4) Risk characterization: gives a probability of occurrence of the disease (e.g.
anisakiasis) and estimates the disease burden in a given population.
Quantitative microbiological risk assessment has been performed to estimate health risk
in humans from exposure to microbes, e.g. Escherichia coli O157 from eating beef
burgers (Cassin et al., 1998) and from recreational use of animal pasture (Strachan et al.,
2002), Cryptosporidium parvum in drinking water (Gale, 1998) and Listeria
monocytogenes in smoked salmon and trout (Lindqvist and Westöö, 2000). A number of
studies have been carried out to determine the levels of Anisakis spp. infection in fish
species and then assess the food safety implications of these findings (Bao et al., 2015,
2013; Cipriani et al., 2016; Rello et al., 2009; Šimat et al., 2015). However, to date no
QRA for Anisakis spp. (or any other marine zoonotic parasite) in a fish meal is available.
The present study aims to integrate data obtained from social science methods
(questionnaires and economic surveys) and from the natural sciences (fish parasite
sampling surveys and infection rates in humans) in a process-based QRA model (Cassin
et al., 1998). This is then used to determine the probability of disease (i.e. anisakiasis)
142
caused by consumption of untreated (i.e. not previously frozen) raw and marinated
anchovy meals prepared at home in Spain (hereinafter: untreated anchovy meals). The
burden of disease for the whole Spanish population will then be estimated, and the effects
of hypothetical scenarios, i.e. factors that can increase risk (post-mortem migration of
Anisakis spp. from fish viscera to muscle) and interventions to decrease risk (public health
education campaign).
Since this is a QRA study it does not follow the typical steps of a scientific paper (Cassin
et al., 1998; Lindqvist and Westöö, 2000; Strachan et al., 2002), as it gives methods,
results and discussion through the steps of the QRA section. This is done for ease of
interpretation of the QRA model.
6.3 QRA Section
6.3.1 Materials and methods
6.3.1.1 Value chain of anchovies
The value chain for anchovies, from sea to consumption, was characterised and from this
a process model was generated which incorporated the four steps of a risk assessment,
i.e. hazard identification, exposure assessment, hazard characterization and risk
characterization (Figure 6.1). The QRA model was implemented in Microsoft Excel™
using @RISK software (Palisade, UK) and parameterized as detailed in Tables 6.1 and
6.2.
6.3.1.2 Data collection
6.3.1.2.1 Anisakis spp. infection descriptors in European anchovy and anchovy
biometrics
Parasite infection parameters (i.e. prevalence and number of Anisakis spp. in anchovy
muscle (intensity of infection)) and other data on the fish (i.e. fishing area and total weight
of fish caught) were obtained from our own sampling and from the literature (Rello et al.,
2009) (see Table 6.3 for further details):
European anchovies (n=3,799) caught in FAO areas 37 (Mediterranean and Black Sea)
and 27 (Atlantic, Northeast) (areas 1 and 2 respectively in the model) were immediately
frozen after capture and later inspected for Anisakis spp. in muscle. The methodology of
parasite inspection followed Karl and Leinemann (1993). Briefly, after thawing, anchovy
were filleted and the fillets were pressed and then frozen prior to further examination.
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Visual inspection was then performed on thawed anchovy fillets using a 366 nm UV-light
source, which caused dead Anisakis spp. larvae present in the fillets to fluoresce.
Data on Anisakis spp. infection of anchovy from FAO area 34 (Atlantic, Eastern Central
(area 3 in the model)) were obtained from Rello et al. (2009) (Table 6.3). The number of
Anisakis spp. larvae found in the muscle of each fish was estimated from Rello et al.
(2009) as follows: the prevalence of infection of the 396 anchovies inspected was 13.13%,
hence 52 anchovies were infected. The number of larvae in the muscle of fish was
calculated to be 67 by multiplying the number of anchovies with muscle infection (52)
by the mean intensity (1.28). Since the intensity range was from 1 to 4 larvae (see Table
1 Rello et al. (2009)), it was then possible to estimate the numbers and hence the
distribution of Anisakis spp. in the flesh of each fish (Table 6.4). Of the 396 anchovies
inspected, 344 anchovies had no Anisakis spp. larva in the muscle, 42 had 1 larva, 6 had
2, 3 had 3 and 1 had 4 (Table 6.4).
6.3.1.2.2 Anchovy consumption habits of the Spanish population
Consumption data from questionnaires: Online questionnaires (see supplementary
materials) were made available to the Spanish population in order to gather information
about anchovy consumption habits (questionnaire 1), and to estimate the number of fillets
eaten by Spanish consumers in each anchovy in vinegar meal (questionnaire 2).
Questionnaire 1 was advertised on social media, the website of the EU FP7 PARASITE
project (GA no. 312068), websites of research institutions and regional government of
the autonomous community of Galicia (NW Spain); in the local press - “Faro de Vigo”
located at the NW of Spain, a fish industry journal - “Industrias pesqueras”, local and
national radio programmes, by emails sent to research, food safety and consumption
institutions, personal/professional contacts, and “word of mouth”. The second
questionnaire was disseminated by emails sent to personal or professional contacts and
by “word of mouth”.
The data collected were used in the risk model (see “Risk assessment” section) to
determine the total numbers of anchovy meals and untreated anchovy meals consumed
by survey respondents, and to then estimate the number of untreated anchovy meals
consumed by the Spanish population. This was used in risk characterization methods 1
and 2 (details in “RC scenarios” section), as well as in the dose response calculations
(details in Hazard characterization” section). Respondents with allergy to Anisakis spp.
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were also identified and this information was used in dose-response method 3 (details in
“Dose-response method 3” section).
Consumption data from governmental estimates: The total mass of fresh anchovies
consumed at home in 2013 by the Spanish population was available from governmental
estimates (MAGRAMA, 2016), and used to determine the total number of untreated
anchovy meals consumed in Spain and in each of its autonomous communities in risk
characterization method 2 (Community scenario) (details in “RC scenarios” section), and
in dose-response method 2 (details in “Dose-response method 2” at “Hospital studies”
section).
6.3.1.2.3 Landing statistics and trade data
Spanish global fishery production statistics and trade (i.e. imports and exports) by
supplier countries of European anchovy were obtained from FAO (2016) and
MAGRAMA (pers. comm.), respectively. These data were used to determine the fishing
area (i.e. origin) of the anchovies consumed in Spain, since Anisakis spp. infection rates
in anchovy vary with area (details in “Fishing area” section).
6.3.1.3 QRA methodology
A process-based risk assessment model was implemented in Microsoft Excel™ using
@RISK software (Palisade, UK). This incorporated variability in input data through the
use of appropriate probability distributions in simulations (n=100,000) using the Monte
Carlo technique (Vose, 2000). The QRA model was performed for the risk assessment
process detailed in Figure 6.1 and parameterised according to Tables 6.1 and 6.2. Each
Monte Carlo iteration determined the probability of disease from consuming one
untreated anchovy meal.
6.3.1.4 Risk characterization and hypothetical scenarios
The burden of disease (i.e. total number of anisakiasis cases and anisakiasis incidence per
year) was estimated by two risk characterization methods:
Method 1 or Base scenario: the number of untreated anchovy meals consumed by the
Spanish population was calculated using questionnaire 1 data.
Method 2 or Community scenario: the number of untreated anchovy meals consumed by
Spaniards was calculated using data on the mass of fresh whole anchovy home-consumed
in Spain available from MAGRAMA (2016).
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The QRA model was modified to determine the potential increase in risk (i.e. increase in
number of anisakiasis cases per year) considering a worst case scenario of post-mortem
migration of Anisakis spp. from fish viscera (where the majority of worms are found while
the fish is alive) to muscle; such post-mortem migration of Anisakis spp. is well-
documented in anchovy (Cipriani et al., 2016; Šimat et al., 2015). The QRA model was
also modified to determine the efficacy of a risk mitigation scenario i.e. education
campaign to freeze anchovies to reduce the number of diseases per year. The latter follows
Spanish legislation which came into effect in 2006 (Royal Decree RD 1420/2006)
(Ministerio de Sanidad y Consumo, 2006) implemented to improve the risk control of
anisakiasis in Spain. Consumers’ attitudes were investigated from questionnaire
responses to determine the likelihood they would respond to an education campaign. Both
hypothetical scenarios were determined using risk characterization method 1 (Base
scenario).
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Figure 6.1: Value chain and process model outline of untreated raw or marinated home-
prepared anchovy.
147
Table 6.1. QRA model variables used to determine the probability of anisakiasis per untreated anchovy meal.
Short description Description in paper Variable Units Distribution, fixed value or selection Data
source
EXPOSURE ASSESSMENT
Selecting Sea area Section “Fishing area” Area Number RiskDiscrete(1,2,3,.55,.43,.03) Table S2
Prevalence in muscle fishing area 1 “ “Prevalence & Intensity” PArea1 Proportion 137/2,913 (approximately 0.05) Table 6.3
“ “ “ “ “ 2 “ “ PArea2 “ 136/886 (approximately 0.15) “
“ “ “ “ “ 3 “ “ PArea3 “ Fixed value at 0.1313 “
Prevalence in muscle of “Area” selected “ “ Pselected “ By IFs from “Area” selected
Number of Anisakis in flesh per infected
anchovy from area 1
“ “ NparasitesArea1 Number RiskDiscrete(1,2,3,4,.825,.139,.029,.007) Table 6.4
“ “ “ “ “ “ “ 2 “ “ NparasitesArea2 “ RiskDiscrete(1,2,3,4,.801,.147,.022,.029) “
“ “ “ “ “ “ “ 3 “ “ NparasitesArea3 “ RiskDiscrete(1,2,3,4,.808,.115,.058,.019) “
Number of fillets in a meal “ “Consumption” Nfillet “ RiskDiscrete(1:39,0.00462: 0.00003) “Anchovy
meal size”
sub-model;
Table S8
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Number of fish in the meal “ “ Nfish “ Obtained from Nfillet but corrects for 2 fillets
per fish
Number of fish with at least one parasite
present
“ “ Nfishinf “ RiskBinomial(Nfish,Pselected)
Number of Anisakis in meal “ “ Nparasite “ IF(Area=1,NparasitesArea1)*Nfishinf
Select NParasitesArea1 if Area1, do likewise if
Area2 or Area3.
Proportion of Anisakis that are viable “ “Viability” Propviable Proportion Set at 1 (100% viable). Also, 0.5 and 0.1.
Number of viable Anisakis in meal “ “ Dose Number ROUND(Nparasite*Propviable,0)
HAZARD CHARACTERIZATION
Dose of parasite required to produce
disease in 50 percent of subjects
“ “Hazard characterization” ID50 Number RiskDiscrete(5018,24897,8153,15737,10439,
45594,10276,32737,828,.11,.11,.11,.11,.11,.
11,.11,.11,.11)
Table 6.5
Probability of one parasite surviving in
host to cause disease
“ “ R “ R=-Ln(0.5)/ID50
RISK CHARACTERIZATION
Probability of disease per untreated
anchovy meal
“ “Risk calculation” Pdisunt 1-EXP(-R*Dose)
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Table 6.2. QRA Risk Characterization model variables to determine the number of anisakiasis in Spain (method 1 or Base scenario) and in Spain
and its Autonomous Communities (method 2 or Community scenario).
RISK CHARACTERIZATION SCENARIOS
METHOD 1 (Base scenario) estimates the number of anisakiasis per year in Spain using anchovy consumption data from questionnaire 1
Short description Description in paper Variable Units Fixed value Data source
Number of questionnaire respondents Section “RC Scenarios” Popsize Number 716 Questionnaire 1
Untreated anchovy meals eaten by
questionnaire respondents per year
“ “ Mealsyear “ 1,508 Table S4
Spanish population aged 18 and over (at
01_07_13)
“ “ Spanishpop “ 38,241,133 INEa
Untreated anchovy meals eaten by Spanish
population per year
“ “ Mealsyearspain “ Mealsyear*Spanishpop/Popsize=80,541,619
Mean number of anisakiasis in Spain per
year caused by untreated anchovy meals
“ “ Anispain “ Mealsyearspain*Pdisuntb
Standard deviation of Anispain “ “ Anispainsd “ (Mealsyearspain*Pdisunt*(1-Pdisunt))^0.5
Normal approximation of the binomial
distribution
RiskNormal(Anispain, Anispainsd)
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METHOD 2 (Community scenario) utilises anchovy consumption data from MAGRAMA (2016), and questionnaire 1 information (i.e. proportion of untreated anchovy
meals, see “Propunt” in Table S4) for calculations
Short description Description in paper Variable Units Fixed value Data source
Untreated anchovy meals eaten in Spain
and its autonomous communities per year
“ “RC Scenarios” Nmunt Number Values of
“Nmunt” in
Table S5
Mean number of anisakiasis in Spain and its
autonomous communities per year caused
by untreated anchovy meals
“ “ Anispain2 “ Nmunt*Pdisunt
Standard deviation of Anispain2 “ “ Anispain2sd “ (Nmunt*Pdisunt*(1-Pdisunt))^0.5
Normal approximation of the binomial
distribution
“ RiskNormal(Anispain2,Anispain2sd)
a INE (Spanish Statistical Office). Available at: http://www.ine.es/. b Pdisunt: Probability of disease per untreated anchovy meal, see Table 6.1 at Risk Characterization.
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Table 6.3. Descriptors of Anisakis spp. infection in European anchovy and fish biometrics. Fishing area (n), area of capture and number of
anchovies analysed; year; TW ± SD (range), total mean weight and standard deviation (minimum – maximum) (g); Ninf, number of anchovies
with infection in muscle; Ptotal, prevalence of infection in muscle; Im (range), mean intensity of infection in muscle (minimum – maximum).
Fishing area (n) Year TW ± SD (range) Ninf Ptotal Im (range) Source of data
1: FAO 37
(2,913)
2013,
2014,
2015
14.90 ± 4.16 (4-31)** 137 5% 1.22 (1-4) PARASITE project*
2: FAO 27 (886) 2014,
2015 21.54 ± 4.83 (12-42) 136 15% 1.28 (1-4) PARASITE project*
3: FAO 34 (396) 1998,
1999 Not available 52 13% 1.28 (1-4)
Estimated from Rello
et al. (2009)
*Data generated within the EU FP7 PARASITE project (GA no. 312068).
** The TW of 501 anchovies was not available and therefore not included in calculations.
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Table 6.4. Distribution of the number of parasites in the muscle of infected anchovies by
fishing area. N, number of Anisakis spp. in muscle per infected anchovy; Frequency,
number of fish infected; Proportion, proportion of fish with Anisakis spp. infection.
Fishing area 1 Fishing area 2 Fishing area 3
N Frequency (proportion) Frequency (proportion) Frequency (proportion)
1 113 (0.825) 109 (0.801) 42 (0.808)
2 19 (0.139) 20 (0.147) 6 (0.115)
3 4 (0.029) 3 (0.022) 3 (0.058)
4 1 (0.007) 4 (0.029) 1 (0.019)
Total 137 (1) 136 (1) 52 (1)
6.3.2 Risk assessment
6.3.2.1 Hazard identification
The first probable case of anisakiasis was reported in 1876 in Greenland (EFSA-
BIOHAZ, 2010). In the Netherlands, during the 1960s, Van Thiel described anisakiasis
as “worm-herring disease”(Van Thiel, 1960). Currently, thousands of cases are diagnosed
every year, particularly in Japan (approximately 2,000 to 3,000 cases (EFSA-BIOHAZ,
2010; Yorimitsu et al., 2013)), Spain, the Netherlands, Germany (EFSA-BIOHAZ, 2010),
Korea (Sohn et al., 2015) and Italy (Mattiucci et al., 2013) where eating raw or marinated
fish is common. Recently, cases were reported in Croatia (Mladineo et al., 2016), China
(Qin et al., 2013) and Taiwan (Li et al., 2015). Anisakis simplex s.s. and A. pegreffii are
the zoonotic species involved. Anisakiasis is therefore a human zoonotic disease caused
by certain species of nematodes belonging to genus Anisakis (Audicana and Kennedy,
2008). It is an increasing health problem worldwide (Baird et al., 2014; EFSA-BIOHAZ,
2010; Llarena-Reino et al., 2015) and its severity may vary from mild to severe
gastrointestinal and ectopic disorders as well as allergy (Audicana and Kennedy, 2008;
Baird et al., 2014; EFSA-BIOHAZ, 2010; Pravettoni et al., 2012). The work in this paper
will concentrate on the gastric, intestinal and gastroallergic anisakiasis forms of disease.
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In Spain, European anchovy is traditionally consumed marinated as “anchovies in
vinegar” and is the main fish specialty implicated as the cause of human anisakiasis
(Alonso-Gómez et al., 2004; Audicana and Kennedy, 2008; Carrascosa et al., 2015;
Puente et al., 2008; Rello et al., 2009; Valiñas et al., 2001). The hazard associated with
this source of infection is the zoonotic nematode Anisakis spp. that can survive in
marinated and raw fish products for a sufficient period of time before being ingested
(EFSA-BIOHAZ, 2010). Since Spanish legislation requires restaurants to freeze the fish
presented as raw or almost raw, before serving (Ministerio de Sanidad y Consumo, 2006),
following the guideline previously established in Regulation (EC) 853/2004 (EC, 2004),
the main risk of disease is from the consumption of untreated raw or marinated anchovy
at home. Freezing or thorough cooking are considered as preventive methods to inactivate
any possible Anisakis spp. present in fishery products (EFSA-BIOHAZ, 2010). Other
anchovy species (e.g. Engraulis anchoita and E. ringens) can be consumed in Spain, but
usually are imported as frozen, salted or canned (MAGRAMA, 2013) and were therefore
excluded from this study.
Hence, Anisakis spp. is considered as the hazard and untreated raw or marinated European
anchovies as the main etiological vehicle involved in anisakiasis in Spain.
6.3.2.2 Exposure assessment
In order to determine the risk of anisakiasis caused by consumption of untreated anchovy
meals, the potential exposure to the parasite from each meal needs to be determined. This
depends on: 1) the fishing area of anchovies consumed in Spain (see section “Fishing
area” below); 2) the prevalence and intensity of Anisakis spp. in anchovy muscle for each
fishing area (see section “Prevalence & Intensity” below); 3) the consumption of
untreated anchovy meals by the Spanish population (see section “Consumption” below)
and 4) viability of Anisakis spp. in untreated anchovy meals (see section “Viability”
below).
6.3.2.2.1 Fishing area - the fishing area of anchovies consumed in Spain
Anchovies from different fishing areas are expected to have different Anisakis spp.
burdens and therefore, the risk for consumers will vary. Considered first are the anchovy
imported into Spain and then anchovy caught by Spanish vessels.
Fishing area of anchovy imported into Spain: Fresh anchovies are imported every year
into Spain from supplier countries (Table S1) and exports also occur (Export, Table S2)
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(MAGRAMA, pers. comm.). The quantities and countries involved in anchovy
transactions are known. To determine the mass, per fishing area, of anchovy imports
(Assumed imports, Table S1), it was assumed that the imports per fishing area are in the
same proportions as seen in landings of the supplier country (Production, Table S1)
(FAO, 2016).
Total exports from Spain are known, but the fishing ground of exported anchovies was
unknown. It was assumed that exports comprised fish from the three different fishing
areas considered in the present study in proportion to the amount caught (Table S2).
Only fresh imported and exported anchovies were considered, since other anchovy
products (i.e. frozen, canned, salted or semi-preserved, etc.) were assumed not to contain
viable Anisakis spp. The trade (i.e. imports and exports) data were provided for fresh
anchovy, identified as Engraulis spp. by MAGRAMA (pers. comm.). However, it was
assumed these data refer to European anchovy (E. encrasicolus) since it is the only
anchovy species fished by supplier countries (FAO, 2016).
Fishing area of anchovy fished by Spanish vessels: The Spanish production of European
anchovy by fishing area was directly available from FAO (2016) (Table S2).
The total quantities of anchovies from each fishing area (Table S2) were the result of
summing “Spanish Production” and “Imports” and then subtracting “Exports”. The
proportion of estimated consumption for each fishing area was then calculated (Table S2).
This was then used in the QRA model to determine the fishing area of each untreated
anchovy meal by randomly sampling the RiskDiscrete() distribution (the first argument
of this @RISK distribution is the set of possible values (i.e. fishing areas), and the second
is the set of corresponding probabilities (the probability of the fish originating from a
particular area)) (Area, Table 6.1). Thus, the QRA model starts by selecting the fishing
area of origin for each anchovy meal (1, 2 or 3).
6.3.2.2.2 Prevalence & Intensity - Prevalence and intensity of infection of Anisakis
spp. in anchovy muscle for each fishing area
The prevalence of Anisakis spp. in the muscle of anchovy for each of the three fishing
areas is given by the variables “Parea1”, “Parea2” and “Parea3” (Table 6.1). The
corresponding numbers of Anisakis spp. in anchovy muscle in each fishing area
(NparasitesArea1, 2 and 3, Table 6.1) are described by a discrete distribution using the
data from Table 6.4.
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The fishing area is selected by sampling the RiskDiscrete variable “Area” whose output
“1”, “2” or “3” selects the corresponding prevalence “Pselected” and also the number of
Anisakis spp. in muscle per infected anchovy (NparasitesArea1, 2 and 3).
The QRA model was performed for 2013, the most recent year for which production and
trade data were available. Anchovy infection data (Table 6.3 and 6.4) were assumed to be
representative for 2013, even though samples were obtained from different years (Table
6.3).
6.3.2.2.3 Consumption - consumption of untreated raw and marinated anchovy
meals by the Spanish population
Estimating the fraction of untreated anchovy meals eaten: A total of 729 completed
questionnaires was obtained from questionnaire 1. Those respondents (n=13) who did not
respond to question 18 (i.e. Do you usually eat raw or undercooked fish?) were removed.
The remaining respondents (n=716) consumed a total of 5,767 anchovy meals per year
(question 16). It was assumed that a respondent consumed untreated anchovy meals when
s/he gave all of the following responses (Table S3): (1) answer “yes” to eating raw fish
in question 18; (2) eating raw fish at their home (question 18A); (3) utilising fresh fish
for raw/undercooked consumption (question 18A2); (4) confirming raw anchovy as the
consumed species (question 18B), and finally (5) indicating the specific kind of raw
and/or marinated anchovy preparation that they consumed (question 18C1). These “at
risk” respondents (n= 46) ate a total of 987 untreated home-prepared raw or marinated
anchovy meals per year (i.e. anchovies in vinegar or lemon (472), marinated anchovies
(280), sushi or sashimi (106), ceviche (80) and carpaccio (49)) (question 18C1). It was
assumed that a respondent ate 1, 12 or 52 meals per year when s/he answered annually,
monthly or weekly anchovy consumption in questionnaire 1 (questions 16 and 18C1).
The probability of an untreated anchovy meal being consumed was 0.17 (=987/5,767).
Finally, it was assumed that these meals were the main vehicle of anisakiasis in Spain
(see section “Hospital studies” and “Anchovy main species causing anisakiasis” at the
“General discussion” section for further details).
Questionnaire 1 was over-represented by respondents from Galicia (62%, n=447), whose
population is approximately 6% of Spain (Spanish Statistical Office - available at:
http://www.ine.es/ accessed 05th Dec 2016). The questionnaire reported that the number
of untreated anchovy meals consumed per Galician respondent per year (0.74=332/447)
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was a third of that from other communities in Spain (2.48=653/263). To correct for this
bias the respondents were grouped according to geographical location (i.e. Galicia,
Cantabrian Sea, Central Spain and Mediterranean Sea communities, Andalucía and
Canary Islands) and their responses were weighted based on the population of each
region, to provide representative results for Spain, (see Table S4). As a result, the number
of untreated meals consumed per Spanish person per year was estimated to be 2.106
(Amealpy, Table S4) and the total number of untreated meals consumed by respondents
was 1,508 (i.e. 716×2.106= 1,508) (Table S4). Hence, the proportion of untreated meals
consumed per person was 0.175 (Propunt, Table S4). These figures were used when
calculating the dose-response for Anisakis spp. (see “Hazard characterization” section)
and during Risk Characterization (see “Risk characterization” section)
Estimating a probability distribution for the number of fillets consumed in an anchovy
meal: A separate model (i.e. “anchovy meal size” sub-model) was built to estimate the
number of anchovy in vinegar fillets consumed per meal (NFillet, Table 6.1) using data
from questionnaire 2. Detailed information of the sub-model is provided in the
supplementary material.
In the QRA model, the number of fish in a meal was determined knowing that there are
two fillets per fish. The number of fish with at least one parasite present was then
calculated using the RiskBinomial() distribution (Nfishinf, see Table 6.1).
The exposure to parasites (i.e. number of Anisakis spp. consumed per untreated anchovy
meal) (Nparasite, Table 6.1) was then determined by multiplying the number of parasites
in muscle of an infected anchovy (NparasitesArea1, 2 and 3, Table 6.1) by the number of
infected anchovies consumed per meal (Nfishinf, Table 6.1). This approximation was
required for the model to be readily implemented in Excel using @RISK. However,
Anisakis spp. exposure was also determined by sampling each infected anchovy
consumed in the meal and using the Monte Carlo method to generate a distribution of the
infected anchovies in each meal. This distribution was compared with the approximate
method described above and was found to have virtually identical mean and variance
(data not presented).
6.3.2.2.4 Viability - viability of Anisakis spp. in untreated anchovy meals
It has been reported that Anisakis spp. can survive the typical conditions experienced in
the traditional preparation of anchovies in vinegar (Sánchez-Monsalvez et al., 2005), even
157
though it is unknown what proportion of parasites is viable in freshly caught anchovy. In
the first instance, the QRA model assumed that all Anisakis spp. present in untreated
anchovy meals had 100% viability (Propviable, Table 6.1). However, to investigate the
effect of reduced viability, simulations were also carried out with viabilities of 50% and
10% in determination of the dose response.
The number of viable Anisakis spp. in the untreated anchovy meal (Dose, Table 6.1) was
then calculated by multiplying “Nparasite” by “Propviable” (Table 6.1).
6.3.2.3 Hazard characterization
No dose-response model has been developed previously for Anisakis spp. in humans.
Typically, if it is assumed that the distribution of the pathogen (i.e. Anisakis spp.) within
a fish is Poisson, that one organism is sufficient to have the potential to cause disease and
that each organism has an equal and identical survival probability (R) (i.e. the probability
of colonising and causing disease), then the form of the dose response is exponential
(Haas et al., 2014):
𝑃 = 1 − 𝑒−𝑅𝑁 (1)
where P, is the probability of disease following consumption of a dose of N pathogens
(e.g. Anisakis spp.). The ID50 (the dose of an infectious organism required to produce
disease in 50 percent of subjects challenged) is given by:
𝐼𝐷50 = −(𝐿𝑛(0.5)/𝑅) (2)
Here, data from four studies performed in Spanish hospitals, our own sampling,
questionnaires and MAGRAMA (2016) were used to estimate R and the ID50 using two
different methods. A third independent method for determining the dose-response using
questionnaire 1 information (e.g. respondents with self-reported allergy to Anisakis spp.)
was also performed. These data were then used in the QRA model to convert the number
of parasites ingested in a meal to the probability of disease from consuming an untreated
anchovy meal (Pdisunt, Table 6.1).
6.3.2.3.1 Hospital studies
In 1997, 96 patients (incidence of 19.2 cases per 100,000 inhabitants/year, Table S9) were
diagnosed with gastroallergic anisakiasis in “La Paz” hospital in Madrid (central part of
Spain) (Alonso-Gómez et al., 2004). Seventy-eight of these patients confirmed ingestion
of raw anchovies in vinegar (n=76) or raw anchovies (n=2) prior to falling ill (Alonso-
158
Gómez et al., 2004). A retrospective study performed in “Virgen de la Salud” hospital in
Toledo (central part of Spain) reported 25 cases (3.87 cases per 100,000 inhabitants/year,
Table S9) of gastrointestinal anisakiasis over two years (December 1999 to January 2002)
(Repiso Ortega et al., 2003). All these patients confirmed ingestion of raw anchovies prior
to falling ill (Repiso Ortega et al., 2003). A third study, performed in “Antequera” hospital
in Málaga (south of Spain) reported 52 patients (11.82 cases per 100,000 inhabitants/year,
Table S9) with anisakiasis over a four-year period (summer 1999 to summer 2003) (Del
Rey Moreno et al., 2013, 2008). Of these, 50 patients confirmed the consumption of fresh,
raw or in-vinegar anchovies (Del Rey Moreno et al., 2008). Lastly, the “Carlos III”
hospital in Madrid reported approximately 30 cases (6.12 cases per 100,000
inhabitants/year, Table S9) of allergy to Anisakis spp. per year (González-Muñoz,
unpublished data). Although, the numbers of patients reporting anisakiasis was unknown
in the latter study, it was assumed, based on general agreement, that sensitization occurs
via infection by live Anisakis spp. larvae (EFSA-BIOHAZ, 2010) so that the number of
allergy cases indicates the incidence of anisakiasis.
The values of R and ID50 were determined using these hospital incidence data by the
following two methods:
Dose-response method 1: calculation of ID50 in hospitalised anisakiasis cases assuming
prior exposure to untreated anchovy meals as reported in questionnaire 1:
Determining the probability of disease: The anisakiasis incidence, number of anisakiasis
cases and duration of study for each hospital are presented in Table S9. The total number
of untreated anchovy meals consumed per person each year in Spain (Amealpy, Table
S9) was calculated from questionnaire 1 and used to estimate the total number of meals
consumed by the catchment population of each hospital (Auntmeal, Table S9). The
probability of disease from an untreated anchovy meal (Pdisease, Table S9) was
determined by dividing the number of anisakiasis cases per year in that population by the
total untreated anchovy meals consumed.
Determining the dose: The average number of viable parasites ingested per meal was
determined by running the QRA model (Dose, Table 6.1). This was repeated for different
viabilities (100%, 50% and 10%) (Dose1, Dose0.5 and Dose0.1, Table S9).
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Determining the ID50: Since both the dose and the probability of disease are now known,
then R and ID50 can be obtained from equation 1 and 2 (Table S9). This was repeated
using data from each of the four study hospitals.
Dose-response method 2: calculation of ID50 in hospitalised anisakiasis cases assuming
prior exposure to untreated anchovy meals calculated using governmental estimates of
anchovy consumption:
Determining the probability of disease: this method used the reported mass of fresh whole
anchovy that was used to prepare meals at home per capita (Capita, Table S9), rather than
using data from the questionnaire. These data were available from MAGRAMA (2016).
For the four hospitals (“Virgen de la Salud”, “Antequera”, and “Carlos III” and “La Paz”),
the consumption per capita of fresh anchovies was assumed to be the same as for the
autonomous communities of Castilla la Mancha, Andalucía, and Madrid in 2013,
respectively (data available from MAGRAMA (2016) and provided in Table S10).
To determine the mass of anchovy muscle eaten, the total mass of whole anchovies eaten
per study population was determined (Cyear, Table S10). This was multiplied by the yield
of skinless fillets obtained from European anchovy (0.51, from FAO (1989)) to determine
the mass of muscle eaten (Cfyear, Table S10). On average a meal consisted of 11 fillets
(Anfillet, Table S10) (see section “Consumption” above). The average mass of muscle
from a European anchovy was determined (8.51 g, n = 3,298) by multiplying the average
total weight of the sampled anchovy (16.69 g, n=3,298) (Table 6.3) by 0.51 (i.e. yield of
skinless anchovy fillet (FAO, 1989)). Since two fillets are obtained from each fish the
average fillet mass is therefore 4.25 g (Amfillet, Table S10). The average mass of a meal
was calculated (Ammeal, Table S10) and from this the total number of anchovy meals
consumed per year was obtained (Nmealyear, Table S10). Multiplying this by the
proportion of anchovy meals that were untreated (Propunt, Table S4 and S10), the total
number of untreated anchovy meals consumed per year was obtained (Auntmeal, Table
S10).
Determining the dose and ID50: the dose, R and ID50 were calculated as in method 1.
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6.3.2.3.2 Dose-response method 3: determination of ID50 from questionnaire 1
respondents reporting allergy
Determining the probability of disease: there were 716 respondents to questionnaire 1 but
only 701 answered question 13S4 (Do you have allergy to Anisakis?). Of these, 9
confirmed they had allergy to Anisakis spp. It was assumed that these persons suffered an
anisakiasis before the development of the allergy and that the other respondents had not
suffered anisakiasis. Following correction of questionnaire 1 responses due to over-
representation of respondents from Galicia the proportion of the Spanish population with
allergy to Anisakis spp. was 0.028, and the number of allergic respondents increased to
19 persons (701×0.028= 19) (Table S4).
To estimate exposure in the 701 respondents it was necessary to know the number of
untreated anchovy meals they had consumed during their life. This was obtained from
their age (question 2) and the number of untreated anchovy meals consumed each year
by respondents. The mean age of the 701 respondents was 41.81 years. It was assumed
that respondents started to eat untreated anchovy meals from the age of 18 (average 23.81
years of consumption). The number of anisakiasis per year and the incidence of the
disease were finally determined using the corrected data (Casesyear and Incidence Table
S9). Specifically, 1,476 untreated meals (i.e. 701×2.106= 1,476) were consumed by the
respondents (Auntmeal, Table S4 and S9). Finally, the probability of disease (Pdisease,
Table S9) was determined as before.
Determining the dose and ID50: the dose, R and ID50 were calculated as in method 1.
6.3.2.3.3 ID50 results and discussion
Table 6.5 presents the results from the 3 different methods. Method 2 yielded higher ID50
values than method 1. This is because method 2 estimates higher numbers of untreated
anchovy meals consumed by the population. ID50 values also varied depending on which
hospital study was used, from 5,018 to 45,594 (with viability 100%). The ID50 estimate
depends on the number of anisakiasis cases and the number of untreated anchovy meals
consumed by the population. However, since the literature (EFSA-BIOHAZ, 2010)
indicates that there is underreporting of anisakiasis and since hospitalised cases are likely
to be only the most severe forms of the disease, it is likely that the value for the ID50 will
be overestimated. The lower ID50 value determined in method 3 might be explained by
overestimation of anisakiasis incidence from questionnaire 1. Therefore, it is possible that
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methods 1 and 2 overestimated the value of ID50 because they were based on hospital
studies, whilst method 3 probably underestimated the ID50 because people with allergy
to Anisakis spp. may have been more likely to respond to the questionnaire. The decrease
of Anisakis spp. viability resulted in decreasing ID50 values, as expected since ID50
depends on the dose of exposure (which depends on viability), decreasing when the dose
decreased. There is no strong evidence to adopt one method over another for selecting the
ID50. Hence, all of the 9 ID50’s (with viability 100%) were used in the QRA (Table 6.5)
utilizing the RiskDiscrete() distribution (Table 6.1).
Table 6.5. Dose response ID50 values determined for hospital populations (H1, “La Paz”;
H2, “Virgen de la Salud”; H3, “Antequera” and H4, “Carlos III”) using dose response
methods 1 and 2, and for the questionnaire population (Qaire) using method 3. Results
for different viabilities of Anisakis spp. are also presented (100% (Base scenario), 50%
and 10%).
Method 1 Method 2 Method 3
Viability H1 H2 H3 H4 H1 H2 H3 H4 Qaire
100% 5,018 24,897 8,153 15,737 10,439 45,594 10,276 32,737 828
50% 3,421 16,975 5,559 10,730 7,117 31,087 7,006 22,321 565
10% 76 377 124 238 158 691 156 496 13
6.3.2.4 Risk characterization
6.3.2.4.1 Risk calculation (calculation of probability of anisakiasis per untreated
anchovy meal)
The probability of an individual contracting anisakiasis from an untreated anchovy meal
(Pdisunt, Table 6.1) was calculated using equation 1, for the 9 ID50’s described above.
6.3.2.4.2 RC scenarios - risk characterization using methods 1 and 2, calculation
of the number of anisakiasis cases in 2013
Two methods were used to estimate the number of anisakiasis cases for Spain in 2013.
Method 1 (or Base scenario) used anchovy consumption data from questionnaire 1
(method 1, Table 6.2). The survey size, number of untreated anchovy meals consumed by
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questionnaire respondents and the total number of people living in Spain aged 18 and
over (Popsize, Mealsyear and Spanishpop in Table 6.2, respectively) were used to
calculate the total number of untreated anchovy meals consumed by the Spanish
population in 2013 (Mealsyearspain, Table 6.1).
Method 2 (or Community scenario) utilised home-consumed anchovy data from
MAGRAMA (2016), and questionnaire 1 information (i.e. proportion of untreated
anchovy meals (i.e. 0.175, see “Propunt” in Table S4 and S5)) for calculations (method
2, Table 6.2). The total number of untreated anchovy meals consumed in Spain and its
autonomous communities in 2013 was calculated as follows: the total kilograms of fresh
anchovy consumed at home in 2013 (Totalmass, Table S5) was available from
MAGRAMA (2016). The mass of muscle of anchovies (Musclemass, Table S5) was
calculated multiplying “Totalmass” by the yield of skinless fillet of anchovy (0.51) (FAO,
1989). The number of fillets consumed (Numberfillets, Table S5) was calculated by
dividing “Musclemass” by the average weight of an anchovy fillet (4.25 g). The number
of meals (Numbermeals, Table S5) was calculated by dividing “Numberfillets” by the
average number of fillets per meal (i.e. 11 fillets). Finally, the total number of untreated
anchovy meals consumed at home in 2013 by the Spanish population (Numbermealsunt,
Table S5) was calculated by multiplying “Numbermeals” by the proportion of untreated
anchovy meals estimated from the questionnaire (0.175, “Propunt”, Table S4 and S5)).
The average numbers of anisakiasis cases in Spain (methods 1 and 2) and its autonomous
communities (method 2) and the corresponding standard deviation (Anispain and
Anispainsd in Table 6.2, respectively) were determined using the method of Lindqvist
and Westwoo (2000). Briefly, the mean and the standard deviation of the probability of
disease for 100,000 meals were calculated using the QRA model. The average number of
cases was then obtained by multiplying the number of untreated anchovy meals consumed
by the Spanish population by the mean probability of disease. The associated standard
deviation, and 2.5th and 97.5th percentiles were calculated assuming the normal
approximation of the binomial distribution (Table 6.2).
6.3.2.4.3 Hypothetical scenarios
The QRA can be used to determine the change in predicted risk if any of the model
parameters change. This procedure was tested in two ways which could result in a
behaviour change in the way the anchovy are prepared. The first was to determine the
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increase in risk caused by post-mortem migration of Anisakis spp. from the fish viscera
to the muscle (worst case scenario). The second was a risk mitigation scenario (hence
targeted to reduce risk) that involved an educational campaign.
Worst case scenario - post-mortem migration of Anisakis spp. from fish viscera to the
muscle: Post-mortem migration of Anisakis spp. from viscera to muscle has been reported
in European anchovy (Cipriani et al., 2016; Šimat et al., 2015). The value chain of
European anchovy (Figure 6.1) starts with fish being caught at sea, stored in ice and
landed within one to two days. Then, the anchovies are transported in boxes with ice to
retail (supermarket, fish market, etc.), where they are purchased by the consumer who
carries the fish in plastic bags to home at ambient temperature where they are then
refrigerated. Only then are the fish cleaned and eviscerated (usually no evisceration
occurs before this step). This usually takes place on the day of purchase. Thus, migration
of Anisakis spp. into muscle can occur at any point prior to evisceration.
The QRA model was modified to simulate the worst case scenario (i.e. anisakiasis caused
by inadequately stored anchovies that allow Anisakis spp. post-mortem migration from
viscera to muscle) as follows:
Cipriani et al. (2016) found that in batches of 100 anchovy immediately frozen after
capture, 20% of the fish had Anisakis spp. in the muscle with a total of 28 larvae.
However, if the anchovies were left for 72 hours at 7ºC (temperature typical of a fridge
in Spanish household (Garrido et al., 2010)) that muscle prevalence increased to 57%
with 117 larvae being present. Additional parameters were incorporated into the QRA
model to determine changes in prevalence, number of parasites consumed per meal,
probability of anisakiasis and finally the number of anisakiasis cases in Spain when this
worst case scenario is considered, as follows: The prevalence of the worst case scenario
(Ptotalmigration) was determined by multiplying the parameter “Pselected” (Table 6.1)
by the increase in prevalence (i.e. 57/20). The number of infected fish per meal
(Nfishinfmigration) was determined as the parameter “Nfishinf” (Table 6.1), using the
RiskBinomial distribution (i.e. RiskBinomial (Nfish,Ptotalmigration)). The number of
ingested Anisakis spp. per meal (Nparamigration) was determined similarly to the
variable “Nparasite” (Table 6.1), but multiplying the intensity of infection (i.e.
NparasitesArea1, 2 and 3, Table 6.1) by “Nfishinfmigration”, and by the increase in the
number of Anisakis spp. per fish (i.e. 117/28). The probability of anisakiasis
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(Pdisuntmigration) was determined as the parameter “Pdisunt” (i.e. 1-EXP(-
R*Nparamigration). Finally, the number of anisakiasis predicted was calculated as the
parameter “Anispain” (Table 6.2) (i.e. Mealsyearspain*Pdisuntmigration).
Risk mitigation scenario – public health education campaign: Del Rey Moreno et al.
(2013) reported that in Antequera, human anisakiasis was reduced from 10-16 to 0-2 cases
per year, following a public health education campaign and implementation of RD
1420/2006 (Ministerio de Sanidad y Consumo, 2006). This recommended consumers to
freeze fish prior to raw consumption and also obliged sellers to freeze fish that would be
served raw or partially raw. The QRA model was modified to simulate this education
campaign. The number of untreated meals consumed by the Spanish population after the
education campaign (“Mealsmitigation”) was calculated multiplying 2/10 (i.e. most
conservative ratio of number of cases post-intervention to those prior to the intervention)
by the total untreated meals eaten by the Spanish population before the education
campaign (Mealsyearspain, Table 6.1). Finally, the effect of the mitigation strategy on
the number of anisakiasis was determined (i.e. Mealsmitigation*Pdisunt).
6.3.3 Results
6.3.3.1 Base results of the QRA model
6.3.3.1.1 Number of viable Anisakis spp. consumed per untreated anchovy meal
(i.e. dose of exposure)
The QRA model estimated the average number of viable Anisakis spp. consumed per
untreated anchovy meal to be 0.66 with standard deviation (SD) 1.15. This distribution
was right-skewed (Figure 6.2), with 38.5% of meals containing at least one viable
Anisakis spp.
6.3.3.1.2 Probability of anisakiasis (disease) per untreated anchovy meal
The dose response for Anisakis spp. generated by applying equal weighting to the nine
ID50 estimates is presented in Figure 6.3. The resulting mean ID50 was 17,000 with SD
14,000. Using this dose response, the probability density function of the probability of
disease per untreated anchovy meal was obtained (Figure 6.4). From this the average
probability of humans contracting anisakiasis per untreated anchovy meal was found to
be 9.56×10-5 with SD of 3.68×10-4. Thus, on average for every 10,456 meals consumed
there was 1 anisakiasis case (since 1/10,456 is 9.56×10-5).
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Figure 6.2. Probability density function of the number of viable Anisakis spp. consumed
per untreated anchovy meal with 100% viability (mean 0.66, SD 1.15).
Figure 6.3. i) Dose response for Anisakis spp. (ID50, mean 17,000, SD 14,000), ii) inset
of dose response at higher resolution for low doses (i.e. exposure between 1 and 10
parasites).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Pro
bab
ilit
y
Number of Anisakis per meal
0.0
0.2
0.4
0.6
0.8
1.0
1 10 100 1000 10000 100000 1000000
Pro
ba
bil
ity
of
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isa
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sis
Anisakis dose
Average 5th percentile 95th percentile
i) ii)
0
0.002
0.004
0.006
0.008
0.01
1 10
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Figure 6.4. Probability density function of the probability of disease (anisakiasis) per
untreated anchovy meal (mean 9.56×10-5, SD 3.68×10-4).
6.3.3.2 Risk characterization results - methods 1 (Base scenario) and 2 (Community
scenario)
The QRA model predicted an average annual number of anisakiasis cases of
approximately 7,700 ± 90 (SD), 2.5th and 97.5th percentiles [7,560 – 7,850] for method 1
and 8,320 ± 90 (SD), 2.5th and 97.5th percentiles [8,170 – 8,470] for method 2 in Spain
(Table 6.6). The anisakiasis incidence varied between 18 and 20 cases per 100,000
inhabitants/year for method 2 and method 1, respectively.
The average number of anisakiasis cases was also determined for Spain and its Spanish
autonomous communities using method 2 (Table 6.7). The largest number of anisakiasis
cases was estimated to occur in Andalucía (n= 2,220 ± 50 (SD)), followed by Madrid (n=
1,280 ± 40 (SD)). In terms of anisakiasis incidence, Cantabria (35 cases per 100,000
inhabitants/year) followed by País Vasco (31 cases per 100,000 inhabitants/year) had the
highest values.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.01 0.011 0.012 0.013 0.014
Pro
ba
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ensi
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un
ctio
n
Probability of anisakiasis per untreated meal
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6.3.3.3 Hypothetical scenarios results (estimated using Risk characterization method 1)
6.3.3.3.1 Worst case scenario – post-mortem migration of Anisakis spp. from fish
viscera to the muscle
The QRA model predicted an annual average number of anisakiasis cases when Anisakis
spp. migration was considered of approximately 91,100 ± 300 (SD) in Spain (see “worst
case scenario” in Table 6.6). This resulted in an increase of >1,000% in the number of
anisakiasis predicted by the base scenario. The base scenario assumes that there is no
migration of Anisakis spp. larvae from the viscera to the muscle.
6.3.3.3.2 Risk mitigation scenario – public health education campaign
The QRA model estimated an annual average number of anisakiasis cases after an
education campaign of approximately 1,540 ± 40 (SD) in Spain (Table 6.6). This was a
reduction of 80% compared with the Base scenario which is as expected. However, the
responses to the questionnaire suggest that the scenario is more complex due to human
behaviour. Notably, it was found that among those respondents (n=46) who consumed
untreated anchovy meals, 89% (41 out of 46 respondents) answered correctly that
marinating or smoking the fish does not prevent Anisakis spp. (question 20A). Moreover,
89% (40 out of 46 respondents) answered correctly that freezing prevents disease
(question 20A) but that they continue to eat raw untreated meals.
Table 6.6. QRA model estimated anisakiasis cases and incidence of disease in Spain
during 2013.
Risk characterization Anisakiasis ± SD Percentagea Incidence (cases per
100,000 inhabitants/year)
RC method 1 (Base scenario) 7,700 ± 90 20b
RC method 2 (Community
scenario)
8,320 ± 90 7% 18c
Worst case scenario (migration) 91,100 ± 300 >1,000%
Risk mitigation scenario
(education)
1,540 ± 40 -80%
a Percentage increase/decrease compared to risk characterization method 1. b Incidence of anisakiasis for Spanish population aged 18 and over (i.e. 38,241,133
inhabitants) (at 01_07_13) (Source: INE (Spanish Statistical Office) available from:
http://www.ine.es/.).
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c Incidence of anisakiasis in the whole Spanish population (i.e. 46,593,236 inhabitants)
(at 01_07_13) (Source: INE (Spanish Statistical Office) available from:
http://www.ine.es/.).
Table 6.7. Estimated cases of anisakiasis and its incidence (i.e. cases per 100,000
inhabitants/year) in Spain and its autonomous communities in 2013 using risk
characterization method 2 (Community scenario).
Community Anisakiasis ±
SD
Incidence Community Anisakiasis ±
SD
Incidence
Total Spain 8,320 ± 90 18 Cataluña 1,140 ± 30 15
Andalucía 2,220 ± 50 27 Extremadura 160 ± 10 14
Aragón 190 ± 10 14 Galicia 160 ± 10 6
Asturias 190 ± 10 18 La Rioja 70 ± 10 21
Baleares 90 ± 10 8 Madrid 1,280 ± 40 20
Canarias 20 ± 4 1 Murcia 210 ± 10 14
Cantabria 210 ± 10 35 Navarra 110 ± 10 16
Castilla la Mancha 430 ± 20 21 País Vasco 670 ± 30 31
Castilla y León 460 ± 20 18 Valenciana 730 ± 30 15
6.4 General discussion
6.5.1 Estimating the number of anisakiasis cases in Spain
The results generated by the QRA suggest that Spain has between 7,700 and 8,320
anisakiasis cases annually. Since these data are predominantly based on hospitalised cases
to determine the ID50, it is likely that the actual number of cases across Spain will be
higher, when mild infections are included. The QRA estimates apparently suggest a
higher incidence of anisakiasis in Spain than previously reported for Japan (about 2,000-
3,000 cases per year (EFSA-BIOHAZ, 2010; Yorimitsu et al., 2013)), but it has to be
borne in mind that the methodology was different (Japanese estimations are based on
annual diagnosed cases and it is likely that the efficacy of the reporting systems are
different (EFSA-BIOHAZ, 2010; Yorimitsu et al., 2013)) and the comparison is therefore
not valid. However, it is clear that published estimates of 500 anisakiasis cases in Europe
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per year (Arizono et al., 2012) or 20 cases on average per European country per year
(D’amico et al., 2014) are very likely to be a considerable underestimation.
It is frequently claimed that the number of anisakiasis cases is underdiagnosed and
misdiagnosed for three reasons. First, due to the non-specific symptoms of the disease
and lack of clinical investigation (EFSA-BIOHAZ, 2010), second confounding with other
gastrointestinal conditions (EFSA-BIOHAZ, 2010; Kim et al., 2013; Toro et al., 2004)
and third it may remain undiagnosed if ingestion of raw or undercooked fish is not
considered during anamnesis (Del Rey Moreno et al., 2013). Although symptomatic
anisakiasis has been reported, asymptomatic intestinal anisakiasis (Mattiucci and
D’Amelio, 2014) or symptomless clinical presentation (Carrascosa et al., 2015) have also
been observed. Toro et al. (2004) suggested that human anisakiasis might be
underdiagnosed in Spain because only the most severe cases that required intervention
are being diagnosed. EFSA-BIOHAZ (2010) suggested that the 96 gastroallergic
anisakiasis cases reported from the “La Paz” area of Madrid (Alonso-Gómez et al., 2004)
could be an underestimation since many patients with mild symptoms were unlikely to
seek medical attention. Hence, in reality it appears that the true burden of disease in the
Spanish population is likely to exceed the estimates based on the risk assessment model.
6.5.2 Comparison of the two Risk Characterization methods and potential biases
The total number of anisakiasis estimated in Spain in 2013 was calculated by two RC
methods. The total number of untreated anchovy meals eaten annually as estimated by
RC method 2 is more than 6 million greater than estimated by RC method 1, resulting in
approximately 7% more anisakiasis cases. This difference reflects the different source of
data used, namely questionnaires for RC method 1 and governmental estimates for RC
method 2, as well as the methodology used during calculations of the number of untreated
meals consumed by the population. In addition, RC method 1 assumed that only adults
>18 years old consume untreated anchovy meals. Hence, RC method 1 may underestimate
the total number of untreated meals consumed by the population and, therefore, the total
annual number of anisakiasis cases occurring in Spain for this reason, since people under
18 may also consume such meals.
Correcting for the over-representation of respondents from Galicia in questionnaire 1
made a significant difference to the number of cases estimated by RC method 1. When
the model is run without the correction it predicted 4,650 cases (data not presented)
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instead of 7,700. This is explained by the difference in the number of untreated anchovy
meals consumed which is lower in Galicia compared with the rest of Spain. These
findings emphasise the importance of having a representative sample of the Spanish
population when conducting a questionnaire survey.
The regional differences found in response to questionnaire 1 are supported by published
information. In mainland Spain, Galicia was estimated to be the region with the lowest
anisakiasis incidence (5 cases per 100,000 inhabitants/year, Table 6.7), and is the
community with lowest consumption of fresh anchovies at home per capita (i.e. 0.31 kg
per year) (MAGRAMA, 2016). Valiñas et al. (2001) reported that 12.8% (n=13/101) of
Galician individuals (control subjects seronegative to Anisakis) prepared untreated raw
anchovies at home. This figure is over three times higher than that found in the current
study in Galicia (3.8%, n=17/447) but similar to that found in the rest of Spain (10.6%,
28/263). It is unclear which study is the most representative, but the current work does
have a larger sample size.
The ID50 for Anisakis spp. was determined using 3 dose-response methods. Method 1
and 2 may have overestimated the value of the ID50 because they were based on hospital
studies (i.e. it is likely that only the most severe cases were diagnosed). Whilst method 3
may have underestimated the ID50 because it is based on respondents reporting allergy
to Anisakis spp. in questionnaire 1 and it is possible that individuals with allergy were
more likely to respond to the questionnaire. It is also worth noting that when correcting
for the over-representation of Galicia in questionnaire 1, the final dose response did not
change significantly (corrected ID50 was 17,000 with SD 14,000 and uncorrected ID50
was 15,000, SD 14,000).
Questionnaire 1 had 716 respondents which is only 0.002% of the population of Spain
aged 18 and over. Bootstrapping of the raw questionnaire responses was used (results not
presented) to incorporate variation (i.e. generate distributions in the number of responses
by community and untreated anchovy meals consumed) (Manly, 2006). This leads to a
change in the variance but not a change in the mean values of the variables. Re-running
the model with these distributions rather than fixed values as done in the QRA model
provided virtually identical results for the number of cases and associated standard
deviations for RC methods 1 and 2.
171
It was assumed that a respondent ate 1, 12 or 52 meals per year when the respondent
answered annually, monthly or weekly anchovy consumption when completing the
relevant questions of questionnaire 1 and 2. It is likely that respondents may not have
remembered exactly what they had eaten in the previous year and this categorisation of
responses would have introduced some variation into these data.
6.5.3 Anchovy is the main fish species causing anisakiasis
The QRA model was built assuming that all anisakiasis cases predicted in Spain in 2013
were caused by home-prepared untreated anchovy meals. While other fish species and
recipes have also been implicated, the great majority of patients for which data is available
had eaten anchovies. For instance, in the “Antequera” hospital study, 50 of 52 anisakiasis
patients consumed fresh, raw or in vinegar anchovies prior to falling ill, but one patient
consumed raw sardine (and there was no food consumption information for the remaining
patient) (Del Rey Moreno et al., 2013, 2008). Also 18 of 96 gastroallergic anisakiasis
cases in the “La Paz” hospital study consumed fish species other than anchovy (9 patients
consumed undercooked hake, 2 patients consumed raw cod and 7 patients consumed other
cooked fish) prior to falling ill (Alonso-Gómez et al., 2004). In the Repiso Ortega et al.
(2003) (i.e. “Virgen de la Salud” hospital study) all 25 anisakiasis patients had a history
of raw anchovy ingestion. Thus, raw or marinated anchovies appear to be the dominant
vehicle of anisakiasis in Spain but it should be remembered that other fish species (and
recipes) can contribute to the disease burden in humans.
6.5.4 Home-prepared raw and marinated anchovies are the main fish specialties
causing anisakiasis
It was assumed that untreated home-prepared raw and marinated anchovies was the fish
specialty involved in parasite transmission to humans. However, raw fish ingestion at
restaurants and bars may also be implicated as a vehicle of disease, especially for the
hospital studies (i.e. “La Paz”, “Virgen de la Salud” and “Antequera”) performed before
the implementation of the Royal Decree RD 1420/2006 (Ministerio de Sanidad y
Consumo, 2006) which requires freezing prior to raw consumption. It was therefore
possible that the estimated ID50 values for the latter hospital studies could be low due to
underestimation of the number of untreated anchovy meals consumed by the catchment
population of each hospital. However, considering that anchovies consumed in
restaurants and bars are often of industrial origin (i.e. prepared from frozen fish) (Valiñas
et al., 2001), and since the Carlos III hospital study was performed after the
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implementation of RD 1420/2006 (Ministerio de Sanidad y Consumo, 2006) (“Carlos III”
ID50 values were not the highest), this may not be a significant issue.
6.5.5 Viability of Anisakis spp. in untreated anchovy meals and their inactivation in
the human stomach
It has been demonstrated that the marinating process does not inactivate all Anisakis spp.
larvae present (EFSA-BIOHAZ, 2010). However, the action of ingredients (e.g. vinegar,
lemon, olive oil, garlic, parsley, salt, etc., usually present in some raw or marinated
specialties) and storage time may reduce the viability or alternatively reduce the
pathogenic potential of larvae. Anisakis spp. can survive the traditional Spanish
marinating procedure, even though its survival can be compromised by increasing the
concentration of salt, acetic acid and storage time in brine (Sánchez-Monsalvez et al.,
2005). In vitro studies have demonstrated that saline extracts from garlic can be
destructive to Anisakis spp. (Valero et al., 2015). The QRA model was run considering
viability of 100%, even though viability could be lower, and ID50 values for viabilities
of 50% and 10% were therefore also generated.
6.5.6 Implementation and validity of the exponential dose response model for
Anisakis spp.
It has been reported that infection with a single parasite may cause severe health problems
that may require surgical treatment (Alonso-Gómez et al., 2004; Del Rey Moreno et al.,
2013; Repiso Ortega et al., 2003) and hence a single hit dose-response, where one
organism has a finite probability of causing disease, is appropriate. The two main forms
of such a model are the exponential, that is used here, and the beta-Poisson (Haas et al.,
2014). The beta-Poisson model incorporates heterogeneity in both the parasite and the
host, whereas the exponential model assumes that all parasites and hosts are identical.
The ID50´s were determined by estimating exposure across a population that had a
specific number of cases reported to the local hospital or from questionnaire 1 respondents
reporting allergy to Anisakis spp. These are novel approaches but could be biased because
of heterogeneity in host immune response and heterogeneity in Anisakis spp.
pathogenicity (see next section for further discussion). Since four different hospital
studies were included, it is anticipated that this will incorporate some of the uncertainty
in the data from across Spain but it should be noted that these hospitals only serve 3% of
the total population (Spanish Statistical Office - available at: http://www.ine.es/ accessed
3rd May 2016). It is also worth noting that these methods of estimating the dose-response
173
are advantageous ethically as they provide an alternative to carrying out animal and
human studies.
6.5.7 Pathogenicity of Anisakis spp. species and susceptibility of humans
The QRA model was performed considering Anisakis spp. as a single pathogen even if A.
pegreffii and presumably A. simplex s.s. have been identified in anchovies (Cipriani et al.,
2016; Rello et al., 2009; Šimat et al., 2015). Some studies have suggested differences
between A. pegreffii and A. simplex s.s. in terms of immunopathogenicity capacity against
humans (Arcos et al., 2014; Arizono et al., 2012; Jeon and Kim, 2015). Humans may also
have different susceptibilities to these zoonotic nematodes (EFSA-BIOHAZ, 2010).
Caballero et al. (2013) found differences in clinical and immunological symptoms
between Italian and Spanish Anisakis spp. allergic patients. Moreover, the presence of
high risk population groups (e.g. elderly population) and major risk factors (e.g. raw fish
consumption) for anisakiasis is also important. Further improvement of the model could
be implemented if heterogeneity of human host and parasite species is confirmed and data
become available.
6.5.8 Sensitization and allergy to Anisakis spp.
Allergy to Anisakis spp. is relatively common in some Spanish regions (Audicana and
Kennedy, 2008; EFSA-BIOHAZ, 2010) and subclinical sensitization (i.e. Anisakis-
specific IgE detection in individuals who do not show allergic manifestations) may also
occur (Del Rey Moreno et al., 2006; Fernández de Corres et al., 2001; Moneo et al., 2000;
Puente et al., 2008; Valiñas et al., 2001). For instance, Del Rey Moreno et al. (2006)
reported seroprevalence to A. simplex of 22.1% (n= 17 out of 77 random blood donors)
tested by CAP-FEIA in a healthy Spanish population (“Antequera” hospital region).
Other studies in healthy populations showed seroprevalence ranging between 6.6 and
27.5% (Del Rey Moreno et al., 2006; Puente et al., 2008). Valiñas et al. (2001) reported
seroprevalence of 0.4% (n= 12 out of 2,801 random blood donors from Galicia) tested by
an antigen-capture ELISA method, and suggested that more than 150,000 individuals may
have IgE sensitization to Anisakis spp. in Spain. It is generally considered that
sensitization with living Anisakis spp. larvae is required prior to development of the
clinical allergic responses (EFSA-BIOHAZ, 2010; Pravettoni et al., 2012). Therefore,
these data suggest that a considerable proportion of the Spanish population is sensitized
by Anisakis spp. infection at some point in their life, even though the disease (anisakiasis)
was not diagnosed or asymptomatic, and therefore remains underreported. However, it
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has been suggested that ingestion (and inhalation) of dead larvae or their related allergens
might also initiate allergic problems (EFSA-BIOHAZ, 2010). In addition, cross-reactivity
with other parasites, insects and shellfish has also been suggested (EFSA-BIOHAZ,
2010), so the allergy debate is still open.
A number of studies have suggested that differences in seroprevalence to Anisakis spp. in
different Spanish regions can be explained by differences in raw fish consumption habits
of the population (Fernández de Corres et al., 2001; Moneo et al., 2000; Puente et al.,
2008). The results presented here are in accordance with this hypothesis, since the
communities (Andalucía and Madrid) with the highest consumption of untreated anchovy
meals, presented the highest numbers of anisakiasis cases, whilst the communities
(Cantabria and País Vasco) with highest consumption of untreated meals per capita,
presented the highest incidence of disease. In addition, a multicentre study found that 8%
of Spanish patients presented with some form of allergic reaction, of which 38.1% were
sensitized and 19.2% were allergic to Anisakis spp., had reported consumption of non-
cooked fish at least once a week, with anchovies in vinegar being the most frequent meal
(Fernández de Corres et al., 2001).
6.5.9 Post-mortem migration of Anisakis spp. from fish viscera to the muscle
The QRA results showed that the number of parasites consumed per meal and the total
number of anisakiasis cases would increase considerably following inadequate storage of
whole anchovies at 7°C during 72 hours pre-evisceration. Typical temperatures of 7°C
have been reported in food stored in Spanish domestic refrigerators (Garrido et al., 2010).
This enables migration of Anisakis spp. from fish viscera to the muscle if the fish are
stored whole. Cipriani et al. (2016) reported no significant increase in the A. pegreffii
infection rates in anchovy muscle when fish were refrigerated at 2°C when examined 24,
48 and 72 h after capture in the Mediterranean Sea. However, significant increases in
infection rates in muscle were observed when fish were refrigerated at 5°C and 7°C at all
storage times. In addition, Šimat et al. (2015) similarly studied post-mortem A. pegreffii
migration from anchovy viscera to muscle when fish were refrigerated at 0°C and 4°C
after zero, three, five and seven days after capture in the Mediterranean Sea. Post-mortem
migration of A. pegreffii was observed in anchovy muscle refrigerated at 0°C and 4°C
after five and three days post capture, respectively (Šimat et al., 2015). Thus, temperatures
of 2°C appears therefore to be sufficient through the anchovy value chain prior to retail
sale to prevent post-mortem migration of A. pegreffii. Further work is required to
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determine if higher temperatures such as 3°C or 4°C would also be sufficient to prevent
A. pegreffii post-mortem migration for at least 48 hours (anchovies should be at retail for
less than two days to ensure organoleptic quality). Further work is also needed to
determine what temperatures and storage times would prevent A. simplex s.s. post-mortem
migration in European anchovies from Atlantic waters. It is also important for consumers
to know that fresh anchovies should be eviscerated as soon as possible after purchase and
frozen. It is likely that this could be achieved through an education campaign.
6.5.10 Risk mitigation strategies
The QRA simulation demonstrates the trivial result that if an education campaign results
in an 80% increase in the number of anchovy meals that are frozen then this results in an
80% reduction in the incidence of anisakiasis in the human population. However, the
finding from questionnaire 1 that 89% of those consuming untreated anchovy meals knew
that freezing was required to prevent anisakiasis but currently do not carry this out
suggests that it is important to target this group of individuals. Further, it is necessary to
understand why they do not freeze anchovy and what motivation is required for them to
change their behaviour. This should be addressed in order to inform targeted public health
education campaigns in Spain. Further, an educational campaign using the media (e.g.
press, television, etc.) may be best targeted in the spring and summer months when
anchovies in vinegar are most frequently consumed, and especially in those communities
with higher numbers of anisakiasis cases (Andalucía and Madrid) and higher incidence
of the disease (Cantabria and País Vasco). Other mitigation strategies, such as removal of
anchovy viscera by retailers to prevent parasite migration may also reduce disease
incidence and it is also important to find out how retailers and/or consumers can be
persuaded to do this (D’amico et al., 2014).
6.5.11 Extending the QRA method to other countries and other parasites
The QRA model focussed on the anchovy value chain from the sea to the consumer. The
model has the potential to be applied to other countries that also have anisakiasis (e.g.
Japan and Italy). The model could be parameterised to include other fish species (e.g.
hake, herring) and other methods of preparing fish (e.g. under or lightly cooked fish).
Moreover, anisakidosis caused by other zoonotic anisakids (e.g. Pseudoterranova spp.,
Contracaecum spp.) or other fish-borne parasitic zoonoses (e.g. ophistorchiasis,
clonorchiasis, intestinal trematodiasis and diphyllobothriasis) may be assessed for risk.
In the first instance, the Anisakis spp. dose-response model in this paper could be used as
176
a surrogate as has been done previously (e.g. using Shigella spp. dose response for E. coli
O157 (Cassin et al., 1998)). However, if data are available then dose response models for
these other parasites could also be developed.
In the QRA model the inclusion of economic data on fish catches and imports, fish
parasite abundance surveys, questionnaire information on consumption habits and
preferences can be updated over time. This will enable temporally dynamic estimates of
the disease burden of this zoonosis to be performed and also identify which factors are
important in its ongoing emergence. These techniques are readily applicable to a number
of other infectious diseases including gastrointestinal pathogens (Cassin et al., 1998;
Gale, 1998; Lindqvist and Westöö, 2000; Strachan et al., 2002).
Finally, the results of the QRA model are only accurate to the extent that the input data
are valid and the model variables represent the process. The results seem plausible, even
though they were estimated based on a number of assumptions that are described above.
Further research is recommended to validate the findings and this could involve an
intensive study of consumption patterns and epidemiological investigations in
representative communities within Spain. The model incorporates the variation in the
datasets that were obtained. However, there are uncertainties in whether some of these
data are properly representative across Spain. Further work needs to be done to include
this uncertainty into the model (e.g. via a second-order Monte Carlo model) to determine
the effect this has on model outputs and identify which model parameters are most
uncertain.
6.5 Conclusions
This is the first time that a QRA study of anisakiasis caused by fish meals has been
performed and it integrates data obtained from natural and social science methods. The
results indicate that anisakiasis is a highly underreported disease (e.g. by misdiagnosis,
undiagnosed and unreported cases). The annual number of anisakiasis cases that required
medical attention in Spain is estimated to be between 7,700 and 8,320 using the two risk
characterisation methods, with 42% occurring in the Spanish communities of Andalucía
and Madrid (RC method 2). These results suggest that Spain has a high anisakiasis
incidence, compared with other countries, but this needs verified by implementation of
comparable methods of analysis between countries. The dose response for Anisakis spp.
and corresponding ID50 (mean 17,000, SD 14,000) was reported for the first time. On
177
average, the QRA estimates that 0.66 Anisakis spp. are consumed per raw or marinated
anchovy meal in Spain.
This study makes use of the ecological information that is known about Anisakis spp., the
anchovy value chain, as well as human behaviour, and puts this into the context of risk
and what can be done to mitigate the disease. In addition, the methods have the potential
to be applied to other countries that also have anisakiasis, fish-borne zoonosis and
different cooking preparations. The results are of relevance to industry, medical
practitioners and consumers, and can be used to inform policy (e.g. by food safety
authorities) and to reduce risk of disease in two ways. Firstly, by highlighting the need
for adequate cold storage of anchovies, early evisceration, and parasite monitoring (e.g.
improved protocols and technology for parasite detection in fishery products) to control
the pathogen along the value chain, and secondly, identifying the importance of changing
consumer habits, particularly those who currently eat untreated anchovy meals, by
education campaigns to encourage freezing of fish prior to consumption. The efficacy of
these strategies can be monitored by observing changes in disease incidence through
improved reporting and by monitoring behavioural changes in anchovy preparation
methods and consumption preferences by questionnaire. Then it will be possible to make
progress in reducing the disease burden of this emerging zoonosis.
6.6 Acknowledgements
The authors sincerely thank the Biobanking platform at the PARASITE project (EU FP7
PARASITE project (GA no. 312068)) for providing host-parasite data. We thank Rosa
Fernández and Cristina Martínez from CETMAR for their help during creation and
divulgation of the questionnaires. We also thank Arturo del Rey Moreno (“Antequera”
hospital) for his helpful comments. We are also grateful to “Subdirección General de
Economía Pesquera” of “Ministerio de Agricultura, Alimentación y Medio Ambiente”
(MAGRAMA) of the Spanish government for providing anchovy trade statistics for 2013.
M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial
support of the contract from the EU Project PARASITE (grant number 312068).
6.7 Authors’ contributions
MB and NS conceived and designed the study, developed the QRA model, analysed the
results and drafted the paper. IM, IB, SM and PC provided and analysed data of Anisakis
spp. incidence in anchovy in the Mediterranean Sea, and SP in the Atlantic Ocean. GJP
178
co-supervised the work and helped draft the paper. All authors (MB, GP, SP, MG-M, SM,
IM, PC, IB and NS) critically reviewed the paper and agreed the final content of the
version to be published.
179
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Chapter 7 - General discussion, future work and conclusions
7.1 Overview
This multidisciplinary thesis focused on providing new insights and methodologies in
relation to the fish-borne zoonotic parasite Anisakis spp. in fish, and associated problems
caused by its presence in fishery products. In particular, the infection levels of Anisakis
spp. in returning anadromous fish (sea lamprey, allis and twaite shads) sampled from
freshwater ecosystems were investigated, and the zoonotic, epidemiological and
ecological implications of the findings were discussed (chapters 2 and 3). The
anatomical location of Anisakis spp. larvae in the viscera of Atlantic herring was studied,
and Magnetic Resonance Imaging was investigated as a new tool for anisakid detection
in the viscera of herring and in fish muscle samples (chapter 4). In addition, the attitudes
of fish consumers in relation to the presence of Anisakis spp. in fishery products and how
they would value fishery products “free” of Anisakis spp. was investigated (chapter 5).
Finally, the risk of anisakiasis caused by consumption of untreated home-prepared raw
and marinated anchovy meals was assessed, and the incidence of disease in population
estimated (chapter 6).
In this chapter, the most important results of this thesis are revisited and the
epidemiological, ecological, technological, socio-economical, legislative and public
health insights and implications of these results to the current knowledge of Anisakis spp.
and associated problems caused by its presence in fishery products are more broadly
discussed. In addition, future studies are suggested.
7.1.1 Anisakis spp. in anadromous fish – potential health risk for humans and
wildlife
The genus Anisakis can be found parasitizing a wide number of marine fish and
cephalopods from all major oceans and seas worldwide (Mattiucci and D’Amelio, 2014;
Mattiucci and Nascetti, 2008). However, little is known about their potential presence in
anadromous fish entering rivers during their reproductive upstream migration phase.
Anisakis spp. have been reported in returning American shads (Alosa sapidissima)
captured from rivers of Pacific Northwest (Shields et al., 2002) and Atlantic Northeast
coasts of Canada (Hogans et al., 1993), as well as in Atlantic salmon (Salmo salar) from
Scottish rivers (Noguera et al., 2015, 2009). Anisakis spp. have been also reported in
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anadromous blueback shad (Alosa aestivalis) and alewife (Alosa pseudoharengus) from
the estuary of the Miramichi river (Canada, Atlantic Northeast) (Landry et al., 1992), in
Japanese grenadier anchovy (Coilia nasus) from the estuary of the Yangtze river (China)
(Li et al., 2011), arctic lamprey (Lethenteron camtschaticum) from Amur river (Russia)
(Appy and Anderson, 1981), and in Atlantic salmon, Arctic char (Salvelinus alpinus) and
Atlantic whitefish (Coregonus huntsmani) from Canadian rivers (Atlantic northeast)
(Pufall et al., 2012). One of the aims of the present thesis was to provide a better
understanding of the ecological and zoonotic importance of the presence of Anisakis spp.
in anadromous fish caught in rivers of Western Iberian Peninsula. This was studied in
three anadromous fish species for the first time, i.e. sea lamprey (chapter 2) and allis and
twaite shads (chapter 3). Results reported the occurrence of zoonotic Anisakis spp. larvae
in these fish species captured from freshwater environments, and highlighted the potential
risk for humans and terrestrial mammals. There may be a risk of anisakiasis if infected
anadromous fish are consumed raw or partially raw, and of allergic symptoms for
Anisakis-sensitized consumers, and this information is of special interest for the European
Food Safety Authority (EFSA) and the Spanish Agency for Consumer Affairs, Food
Safety and Nutrition (AECOSAN), and it should be considered, especially during
elaborations of guidelines for prevention and control of anisakiasis and allergy to Anisakis
spp., and related educational campaigns. It should be remembered here that the EFSA and
AECOSAN consider that fish from continental waters would not act as vehicle for
anisakiasis (see section 1.4 in chapter 1). See also section 7.2.2 for further discussion
and recommendations for future work.
7.1.2 Anisakis spp. in freshwater ecosystems – ecological implications
In the last two decades, anisakids (especially, Anisakis spp.) have been a trending topic
within academia, fish industry and consumers, mainly due to the risk posed by their
presence in fishery products (Llarena-Reino et al., 2015). As a result, the number of
scientific publications increased, some of them focussed on providing better knowledge
of the life cycle, geographical and seasonal distribution, prevalence, intensity and
anatomical location of Anisakis spp. larvae in fish as recommended by EFSA (EFSA-
BIOHAZ, 2010). The results provided in chapter 2 reported for the first time the
epidemiology of infection of A. simplex s.s. in sea lamprey, and discussed the role of sea
lamprey acting as a transport host for A. simplex s.s. from marine to freshwaters
ecosystems. In chapter 3, mixed infections of A. simplex s.s. and A. pegreffii were
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reported for the first time in allis and twaite shads of Western Iberia rivers, and similarly,
discussed the role of anadromous shads as transport hosts for both Anisakis species from
the marine to freshwater ecosystems. Anisakis spp. larvae cannot survive in freshwater
habitat (Højgaard, 1998), hence, the effective expansion of the life cycle of Anisakis spp.
to these ecosystems do not seem possible. However, their transmission through the
trophic chain to other fish host and/or wildlife (aquatic birds (e.g. cormorants) or
terrestrial mammals) cannot be excluded (see also section 7.2.2). Indeed, A. simplex s.s.
has been recently reported in downstream post-metamorphic sea lamprey (Gérard et al.,
2015), probably by acquiring the larvae feeding on parasitized anadromous or estuarine
fish hosts.
7.1.3 Anatomical location of Anisakis spp. in anadromous fish – epidemiological
insights
The study of the infection levels and location of Anisakis spp. in the fish host is important
for a better epidemiological understanding of the infection as well as for obvious food
safety and quality issues. In terms of health risk, the presence of larvae in edible parts (i.e.
muscle, roes) constitutes the main source for human infection. The occurrence of larvae
in the body cavity may also represent a health risk, and affects the quality of the product.
It has been reported that in some fish species, e.g. European hake (Merluccius merluccius)
and blue whiting (Micromesistius poutassou), Anisakis spp. larvae normally occur in
higher levels in the body cavity organs, even though they also occur in high numbers in
muscle (Cipriani et al., 2015; Gómez-Mateos et al., 2016; Tejada et al., 2014). On the
contrary, in wild Alaska salmon, higher numbers of larvae were found in musculature
rather than in visceral organs (Karl et al., 2011). Within the A. simplex complex, A.
simplex s.s. apparently tends to occur in higher rates than A. pegreffii in fish muscle when
cohabiting the same fish host (e.g. European hake, chub mackerel (Scomber japonicus)
and blue whiting) (Cipriani et al., 2015; Gómez-Mateos et al., 2016; Suzuki et al., 2010).
Thus, Anisakis spp. larvae can parasitize the viscera and muscle of a large number of
paratenic or intermediate fish hosts (Mattiucci and D’Amelio, 2014; Mattiucci and
Nascetti, 2008), however knowledge on these epidemiological aspects of this zoonotic
parasite are still insufficient. In this regard, the anatomical location of Anisakis spp. larvae
was studied in the latter anadromous species (chapters 2 and 3). Anisakis simplex s.s.
was found parasitizing the viscera and muscle of sea lamprey in unexpected high
numbers, being more frequent in the viscera of spawning lampreys (Prevalence 44% in
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viscera compared to 38% in muscle), but more frequent in the muscle in post-spawning
individuals (Prevalence 36% in muscle compared to 29% in viscera) (chapter 2). This
difference might be an artefact of sampling effort in post-spawning individuals (only 14
lampreys were sampled), or it might be that some Anisakis spp. larvae could have been
expelled during spawning. Further research would be required to confirm or reject these
differences in muscle infection rates and investigate the causes. In shads, mixed infections
of A. simplex s.s. and A. pegreffii were clearly more abundant on viscera than in muscle
(chapter 4). Only one larva of A. simplex s.s. was found parasitizing the muscle of twaite
shad. Interestingly, four Anisakis spp. larvae were molecularly identified from the flesh
of allis shad, three belonging to A. simplex s.s. and one to A. pegreffii. These results
appeared to be similar to previous studies reporting higher rates in muscle for A. simplex
s.s. (see above).
7.1.4 Anatomical location of Anisakis spp. in clupeids – epidemiological insights
In chapters 3 and 4 the location of Anisakis spp. larvae in the body cavity of allis and
twaite shads and in Atlantic herring was investigated, shifting the focus towards marine
fish (i.e. herring, chapter 4). Findings showed that A. simplex s.l. larvae tend to
accumulate in the visceral cavity at the posterior end of the terminal blind sac of the
stomach, around the ductus pneumaticus, therefore, providing new insights and
hypothesis (in chapter 4) in relation to the preferred habitat of settlement of Anisakis spp.
larvae within fish hosts, which it is still largely unknown. Apparently, during the digestion
process, Anisakis spp. larvae emerge from the stomach to the body cavity at this location,
and in mature clupeids, the gonads may act as physical barriers, therefore, preventing in
vivo migration of larvae from viscera to muscle (Figure 7.1). This information is
important because it helps to understand the epidemiology of Anisakis spp. infection in
clupeid hosts (e.g. preferred encapsulation sites, in vivo migration from fish viscera to
flesh), which is likely associated with the digestion process, internal anatomy of the fish
and physicochemical properties of the fish tissues. In addition, as detailed above, different
Anisakis species may have different preferred habitat of settlement (i.e. A. simplex s.s.
appears to be in higher rates in muscle than A. pegreffii). Further investigations are
required to understand which are the main settlement habitats and the in vivo migration
drivers of these parasites in different fish species and why.
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Figure 7.1. Accumulation of anisakid larvae at the posterior end of the blind sac of an A.
alosa stomach.
7.1.5 Magnetic Resonance Imaging – potential new technology for parasite
investigation and detection
In chapter 4, a summary of the methodologies applied for anisakid detection in fishery
products was provided. In general, invasive and destructive methods, such as artificial
enzymatic digestion (Llarena-Reino et al., 2013b) and UV-press (Karl and Leinemann,
1993), are more accurate for detecting and quantifying whole anisakids embedded in fish
muscle, but more time-consuming and expensive than non-destructive visual inspection
and candling methods (EFSA-BIOHAZ, 2010; Levsen et al., 2005). Visual inspection and
candling may detect the Anisakis spp. larvae on (or embedded near) the surface, and
Pseudoterranova spp. larvae embedded in skinless and white fish muscle. However, these
methods fail to detect and quantify Anisakis spp. larvae embedded deep in thick or skin-
on fillets (EFSA-BIOHAZ, 2010; Karl and Leinemann, 1993; Levsen et al., 2005). Visual
inspection of fish or fishery products, including if necessary, candling of fillets on a light
table in a darkened room, are the parasite detection methods required by EU legislation
to be applied by Food Business Operators (FBOs) during the production of fishery
products (EC, 2005, 2004a). However these methods, as detailed above, are not reliable
for the quantitative determination of anisakids in fish and may depend on inspector
training and skills (Levsen et al., 2005) (further discussion in section 7.1.6).
A better understanding of the epidemiology of infection of Anisakis spp. in fish hosts is
important, as is the control of the parasite in the food chain in order to ensure food safety
and quality. In chapter 4, Magnetic Resonance Imaging (MRI) was utilised for the first
time for the investigation of A. simplex s.l. larvae accumulations in the viscera of whole
herring (which were previously identified by visual inspection), and to detect anisakid
larvae (Pseudoterranova and Anisakis) in fish muscle. The study showed the potential of
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MRI technology to detect anisakids in fish in situ in a 3D environment, thereby showing
capacity to identify the actual site of infection, and in a non-invasive and non-destructive
way, which may be of interest for fundamental (i.e. to investigate Anisakis spp.
anatomical location and migration behaviours within fish hosts) and applied research
(new potential technology for parasite detection in fishery products). In relation to the
latter, MRI has some interesting benefits for its potential use by the industry, i.e. it is a
non-destructive and non-invasive tool, which means that the quality and safety of the
product will not be comprised by the scanning process (Cheng et al., 2013). However, it
has a number of disadvantages that need to be addressed before it can be applied in routine
(automated or not) inspection/control of parasites in fishery products by the industry.
Some disadvantages were summarized in chapter 4, e.g. MRI is expensive, time-
consuming, requires specialised expertise (Cheng et al., 2013; Mathiassen et al., 2011;
Patel et al., 2015; Veliyulin et al., 2007), and cannot be applied in frozen products. Further
evidence and research is required to show if MRI can also be able to quantify nematode
numbers and differentiate nematode species in products. Finally, it has to be efficient,
reliable and feasible technology for parasite detection in fishery products that can be
readily used by the fishing industry.
7.1.6 The problem of Anisakis spp. in fishery products – socioeconomic, legislative
and parasite control issues
7.1.6.1 Socioeconomic aspects
The socioeconomic (and legislative) issues caused by the presence of Anisakis spp. in
fishery products are well recognized in the literature (D’amico et al., 2014; Llarena-Reino
et al., 2015, 2013a, 2013b, 2012; Pascual et al., 2010). Anisakis spp. larvae compromise
the quality and safety of fishery products, therefore becoming a considerable concern for
consumers, official control authorities and FBOs in the fishery value chain (D’amico et
al., 2014). Indeed, Anisakis spp. can provoke rejections and claims of fishery products by
fish sellers and consumers (Llarena-Reino et al., 2015), and notable negative effects on
fish marketability (Abollo et al., 2001; Karl, 2008; Mattiucci and D’Amelio, 2014), which
may result in economic losses for the industry, loss of fishery jobs, and an increasing lack
of confidence in fishery products by consumers (D’amico et al., 2014; Karl, 2008;
Llarena-Reino et al., 2015; Pascual et al., 2010). In chapter 5, a survey-based contingent
valuation study was performed to investigate the attitudes and opinions of fish consumers
in relation to the presence of Anisakis spp. in fish and associated diseases (i.e. anisakiasis
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and allergy), and how much they would value the removal of Anisakis spp. from fishery
products for the first time. Results identified that a majority of consumers (77%) were
willing to pay for Anisakis-free fish product, and that over a quarter of consumers (>25%)
had previously avoided purchasing/consuming fishery products due to the presence of
Anisakis spp. Moreover, almost one third of consumers (29%) would always avoid (in the
future), or would avoid if there were a high chance of Anisakis ssp. in their fish (31%),
purchasing/consuming fishery products due to this parasite. These findings provided
further evidence to support the premise that Anisakis spp. is an important health and
aesthetic issue for consumers and to highlight the potential monetary losses for the
industry and also revealed that currently parasite control through the fish value chain is
not efficient.
7.1.6.2 Parasite risk control by FBOs in the fishery chain and legislative implications
The problem caused by the presence of Anisakis spp. in fishery products was recently
examined in the European context, with particular focus on the risk management issues
that are of interest for FBOs in the fish value chain of Italy (D’amico et al., 2014) and
Spain (Llarena-Reino et al., 2015, 2013a). After the “European Hygiene Package” (EC,
2005, 2004a, 2004b, 2004c, 1996, 1993, 1991) and its modifications (EC, 2006a, 2006b),
FBOs at any stage of the fishery chain (on shore and on board vessels) have become
responsible for ensuring consumer protection with regard to food safety, and they must
ensure that fishery products have been subjected to a visual examination to detect visible
parasites, and ensure that no obviously contaminated fish product reaches the consumer
(EC, 2004a). In particular, Commission Regulation (EC) No. 2074/2005 laid down the
rules to be applied by FBOs relating to visual inspections to detect parasites in fishery
products, specifying that “Visible parasite means a parasite or group of parasites which
has a dimension, colour or texture which is clearly distinguishable from fish tissues”, that
“Visual inspection means non-destructive examination of fish or fishery products with or
without optical means of magnifying and under good light conditions for human vision,
including, if necessary, candling”, and that “Candling means, in respect of flat fish or fish
fillets, holding up fish to a light in a darkened room to detect parasites” (EC, 2005). The
use of confusing terminology such as “visible parasites” and “obviously infected with
parasites” as established in the Regulations, and the lack of standardized and efficient
methods and procedures of parasite detection during fish inspections by the fishing
industry, represent a source of uncertainty in hazard analysis during parasite self-controls
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(D’amico et al., 2014; Llarena-Reino et al., 2015, 2013a, 2013b, 2012). Hence, visual
inspection on liver, gonads and visceral cavity of eviscerated fish, and of fish fillets
(where necessary, candling must be included in the sampling plan) are required by
legislation (EC, 2005) but these visual methods are not efficient for quantitative
determination of parasites in fish (see section 7.1.5). In addition, Llarena-Reino et al.
(2012) evidenced the low efficiency of visual inspection of visceral nematodes as a
predictor of the number of parasites in flesh, therefore, recommending invasive inspection
of flesh for accurate detection of parasitized fish (Llarena-Reino et al., 2012). The results
of the contingent valuation study (chapter 5) revealed that more than a quarter (27%) of
respondents (Spanish fish consumers) had avoided purchasing or consuming fish due to
the presence of Anisakis spp. in fishery products, thus emphasising the inefficiency of the
risk management procedures carried out by FBOs, and that fish contaminated with visible
Anisakis spp. are actually reaching the market (Figure 7.2).
Figure 7.2. Ventral fillet of European hake purchased in a local supermarket (Aberdeen)
clearly parasitized by Anisakis spp.
In addition to EU regulations, the Codex Alimentarius of the Food and Agriculture
Organization of the United Nations (FAO) and World Health Organization (WHO),
through its International Food Standards, guidelines and codes of practice, also
contributes to ensure the safety and quality of fishery products, and although it does not
impose a legal obligation for FBOs, these are internationally accepted recommendations
(Codex Alimentarius, 2013, 2004, 1995, 1989a, 1989b). These standards set out the
procedures for detection of parasites in certain fishery products and recommend in which
cases a fish lot should be rejected as being considered defective. The standard for quick
frozen fish fillets (CODEX STAN 190-1995) recommends the candling method for the
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detection of parasites in skinless fillets, and that a sample unit shall be considered as
defective when the presence of two or more parasites with a capsular diameter greater
than 3 mm or a parasite not encapsulated and greater than 10 mm in length, per kg of
sample unit, is detected (Codex Alimentarius, 1995). The standards for salted herring and
sprat (CODEX STAN 244-2004) and for smoked fish (CODEX STAN 311-2013)
recommend that the flesh shall not contain living nematodes, the viability of which shall
be examined by means of artificial peptic digestion (i.e. nematodes survive the digestion
process) (Codex Alimentarius, 2013, 2004). If living nematodes are present, the accepted
freezing procedures to kill the nematodes must be applied (e.g. Regulation EC No
853/2004 requires fishery products intended to be consumed raw, almost raw, marinated,
salted or cold-smoked must be frozen at -20°C or less, for not less than 24 hours in all
parts of the product (EC, 2004a)) prior to placing the product on the market for
consumption. Salted fish should be examined for visible parasites by normal visual
inspection (Codex Alimentarius, 2004) and smoked fish by candling (Codex
Alimentarius, 2013), and the sample shall be considered defective as explained above
(see CODEX STAN 190-1995) for salted fish, and if readily visible parasites are detected
in the sample for smoked fish. FBOs comply with the legislation and these internationally
accepted standards, to carry out parasite control on their facilities and products (Llarena-
Reino et al., 2013a), but again this may not guarantee the quality and safety of fishery
products.
7.1.6.3 How to improve parasite control by FBOs in the fishery chain?
Llarena-Reino et al. (2013a) proposed a scoring system approach to categorize parasite
infection in fish lots, therefore, helping the FBOs to the use of a standardized protocol to
ensure food quality and safety during parasite self-control. The scoring system ranked the
parasite risk from 0 to 10 (from high to lower risk) depending on four different criteria:
site of infection (e.g. visceral body cavity, hypaxial and epaxial flesh), quality (i.e. visual
parasite problems), demography of infection (density of parasites per Kg of flesh
(determined by a combination of visual followed by peptic digestion methods)) and
epidemiology relevance (i.e. zoonotic or non-zoonotic parasite). In terms of demography
of infection, samples were divided into three groups, D0: density > 5 parasites/Kg, D1:
density 2-5 parasites/Kg, D2: density < 2 parasites/Kg. The scoring system would
probably achieve better levels of food quality compared to the current legislation criteria
followed by FBOs, but further work is definitely required to also ensure food safety of
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fishery products. Further work is required to determine what number of parasites (and
their allergens) per Kg of fish flesh would represent an 1) acceptable, 2) tolerable or 3)
unacceptable health risk for human consumption (see section 7.2.4). Thus, improvements
in the parasite control legislation of fishery products are recommended in order to
establish standardized, efficient, fast, low cost, and research-based (e.g. dose-response
relationship modelling) parasite detection procedures, which can then be implemented by
FBOs in the fishery chain to protect consumers by ensuring the quality and safety of
fishery products, as well as to protect the fishing and food industries by minimizing the
parasite-associated economic losses (see future work section).
7.1.7 Assessing the risk of anisakiasis and its incidence in population – public health
insights
Anisakiasis is considered an emerging zoonosis of worldwide concern (EFSA-BIOHAZ,
2010; McCarthy and Moore, 2000). Over the last decades, there has been a marked
increase in the reported prevalence and geographic range of anisakiasis throughout the
world (EFSA-BIOHAZ, 2010; Mattiucci et al., 2017). Approximately 20,000 cases of
anisakiasis were reported worldwide up to 2010, of which over 90% were from Japan,
where it is estimated that around 2,000 cases are diagnosed annually (EFSA-BIOHAZ,
2010). Previously, Ishikura et al. (1998) estimated that the total number of anisakidosis
(including zoonosis caused by Anisakis spp. and other Anisakidae family members (i.e.
Pseudoterranova and Contracaecum)) in the world up to 1997 was around 35,000 cases,
of which 97.11% (32,300 cases) were from Japan (Ishikura et al., 1998). It is considered
that the anisakid most frequently involved with human infection is Anisakis spp.
(Buchmann and Mehrdana, 2016; EFSA-BIOHAZ, 2010), therefore, it appears that the
estimations reported by EFSA-BIOHAZ may be underestimated, if Ishikura et al. (1998)
estimates are accurate. Sohn and Chai (2011) estimated later that over 50,000 anisakidosis
cases have been reported worldwide, increasing more than two times the estimations by
EFSA-BIOHAZ (Sohn and Chai, 2011). Recently, Yorimitsu et al. (2013) and references
therein (in Japanese) estimated approximately 3,000 cases of anisakiasis in Japan per year
(Yorimitsu et al., 2013). New cases of anisakiasis have been reported in several countries
where they were not previously reported, such as Croatia (Juric et al., 2013; Mladineo et
al., 2016), Taiwan (Li et al., 2015), China (Qin et al., 2013) and Malaysia (Amir et al.,
2016), as well as in endemic countries of Europe, including Spain (Baeza-Trinidad et al.,
2015; Menéndez et al., 2015; Sánchez Justicia et al., 2017), Italy (Piscaglia et al., 2014),
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France (Dupouy-Camet et al., 2016) and Belgium (Özcan et al., 2012); Asia, including
Japan (Matsuura and Moritou, 2017; Shimamura et al., 2016a) and South Korea (Sohn et
al., 2015); and North America, including United States (Ahmed et al., 2016; Shweiki et
al., 2014) and Canada (Muwanwella et al., 2016; Shimamura et al., 2016b). This increase
in the number of reported anisakiasis cases and geographical range observed over the last
three decades is probably due to changes in dietary behaviour (increasing global demand
for seafood and growing interest for raw or lightly cooked fish) as well as to improved
techniques and knowledge/expertise to diagnose infection (Audicana and Kennedy, 2008;
EFSA-BIOHAZ, 2010; McCarthy and Moore, 2000). However, in spite of the increasing
medical reports worldwide, the actual incidence of disease in each country is still poorly
estimated or unknown, and anisakiasis (and anisakidosis) is believed to be an
underestimated zoonosis globally (Baird et al., 2014; Buchmann and Mehrdana, 2016;
Del Rey Moreno et al., 2013; EFSA-BIOHAZ, 2010; Hernandez-Prera and Polydorides,
2012; Hochberg and Hamer, 2010; Mattiucci et al., 2013; Mladineo et al., 2016; Pantoja
et al., 2016; Shamsi, 2016; Shimamura et al., 2016b; Wiwanitkit and Wiwanitkit, 2016).
There is therefore a need for a better understanding and quantitative determination of the
burden of disease of this emerging and hidden fish-borne zoonosis globally, as well as to
identify strategies to help to reduce its incidence.
In chapter 6, using a probabilistic quantitative risk assessment (QRA) approach, it was
estimated for the first time that the total number of anisakiasis cases requiring medical
attention in Spain was approximately 8,000 annually (20 cases per 100,000
inhabitants/year), even though these figures might be even higher when mild anisakiasis
cases are reported and included in the QRA model. Thus, the present thesis provides
further evidence to conclude that anisakiasis is a highly underestimated and
underdiagnosed fish-borne parasitic zoonosis in Spain, basically due to misdiagnosis (i.e.
disease symptoms are not specific and may be confound with other gastrointestinal
diseases), undiagnosed (e.g. lack of clinical investigation during anamnesis, mild and
asymptomatic cases may occur) and not reported cases (anisakiasis is not a notifiable
disease) (Del Rey Moreno et al., 2013; EFSA-BIOHAZ, 2010; Kim et al., 2013; Toro et
al., 2004). These figures will inform fishing and food industry, academia, consumers and
health professionals about this underestimated zoonosis, and can be used to inform policy
(e.g. by food safety authorities) and to reduce risk of anisakiasis in a number of ways.
Firstly, anchovies (or any fish intended to be consumed as raw or lightly cooked) should
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be eviscerated as soon as possible, and low temperatures (e.g. 2°C) should be maintained
throughout the whole fish value chain from the sea to the plate (if fish remains ungutted)
in order to prevent post-mortem migration of Anisakis spp. from fish viscera to flesh (this
parasite behaviour might result in an increase of >1,000% in the number of predicted
anisakiasis cases (chapter 6)). Secondly, changing consumer habits, particularly in those
people who currently eat untreated anchovy meals (or any other raw, marinated or lightly
cooked fish meal), by education campaigns to encourage freezing fish prior to
consumption. Finally, improvements in parasite control legislation of fishery products
and parasite inspection and risk management by FBOs in the fishery chain would also
help to reduce the incidence of disease (section 7.1.6.3). The efficacy of these strategies
to reduce disease burden could then be monitored by observing changes in disease
incidence through improved reporting and by monitoring behavioural changes in anchovy
preparation methods and consumption preferences by questionnaire. Then will it be
possible to make progress in reducing the disease burden of this emerging zoonosis in
Spain.
7.2 Future work
7.2.1 QRA for allergy to Anisakis spp.
Anisakis spp. can infect humans through consumption of parasitized raw or lightly cooked
fishery products, manifesting symptoms such as abdominal pain, nausea, diarrhoea, mild
fever, etc., which may be associated with allergic reactions (Audicana and Kennedy,
2008). It is therefore considered that anisakiasis can have ectopic, gastric, intestinal and
gastroallergic clinical forms, and, additionally, allergy to Anisakis spp. can also occur
(Audicana and Kennedy, 2008). It is generally believed that Anisakis spp. infection must
occur to initiate allergic sensitivity to Anisakis spp. in humans (EFSA-BIOHAZ, 2010).
In gastroallergic anisakiasis, an acute parasite infection with live Anisakis spp. larva can
provoke gastric symptoms together with an immunoglobulin E (IgE)-mediated immune
response, which results in the presentation of allergic symptoms, such as urticaria,
angioedema or anaphylaxis (Daschner et al., 2000). However, allergy to Anisakis spp. has
been also described in sensitized patients after consumption of fish contaminated with
live or dead larvae, or with their allergens (Audicana and Kennedy, 2008). This implies
that some allergic individuals can have allergic symptoms even when the fish meal was
properly cooked or previously frozen (Audicana and Kennedy, 2008; Carballeda-Sangiao
et al., 2016b; EFSA-BIOHAZ, 2010). Other allergic manifestations, such as chronic
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urticaria, rhinitis, conjunctivitis, contact dermatitis, asthma, rheumatologic disorders, etc.
were also reported (Audicana and Kennedy, 2008; EFSA-BIOHAZ, 2010). Occupational
allergy has also been described in fishmongers and fishermen (Añíbarro and Seoane,
1998; Armentia et al., 1998; Nieuwenhuizen et al., 2006) and, recently, a case of non-
occupational airborne-induced anaphylaxis caused by Anisakis spp. has been described in
Spain (Barbarroja-Escudero et al., 2016). To date, thirteen Anisakis spp. allergens have
been described, some of them being heat and/or pepsin resistant (Baird et al., 2014;
Caballero and Moneo, 2004; Carballeda-Sangiao et al., 2016a; Moneo et al., 2005;
Nieuwenhuizen and Lopata, 2014). Allergy to Anisakis spp. has been reported to be
relatively common in some areas of Spain (Audicana and Kennedy, 2008; Fernández de
Corres et al., 2001). In addition, subclinical sensitization to Anisakis spp. allergens in
population also occur. Studies carried out in Spain showed that subclinical sensitization
in blood donors and healthy populations ranged from 0.4% to 22% (Fernández de Corres
et al., 2001; Puente et al., 2008; Valiñas et al., 2001). Thus, a probabilistic quantitative
risk assessment study should be performed to assess the risk posed by the presence of
Anisakis spp. allergens in fish meals to sensitized and healthy fish consumers.
7.2.2 QRA for human/wildlife disease from Anisakis spp. in anadromous fish
In Western Iberia (Galician and Portuguese rivers), anadromous fish species are usually
consumed well-cooked locally (in the vicinity of the rivers), therefore, the risk for parasite
live infection seems unlikely. However, during a show recently performed by a cooking
school of the region, the specialty “sushi of sea lamprey” was presented (Dr. Carlos
Antunes, pers. comm.). Such innovations in food gastronomy and subsequent new food
habits in population may imply a potential risk of anisakiasis that should be investigated
and evaluated if appropriate. In relation to this, it has been previously reported a case of
gastric anisakiasis in Canada following the consumption of raw arctic char from a local
river (Bhat and Cleland, 2010), implying that anadromous fish captured in rivers can
actually act as vehicle for anisakiasis. Thus, a QRA study would be recommended to
investigate potential risky habits of consumption of infected anadromous fish in these
regions. Moreover, the potential presence of thermostable gastro-allergens in anadromous
fish may also pose a threat to Anisakis spp. sensitized individuals that should be
investigated.
The presence of Anisakis spp. in anadromous fish entering rivers may also represent a
risk of anisakiasis for terrestrial mammals. European otter (Lutra lutra) (or other
197
mammals (e.g. European mink or invasive American mink)) inhabiting these rivers that
sustain populations of anadromous fish, may well feed on these parasitized species,
therefore, there may be a risk for accidental anisakiasis for these mustelids that should be
investigated. Indeed, European otter has been previously reported as an accidental host
of Anisakis spp. in one Spanish river (Torres et al., 2004).
7.2.3 Assessing the risk of anisakiasis in other fish preparations/species and
countries
In chapter 6 the risk of anisakiasis caused by consumption of untreated raw and
marinated anchovy meals was assessed, and the burden of disease in the Spanish
population estimated. This was performed assuming (based on literature) that all
anisakiasis cases occurring in Spain in 2013 were caused by home-prepared raw and
marinated anchovy meals. However, consumption of raw and marinated anchovies at
restaurants, or other fish species and recipes at home or at restaurants may also represent
a risk for disease. After implementation of the Spanish Royal Decree 1420/2006
establishments that offer raw and marinated fish meals are obliged to freeze the fish prior
to customer consumption (Ministerio de Sanidad y Consumo, 2006). However, this may
not always be the case, perhaps for lack of awareness of sellers or unknown reasons.
Considering that anchovies in vinegar are highly consumed in Spain, and that home-
consumed meals were suggested to be the food vehicle for thousands of anisakiasis cases
each year (chapter 6), the risk of anisakiasis due to consumption of marinated anchovies
at bars/restaurants should be assessed. Undercooked hake has been implicated in a
number of anisakiasis cases reported in Spain (Alonso-Gómez et al., 2004; Jurado-
Palomo et al., 2010). Cooked and non-cooked hake has been also implicated in Anisakis-
associated anaphylaxis cases in Spain (Audicana and Kennedy, 2008). Bearing in mind,
the high levels of Anisakis spp. infestation in the viscera and muscle of hake, as well as
the high levels of consumption of hake in Spain (Tejada et al., 2014), and also supported
by the responses about frequency and habits of hake consumption provided by consumers
in questionnaire 1 (chapter 5), a QRA study to assess the risk of anisakiasis caused by
undercooked hake in Spain is recommended. Anisakiasis cases have been also described
in Italy (Andrisani et al., 2014; Mattiucci et al., 2013; Piscaglia et al., 2014) and in Croatia
(Juric et al., 2013; Mladineo et al., 2016). In addition, raw and marinated anchovies are
also frequently consumed in Italy and Croatia. As a part of the EU FP7 PARASITE
project, questionnaire 1 (see appendix) was used in chapter 5 and 6 to gather information
198
about the frequency and habits of fish consumption of Spanish individuals. Similarly, the
questionnaire was adapted and disseminated in Croatia, and a simplified version of the
questionnaire was disseminated in Italy. The QRA model used in chapter 6 was adapted
for the Italian and Croatian cases using results from their questionnaires as well as
Anisakis spp. epidemiology data in anchovies from the Mediterranean Sea. Monte Carlo
simulations estimated that raw and marinated anchovies would also cause approximately
3,500 and 170 anisakiasis cases in Italy and Croatia, respectively (Bao et al., 2016),
however a robust QRA study would be needed and recommended to confirm these
preliminary analyses for both countries.
7.2.4 Categorizing the health risk posed by parasitized fishery products
FBOs are required to inspect for “visible parasites” and fishery products “obviously
contaminated” must not reach the market for human consumption (EC, 2004a). Thus, it
appears that the potential presence of non-visible parasites (even though these are
zoonotic) does not imply a food risk for consumers, giving the responsibility to manage
the risk to consumers which should freeze or cook properly the fish to prevent anisakiasis.
However, it is possible that the consumption of parasitized fishery products (even if the
meal was properly cooked or previously frozen) might pose a risk for health (probably
mainly derived from ingestion of Anisakis spp. thermostable allergens, and/or from
bacterial contamination transferred into the fish flesh by muscle-invading Anisakis larvae
(Svanevik et al., 2013)), and it is likely that the risk increases with the intensity of the
infection (e.g. a meal containing a hundred Anisakis spp. larvae may probably pose higher
risk than a meal with only one larva). Moreover, it is likely that a fish meal containing a
hundred larvae contains higher levels of Anisakis spp. thermostable allergens, therefore,
it may pose higher risk for consumers (sensitized or not). Therefore a QRA study should
be performed to determine the possible human health risk implied by the presence of non-
viable Anisakis spp. (and/or its allergens (section 7.2.1)) in fishery products, to then
categorize fish lots depending on this risk. This might be pursued by categorizing the risk
based on dose-response relationships (e.g. number of parasites consumed or (µg of
allergen consumed) versus antibody response in human blood (or allergic response (i.e.
urticaria, etc.) in allergic individuals, or animal studies). Ideally, the risk might then be
categorized into non-tolerable, tolerable, and negligible (parasite/allergen free) risk by
ranking the fish lots depending on potential exposure (i.e. density of muscle infection
(e.g. number of parasites or concentration of allergen per 100 g of fish flesh)). The results
199
of this study might help to mitigate the public health, legislative and socioeconomic issues
caused by the presence of Anisakis spp. in fish by developing a standardized protocol of
parasite inspection in fishery products based on health risk which would benefit policy,
FBOs and consumers.
7.2.5 Extending the contingent valuation study to other countries, and cost-benefit
analysis for Anisakis-free fishery products
In chapter 5, a survey-based contingent valuation study showed that many Spaniard
consumers have avoided fish in the past and would avoid fish in the future due to Anisakis
spp. presence on their fish. This behaviour causes monetary losses to the industry that
should be quantitatively estimated. Anisakis-associated diseases not only compromise
human health and the quality of life of unwell, but also generates a cost for the health
sanitary system that should also be estimated. On the other hand, results of chapter 5
showed that most of consumers were willing to pay for Anisakis-free fish, therefore,
suggesting a potential monetary benefit for the industry for offering such products.
Preliminary results from an equivalent contingent valuation study performed in Croatia
within the EU FP7 PARASITE project also showed a similar distribution of the
willingness to pay for Anisakis-free fish of Croatian consumers (Theodossiou et al.,
2016). These findings should be fully analysed in the future and compared with the
Spanish case. Additionally, similar contingent valuation studies upon the presence of
Anisakis in fishery products are recommended to be performed in other European
countries with high levels of fish consumption and/or history of Anisakis-associated
health problems (e.g. Italy, France, Denmark, Norway, Germany, and Portugal). A cost-
benefit analysis should be also performed to estimate in monetary units (if possible) the
potential benefits (e.g. reduction of the incidence of disease, ensuring food safety and
quality, increase consumer trust in fishery products, increase the value of fishery
products, decrease fish rejections/claims, etc.) versus costs (e.g. purchase/development
of technology for parasite detection, parasite fish sampling, training of employees, etc.)
of implementing measures to reduce Anisakis spp. presence in fishery products.
7.3 Conclusions
This multidisciplinary thesis provides first reports of the presence of Anisakis in sea
lamprey (A. simplex), and in allis and twaite shads (A. simplex and A. pegreffii) captured
in rivers of Western Iberia, demonstrating that anadromous fish can act as transport hosts
of Anisakis from sea to freshwater environments, and therefore highlighting the potential
200
health risk for humans and wildlife. The research presented here shows the potential of
Magnetic Resonance Imaging to detect in situ in a non-invasive and non-destructive way
Anisakis in the viscera of whole Atlantic herring and in fish muscle. In addition, results
suggested that a majority of Spanish fish consumers are willing to pay above the usual
fish price at market for Anisakis-free fishery products, and that many would cease
consuming fish if these are parasitized by Anisakis. These results therefore suggest that
the presence of Anisakis in fish is an important health and aesthetic issue for consumers,
and this is of relevance for both the fishing and food industries as well as for food safety
authorities. Finally, in this thesis, the burden of anisakiasis in Spain has been
quantitatively estimated for the first time. Results estimated that the total annual number
of anisakiasis cases caused by consumption of untreated (non-frozen) home-made raw
and marinated anchovy meals was approximately 8,000 (incidence around 20 cases per
100,000 inhabitants). These figures highlight that anisakiasis is a highly underestimated
and underdiagnosed disease in Spain and these findings will inform food safety
authorities, health professionals, industry, academy and consumers. The information can
be used to reduce the burden of disease in a number of ways. Firstly, by educational
campaigns to encourage freezing of fish prior to raw or lightly cooked consumption,
particularly by targeting those consumers who eat untreated raw and marinated fish meals
and are aware about the prevention methods against Anisakis infection but do not carry
this out. Secondly, by highlighting the need for adequate cold storage of anchovies (or
any other fish intended to be consumed as raw, marinated or undercooked), early
evisceration of fish and fish parasite monitoring (e.g. improved protocols and technology
for parasite detection in fishery products) to control the parasite (and prevent its migration
from fish viscera to flesh) along the value chain.
201
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Noguera, P., Collins, C., Bruno, D., Pert, C., Turnbull, A., McIntosh, A., Lester, K.,
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Pascual, S., Antonio, J., Cabo, M.L., Piñeiro, C., 2010. Anisakis survival in refrigerated
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Puente, P., Anadón, A.M., Rodero, M., Romarís, F., Ubeira, F.M., Cuéllar, C., 2008.
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Pufall, E.L., Jones-Bitton, A., McEwen, S.A., Brown, T.M., Edge, V.L., Rokicki, J.,
Karpiej, K., Peregrine, A.S., Simard, M., 2012. Prevalence of Zoonotic Anisakid
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Qin, Y.H., Zhao, Y.F., Ren, Y.X., Zheng, L.L., Dai, X.D., Li, Y.G., Mao, W.F., Cui, Y.,
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Shamsi, S., 2016. Seafood-borne parasitic diseases in Australia: How much do we know
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Shimamura, Y., Ishii, N., Ego, M., Nakano, K., Ikeya, T., Nakamura, K., Takagi, K.,
Fukuda, K., Fujita, Y., 2016a. Multiple Acute Infection by Anisakis: A Case Series.
Intern. Med. 55, 907–910. doi:10.2169/internalmedicine.55.5628
Shimamura, Y., Muwanwella, N., Chandran, S., Kandel, G., Marcon, N., 2016b.
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Can. J. Gastroenterol. Hepatol. 2016, 1–7. doi:10.1155/2016/5176502
Shweiki, E., Rittenhouse, D.W., Ochoa, J.E., Punja, V.P., Zubair, M.H., Baliff, J.P., 2014.
Acute small-bowel obstruction from intestinal anisakiasis after the ingestion of raw
clams; Documenting a new method of marine-to-human parasitic transmission.
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Sohn, W.-M., Na, B.-K., Kim, T.H., Park, T.-J., 2015. Anisakiasis : Report of 15 Gastric
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Cases. Korean J. Parasitol. 53, 465–470.
Suzuki, J., Murata, R., Hosaka, M., Araki, J., 2010. Risk factors for human Anisakis
infection and association between the geographic origins of Scomber japonicus and
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Svanevik, C.S., Levsen, A., Lunestad, B.T., 2013. The role of muscle-invading anisakid
larvae on bacterial contamination of the flesh of post-harvest blue whiting
(Micromesistius poutassou). Food Control 30, 526–530.
doi:10.1016/j.foodcont.2012.08.003
Tejada, M., Karl, H., de las Heras, C., Vidacek, S., Solas, M.T., Garcia, M.L., 2014. Does
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Theodossiou, I., Bao, M., Pierce, G.J., Hastie, L.C., Strachan, N., MacKenzie, K., Pita,
C., Fernández, R., Martínez, C., González-Muñoz, M., Mattiucci, S. 2016. Analysis
of willingness to pay for an Anisakis-free fish product - a contingent valuation on
Anisakis/anisakiasis/allergy risk. EU FP7 PARASITE project (GA no. 312068),
deliverable D 8.3.
Toro, C., Caballero, M.L., Baquero, M., Garcia-Samaniego, J., Casado, I., Rubio, M.,
Moneo, I., 2004. High prevalence of seropositivity to a major allergen of Anisakis
simplex, Ani s 1, in dyspeptic patients. Clin Diagn Lab Immunol 11, 115–118.
doi:10.1128/CDLI.11.1.115
Torres, J., Feliu, C.C., Fernández-Morán, J., Ruíz-Olmo, J., Rosoux, R., Santos-Reis, M.,
Miquel, J., Fons, R., 2004. Helminth parasites of the Eurasian otter Lutra lutra in
southwest Europe. J. Helminthol. 78, 353–359. doi:10.1079/JOH2004253
Valiñas, B., Lorenzo, S., Eiras, a, Figueiras, a, Sanmartín, M.L., Ubeira, F.M., 2001.
Prevalence of and risk factors for IgE sensitization to Anisakis simplex in a Spanish
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Veliyulin, E., Felberg, H.S., Digre, H., Martinez, I., 2007. Non-destructive nuclear
magnetic resonance image study of belly bursting in herring (Clupea harengus).
Food Chem. 101, 1545–1551. doi:10.1016/j.foodchem.2006.04.007
Wiwanitkit, S., Wiwanitkit, V., 2016. Anisakiasis in Southeast Asia: A story of new
tropical disease? Asian Pac. J. Trop. Med. 6, 382–383.
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Yorimitsu, N., Hiraoka, A., Utsunomiya, H., Imai, Y., Tatsukawa, H., Tazuya, N.,
Yamago, H., Shimizu, Y., Hidaka, S., Tanihira, T., Hasebe, A., Miyamoto, Y.,
Ninomiya, T., Abe, M., Hiasa, Y., Matsuura, B., Onji, M., Michitaka, K., 2013.
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List of publications
Peer reviewed papers
1. Bao, M., Pierce, G.J., Pascual, S., González-Muñoz, M., Mattiucci, S., Mladineo, I.,
Cipriani, P., Bušelić, I., Strachan, N.J.C. 2017. Assessing the risk of an emerging
zoonosis of worldwide concern: anisakiasis. Scientific Reports, 7, 43699. Chapter 6.
2. Bao, M., Strachan, N.J.C., Hastie, L.C., MacKenzie, K., Seton, H.C., Pierce, G.J.
2017. Employing visual inspection and magnetic resonance imaging to investigate
Anisakis spp. infection in herring viscera. Food Control, 75: 40-47. Chapter 4.
3. Cosgrove, P., Watt, J., Hastie, L., Shields, D., Cosgrove, C., Brown, L., Isherwood,
I., Bao, M. 2016. The status of the freshwater pearl mussel Margaritifera
margaritifera in Scotland: extent of change since 1990s, threats and management
implications. Biodiversity and Conservation, 25(11), 2093-2112.
4. Bao, M., Costal, D., Garci, M.E., Pascual, S., Hastie, L.C. 2016. Sea lice
(Lepeophtheirus salmonis) and anchor worms (Lernaea cyprinacea) found on sea
trout (Salmo trutta) in the River Minho catchment, an important area for conservation
in NW Spain. Aquatic Conservation: Marine and Freshwater Ecosystems, 26(2): 386-
391.
5. Bao, M., Roura, A., Mota, M., Nachón, D.J., Antunes, C., Cobo, F. 2015.
Macroparasites of allis shad (Alosa alosa) and twaite shad (Alosa fallax) of the
Western Iberian Peninsula Rivers: ecological, phylogenetic and zoonotic insights.
Parasitology Research, 114(10): 3721-3739.
6. Bao, M., Mota, M., Nachón, D.J., Antunes, C., Cobo, F., Garci, M.E., Pierce, G.J.,
Pascual, S. 2015. Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and
twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers:
zoonotic and ecological implications. Parasitology Research, 114(6): 2143-2154.
Chapter 3.
7. Mota, M., Bio, A., Bao, M., Pascual, S., Rochard, E., Antunes, C. 2015. New insights
into biology and ecology of the Minho River Allis shad (Alosa alosa L.): contribution
to the conservation of one of the last European shad populations. Reviews in Fish
Biology and Fisheries, 25(2): 395-412.
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8. Silva, S., Araújo, M.J., Bao, M., Mucientes, G., Cobo, F. 2014. The haematophagous
feeding stage of anadromous populations of sea lamprey Petromyzon marinus: low
host selectivity and wide range of habitats. Hydrobiologia, 734(1): 187-199.
9. Bao, M., Garci, M.E., Antonio, J.M., Pascual, S. 2013. First report of Anisakis
simplex (Nematoda, Anisakidae) in the sea lamprey (Petromyzon marinus). Food
Control, 33: 81-86. Chapter 2.
Papers under review
1. Bao, M., Pierce, G. J., Strachan, N. J. C., Martínez, C., Fernández, R., Theodossiou,
I. Consumers’ attitudes and willingness to pay for Anisakis-free fish. Fisheries
Research, under review. Chapter 5.
2. Pierce, G. J., Bao, M., MacKenzie, K., Hastie, L. Anisakid nematode infections in
haddock (Melanogrammus aeglefinus) and whiting (Merlangius merlangus) in the
Northeast Atlantic. Fisheries Research, under review.
3. Gay, M., Bao, M., MacKenzie, K., Pascual, S., Buchmann, K., Bourgau, O., Couvreur
C., Mattiucci., S., Paoletti, M., Hastie, L. C., Levsen, A., Pierce, G. J. Prevalence,
abundance, intensity and species diversity of ascaridoid nematode in Atlantic cod are
correlated with geographic area and fish size. Fisheries Research, under review.
4. Mattiucci, S., Giulietti, L., Paoletti, M., Cipriani, P., Gay, M., Levsen, A., Klapper,
R., Karl, H., Bao, M., Pierce, G. J., Nascetti, G. Population genetic structure of the
parasite Anisakis simplex (s.s.) collected in Clupea harengus L. from North East
Atlantic fishing grounds. Fisheries Research, under review.
Other scientific output
1. Bao, M., Strachan, N., Pierce, G.J., Hastie, L.C., MacKenzie, K., Pascual, S.,
González, A.F., Fernández, R., Martínez, C. 2016. Quantitative risk assessment for
Anisakis. EU FP7 PARASITE project (GA no. 312068), deliverable D 8.2
2. Theodossiou, I., Bao, M., Pierce, G.J., Hastie, L.C., Strachan, N., MacKenzie, K.,
Pita, C., Fernández, R., Martínez, C., González-Muñoz, M., Mattiucci, S. 2016.
Analysis of willingness to pay for an Anisakis-free fish product – a contingent
valuation study on Anisakis/anisakiasis/allergy risk. EU FP7 PARASITE project (GA
no. 312068), deliverable D 8.3.
211
3. Pierce, G.J., Hastie, L.C., González, A.F., Levsen, A., Mattiucci, S., Cipriani, P., Bao,
M., Dunser, A., Paoletti, M., Acerra, V. 2015. Statistical modelling and inference. EU
FP7 PARASITE project (GA no. 312068), deliverable D 8.1.
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Overview of completed training activities
Discipline specific activities
Courses
Statistical modelling with R, University of Aberdeen (UoA), 2015.
Introduction to R, UoA, 2015.
Postgraduate basic statistics, UoA, 2015.
Conferences/meetings
EU FP7 PARASITE project meeting, Vigo (Spain), 2015 (oral presentation).
9th International Symposium of Fish Parasites, Valencia (Spain), 2015 (poster
presentations).
EU FP7 PARASITE project meeting, Hamburg (Germany), 2015 (oral presentation).
EU FP7 PARASITE project meeting, Madrid (Spain), 2014 (oral presentation).
General courses
CV and Cover letters for Life and Physical Sciences, UoA, 2016.
Exceptional Conference Presentations, UoA, 2016.
Presentations using PowerPoint 2013, UoA, 2016.
Creating Posters using PowerPoint 2013, UoA, 2016.
A bioscientist’s guide to Research Integrity, UoA, 2016.
Skye for Business: messaging and more, UoA, 2016.
Become a published researcher, UoA, 2016.
Writing in the Second and Third Year of Your PhD, UoA, 2016.
Improving your presentation skills workshop, UoA, 2016.
Writing in the second year of your PhD, UoA, 2016.
A punctuation guide for academic writing, UoA, 2016.
Excel 2013 Further II: Working with Lists, UoA, 2016.
Excel 2013 Further I: Functions and Formulas, UoA, 2016.
Word 2013: Working with Long Documents, UoA, 2015.
Excel 2013 Introduction II: Formulas, Functions, Charting, UoA, 2015.
Quotes, Referencing and Bibliographies, UoA, 2015.
Improving Your Writing: Grammar & Punctuation, UoA, 2015.
213
Other activities
Demonstrator in Diversity of Life II, UoA, 2017.
Preparation of Post-doc Marie Curie research proposal, 2016.
Research stay at IIM-CSIC (Spain), two weeks, 2015.
Research survey IBTS Q3 in the North Sea on board “Scotia” vessel, 2015.
“Personal Survival Techniques” and “Medical” certificates, Falck Safety services,
Aberdeen, 2015.
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Abstract (Spanish)
Los parásitos nematodos del género Anisakis tienen ciclos de vida complejos en el medio
marino, incluyendo en los mismos a una gran variedad de organismos acuáticos presentes
en todos los océanos del mundo. Durante su ciclo de vida, los mamíferos marinos (en
concreto los cetáceos) sirven como hospedadores definitivos o finales, los pequeños
crustáceos como hospedadores intermediarios y los peces y cefalópodos como
hospedadores intermediarios o paraténicos (también conocidos como hospedadores
transporte). Estos parásitos son fundamentalmente conocidos por ser los agentes
causantes de una enfermedad zoonótica denominada anisakiasis que se adquiere a través
de los alimentos (es decir, los productos de la pesca parasitados por Anisakis), así como
de reacciones alérgicas en personas sensibilizadas. Los seres humanos pueden infectarse
y desarrollar la enfermedad mediante el consumo de productos pesqueros crudos o poco
cocinados que estén parasitados por larvas vivas de Anisakis. El parásito Anisakis también
ocasiona pérdidas económicas a la industria pesquera y provoca la desconfianza de los
consumidores hacia los productos de la pesca. Por todos estos motivos, el Anisakis ha
generado un creciente interés y preocupación dentro del mundo académico, la industria
pesquera y de alimentación, los profesionales de la salud, las autoridades sanitarias y los
consumidores.
Mediante el uso de metodologías multidisciplinares, esta tesis se centró
fundamentalmente en la generación de nuevo conocimiento, entendimiento y métodos
relacionados con la epidemiología y ecología del ciclo de vida del Anisakis en algunas
especies de peces (en concreto, tres especies anádromas como la lamprea, el sábalo y la
saboga, y una especie marina como el arenque), y con la problemática socioeconómica y
de salud pública originada por su presencia en los productos pesqueros.
Se estudió la epidemiología del Anisakis en peces anádromos capturados en ríos del oeste
de la Península Ibérica. La presencia de A. simplex s.s. en la lamprea marina, y de
infecciones mixtas de A. simplex s.s. y A. pegreffii en el sábalo y en la saboga fue
reportada por primera vez. Los resultados destacaron el papel desempeñado por los peces
anádromos como transportadores de Anisakis del ecosistema marino al fluvial, así como
la importancia zoonótica de los hallazgos debido a los riesgos gastro-alérgicos que supone
la presencia de estos parásitos en el pescado.
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Trasladando el foco a los peces marinos, también fueron investigados los niveles de
parasitación por Anisakis en arenques capturados en el Mar del Norte, así como la
localización anatómica del parásito dentro de la cavidad visceral del arenque. Este estudio
formó parte del proyecto Europeo FP7 PARASITE (GA no. 312068), con el objetivo de
aportar nueva información epidemiológica sobre el Anisakis en las especies de peces
comerciales más importantes de los caladeros europeos. El método de inspección visual
mostró que las larvas de Anisakis tienden a acumularse en la víscera del arenque, sábalo
y saboga (todos miembros de la familia Clupeidae), allí dónde termina el saco ciego del
estómago, alrededor del ductus pneumaticus. Posiblemente, esto pueda ser debido a la
forma en “Y” del estómago de estos clupeidos, de manera que los Anisakis ingeridos en
la dieta son dirigidos a esta zona terminal del estómago durante el proceso de digestión,
para después éstos atravesar la pared estomacal y emerger a la cavidad visceral. En
aquellos peces dónde la gónadas se encuentren muy desarrolladas, éstas podrían actuar
como barreras o trampas para el Anisakis, de manera que impedirían la migración de los
parásitos hacia el músculo u otros órganos, y favorecería la formación de acumulaciones
de Anisakis en la arriba citada región visceral. En esta tesis se investigó por primera vez
el potencial de la técnica de imagen por resonancia magnética (IRM) como método de
detección de larvas de Anisakis en el pescado. Los resultados demostraron la capacidad
del IRM para detectar acumulaciones de Anisakis en la cavidad visceral de un arenque
entero in situ, en 3D, y de una forma no invasiva y no destructiva. Además, los resultados
también mostraron la capacidad del IRM para detectar larvas de Anisakis y
Pseudoterranova en el músculo del pescado.
Mediante el uso de cuestionarios, se utilizó por primera vez el método de valoración
contingente con la finalidad de averiguar la máxima disposición de pago de los
consumidores de pescado españoles por la compra de un pescado “libre” de Anisakis.
Además, se investigaron las actitudes de los consumidores respecto a la presencia de
Anisakis en el pescado, centrándose especialmente en aquellos pescados más
frecuentemente consumidos por los encuestados. Los resultados indicaron que una
mayoría de consumidores estaban dispuestos a pagar un precio extra por dicho producto.
La moda de la disposición a pagar fue del 10% extra por encima del típico precio de un
pescado en el mercado (es decir, 6,60 €/kg de pescado “libre” de Anisakis, teniendo en
cuenta que 6 €/kg es el precio de un pescado normal). Los resultados también indicaron
que más de un cuarto de los consumidores ya habían evitado consumir pescado (sobre
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todo merluza), y que una mayoría de ellos evitarían consumir pescado en el futuro debido
a la presencia de Anisakis en el pescado. En general, este estudio proporcionó nuevas
evidencias para concluir que la presencia de Anisakis en los productos de la pesca es un
importante problema estético y de salud para los consumidores españoles.
Por último, se empleó el método de evaluación del riesgo cuantitativo para determinar
cuál era la probabilidad de enfermar de anisakiasis debido al consumo en el hogar de
boquerones crudos o marinados que no habían sido previamente congelados (congelar los
filetes de boquerón a una temperatura de ≤20 °C durante al menos 24 horas mata cualquier
Anisakis que pudiese estar presente, y así previene la parasitación), para posteriormente
calcular cuál era la carga de esta enfermedad zoonótica en España. Asimismo, la
frecuencia de consumo en el hogar de boquerones crudos o marinados no precongelados
fue determinada mediante el uso de cuestionarios. La relación dosis-respuesta para el
Anisakis fue determinada por primera vez y la probabilidad de padecer anisakiasis fue
estimada en 9,56×10-5 por menú de boquerones. Los resultados sugirieron que
aproximadamente ocurren entre 7.700 y 8.320 casos de anisakiasis que requieren atención
médica cada año en España. Este hallazgo sugiere que la anisakiasis es una enfermedad
altamente infraestimada e infradiagnosticada, probablemente debido a los siguientes
motivos: diagnóstico erróneo (los síntomas de la anisakiasis no son específicos por lo que
dificulta su diagnosis (puede ser confundida con otras enfermedades gastrointestinales)),
a un no diagnóstico (por falta de investigación clínica o casos asintomáticos) y a que la
enfermedad no es reportada por el médico (la anisakiasis no es una enfermedad de
declaración obligatoria). Esta información es de gran interés para la industria pesquera y
de la alimentación, profesionales de la medicina, mundo académico y consumidores, y
puede ser utilizada por las agencias de seguridad alimentaria para reducir la incidencia de
la anisakiasis de la siguiente manera: En primer lugar, destacando la importancia de
mantener la cadena de frío del boquerón y de una evisceración temprana de los mismos,
con el fin de evitar la migración post-mortem del parásito de las vísceras al músculo, lo
cual podría incrementar el riesgo de anisakiasis. Se recomienda también la monitorización
del parásito a lo largo de la cadena de valor del pescado para mejorar su control y
minimizar los riesgos de contraer anisakiasis. Finalmente, se recomienda llevar a cabo
campañas de educación para concienciar a los consumidores de la necesidad de congelar
previamente todo aquel pescado que se pretenda consumir crudo o poco cocinado. En
especial, la campaña debe también dirigirse a aquellos consumidores que a pesar de ser
217
conocedores de los métodos de prevención de Anisakis optan por no congelar el pescado
que van a consumir crudo o marinado.
En conclusión, esta tesis contribuye con un mejor conocimiento sobre 1) la ecología del
ciclo de vida y la epidemiología del Anisakis en peces anádromos y marinos, 2) las
actitudes de los consumidores respecto a los productos de la pesca parasitados por
Anisakis y cómo valorarían la posibilidad de comprar un producto “libre” de Anisakis, y
3) cómo un hábito de consumo de pescado peligroso puede resultar en zoonosis (es decir,
anisakiasis), y cómo la incidencia de esta enfermedad oculta y emergente puede ser
estimada para el total de la población. Esta tesis aporta nuevas evidencias sobre la
problemática socioeconómica, legislativa y de salud pública originada por la presencia de
Anisakis en los productos pesqueros, y puede ser utilizada para mejorar la calidad y
seguridad alimentaria de los mismos, y para reducir la incidencia de la anisakiasis en la
población. Por último, se recomiendan futuras investigaciones para evaluar el riesgo
potencial para la salud humana que plantea la presencia de alérgenos de Anisakis en los
productos pesqueros.
218
Abstract (Galician)
Os parasitos nematodos do xénero Anisakis teñen ciclos de vida complexos no ecosistema
mariño, englobando a unha gran variedade de organismos acuáticos presentes en tódolos
grandes mares do mundo. Durante o seu ciclo de vida, os mamíferos mariños (en concreto,
os cetáceos) serven como hóspedes finais ou definitivos, os pequenos crustáceos como
hóspedes intermediarios, e os peixes e cefalópodos como hóspedes intermediarios ou
paraténicos (tamén coñecidos como hóspedes transporte). Estes parasitos son
principalmente coñecidos por selos axentes causantes dunha enfermidade zoonótica que
se transmite a través dos produtos da pesca denominada anisakiase, así como de ocasionar
reaccións alérxicas en persoas sensibilizadas. Os seres humanos poden infectarse e
padecer anisakiase a través do consumo de pescado cru ou pouco cociñado parasitado por
larvas vivas de Anisakis. Tamén poden causar perdas económicas á industria pesqueira e
xerar desconfianza nos consumidores cara os produtos pesqueiros. Por tódolos anteriores
motivos, o Anisakis xerou un crecente interese e preocupación no mundo académico,
industrias pesqueiras e da alimentación, profesionais da medicina, axencias de seguridade
alimentaria e consumidores.
Mediante o uso de metodoloxías multidisciplinares, a presente tese centrouse
principalmente na xeración de novo coñecemento, entendemento e novos métodos
relacionados coa epidemioloxía e ecoloxía do ciclo de vida do Anisakis nos peixes
hóspedes (tres peixes anádromos (lamprea mariña, sábalo e sabela) e un mariño
(arenque)), e sobre a importancia socioeconómica e de saúde pública ocasionada pola súa
presenza nos produtos da pesca.
A epidemioloxía do Anisakis estudouse en peixes anádromos capturados en ríos do oeste
da Península Ibérica (Galicia e Portugal). Por primeira vez, reportouse a presenza de A.
simplex s.s. na lamprea mariña, e de infeccións mixtas de A. simplex s.s. e A. pegreffii no
sábalo e na sabela. Os resultados destacaron o rol destes peixes anádromos como medio
de transporte do Anisakis do ecosistema mariño o fluvial, e destacaron a importancia
zoonótica do descubrimento debido o potencial risco gastroalérxico asociado con estes
parasitos.
Os niveis de infección e a localización anatómica do Anisakis na cavidade visceral no
arenque do Mar do Norte foron investigados, desprazando o foco do estudo cara os peixes
mariños. Este estudo formou parte do proxecto europeo FP7 PARASITE (GA no.
219
312068) coa finalidade de xerar nova información epidemiolóxica sobre o Anisakis en
varias especies de peixe de gran valor comercial presentes nos caladoiros europeos. A
inspección visual da cavidade visceral do arenque, sábalo e sabela mostrou que os
Anisakis tenden a acumularse na parte terminal do saco cego do estómago, ó redor do
ductus pneumaticus. Isto probablemente sexa ocasionado pola forma en “Y” do estómago
destes peixes clupeidos. Durante o proceso de dixestión os Anisakis son transportados
xunto coa comida á parte terminal do saco cego estomacal, dende onde atravesan a parede
estomacal para emerxer e asentarse nesta rexión anatómica da cavidade visceral. Nos
peixes maduros con grandes gónadas, estas poden actuar como barreiras ou trampas
físicas para os Anisakis, impedindo deste xeito a súa migración a outros órganos ou o
músculo e favorecendo a formación de acumulacións de parasitos nesta rexión. Nesta tese
investigouse por primeira vez o potencial da técnica de imaxe por resonancia magnética
(IRM) como método de detección de larvas de Anisakis no pescado. Os resultados
demostraron a capacidade da IRM para detectar acumulacións de Anisakis na cavidade
visceral dun arenque enteiro in situ, en 3D, e dun xeito non invasivo e non destrutivo.
Mediante o uso de cuestionarios, empregouse por primeira vez o método de valoración
continxente coa finalidade de determinar a máxima disposición ó pago dos consumidores
de pescado españois pola compra dun pescado “libre” de Anisakis. Ademais,
investigáronse as actitudes dos consumidores respecto á presenza de Anisakis no pescado,
centrándose especialmente naqueles pescados máis frecuentemente consumidos polos
enquisados. Os resultados indicaron que unha maioría de consumidores estaban dispostos
a pagar un prezo extra por dito produto, presentando unha moda dun 10% extra por riba
do prezo dun pescado normal no mercado (é dicir, 6,60 €/kg de pescado “libre” de
Anisakis, tendo en conta que 6 €/kg é o prezo dun pescado normal). Os resultados tamén
indicaron que máis dun carto de consumidores xa evitaran consumir pescado (sobre todo
pescada), e que unha maioría deles evitarían consumir pescado no futuro, por mor da
presenza de Anisakis no pescado. En xeral, este estudo proporcionou novas evidencias
para concluír que a presenza de Anisakis nos produtos pesqueiros é un importante
problema estético e de saúde para os consumidores españois.
Por último, empregouse o método de avaliación do risco cuantitativo para determinar cal
era a probabilidade de enfermar de anisakiase debido ó consumo no fogar de bocareus
crus ou mariñados que non foron previamente conxelados (a conxelación dos filetes a
unha temperatura de ≤20 °C durante polo menos 24 horas mata calquera Anisakis que
220
puidese estar presente, e deste xeito previndo a anisakiase). Posteriormente, calculouse
cal era a carga desta enfermidade zoonótica en España. A frecuencia de consumo no fogar
de bocareus crus ou mariñados non preconxelados determinouse mediante o uso de
cuestionarios. A relación dose-resposta para o Anisakis determinouse por primeira vez e
a probabilidade de padecer anisakiase estimouse en 9,56×10-5 por menú de bocareus. Os
resultados suxeriron que aproximadamente ocorren entre 7.700 e 8.320 casos de
anisakiase que requiren atención médica cada ano en España. Esta estimación suxire que
a anisakiase é unha enfermidade altamente infraestimada e infradiagnosticada en España,
probablemente debido as seguintes causas: 1) unha diagnose errónea (os síntomas da
anisakiase son inespecíficos, polo que poden confundirse con outras enfermidades
gastrointestinais), 2) a non diagnose (por unha falla de investigación clínica, ou pola
carencia de síntomas da enfermidade ou sintomatoloxías leves) e 3) a enfermidade non é
reportada polo médico ( a anisakiase non é unha enfermidade de declaración obrigatoria).
Esta información é de gran interese para a industria pesqueira e da alimentación,
profesionais da medicina, mundo académico e para os consumidores, e pode ser
empregada polas axencias de seguridade alimentaria para reducila incidencia da
anisakiase na poboación do seguinte xeito: En primeiro lugar, destacando a importancia
de manter a cadea do frío do bocareu e dunha pronta evisceración dos mesmos, coa
finalidade de evitar a migración post-mortem do parasito das vísceras ó músculo, o cal
podería aumentar o risco de anisakiase. Tamén recoméndase a mostraxe do parasito ó
longo da cadea de valor do peixe para mellorar o seu control e minimizar o risco de
anisakiase. Finalmente, recoméndase levar a cabo campañas de educación para
concienciar ós consumidores da necesidade de conxelar previamente todo aquel pescado
que quéirase consumir como cru ou pouco cociñado. En particular, a campaña debe
prestar especial atención a aqueles consumidores que a pesares de coñecer os métodos de
prevención do Anisakis optan por non conxelalo peixe que van a consumir cru ou pouco
cociñado, aumentando deste xeito o risco de padecer anisakiase.
En conclusión, esta tese contribúe cun mellor coñecemento sobre 1) a ecoloxía do ciclo
de vida e a epidemioloxía do Anisakis en peixes anádromos e mariños, 2) as actitudes dos
consumidores cara os produtos da pesca parasitados por Anisakis e cómo valorarían tela
posibilidade de mercar un produto “libre” de Anisakis, e 3) cómo un hábito de consumo
perigoso pode resultar en zoonose (é dicir, anisakiase), e cómo a incidencia desta
enfermidade oculta e emerxente pode estimarse para o conxunto da poboación. Esta tese
221
aporta novas evidencias sobre a problemática socioeconómica, lexislativa e de saúde
pública orixinada pola presenza dos Anisakis nos produtos da pesca, e pode ser empregada
para mellorala calidade e seguridade alimentaria dos mesmos, e para reducila incidencia
da anisakiase na poboación. Por último, recoméndanse o desenrolo de futuros estudos
para avaliar o risco potencial para a saúde humana causado pola presenza de alérxenos de
Anisakis nos produtos pesqueiros.
222
Appendices
Supplementary material of Chapter 4
Diagram. Taxonomic classification of the ascaridoid nematodes in this study (source:
Horton et al., 2016; Mattiucci & Nascetti, 2008).
References
Horton et al. 2016. World Register of Marine Species (WoRMS). doi:10.14284/170
Mattiucci, S., Nascetti, G., 2008. Chapter 2 Advances and Trends in the Molecular
Systematics of Anisakid Nematodes, with Implications for their Evolutionary Ecology
and Host-Parasite Co-evolutionary Processes. Adv. Parasitol. 66, 47–148.
doi:10.1016/S0065-308X(08)00202-9
223
Figure. Time series images showing A. simplex s.l. larvae moving inside the fish muscle.
224
Supplementary material of Chapter 5
In the current section is supplementary table S1. The questionnaire 1 (Spanish version),
and the English version of questionnaire 1 (disseminated in Scotland) are provided in
“Supplementary material of Chapter 6” section.
225
Table S1. Response and explanatory variables parameterized in the generalized additive
model.
Variable definition Code – question choices or recoded
responses from questionnaire
Variable or question code
number
Response variable
Willingness to pay 0 – 0% extra: 6 €/Kg (I would buy it at the
stated price (6€))
1 – 10% extra: 6.60 €/Kg
2 – 20% extra: 7.20 €/Kg
3 – 30% extra: 7.80 €/Kg
4 – 40% extra: 8.40 €/Kg
5 – 50% extra: 9 €/Kg
6 – 60% extra: 9.60 €/Kg
7 – 70% extra: 10.20 €/Kg
8 – 80% extra: 10.80 €/Kg
9 – 90% extra: 11.40 €/Kg
10 – 100% extra: 12 €/Kg or more
23
Explanatory variables
Gender (categorical) 1 – Female
2 – Male
NA – Not available
1
Age (ordinal treated as
numerical)
23.5 (average value) – (18-29 years old)
39.5 (average value) – (30-49 years old)
57 (average value) – (50-64 years old)
68 (assumed value) – (65 years and over)
NA - Not available
2
Nationality
(categorical)
1 – Spanish
2 – Other
3
226
Area of residence
(categorical)
1 – Centre of Spain
2 – Galicia
3 – Cantabrian Sea region
4 – Mediterranean Sea region, Andalucía &
Canary Islands
NA – Not available
4
Marital status
(categorical)
1 – Married or cohabiting
2 – Single
NA – Not available
5
Adults in household
(numerical treated as
categorical)
1 – Respondents who answered 0 and 1 adult
2 – Respondents who answered 2, 4, 5, 6, 21,
30 adults
NA – Not available
6
Children in household
(numerical treated as
categorical)
0 – Respondents who answered 0 children
1 – Respondents who answered 1, 2, 3 and 4
children
NA – Not available
7
Education (categorical) 1 – Primary education
2 – Secondary education
3 – University degree
NA – Not available
8
Employment status
(categorical)
1 – Respondents who answered:
Employed, and
Self-employed
2 – Respondents who answered:
Unemployed, and
Labour market inactive (e.g. students,
domestic activity, child rearing)
9
227
3 – Retired.
NA – Not available
Occupation
(categorical)
1 – Respondents who answered:
Extractive fishing sector (fisherman, owner,
etc.), and
Processing sector of fishery products
2 – Respondents who answered:
Science and Development,
Human Health activities, and
Education and Training (including students
without work experience)
3 – Respondents who picked any other
occupation category.
NA – Not available
10
Income (ordinal treated
as numerical)
6,000 (average value) – (0 – 12,000€)
18,000.5 (average value) – (12,001€ -
24,000€)
30,000.5 (average value) – (24,001€ -
36,000€)
42,000.5 (average value) – (36,001€ -
48,000€)
54,000.5 (average value) – (48,001€ -
60,000€)
80,000.5 (average value) – (60,001€ -
100,000€)
120,000.5 (assumed value) – (100,001€ and
above)
NA – Not available
11
Health status
(categorical)
1 – Respondents who answered:
Poor, and
12
228
Fair
2 – Respondents who answered:
Good, and
Excellent
Allergy (categorical) 1 – Yes
2 – No
NA – Not available
13
Frequency of fish
consumption (ordinal
treated as numerical)
0.23 (calculated fish consumption per week
(i.e. 365/7 days/12 months= 4.34
week/month; 1 per month/4.34 week/month=
0.23 per week) – 1 per month
1 – 1 per week
2.5 (average value) – 2-3 times per week
5.5 (assumed value) – 4 times or more per
week
NA – Not available
14
Consumption of fresh
fish
1 – Yes
2 – No
15_1
Consumption of frozen
fish
1 – Yes
2 – No
15_2
Consumption of surimi
products
1 – Yes
2 – No
15_3
Consumption of other
(e.g. canned)
1 – Yes
2 – No
15_4
Frequency of hake
consumption
(categorical)
0 – No hake consumption
1 – Respondents who answered:
Yearly,
Monthly, and
16_1
229
Weekly
NA – Not available
Frequency of anchovy
consumption
(categorical)
0 – No anchovy consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_2
Frequency of monkfish
consumption
(categorical)
0 – No monkfish consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_3
Frequency of megrim
consumption
(categorical)
0 – No megrim consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_4
Frequency of blue
whiting consumption
(categorical)
0 – No blue whiting consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_5
230
Frequency of horse
mackerel consumption
(categorical)
0 – No horse mackerel consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_6
Frequency of cod
consumption
(categorical)
0 – No cod consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_7
Frequency of pollack
consumption
(categorical)
0 – No pollack consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_8
Frequency of mackerel
consumption
(categorical)
0 – No mackerel consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_9
Frequency of sardine
consumption
(categorical)
0 – No sardine consumption
1 – Respondents who answered:
Yearly,
16_10
231
Monthly, and
Weekly
NA – Not available
Frequency of tuna
consumption
(categorical)
0 – No tuna consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_11
Frequency of salmon or
trout consumption
(categorical)
0 – No salmon or trout consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_12
Frequency of John dory
consumption
(categorical)
0 – No John dory consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_13
Frequency of
consumption of other
fish species
(categorical)
0 – No other fish consumption
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
NA – Not available
16_14
232
Where do you eat fish?
(categorical)
1 – Home
2 – Other (e.g. restaurant)
3 – Both
NA – Not available
17
Raw fish consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18
Frequency of raw fish
consumption at home
(categorical)
0 – No raw fish consumption at home
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
18A1_1
Frequency of raw fish
consumption at
restaurant (categorical)
0 – No raw fish consumption at home
1 – Respondents who answered:
Yearly,
Monthly, and
Weekly
18A1_2
Consumption already
prepared fish at home
(categorical)
0 – Yes
1 – No
NA – Not available
18A2_1
Consumption raw fresh
fish at home
(categorical)
0 – Yes
1 – No
NA – Not available
18A2_2
Consumption raw
frozen fish at home
(categorical)
0 – Yes
1 – No
NA – Not available
18A2_3
233
Raw anchovy
consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_1
Raw sardine
consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_2
Raw hake consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_3
Raw mackerel
consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_4
Raw salmon or trout
consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_5
Raw tuna consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_6
Raw monkfish
consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_7
Raw cod consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_8
Raw megrim
consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_9
234
Raw squid consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_10
Raw other fish
consumption
(categorical)
1 – Yes
2 – No
NA – Not available
18B_11
Awareness of Anisakis
(categorical)
1 – Yes
2 – No
NA – Not available
19
Knowledge of Anisakis
prevention methods
(categorical)
1 – Yes
2 – No
NA – Not available
20
Historical fish
avoidance due to
Anisakis (categorical)
1 – Yes
2 – No
NA – Not available
21
Historical hake
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_1
Historical cod
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_2
Historical Atlantic
pomfret avoidance
(categorical)
1 – Yes
2 – No
NA – Not available
21A_3
Historical sardine
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_4
235
Historical salmon or
trout avoidance
(categorical)
1 – Yes
2 – No
NA – Not available
21A_5
Historical monkfish
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_6
Historical tuna
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_7
Historical blue whiting
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_8
Historical megrim
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_9
Historical mackerel
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_10
Historical anchovy
avoidance (categorical)
1 – Yes
2 – No
NA – Not available
21A_11
Future fish avoidance
due to Anisakis
(categorical)
1 – Yes, always
2 – Yes, but only if there is high chance of
worms
3 – No, cooking or freezing kill any possible
worm
NA – Not available
24
236
Reasons future
avoidance due to
Anisakis (categorical)
1 – Health risk
2 – Unappetising
3 – Both
4 – Other
5 – No
NA – Not available
24A
Self-perceived risk of
future development of
anisakiasis or allergy
(ordinal treated as
numerical)
0 – no chance
1 – slight chance
2 – moderate chance
3 – high chance
4 – absolute chance
NA – Not available
25
237
Supplementary material of Chapter 6
Models
The quantitative risk assessment model (QRA model) and the “anchovy meal-size” sub-
model are available by request from the authors.
In the current section are supplementary tables S1-S10, questionnaire 1 (Spanish version)
and questionnaire 2. In addition, provided is an English version of questionnaire 1
disseminated in Scotland. Please, note that there are some minor differences in the
questionnaires because some questions were adapted to each country.
238
References
1. FAO, 2016. Fishery Statistical Collections - Global Production Statistics [WWW
Document]. URL http://www.fao.org/fishery/statistics/global-capture-production/en
(accessed 2.3.16).
2. MAGRAMA, 2016. Database consumption in households [WWW Document]. URL
http://www.magrama.gob.es/es/alimentacion/temas/consumo-y-comercializacion-y-
distribucion-alimentaria/panel-de-consumo-alimentario/base-de-datos-de-consumo-
en-hogares/consulta10.asp (accessed 2.3.16).
3. FAO, 1989. Yield and nutritional value of the commercially more important fish
species. Rome. Available at: http://www.fao.org/docrep/003/t0219e/t0219e00.htm
(accessed 2.3.16).
239
Table S1. Global production statistics (production) of European anchovy from supplier
countries in 2013 (FAO)1. Total imports of fresh anchovy from supplier countries
(“Ministerio de Agricultura, Alimentación y Medio Ambiente – Spanish government”,
MAGRAMA, pers. comm.). The estimated imports per sea area were calculated
multiplying the total imports by the proportion per fishing area. Quantities presented in
tonnes (t).
Country and fishing area Production (t) Proportion Total imports (t) Estimated
imports (t)
Morocco
Area 3 (FAO 34) 34,611 0.99
1086
1,072
Area 1 (FAO 37) 454 0.01 14
Total 35,065 1 1,086
France
Area 2 (FAO 27) 2,636 0.52
3289
1,725
Area 1 (FAO 37) 2,389 0.48 1,564
Total 5,025 1.00 3,289
Greece
Area 3 (FAO 37) 8,756 109
109
Total 8,756 109
Italy
Area 1 (FAO 37) 29,664 3,872
3,872
Total 29,664 3,872
Portugal
Area 2 (FAO 27) 387 359
359
Total 387 359
United Kingdom
Area 2 (FAO 27) 10
6
6
Area 3 (FAO 34) 0 0
Total 10 6
Croatia
Area 1 (FAO 37) 8,904 34
34
Total 8,904 34
Norway
Area 2 (FAO 27) NA* 1
1
Total NA* 1
240
* No reported captures of anchovies from Norway were available at FAO1. Therefore, it
was assumed that the imported tonne of anchovy fished by Norway came from FAO 27.
Total Area 1 (FAO 37) 5,593
Total Area 2 (FAO 27) 2,091
Total Area 3 (FAO 34) 1,072
TOTAL 8,756 8,756
241
Table S2. Spanish global production (production) of European anchovy1, imports
(import) (determined in Table S1) and exports (export) of fresh anchovy in tonnes (t) for
2013, estimated consumption and resulting proportions (Proportion) per fishing area
(Area).
* Total exports were provided by MAGRAMA (MAGRAMA, pers. comm.). The exports
per fishing area were assumed in the same proportion to what was produced.
Area Production (t) Import (t) Export (t)* Estimated consumption
(t) Proportion
1 18,865 5,593 1,539 22,918.696 0.546
2 17,255 2,091 1,408 17,938.649 0.428
3 28 1,072 2 1,097.655 0.026
Total 36,148 8,756 2,949 41,955 1
242
Table S3. The following questions and subsequent answers from questionnaire 1 were
used to identify respondents at risk of anisakiasis caused by ingestion of raw and
marinated anchovies at home.
Code Question Response(s)
18 Do you usually eat raw or undercooked fish? Yes
18A Where and how often? Home (yearly, monthly, weekly)
18A2
What fish products do you use when preparing food
containing raw or light cooked fish?
Fresh fish
18B What fish? Anchovy
18C1 What type of anchovy recipe or specialty? In vinegar or lemon
Pickled or marinated (eg.
“escabeche”)
Sushi or sashimi
Ceviche
Carpaccio
243
Table S4. Correction of questionnaire 1 by geographical location and population size. n: number of respondents; Nallmeal: number of anchovy
meals consumed by respondents per year; Nuntmeal: number of untreated anchovy meals consumed by respondents per year; Allergy: Number of
respondents (out of those (n= 701) answering question 13S4 (Do you have allergy to Anisakis?)) with allergy to Anisakis spp.
Region na Nallmeal Nuntmeal Allergy Populationb Nuntmeal/n (Nuntmeal/n)
*Population (Nallmeal/n)*Population Allergy/n
Allergic
Population
Galicia 447 2,267 332 0 2,362,657 0.74 1,754,814 11,982,423 0 0
Central Spain 119 1,694 348 8 11,847,502 2.92 34,646,477 168,652,678 0.067 796,471
Mediterranean
Sea 47 545 102 1 12,194,004 2.17 26,463,584 141,398,560 0.021 259,447
Cantabrian Sea 59 875 178 0 3,233,307 3.02 9,754,721 47,951,580 0 0
Andalusia 22 237 25 0 6,740,745 1.14 7,659,938 72,616,209 0 0
Canary Islands 16 147 0 0 1,738,480 0 0 15,972,289 0 0
Total 710a 5,765 985 9 38,116,695 NA 80,279,534 458,573,739 NA 1,055,918
Key variables that are used in the dose response and risk characterisation methods calculated from the corrected variables above.
Amealpy= Number of
untreated anchovy meals
eaten by Spanish person
per year
Number of untreated meals
eaten by respondents
(n=716)
Propunt= Proportion of
untreated anchovy meals
consumed per person
Proportion allergic=
Proportion of the Spanish
population with allergy to
Anisakis spp.
Allergic
respondents=
number of
respondents with
Auntmeal= Number of
untreated meals eaten by
allergic respondents
244
allergy to Anisakis
spp.
80,279,534/38,116,695=
2.106
716*2.106=
1,508
80,279,534/458,573,739=
0.175
1,055,918/38,116,695=
0.028
701*0.028=
19
701*2.106=
1,476
a The region of some respondents (n=6) was unknown, so they were omitted from the analysis.
b Spanish population aged 18 and over (at 01_07_13) (Source: INE (Spanish Statistical Office) available from: http://www.ine.es/.)
245
Table S5. Calculation of the number of untreated anchovy meals eaten in 2013 utilised in risk characterization method 2 (Community scenario).
Totalmass: kg of fresh anchovies consumed at home2; Musclemass: grams of muscle; Numberfillets: anchovy fillets; Numbermeals: number of
anchovy meals; Nmunt: number of untreated anchovy meals.
RC method 2: Calculation of the anchovy meals consumed in Spain and the autonomous communities in 2013.
Variable Totalmass (Kg) Musclemass (g) Numberfillets Numbermeals Nmunt
Formula =Totalmass*1000*0.51a =Musclemass/4.25b =Numberfillets/11c =Numbermeals*(0.175)d
Total Spain 45,542,480 23,226,664,800 5,465,097,600 496,827,055 86,976,294
Andalucía 12,177,090 6,210,315,900 1,461,250,800 132,840,982 23,255,610
Aragón 1,044,430 532,659,300 125,331,600 11,393,782 1,994,636
Asturias 1,026,890 523,713,900 123,226,800 11,202,436 1,961,138
Baleares 511,310 260,768,100 61,357,200 5,577,927 976,492
Canarias 100,910 51,464,100 12,109,200 1,100,836 192,716
Cantabria 1,140,110 581,456,100 136,813,200 12,437,564 2,177,364
Castilla la Mancha 2,351,520 1,199,275,200 282,182,400 25,652,945 4,490,895
Castilla y León 2,499,090 1,274,535,900 299,890,800 27,262,800 4,772,722
Cataluña 6,213,760 3,169,017,600 745,651,200 67,786,473 11,866,939
Extremadura 871,200 444,312,000 104,544,000 9,504,000 1,663,804
Galicia 879,230 448,407,300 105,507,600 9,591,600 1,679,139
246
La Rioja 356,110 181,616,100 42,733,200 3,884,836 680,093
Comunidad de Madrid 7,017,070 3,578,705,700 842,048,400 76,549,855 13,401,087
Murcia 1,145,980 584,449,800 137,517,600 12,501,600 2,188,574
Navarra 574,600 293,046,000 68,952,000 6,268,364 1,097,362
País Vasco 3,650,260 1,861,632,600 438,031,200 39,821,018 6,971,208
Comunidad Valenciana 3,982,930 2,031,294,300 477,951,600 43,450,145 7,606,536
a Yield of a skinless fillet of European anchovy (i.e. 0.51)3.
b Average mass of an anchovy fillet in grams (i.e. 4.25 g) (see subsection “Hospital studies” at “Hazard characterization” section).
c Average number of anchovy in vinegar fillets consumed per meal (11 fillets) (see subsection “RC scenarios” at “Risk characterization” section).
d Proportion of untreated anchovy meals calculated from corrected questionnaire 1 (see subsection “Consumption” at “Exposure assessment”
section, and “Propunt” in Table S4).
247
“Anchovy meal size” sub-model.
This sub-model was implemented in Microsoft Excel™ using @RISK software (Palisade,
UK) for estimating a probability distribution for the number of anchovy fillets consumed
in a meal.
The second questionnaire (see supplementary materials) determined the number of fillets
consumed at home per anchovy in vinegar meal and the frequency of consumption. A
total of 35 respondents answered what was their minimum, most likely and maximum
number of fillets consumed per meal (question 3) and their frequency of consumption
(i.e. weekly, monthly, yearly) (question 4) (Table S6). It was assumed that a respondent
ate 1, 12 or 52 meals per year when s/he answered annually, monthly or weekly anchovy
consumption to question 4.
The number of fillets consumed per meal by a respondent was selected using a
RiskTriang() distribution, in which the minimum, most likely and maximum number of
“Fillets” consumed per meal were the parameters of the distribution (Table S7). The
frequency of meals consumed per year by each respondent was stored in the variable
“Fweek” (Table S6). A total of 229 anchovy in vinegar meals (Fwcum, Table S6) were
eaten each year by the 35 respondents.
A random number (Rand, Table S7) was generated between 1 and 229 in order to select
a particular anchovy in vinegar meal from a particular respondent (Sresp, Table S7) and
number of fillets (Sfil, Table S7). Finally, the number of fillets consumed per meal was
recorded (Mfillets, Table S7). This was repeated for 100,000 iterations and the output
distribution is presented in Table S8. As a result, a meal can have from one to 39 anchovy
fillets with an average of 11 anchovy fillets consumed per meal. These data were then
used in the QRA model via the use of the RiskDiscrete() distribution (Nfillet, Table 1).
248
Table S6. Input data for sub-model “anchovy meal size”. Responses from questionnaire
2 are listed below. Respondent: survey respondents. Fweek: number of anchovy meals
eaten per year. Fwcum: cumulative of Fweek. Min., mlik. and max.: minimum, most
likely and maximum number of anchovy fillets consumed by respondent per meal.
Respondent Fweek Fwcum min mlik max
1 12 12 6 12 25
2 1 13 4 7 10
3 12 25 10 15 20
4 12 37 5 9 15
5 12 49 4 8 12
6 1 50 10 10 10
7 12 62 5 8 12
8 1 63 5 7 10
9 1 64 5 10 15
10 1 65 1 10 15
11 1 66 2 3 5
12 12 78 7 20 30
13 1 79 4 6 8
14 1 80 1 1 1
15 12 92 5 12 20
16 1 93 7 8 10
17 1 94 2 4 7
18 1 95 1 3 5
19 1 96 10 10 20
20 1 97 8 20 40
21 1 98 4 7 10
22 1 99 4 6 6
23 1 100 5 10 15
249
24 12 112 5 10 20
25 1 113 6 12 20
26 12 125 5 7 10
27 1 126 2 10 15
28 1 127 4 4 4
29 1 128 6 6 8
30 12 140 10 12 20
31 1 141 5 5 7
32 12 153 9 12 15
33 52 205 6 8 10
34 12 217 10 10 30
35 12 229 10 12 16
250
Table S7. Sub-model “anchovy meal size”: Calculations to obtain the distribution that
defines the number of anchovy in vinegar fillets consumed per meal and its frequency of
consumption using information from questionnaire 2 provided in Table S6. Model
variables: Fillets: selecting number of fillets per meal by respondent using the
RiskTriang() function. Rand: random number generated between 1 and 35 (i.e.
respondents). Sresp: selecting one respondent per iteration. Sfil: selecting the number of
fillets consumed by the selected respondent. Mfillets: distribution of the number of meals
consumed by respondents (the average number of anchovy fillets consumed by
respondent can be also obtained (“Anfillet” in Table S10)).
Model variables
Fillets Rand Sresp Sfil Mfillets
Round(RiskTriang(min
1,mlik1,max1),0)
INT(RAND()*
Fwcum35)
IF(Rand>-
0.1,IF(Rand<(Fwcum+1),1
,0),0)
Srep1*
Fillets1
Sum(Sfil1:Sfil35)
Round(RiskTriang(min
2,mlik2,max2),0)
IF(Rand>Fwcum1,IF(Ran
d<(Fwcum2+1),1,0),0)
Srep2*
Fillets2
… … …
Round(RiskTriang(min
35,mlik35,max35),0)
IF(Rand>Fwcum34,IF(Ra
nd<(Fwcum35+1),1,0),0)
Srep35
*Fillets
35
251
Table S8. Output distribution from Monte Carlo “anchovy meal size” sub-model
(100,000 iterations) of the frequency of the number of fillets consumed per anchovy in
vinegar meal.
Nfillets Frequency Fraction
1 462 0.00462
2 149 0.00149
3 558 0.00558
4 979 0.00979
5 1,445 0.01445
6 4,657 0.04657
7 11,593 0.11593
8 16,456 0.16456
9 11,275 0.11275
10 6,631 0.06631
11 6,786 0.06786
12 7,845 0.07845
13 6,864 0.06864
14 5,460 0.0546
15 4,265 0.04265
16 3,210 0.0321
17 2,688 0.02688
18 2,031 0.02031
19 1,555 0.01555
20 1,062 0.01062
21 890 0.0089
22 759 0.00759
23 603 0.00603
252
24 495 0.00495
25 401 0.00401
26 315 0.00315
27 256 0.00256
28 155 0.00155
29 77 0.00077
30 26 0.00026
31 6 0.00006
32 6 0.00006
33 14 0.00014
34 8 0.00008
35 5 0.00005
36 5 0.00005
37 3 0.00003
38 2 0.00002
39 3 0.00003
253
Table S9. Calculation of ID50 and R by method 1 (hospital studies) and 3 (questionnaire 1 (Qaire 1)). Variables; considered variables: Population,
number of persons in the hospital influence area; Cases, number of anisakiasis cases in the studied population; Year, duration of the study in years;
Casesyear, number of anisakiasis per year; Incidence, number of anisakiasis cases per 100,000 inhabitants/year; Amealpy, number of untreated anchovy
meals consumed per person and year; Auntmeal, number of untreated anchovy meals consumed by study population; Pdisease, probability of disease
caused by untreated anchovy meals; Dose1, Dose0.5, Dose0.1; average number of viable parasites in untreated anchovy meals when viability of Anisakis
was 100%, 50% and 10%; R1, R0.5, R0.1: R value when Anisakis viability was 100%, 50% or 10%; ID50_1, ID50_0.5, ID50_0.1: ID50 value when
Anisakis viability was 100%, 50% or 10%.
Method 1 (Hospital studies) Method 3
(Qaire 1)
La Paz Virgen de la Salud Antequera Carlos III Qaire 1
Variables Distribution, fixed
number or calculation
Value Reference Value Reference Value Reference Value Reference Value
Population Number 500,000 BIOHAZ
(2010)
323,000 Repiso
Ortega et
al., 2003
110,000 Del Rey
Moreno pers.
comm.
490,000 González
Muñoz pers.
comm.
701
Cases Number 96 Alonso
Gómez et
al., 2004
25 Repiso
Ortega et
al., 2003
52 Del Rey
Moreno et al.,
2008
30 González
Muñoz pers.
comm.
19c
254
Year Number 1 Alonso
Gómez et
al., 2004
2 Repiso
Ortega et
al., 2003
4 Del Rey
Moreno et al.,
2008
1 González
Muñoz pers.
comm.
23.81
Casesyear =Cases/Year 96 NAa 12.5 NA 13 NA 30 NA 0.8
Incidence =(Cases/Year)*100000/
Population
19.2 NA 3.87 Repiso
Ortega et
al., 2003
11.82 NA 6.12 NA 116.33
Amealpy NA 2.106b Table S4 2.106b Table S4 2.106b Table S4 2.106b Table S4 NA
Auntmeal =Amealpy*Population 1,053,076 NA 680,287 NA 231,677 NA 1,032,014 NA 1,476d
Pdisease =Casesyear/Auntmeal 0.0000912 NA 0.0000184 NA 0.0000561 NA 0.0000291 NA 0.000552
Dose1 Average 0.66 QRA
model
0.66 QRA
model
0.66 QRA model 0.66 QRA model 0.66
R1 =-(1/Dose1)*LN(1-
Pdisease)
0.0001381 NA 0.0000278 NA 0.0000850 NA 0.0000440 NA 0.0008371
ID50_1 -(LN(0.5)/R1) 5,018 NA 24,897 NA 8,153 NA 15,737 NA 828
255
Dose0.5 Average 0.45 QRA
model
0.45 QRA
model
0.45 QRA model 0.45 QRA model 0.45
R0.5 =-(1/Dose0.5)*LN(1-
Pdisease)
0.000203 NA 0.0000408 NA 0.0001247 NA 0.0000646 NA 0.0012277
ID50_0.5 -(LN(0.5)/R0.5) 3,421 NA 16,975 NA 5,559 NA 10,730 NA 565
Dose0.1 Average 0.01 QRA
model
0.01 QRA
model
0.01 QRA model 0.01 QRA model 0.01
R0.1 =-(1/Dose0.1)*LN(1-
Pdisease)
0.0091166 NA 0.0018375 NA 0.0056114 NA 0.0029070 NA 0.0552471
ID50_0.1 -(LN(0.5)/R0.1) 76 NA 377 NA 124 NA 238 NA 13
a NA - not applicable.
b Number of untreated anchovy meals eaten by Spanish person per year (see “Amealpy” in Table S4).
c Number of respondents with allergy to Anisakis spp. (see “Allergic respondents” in Table S4).
d Number of untreated meals eaten by allergic respondents (see “Auntmeal” in Table S4).
256
Table S10. Calculation of ID50 and R by method 2 (hospital studies). Variables; considered variables: Capita, consumption per capita of fresh anchovies
at home (available at MAGRAMA (2016)); Cyear: kilograms of fresh anchovy consumed by studied population; Yield, yield of skinless anchovy fillets
(FAO); Cfyear, kilograms of flesh consumed by population; Anfillet, average number of fillets consumed per meal by Spanish respondents; Amfillet,
average weight of muscle of an anchovy fillet. Ammeal, average mass in kilograms of an anchovy meal; Nmealyear, number of anchovy meals consumed
per year by population. Auntmeal: number of untreated anchovy meals consumed by population. Pdisease, R1, R0.5, R0.1, ID50_1, ID50_0.5, ID50_0.1
as described in Table S8.
Method 2 (Hospital studies)
La Paz Virgen de la Salud Antequera Carlos III Source of data
Variable Distributional assumption Value Value Value Value
Capita Number 1.17 1.03 1.39 1.17 MAGRAMA2
Cyear Capita*Population 585,000 332,690 152,900 573,300
Yield Number 0.51 0.51 0.51 0.51
Cfyear Cyear*Yield 298,350 169,672 77,979 292,383
Anfillet Average 11 11 11 11
Amfillet Average 0.00425 0.00425 0.00425 0.00425
Ammeal Anfillet*Amfillets 0.04675 0.04675 0.04675 0.04675
257
Nmealyear Cyear*Ammeal 12,513,369 7,116,364 1,668,000 12,263,102
Propunt NAa 0.175 0.175 0.175 0.175 Table S4
Auntmeal =Propunt*Nmealyear 2,190,634 1,245,816 292,006 2,146,822
Pdisease Casesyear/Auntmeal 0.0000438 0.0000100 0.0000445 0.0000140 Casesyear in Table S9
R1 =-(1/Dose1)*LN(1- Pdisease) 0.0000664 0.0000152 0.0000675 0.0000212 Dose1 in Table S9
ID50_1 -(LN(0.5)/R1) 10,439 45,594 10,276 32,737
R0.5 =-(1/Dose0.5)*LN(1- Pdisease) 0.0000974 0.0000223 0.000099 0.000031 Dose0.5 in Table S9
ID50_0.5 -(LN(0.5)/R0.5) 7,117 31,087 7,006 22,321
R0.1 =-(1/Dose0.1)*LN(1- Pdisease) 0.0043824 0.0010034 0.004452 0.001397 Dose0.1 in Table S9
ID50_0.1 -(LN(0.5)/R0.1) 158 691 156 496
a NA - not applicable.
258
Questionnaire 1 (Spanish version).
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Seguridad alimentaria en productos de la pescaEsta encuesta forma parte del proyecto 'PARASITE', queestudia la incidencia, los efectos y el control de gusanosnematodos en productos de la pesca. Se trata de untrabajo de gran importancia con el que se pretendeaumentar la calidad y seguridad de los productospesqueros. Sus respuestas serán estrictamenteconfidenciales y anónimas. Por favor, si usted es mayorde edad y vive en España, responda a las siguientespreguntas. Muchas gracias por su colaboración.Hay 46 preguntas en esta encuesta
País de residencia
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Preguntas generales
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4) ¿En qué localidad vive actualmente? Indique también la provincia.
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Ej.: Vigo (Pontevedra).
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5) Estado civil
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6) ¿Cuántos adultos viven en su hogar?
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7) ¿Cuántos menores de 18 años viven en su hogar?
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9) Indique su situación laboral
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11) ¿Cuál es el ingreso bruto anual de su hogar?
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Alergias
12) ¿Cómo calificaría su estado de salud?
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13) ¿Tiene alguna alergia?
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13. A) Si ha contestado afirmativamente a la anterior cuestión, por favorindique el o los tipos de alergias:
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'Sí' en la pregunta '14 [A13]' (13) ¿Tiene alguna alergia?)
Por favor, marque las opciones que correspondan:
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Anisakis
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Consumo de pescado
14) ¿Con qué frecuencia come pescado?
Por favor seleccione sólo una de las siguientes opciones:
Nunca
1 vez al mes
1 vez a la semana
2 3 veces por semana
4 veces o más por semana
15) ¿Cómo suele consumir el pescado?
Por favor, marque las opciones que correspondan:
Fresco
Congelado
Productos surimi
Otros (e.j. conservas)
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16) ¿Qué especies de pescado suele consumir?
Por favor, indique la frecuencia de consumo de los pescados que consuma: Anual, Mensualo Semanal
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmenteMerluza,pescadilla, lluço legatzaBoquerón, anchoa,seitó o bokarteRape, peixe sapo,rap o zapo zuria (osapo baltza)Gallo, rapante,bruixa o itxasoillarraBacaladilla, lirio,maire o perlitaJurel o chicharro,xurelo, sorell otxitxarro baltzaBacalao, bacallao,bacallá o bakailaoaAbadejo, abadexo oabadiraCaballa, xarda,verat o berdelaSardina, sardiña oparrotxaAtúnSalmón o truchaPez de San Pedro,San martiño, gall omuxumartinOtros
17) ¿Dónde suele comer pescado?
Por favor seleccione sólo una de las siguientes opciones:
Casa
Otros (e.j. Restaurante)
Ambos lugares
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Consumo de pescado crudo
18) ¿Suele comer pescado crudo o poco cocinado?
Por favor seleccione sólo una de las siguientes opciones:
Sí
No
Las recetas de pescado crudo o poco cocinado incluyen:
Pescado poco hecho. (e.j. cocinado por fuera pero poco hecho por dentro).Pescado crudo macerado en limón o vinagre (ej. boquerones en vinagre).Pescado encurtido o marinado.Pescado ahumado (ej. salmón).Pescado en salmuera o salazón.Pescado Seco.Sushi o sashimi.Ceviche.
Conteste a estas preguntas si consume pescado crudo o ligeramentecocinado:
18. A) ¿Dónde y con qué frecuencia? Puede seleccionar una o dosopciones:
Por favor marque la casilla por frecuencia de consumo en los distintoslugares.
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'Sí' en la pregunta '20 [A18]' (18) ¿Suele comer pescado crudo o poco cocinado?)
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmenteHogarOtros sitios (ej.restaurantes)
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18. A.2 ¿Qué tipo de pescado utiliza para comer crudo o poco cocinado?
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'Semanalmente' o 'Mensualmente' o 'Anualmente' en la pregunta '21 [A18A]' ( Conteste a estaspreguntas si consume pescado crudo o ligeramente cocinado: 18. A) ¿Dónde y con qué frecuencia? Puedeseleccionar una o dos opciones: Por favor marque la casilla por frecuencia de consumo en los distintos lugares.(Hogar))
Por favor, marque las opciones que correspondan:
Pescado crudo ya preparado (ej. salmón ahumado envasado, sushi, etc.)
Pescado fresco
Pescado congelado
Si come pescado crudo o ligeramente cocinado:
18. B) ¿Con qué tipo de pescado?
Por favor, marque sólo el tipo de producto pesquero que consuma demodo crudo o ligeramente cocinado.
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'Sí' en la pregunta '20 [A18]' (18) ¿Suele comer pescado crudo o poco cocinado?)
Por favor, marque las opciones que correspondan:
Boquerón, anchoa, seitó o bocarte
Sardina, sardiña o parrotxa
Merluza, pescadilla, lluç o olegatza
Caballa, xarda, verat o berdela
Salmón o trucha
Atún
Rape, peixe sapo, rap, zapo zuria o sapo baltza
Bacalao, bacallao, bacallá, bakailaoa
Gallo, rapante, bruixa o itxas oillarra
Calamares
Otro
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Usted come Boquerón, anchoa, seitó obokarte (crudo o ligeramente cocinado:18. C1) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinado(ej. cocinado porfuera, poco hechopor dentro)En vinagre o allimónEncurtido omarinado (ej. enescabeche)Ahumado en fríoSushi o sashimiCevicheSecoSaladoCarpaccioOtros
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Usted come Sardina, sardiña o parrotxa cruda oligeramente cocinada:
18. C2) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinada(ej. cocinada porfuera, poco hechopor dentro)Al limón o convinagreEncurtida omarinada (ej. enescabeche)Ahumada en fríoSushi o sashimiCevicheSecaSaladaCarpaccioOtra
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Usted come Merluza, pescadilla, lluç oolegatza cruda o ligeramente cocinada:18. C3) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinada(ej. cocinada porfuera, poco hechapor dentro)Al limón o convinagreEncurtida omarinadaAhumada en fríoSushi o sashimiCevicheSecaSaladaCarpaccioOtros
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Usted come Caballa, xarda, verat o berdela cruda oligeramente cocinada:
18. C4) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinada(ej. cocinada porfuera, poco hechapor dentro)Al limón o convinagreEncurtida omarinada (ej. enescabeche)Ahumada en fríoSushi o sashimiCevicheSecaSaladaCarpaccioOtros
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Usted come Salmón o trucha crudo o ligeramente cocinado:18. C5) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinado(ej. cocinado porfuera, poco hechopor dentro)Al limón o convinagreEncurtido omarinadoAhumado en frío(ej. salmón otrucha)Sushi o sashimiCevicheSecoSaladoCarpaccioOtros
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Usted come Atún crudo o ligeramente cocinado:18. C6) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinado(ej. cocinado porfuera, poco hechopor dentro)Atún al limón ocon vinagreEncurtido omarinado (ej. enescabeche)Ahumado en fríoSushi o sashimiCevicheSecoSaladoCarpaccioOtro
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Usted come Rape, peixe sapo, rap o zapo zuria (osapo baltza) crudo o ligeramente cocinado:18. C7) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinado(ej. cocinado porfuera, poco hechopor dentro)Al limón o convinagreEncurtido omarinadoAhumado en fríoSushi o sashimiCevicheSecoSaladoCarpaccioOtro
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Usted come Bacalao, bacallao, bacallá obakailaoa crudo o ligeramente cocinado:18. C8) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinado(ej. cocinado porfuera, poco hechopor dentro)Al limón o convinagreEncurtido omarinado (ej. enescabeche)Ahumado en fríoSushi o sashimiCevicheSecoSaladoCarpaccioOtro
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Usted come Gallo, rapante, bruixa o itxasoillarra crudo o ligeramente cocinado:18. C9) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinado(ej. cocinado porfuera, poco hechopor dentro)Al limón o convinagreEncurtido omarinadoAhumado en fríoSushi o sashimiCevicheSecoSaladoCarpaccioOtro
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Usted come Calamares crudos o ligeramente cocinados:18. C10) ¿Qué tipo de recetas o especialidades consume? Es posibleseleccionar distintas opciones.
Por favor marque la frecuencia de consumo en cada casilla sólo para aquellasrecetas que consuma: anualmente, mensualmente, semanalmente
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
Anualmente Mensualmente SemanalmentePoco cocinado(ej. cocinado porfuera, poco hechopor dentro)Al limón o envinagreEncurtido omarinadoAhumado en fríoSushi o sashimiCevicheSecoSaladoCarpaccioOtro
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione la respuesta apropiada para cada concepto:
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Usted come Otros crudos o ligeramente cocinados:18. C12) Por favor escriba que tipo de pescado consume, y la frecuenciacon la que lo hace (anual, mensual, semanal) en la casillacorrespondiente.
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue en la pregunta '23 [A18B]' ( Si come pescado crudo o ligeramente cocinado: 18. B) ¿Con quétipo de pescado? Por favor, marque sólo el tipo de producto pesquero que consuma de modo crudo o ligeramentecocinado. )
Por favor, seleccione todas las opciones que correspondan y escriba un comentario:
Pescado poco cocinado (ej.
cocinado por fuera, poco
hecho por dentro)
Pescado al limón o con
vinagre (ej. Boquerones)
Encurtido o marinado (ej. en
escabeche)
Ahumado en frío (ej.salmón o
trucha)
Sushi o sashimi
Ceviche
Pescado seco
Pescado salado
Carpaccio
Otro
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Conocimiento sobre Anisakis
19) ¿Ha oído hablar del Anisakis o del “gusano del bacalao”o de “gusanos delpescado”?
Por favor seleccione sólo una de las siguientes opciones:
Sí
No
20) ¿Conoce algún método de prevención contra el Anisakis o los” gusanosdel pescado”?
Por favor seleccione sólo una de las siguientes opciones:
Sí
No
20. A) Si ha contestado que sí a la anterior cuestión: Por favor, marque lacasilla con los métodos que considera recomendados para prevenir lasconsecuencias de la presencia de los Anisakis o gusanos del pescado.
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'Sí' en la pregunta '37 [A20]' (20) ¿Conoce algún método de prevención contra el Anisakis o los”gusanos del pescado”?)
Por favor, marque las opciones que correspondan:
Comer pescado crudo o ligeramente cocinado
Quitar pronto las tripas al pescado
Cocinar el pescado
Marinar o ahumar el pescado
Congelar el pescado
21) ¿Alguna vez ha dejado de comprar o consumir pescado por la presenciade Anisakis?
Por favor seleccione sólo una de las siguientes opciones:
Sí
No
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21. A) Si ha contestado que sí a la anterior cuestión, ¿qué especie ha sido?
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'Sí' en la pregunta '39 [A21]' (21) ¿Alguna vez ha dejado de comprar o consumir pescado por lapresencia de Anisakis?)
Por favor, marque las opciones que correspondan:
Merluza, pescadilla, lluç o olegatza
Bacalao, bacallao,, bacallá o bakailaoa
Palometa o japuta, castañeta, castanyola o papardoa
Sardina, sardiña o parrotxa
Salmón o trucha
Rape, peixe sapo, rap o zapo zuria (o sapo baltza)
Atún
Bacaladilla, lirio, maire o perlita
Gallo, rapante, bruixa o itxas oillarra
Caballa, xarda, verat o berdela
Boquerón, anchoa, seitó o bokarte
Otro:
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Anisakis, anisakiasis y alergia al AnisakisPor favor, lea detenidamente la siguiente información sobre el Anisakis o el “gusano del pescado” antes de responder ala cuestión.
Anisakis o “gusano del pescado” es un gusano presente de manera natural enmuchos pescados marinos.
Cómo puede contraerse: este gusano puede provocar diversasenfermedades en humanos que consuman pescado crudo o ligeramentecocinado sin métodos preventivos.
Gravedad: La enfermedad llamada anisakiasis puede cursar con gravedadleve a severa, incluyendo síntomas asociados a reacciones alérgicas.
Probabilidad: Este parásito se considera un problema de saludemergente a nivel mundial, especialmente en Japón, España e Italiadonde el pescado crudo, ligeramente cocinado o marinado, es parte de ladieta habitual (sushi, sashimi, boquerones en vinagre, ceviche, etc.).Además, en lo concerniente a las alergias alimentarias, datos publicadossugieren que el Anisakis es la mayor causa subyacente de reaccionesalérgicas en España*.
Prevención: Hasta la fecha, sabemos que para matar al gusano Anisakishay que congelar o cocinar correctamente el pescado. Sin embargo, confrecuencia las personas que padezcan alergia a este parásito continuaránmostrando síntomas de alergia después de haber consumido pescadoinfectado, aunque éste haya sido congelado, cocinado o inclusoprocesado adecuadamente.
Nota: Consulte la información disponible en el Panel de Riesgos Biológicos enpágina de la EFSA (European Food Safety Authority) si precisa másinformación sobre el Anisakis y enfermedades asociadas.
* Fuente: Ver el enlace. *
Por favor seleccione sólo una de las siguientes opciones:
Sí
No
Por favor, confirme la lectura de la información previa.
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Disponibilidad de pago
23) Por favor, tenga en cuenta el siguiente escenario o situación y responda ala cuestión:
Suponga que haya sido descubierta una tecnología o un tratamiento quegarantice que el Anisakis y sus características alergénicas puedan sercompletamente eliminadas del pescado, siendo un tratamiento 100% efectivoque además no altere la calidad del pescado.
En ese caso, si su pescado favorito (ej. bacalao) costase 6€ por kilogramo enel supermercado:
¿Cuál sería el precio máximo que estaría dispuesto y podría pagar por elmismo producto libre de Anisakis y de sus características alergénicas?
Por favor seleccione sólo una de las siguientes opciones:
No compraría ese producto
0% extra: 6 €/Kg (Lo compraría al precio establecido de 6€)
10% extra : 6.60 €/Kg
20% extra: 7.20 €/Kg
30% extra: 7.80 €/Kg
40% extra: 8.40 €/Kg
50% extra: 9 €/Kg
60% extra: 9.60 €/Kg
70% extra: 10.20 €/Kg
80% extra: 10.80 €/Kg
90% extra: 11.40 €/Kg
100% extra: 12 €/Kg o más
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23. A) ¿Cuál es la razón por la que no está dispuesto a pagar más por esetratamiento en el pescado?
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'No compraría ese producto' o '0% extra: 6 €/Kg (Lo compraría al precio establecido de 6€)' en lapregunta '42 [A23]' ( 23) Por favor, tenga en cuenta el siguiente escenario o situación y responda a la cuestión:Suponga que haya sido descubierta una tecnología o un tratamiento que garantice que el Anisakis y suscaracterísticas alergénicas puedan ser completamente eliminadas del pescado, siendo un tratamiento 100%efectivo que además no altere la calidad del pescado. En ese caso, si su pescado favorito (ej. bacalao) costase6€ por kilogramo en el supermercado: ¿Cuál sería el precio máximo que estaría dispuesto y podría pagar por elmismo producto libre de Anisakis y de sus características alergénicas? )
Por favor, marque las opciones que correspondan:
El riesgo es demasiado pequeño para justificar la diferencia de precio
El escenario propuesto no es realista
No se debería pagar ningún coste extra por una alimentación segura
Otro:
24) ¿Evitaría comprar o comer pescado por la presencia de Anisakis ogusanos?
Por favor seleccione sólo una de las siguientes opciones:
Sí, siempre
Sí, pero sólo si existiese una alta probabilidad de que el pescado contenga gusanos
No, porque cocinar o congelar el pescado mataría a cualquier gusano
24. A) ¿Por qué evitaría comprar o comer pescado con Anisakis?
Sólo conteste esta pregunta si se cumplen las siguientes condiciones:La respuesta fue 'Sí, siempre' o 'Sí, pero sólo si existiese una alta probabilidad de que el pescado contengagusanos ' en la pregunta '44 [A24]' ( 24) ¿Evitaría comprar o comer pescado por la presencia de Anisakis ogusanos? )
Por favor seleccione sólo una de las siguientes opciones:
Riesgo de salud
Poco apetecible
Ambos motivos
Otro
1/16/2015 Encuestas CETMAR Seguridad alimentaria en productos de la pesca
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Percepción del riesgo
25) Aunque por supuesto nadie puede asegurar qué sucederá en el futuro,nos gustaría saber qué probabilidades considera que tiene usted paradesarrollar Anisakiasis o/y una alergia al Anisakis.
Por favor, use una escala de 04 donde 0 significa ninguna probabilidad y 4absoluta seguridad de desarrollarla.
Por favor seleccione sólo una de las siguientes opciones:
0 (ninguna probabilidad)
1 (escasa probabilidad)
2 (probabilidad moderada)
3 (alta probabilidad)
4 (absoluta seguridad)
1/16/2015 Encuestas CETMAR Seguridad alimentaria en productos de la pesca
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¡Muchas gracias por su colaboración!
Enviar su encuesta.Gracias por completar esta encuesta.
259
Questionnaire 2. Consumption of anchovies in vinegar at Spanish homes.
1) What is your gender?
Please choose only one of the following:
o Female
o Male
2) What is your age?
Please choose only one of the following:
o 18-29 years old
o 30-49 years old
o 50-64 years old
o 65 years and over
3) How many anchovies in vinegar do you consume at home?
Please write your answer here:
o Minimum number of fillets per meal:__
o Most likely number of fillets per meal:__
o Maximum number of fillets per meal:__
4) Please, how often do you consume anchovies in vinegar at home?
Please choose only one of the following:
o Weekly
o Monthly
o Yearly
260
Questionnaire 1 (English version).
4/10/2015 Encuestas CETMAR Food safety in fishery products
http://cetcuestas.cetmar.org/index.php/admin/printablesurvey/sa/index/surveyid/121598/lang/en 1/29
Food safety in fishery productsThis questionnaire is part of the PARASITE Project,funded by the European Commission, which aims toprovide new scientific evidence and technologicaldevelopments to detect, monitor, and mitigate impacts ofparasites occurring in European and imported fisheryproducts, thus addressing concerns identified byEuropean Food Safety Authority (EFSA). The University ofAberdeen (Scotland) is coordinating this survey incollaboration with several European research institutionsand universities “Instituto de Investigaciones Marinas”and “Centro Tecnológico del Mar” (Spain), Institute ofOceanography and Fisheries (Croatia) and University ofCopenhagen (Denmark)). The results of the study will bepublished in the final report of the project and inscientific papers.
Your replies will be treated as completely confidential andthe questionnaire responses are anonymous. Pleaseproceed only if you are currently living Scotland and youare aged 18 or over. Completing the questionnaire will betaken to imply that you provide the researchers with yourinformed consent to use your responses anonymously forthe planned research. If you require any furtherinformation or have any comments or complaints, etc.please contact one of the project leads at the Universityof Aberdeen; Graham Pierce g.j.pierce@abdn.ac.uk orIoannis Theodossiou theod@andn.ac.uk or NorvalStrachan n.strachan@abdn.ac.uk.
For further information about this project please visit theproject website http://parasiteproject.eu/.There are 46 questions in this survey
Country of residence
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Please, confirm your country of residence. *
Please choose only one of the following:
Spain
Croatia
Denmark
Scotland
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General questions
1) What is your gender?
Please choose only one of the following:
Female
Male
2) What is your age?
Please choose only one of the following:
1829 years old
30 49 years old
5064 years old
65 years and over
3) In which country did you grow up?
Please choose only one of the following:
Spain
Croatia
Denmark
Scotland
Other
E.g. Greece
4) In which city/town do you currently live? Including county councils.
Please write your answer here:
E.g.: Inverurie (Aberdeenshire).
4/10/2015 Encuestas CETMAR Food safety in fishery products
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5) Are You?
Please choose only one of the following:
Married or cohabiting
Single
6) How many adults live in your household?
Only numbers may be entered in this field.
Please write your answer here:
7) How many children (<18 years) live in your household?
Only numbers may be entered in this field.
Please write your answer here:
8) What is the highest educational qualification you have obtained?
Please choose only one of the following:
No education
Primary education
Secondary education
College education or university degree
4/10/2015 Encuestas CETMAR Food safety in fishery products
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9) Please indicate your employment status:
Please choose only one of the following:
Employed
Selfemployed
Unemployed
Retired
Labour market inactive (e.g. students, domestic activity, child rearing)
10) Please indicate below your own main occupation or expertise.
Please choose only one of the following:
Extractive fishing sector (fisherman, owner, etc.)
Processing sector of fishery products
Science and Development
Human Health activities
Education and Training (including students without work experience)
Energy & water supplies
Extraction of minerals & ores other than fuels; manufacture of metals, mineral products & chemicals
Metal goods, engineering & vehicles industries
Other manufacturing industries
Construction
Distribution, hotels & catering, restaurants & pubs
Transport & communication
Banking, finance, insurance, business services & leasing
Other
4/10/2015 Encuestas CETMAR Food safety in fishery products
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11) What is your total annual household income before tax?
Please choose only one of the following:
0 £12000
£12001 £24000
£24001 £36000
£36001 £48000
£48001 £60000
£60001 £100000
£100001 and above
4/10/2015 Encuestas CETMAR Food safety in fishery products
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Allergies
12) How would you judge your health?
Please choose only one of the following:
Excellent
Good
Fair
Poor
13) Have you got an allergy?
Please choose only one of the following:
Yes
No
13. A) You said yes to the previous question, please indicate which allergiesyou have:
Only answer this question if the following conditions are met:Answer was 'Yes' at question '14 [A13]' (13) Have you got an allergy?)
Please choose all that apply:
Fish
Shrimp
Other seafood
Anisakis
Other food allergies
House dust mites
Other
4/10/2015 Encuestas CETMAR Food safety in fishery products
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Fish consumption habits
14) How many times do you eat fish?
Please choose only one of the following:
Never
1 per month
1 per week
2 3 times per week
4 times or more per week
15) Which kind of fish do you usually eat?
Please choose all that apply:
Fresh
Frozen
Surimi products
Other (e.g. canned)
4/10/2015 Encuestas CETMAR Food safety in fishery products
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16) Which fish species do you usually eat?
Please tick only the following fish in their corresponding box if you eat them: Yearly, Monthly or Weekly.
Please choose the appropriate response for each item:
Yearly Monthly WeeklyHakeAnchovyHerringFlatfish (Plaice,sole, etc.)Blue whitingHaddockCodPollockMackerelSardineTunaSalmon or troutWhitingOther
17) Where do you usually eat fish?
Please choose only one of the following:
Home
Other (e.g. restaurant)
Both
4/10/2015 Encuestas CETMAR Food safety in fishery products
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Consumption of raw fish
18) Do you eat any of the following raw or lightly cooked fish?
Please choose only one of the following:
Yes
No
Raw or lightly cooked fish include dishes like:
Undercooked fish. (e.g. cooked on outside and raw in middle).Fish with lemon or vinegar.Pickled/marinated fish (e.g. soused herring or rollmops).Coldsmoked fish (e.g. salmon) or gravlax.Brined or salted fish.Dried fish.Sushi or sashimi.Ceviche.
If you eat raw or lightly cooked fish:
18. A) Where and how often? One or two choices are possible:
Please tick the corresponding box in terms of how often you eat them in thedifferent places.
Only answer this question if the following conditions are met:Answer was 'Yes' at question '20 [A18]' (18) Do you eat any of the following raw or lightly cooked fish?)
Please choose the appropriate response for each item:
Yearly Monthly WeeklyHomeOther (e.g.restaurant)
4/10/2015 Encuestas CETMAR Food safety in fishery products
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If you eat raw or lightly cooked fish at home:
18. A.2 Do you use?
Only answer this question if the following conditions are met:Answer was 'Weekly' or 'Monthly' or 'Yearly' at question '21 [A18A]' ( If you eat raw or lightly cooked fish: 18. A)Where and how often? One or two choices are possible: Please tick the corresponding box in terms of how oftenyou eat them in the different places. (Home))
Please choose all that apply:
Already prepared raw fish (e.g. packed smoked salmon, rollmops, sushi, etc.)
Fresh fish
Previously frozen fish
If you eat raw or lightly cooked fish:
18. B) Which fish?
Please tick only the following fish in their corresponding box if you eatthem.
Only answer this question if the following conditions are met:Answer was 'Yes' at question '20 [A18]' (18) Do you eat any of the following raw or lightly cooked fish?)
Please choose all that apply:
Anchovy
Sardine
Herring
Mackerel
Salmon or trout
Tuna
Haddock
Cod
Flatfish (e.g. plaice, sole, etc.)
Squid
Other
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Anchovy:18. C1) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedanchovy (e.g. rawin middle)Anchovies withvinegar or lemonPickled or othermarinatingprocessColdsmokedSushi or sashimiCevicheDriedBrined or saltedCarpaccioOthers
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Sardine:18. C2) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedsardine (e.g. rawin middle)Sardine withlemon or vinegarPickled or othermarinatingprocessColdsmokedSushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Herring:18. C3) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedherring (e.g. rawin middle)Herring withlemon or vinegarPickled or othermarinatingprocess (e.g.soused herringand rollmops)Coldsmoked(e.g. kippers)Sushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Mackerel:18. C4) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedmackerel (e.g.raw in middle)Mackerel withlemon or vinegarPickled or othermarinatingprocessColdsmockedSushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Salmon or trout:18. C5) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedsalmon (e.g. rawin middle)Salmon withlemon or vinegarPickled or othermarinatingprocessColdsmoked(e.g. salmon ortrout)Sushi or sashimiCevicheDriedBrined or salted(e.g. gravlax)CarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Tuna:18. C6) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedtuna (e.g. raw inmiddle)Tuna with lemonor vinegarPickled or othermarinatingprocessColdsmockedSushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Haddock:18. C7) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedhaddock (e.g. rawin middle)Haddock withlemon or vinegarPickled or othermarinatingprocessColdsmokedhaddockSushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Cod:18. C8) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercooked cod(e.g. raw inmiddle)Cod with lemon orvinegarPickled or othermarinatingprocessColdsmoked codSushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Flatfish (e.g. plaice, sole,etc.):18. C9) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedflatfish (e.g. rawin middle)Flatfish withlemon or vinegarPickled or othermarinatingprocessColdsmokedSushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Squid:18. C10) Which type of recipes or specialties? Multiple choices arepossible.
Please tick only the following fish in their corresponding box if you eat them: yearly, monthly; weekly.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
Yearly Monthly WeeklyUndercookedsquid (e.g. raw inmiddle)Squid with lemonor vinegarPickled or othermarinatingprocessColdsmokedSushi or sashimiCevicheDriedBrined or saltedCarpaccioOther
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose the appropriate response for each item:
4/10/2015 Encuestas CETMAR Food safety in fishery products
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You eat raw or lightly cooked Other:18. C12) Please, write what kind of fish you consume and the frequencywith which it does (yearly, monthly, weekly) in the appropriate box.
Only answer this question if the following conditions are met:Answer was at question '23 [A18B]' ( If you eat raw or lightly cooked fish: 18. B) Which fish? Please tick only thefollowing fish in their corresponding box if you eat them. )
Please choose all that apply and provide a comment:
Undercooked fish (e.g. raw in
middle)
Fish with lemon or vinegar
Pickled or other marinating
process (e.g. soused herring
and rollmops)
Coldsmoked (e.g. salmon or
trout)
Sushi or sashimi
Ceviche
Dried
Brined or salted (e.g. gravlax)
Carpaccio
Other
4/10/2015 Encuestas CETMAR Food safety in fishery products
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Knowledge of Anisakis
19) Have you ever heard about Anisakis or “herring or cod worm” or “wormsin fish”?
Please choose only one of the following:
Yes
No
20) Do you know about prevention methods against Anisakis or “herring orcod worm”?
Please choose only one of the following:
Yes
No
20. A) You answered yes to the previous question: Please pick therecommended prevention methods against worms in fish.
Only answer this question if the following conditions are met:Answer was 'Yes' at question '37 [A20]' (20) Do you know about prevention methods against Anisakis or “herring orcod worm”?)
Please choose all that apply:
Eat raw or lightly cooked fish
Gutting the fish as soon as possible
Cooking the fish
Marinating or coldsmoking the fish
Freezing the fish
21) Have you ever avoided buying or eating any fish due to the presence ofAnisakis?
Please choose only one of the following:
Yes
No
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21. A) You answered yes to the previous question, which fish have youavoided?
Only answer this question if the following conditions are met:Answer was 'Yes' at question '39 [A21]' (21) Have you ever avoided buying or eating any fish due to the presenceof Anisakis?)
Please choose all that apply:
Hake
Cod
Herring
Sardine
Salmon or trout
Whiting
Tuna
Blue whiting
Flatfish (Plaice, sole, etc.)
Mackerel
Anchovy
Other:
4/10/2015 Encuestas CETMAR Food safety in fishery products
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Anisakis, anisakiasis and allergy to AnisakisPlease, read carefully the following information regarding Anisakis or “herring or cod worm” in fish products beforeanswering the question.
Anisakis or “herring worm” is a natural worm in many marine fish.
How to contract it: this worm may cause disease in humans who eat rawor lightly cooked infected fish products without prevention methods.
Severity: This disease is called anisakiasis and its severity may vary frommild to severe. Other symptoms associated can be allergic reactions.
Likelihood: This parasite is considered an emerging health problemworldwide, especially in Japan, Spain and Italy; where raw, lightly saltedor marinated fish is part of the regular diet (sushi, sashimi, gravlax,salted or pickled herring, anchovies with vinegar, ceviche, etc.).Moreover, regarding food allergies, published data suggest that Anisakisis the major hidden producer of allergic reactions within Spain*. Only afew cases of human Anisakiasis were reported per year in Scotland,however no data about Anisakis allergy are available
Prevention: To date, we know that freezing and/or cooking properly thefish kills the worm (Anisakis). However, it is common that people withallergy to Anisakis still show allergic symptoms after consuming fish thathas been correctly frozen, cooked, and even processed.
Note: Please visit EFSA (European Food Safety Authority) if you require moreinformation about Anisakis and their related disease.
*Source: See the link. *
Please choose only one of the following:
Yes
No
Please confirm that you have read this information
4/10/2015 Encuestas CETMAR Food safety in fishery products
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Willingness to pay
23) Please consider the following scenario and answer the question:
Suppose that a technology or treatment were discovered that guarantee thatevery Anisakis worms (and their related allergens) would be completelyremoved from fish, so the treatment was 100% effective. Suppose furtherthat there are no side effects on fish quality.
Then, imagine that your favourite fish (e.g. cod) costs £6 per kilogram at thesupermarket.
What is the maximum you would be willing and able to pay for the same fishproduct free of Anisakis fish worms and their allergens?
Please choose only one of the following:
I would not buy this product
0% extra or £6 per kilogram (I will buy it at the stated price (£6))
10% extra or £6.60 per kilogram
20% extra or £7.20 per kilogram
30% extra or £7.80 per kilogram
40% extra or £8.40 per kilogram
50% extra or £9 per kilogram
60% extra or £9.60 per kilogram
70% extra or £10.20 per kilogram
80% extra or £10.80 per kilogram
90% extra or £11.40 per kilogram
100% extra or £12 per kilogram or more
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23. A) If you are not willing to pay for this treatment, why?
Only answer this question if the following conditions are met:Answer was 'I would not buy this product' or '0% extra or £6 per kilogram (I will buy it at the stated price (£6))' atquestion '42 [A23]' ( 23) Please consider the following scenario and answer the question: Suppose that atechnology or treatment were discovered that guarantee that every Anisakis worms (and their related allergens)would be completely removed from fish, so the treatment was 100% effective. Suppose further that there are noside effects on fish quality. Then, imagine that your favourite fish (e.g. cod) costs £6 per kilogram at thesupermarket. What is the maximum you would be willing and able to pay for the same fish product free of Anisakisfish worms and their allergens? )
Please choose all that apply:
The risk is too small to warrant any price difference
The scenario is not realistic
You should not have to pay any premium for having safe food
Other:
24) Would you avoid buying or eating a fish due to the presence of Anisakisor worms in fish?
Please choose only one of the following:
Yes, always
Yes, but only if there is high chance of worms
No, cooking or freezing kill any possible worm
24. A) Why would you avoid the purchase or eating of these fish?
Only answer this question if the following conditions are met:Answer was 'Yes, always' or 'Yes, but only if there is high chance of worms ' at question '44 [A24]' ( 24) Would youavoid buying or eating a fish due to the presence of Anisakis or worms in fish? )
Please choose only one of the following:
Health risk
Unappetising
Both
Other
4/10/2015 Encuestas CETMAR Food safety in fishery products
http://cetcuestas.cetmar.org/index.php/admin/printablesurvey/sa/index/surveyid/121598/lang/en 28/29
Risk perception
25) Of course, no one can know for sure what will happen in the future, butwe would like to know what you think about the following health risk:
Using a scale of 04 which 0 means no chance and 4 means absolutely certain,
what are the chances that you will develop an Anisakiasis and/or an allergy toAnisakis?
Please choose only one of the following:
0 (no chance)
1 (slight chance)
2 (moderate chance)
3 (high chance)
4 (absolutely chance)
4/10/2015 Encuestas CETMAR Food safety in fishery products
http://cetcuestas.cetmar.org/index.php/admin/printablesurvey/sa/index/surveyid/121598/lang/en 29/29
Thank you very much for your kind cooperation!
Submit your survey.Thank you for completing this survey.
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