semi-annuel newsletter - n°8 july 2012 labinfo

Labinfo

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R E F E R E N C ELABORATORIESNRL

Newsletter for the approved food safety laboratories

SEMI-ANNUEL NEWSLETTER - N°8 JULY 2012

FASFCAC-Kruidtuin - Food Safety Center, Kruidtuinlaan 55, 1000 Brussels

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p. 4 Nanoparticles in food

p. 8 Endocrine disruptors : link with obesity ?

p. 12 Validation of the detection of E. coli O104:H4 in vegetables.

p. 15 Emerging contaminants

p. 18 Emerging threats: new GMO and UGM

p. 23 Tick-Borne Encephalitis Virus Seropositive Dog Detected in Belgium: Multi-species Screening of Sentinels for Public Health

p. 27 Schmallenberg virus in Belgium

p. 30 EMERGING MARINE BIOTOXINS report from the seminar 2012

p. 35 Workshops & Symposia

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LabInfoNewsletter for the approved food safety laboratories

Editors’ groupDirk Courtheyn, Mieke De Mits, Conny De Schepper, Alain Dubois, Marc Evrard, Alain Laure, Bert Vandenborre, Mieke Van de Wiele, Eva Wevers and Marie-Christine Wilem

Authors of this issueGeert De Poorter, Jorina Geys, Heidi Demaegdt, Nadine Botteldoorn, Alexandra Duarte, Sarah Denayer, Huig Vanderperren, Sylvia Broeders, Sigrid De Keersmaecker, Nancy Roosens, Sophie Roelandt, Paul Heyman, Marina De Filette, Sirkka Vene, Yves Van der Stede, Ann Brigitte Caij, Paul Tavernier, Alexandre Dobly, Hendrik De Bosschere, Philip Vyt, Carole Meersschaert, Stefan Roels, Nick De Regge, Thierry van den Berg and Mirjana Andjelkovic

TranslationTranslation Service of the AgencyEditors’ group

Photographs and illustrationsSupplied by the laboratories

LayoutGert Van Kerckhove

Editor’s addressLabInfop.a. D. CourtheynFASFCAC-Kruidtuin – Food Safety Center4de verdieping, bureel K04/120218Kruidtuinlaan 551000 BrusselTel.: [email protected]




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Dear Reader,

In a few years’ time Labinfo has become highly valued for providing external food analysis laboratories with information on the rapidly developing laboratory techniques and me-thods. In recent years, the number of approved laboratories has increased. So far 59 labs have been approved, including 9 foreign labs. Even RIKILT, the European Union Reference Laboratory for hormonal substances and sedatives has recently been approved by the FASFC. This is quite remarkable since the Belgian laboratory sector is in the midst of a merger wave in which all possible legal constructions are being set up in order to increase the effectiveness of private laboratories (such as takeovers, all forms of cooperation, joint ventures, partnerships, …).

These labs expect clearness and a stable legal framework from highly efficient authorities. That explains the success of the FASFC approval policy that links quality and environ-mental requirements and hence ensures the customers of FASFC approved labs that the analyses on their samples are performed in a correct manner. Foreign authorities are eager to adopt the good elements of our system; on the other hand, the administrative proce-dures related to the approval of labs must be further simplified. We are now working on that aspect.

The concept of Labinfo has been changed and now each issue is dedicated to one parti-cular subject. The subject of this issue is emerging threats.

I hope you will enjoy reading this issue of Labinfo and wish you all happy summer holi-days.

Geert De PoorterDirector general Laboratories

Editorial

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Nanoparticles in foodIntroduction

Nanotechnology is a fast growing and promising innovation. In this technology, particles (nanoparticles, nano-tubes) with at least one dimension less than 100 nm (see Figure 1) are being developed. Due to their small size, nanoparticles have specific physico-chemical properties, such as strength, chemical reactivity, electrical conduct-ance, magnetism and optical effects; these give rise to new applications. Not only the size, but also the size distri-bution, the shape and the surface reactivity are important. Several applications of nanoparticles in consumer and personal care products are known and yet on the market; such as transparent sunscreens, antibacterial clothing, scratch-resistant paint for cars, self-cleaning windows, etc. However, nanotechnology has also applications in food and beverages. Deliberate uses of nanotechnology in agriculture and food industry are mainly still in research and would not yet occur on the European market.

Figure 1. Comparison size nanoparticles with biological objects.

Figure 1. Comparison size nanoparticles with biological objects.

diameter human hair (80 µm)

0,01 µm 0,1 µm 1 µm 10 µm 10 nm 100 nm 1 000 nm 10 000 nm

virus bacteria

RBC nanoparticles

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Applications of nanotechnology in the food chain

Nanotechnology can be used in all phases of the food production chain (Table 1), from the field with agricultural production till the industrial processing and finally the packaging of foods. Nanoparticles can also be added to food to enhance the nutritional aspects, by means of nanosupplements and nano delivery systems for bioactive compounds.

Table 1. Overview applications of nanoparticles in the food production chain

production toolnanosievesnanosensors

removal of pathogens or contaminantsdetection of micro-organisms, food deterioration

inert nanoparticleswater purification/soil cleaningfood storagefood packaging materialsfood supplements

Al2O3, La, nano-FeAgAg, SiO2, Mg, ZnOcolloidal metals

oxidation of contaminantsanti-bacterialprevention of food deteriorationenhanced uptake

‘nano delivery systems’ nanocapsules

nanocapsules

nanocapsules

pesticides

bioactive compound

nutrients

increased efficacy and water solubility, local and controlled release

local and controlled release, increased absorption and bioavailability

local and controlled release, increased absorption

Roughly there are two groups of application of nanotechnology in food. In a first group, nanotechnology is ap-plied as a production tool, without the addition of nanoparticles to the food. Examples are the use of nanosieves to filter out bacteria and nanosensors for the detection of contaminants or micro-organisms. These sensors can also be incorporated in food packaging materials to detect food deterioration.

In a second group, nanoparticles are introduced into the food during the production. A whole diversity of nanoparticles are being applied, both inert particles and nanocapsules. Inert nanoparticles are used in the food production chain for a variety of purposes. For example, in the process of water purification and soil cleaning, aluminum oxide, lanthanum particles and nanoscale iron powder can be used. In food storage, silver is frequently used and in food packaging materials also silica, magnesium and zinc oxide are sometimes present. Consum-ers can be exposed to nanoparticles by direct application of inert particles in the food; however, as long as the nanoparticles remain bound in the packaging materials, exposure to consumers is low. An important safety issue is the migration of nanoparticles resulting in their appearance in the food. Besides inert nanoparticles, also nano delivery systems are used. Nanocapsules consist of a shell and an internal space in which the desired active compounds are enclosed. The shell is usually built from polymers or lipids. The advantage of nanocapsules is that they can deliver the desired active compound in a targeted way, so less active compound is required for a desired result. Examples of active compounds are pesticides or medicines. The use of nanocapsules leads to increased absorption and bioavailability.

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It is difficult to predict the long-term applications of nanotechnology. Within agriculture, precision farming is a promising objective. Expected applications are smart sensors for early warning of changing conditions, and the use of nanocapsules with pesticides that are able to respond to different conditions. Also the utilization of nanoparticles in food packaging materials as sensor will continue to increase. Finally, the potential applications of nanocapsules will continue to expand. Nanocapsules can be designed with specific chemical receptors that bind only to particular cells; also the release of nutrients or bioactive compounds can be controlled. This gives rise to many possible applications.

Toxicological aspects of nanotechnology in the food chain

It is important to gain insight about possible adverse health effects of nanoparticles. Due to their small size and their large surface area, nanoparticles have specific physico-chemical properties that differ from their conven-tional counterparts. This has consequences for the risk assessment, which is currently based on knowledge of conventional chemicals. Due to the specific physico-chemical properties of nanoparticles, the findings of conven-tional chemicals cannot be extrapolated as such. Also the extrapolation of findings from one product to another is under debate for nanoparticles. Generally, a “case-by-case” approach is recommended for the risk assessment of nanoparticles. At this moment, only few data exist on the exposure and harmful effects of nanoparticles, and the absorption, distribution, metabolism and excretion after oral exposure.

The use of nanoparticles in food needs to be documented, and the associated exposure to the consumers. To do so, methods for detection and characterization of nanomaterials in complex matrices such as foodstuff and biological tissues/fluids are essential. Many nanomaterials are composed of classical chemical elements, which makes is more difficult to distinguish between chemical compounds and nanoparticles. Furthermore, we also have to discriminate between manufactured and naturally occurring nanoparticles; and nanoparticles can occur as aggregates and agglomerates.

The existing tests to evaluate the toxicity of substances are not as such applicable to nanomaterials. Especially the high reactivity and the greater ability to cross barriers can lead to different toxicokinetic and toxicodynamic properties of nanomaterials compared to conventional chemicals. In the toxicological assessment of nanomateri-als, characterization is a critical point. As mentioned above, not only the chemical composition and the size, but also the size distribution, the surface characteristics and the morphology are determining for nanomaterials. For many nanoparticles it is not yet clear which biological barriers in the body they could cross and where they could accumulate. Other issues include the generation of oxidative stress, inflammatory reactions and the interactions with biomolecules such as DNA and proteins.

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Conclusion

Nanotechnology has many interesting applications in the food industry, for example in terms of food safety and quality control. However, this new technology also raises questions about a proper risk assessment. It is crucial to develop reliable tests to detect the presence of nanoparticles in food, and for a proper assessment of possible adverse effects.

Jorina Geys (CODA-CERVA, Tervuren)[email protected]

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Endocrine disruptors : link with obesity ?

Overweight and obesity are defined as a disease in which abnormal excessive body fat accumulation causes ad-verse effects on health. This disease has reached epidemic proportions, with, in Europe alone, half of the popula-tion suffering from overweight and one third being obese. Whereas nutrition (caloric intake) and lifestyle choices (physical activity) are universally considered the most important, other environmental influences can also play a role in the development of obesity. A proposed contributor to the rise in obesity is exposure to endocrine disrupt-ing chemicals (EDC’s). EDC’s are chemicals that alter the normal functioning of hormones or signaling molecules in the body. While much early work on endocrine disruption focused on reproductive effects (cfr the disruption of the normal activity of our sex hormones, the estrogens and androgens, …), the hypothesis that chemicals may affect weight homeostasis (called obesogens or metabolic disruptors) emerged more recently from different lines of research.

Plausible mechanisms

Hormones function mainly through interaction with their receptors, which can be classified in two large groups: membrane bound receptors (primarily respond to peptide hormones) and nuclear receptors (NRs) which are acti-vated by interaction with small lipophilic hormones such as sex steroids. EDCs may possess multiple mechanisms of action, however, because many EDCs are small lipophilic compounds, one privileged route is through their direct interaction with a given NR leading to perturbation or modulation of downstream gene expression.

Several NRs are linked to metabolism and obesity. Firstly, regarding the estrogen receptor, periods of low estrogen levels can lead to an increase in adipose tissue and this seems to be mediated by ERα (an isotype of the estrogen receptor or ER). During development however, estrogens contribute to an increase in adipocyte or fat cell num-bers. Androgen levels on the other hand are related to lower body mass index in men. EDCs known as estrogens are bisphenol A (BPA), diethylstilbestrol, alkylphenols, some pesticides … Several EDCs block the normal function of the androgen receptor, they are called antagonists or antiandrogens. Examples are some phthalates, alkylphe-nols and some pesticides (Casals-casas & Desvergne, 2011; Grün & Blumberg, 2009).

Also, thyroid hormones (THs) and their receptors (TRα en β) are tightly associated with basal metabolism. Besides the receptors, thyroid disruptors can target or interfere with the synthesis of thyrotropin releasing hormone, the TH synthesis and metabolism, iodine uptake, TH transport, … which can lead to a change in TH serum levels. Ele-vated TH levels accelerate metabolism, increase lipolysis and provoke weight loss, the opposite effect is seen with low TH levels. A number of EDCs, including phthalates, BPA, flame retardants, … are suspected thyroid disruptors and may reduce circulating thyroid level (Zoeller et al., 2010; Grün & Blumberg, 2009).

In addition, glucocorticoids acting through glucocorticoid receptors (GR1 and 2) allow an organism to adequately respond to physical or emotional stresses by promoting gluconeogenesis, increasing blood glucose levels, and mobilizing the oxidation of fatty acids. Besides BPA and dicyclohexylphthalate, many pharmaceutical compounds interact with these receptors (Sargis et al., 2010).

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Most importantly, PPARs (peroxisome proliferator activated receptors, the main isotypes being PPARα and PPARγ) play critical roles in adipogenesis and lipid metabolism. PPARα’s primary purpose is the regulation of energy ho-meostasis, PPARα activates fatty acid catabolism and increases gluconeogenesis. PPARα stimulation during devel-opment might lead to obesity in later stages. PPARγ is described as the master regulator of fat cell development, with activation required for adipocyte differentiation and fat storage, it also improves insulin sensitivity. A variety of chemicals have been shown to bind and activate with for example perfluoroalkyl compounds that mostly bind PPARα; and organotins, and some phthalates or their metabolites like MEHP (monoethylhexylphthalate) that mostly bind PPARγ (Hatch et al., 2010; Feige et al., 2007; OECD draft, 2011).

Recently, AhR (aryl hydrocarbon receptor), well known as the receptor for dioxin like compounds, has been impli-cated as another regulator of metabolism. The mechanisms are not yet well described, but cross-talk with ER and PPARy may be involved through influencing their expression (Casals-casas & Desvergne, 2011).

Figure 1: Endocrine disrupting chemicals or EDCs interact with diverse members of the nuclear receptor family and with aryl hydrocarbon receptor (AhR). PFCs: perfluoroalkyl compounds, ER: estrogen receptor, GR: glucocorti-coid receptor, TR: thyroid receptor, PPARα/γ: peroxisome proliferator activated receptors, RXR: retinoid x receptor.

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In vivo and epidemiological evidence: some examples

A number of epidemiological studies have linked pesticide exposure with obesity and metabolic syndrome. For example in utero (or foetal) exposure of dichlorodiphenyldichloroethylene (DDE) and hexachlorobenzene are as-sociated with an increased BMI in women and children. A similar result was seen for diethylstilbestrol in an animal model. A positive correlation with BMI was also seen for nonylphenol measured in adipose tissue of women and BPA blood levels. Several phthalate metabolites, measured in urine, are also linked with abdominal obesity in adult males. (Casals-casas & Desvergne, 2011; Diamanti-Kandarakis et al., 2009)

The most well known pharmaceuticals that are obesogens are thiazolidinediones, used to treat type 2 diabetes. These compounds are linked to weight gain in humans through activation of PPARγ. (Casals-casas & Desvergne, 2011). The organotin tributyltin induces adipogenesis in cell culture models and increases adipose mass in vivo in two vertebrate model organisms, frogs and mice (Grun et al., 2006). However, for tributyltin, no epidemiological data are available concerning human exposure.

Screening for obesogen candidates

Since PPARγ is the most important regulator of adipogenesis and fat metabolism, screening for activity through this receptor is a good way to start. Classically, a transactivation reporter assay can screen for possible binders to PPARγ (both agonists or antagonists). It consists of a cell line in which the human PPARγ receptor and a PPARγ re-sponsive reporter gene (mostly luciferase) are expressed. For possible interactions with the other NRs mentioned similar assays exist (OECD draft, 2011).

However, an effect in a transactivation test does not prove that the particular compound is an obesogen. Addi-tionally, the mouse preadipose cell line, 3T3-L1 cells can be used. This cell line can be induced to differentiate and accumulate fat. For several compounds like 4-nonylphenol, BPA, organotins, … lipid accumulation and adipocyte differentiation was indeed observed in these cells (Li et al., 2011). However, this does not necessarily mean that the changes in differentiation are due to PPARγ activation, since this can be induced by several pathways, for example these cells also contain ER and GR receptors (OECD draft, 2011).

Alternatively, recently the zebrafish obesogenic test was designed; adiposity is measured using a fluorescent probe that stains lipid droplets. Zebrafish is one of the most important models in environmental toxicology and is rapidly becoming a major model for studies on human health and disease. For both BPA and tributyltin an increase in adiposity was seen in this model (Tingaud-Sequeira et al., 2011).

Although all previous tests can also be used to test mixtures or extracts, they can only hint for possible obesogen-ity. Even though there is evidence that EDCs can play a role in obesity and some mechanisms are available, it still remains hard to predict the outcome for human health. Many EDCs can interact with different hormone receptors (for example BPA and phthalates, see Figure 1) and cross-talks between receptors exist. Experiments in mamma-lian models are therefore still needed preferably in combination with human biomonitoring.

Heidi Demaegdt, CODA-CERVA [email protected]

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Interesting lecture

• Casals-Casas, C. and B. Desvergne, Endocrine disruptors: from endocrine to metabolic disruption. Annu Rev Physiol, 2011. 73: p. 135-62.• Diamanti-Kandarakis, E., et al., Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr Rev, 2009. 30(4): p. 293-342.• Feige, J.N., et al., The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis. J Biol Chem, 2007. 282(26): p. 19152-66.• Grun, F., et al., Endocrine-disrupting organotin compounds are potent inducers of adipogenesis in vertebrates. Mol Endocrinol, 2006. 20(9): p. 2141-55.• Grun, F. and B. Blumberg, Minireview: the case for obesogens. Mol Endocrinol, 2009. 23(8): p. 1127-34.• Hatch, E.E., Nelson, J. W., Stahlhut, R. W., Webster, T. F., Association of endocrine disruptors and obesity: perspectives from epidemiological studies. Int J Androl, 2010. 33(2): p. 324-32.• Li, X., J. Ycaza, and B. Blumberg, The environmental obesogen tributyltin chloride acts via peroxisome proliferator activated receptor gamma to induce adipogenesis in murine 3T3-L1 preadipocytes. J Steroid Biochem Mol Biol, 2011.• OECD document, Draft Detailed Review Paper: State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors. 2011.• Sargis, R.M., Johnson, D. N., Choudhury, R. A., Brady, M. J., Environmental endocrine disruptors promote adipogenesis in the 3T3-L1 cell line through glucocorticoid receptor activation. Obesity (Silver Spring), 2010. 18(7): p. 1283-8.• Tingaud-Sequeira, A., N. Ouadah, and P.J. Babin, Zebrafish obesogenic test: a tool for screening molecules that target adiposity. J Lipid Res, 2011. 52(9): p. 1765-72.• Zoeller, T.R., Environmental chemicals targeting thyroid. Hormones (Athens), 2010. 9(1): p. 28-40.

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Validation of the detection of E. coli O104:H4 in vegetables.

N. Botteldoorn, A. Duarte and S. Denayer (WIV-ISP, Brussels)

Shigatoxin producing Escherichia coli (E. coli) O104:H4 in sprouts was at the origin of the outbreak occurred in Ger-many and some other European Union countries’, May-June 2011. It caused a high number of infections (3774), 750 HUS (haemolytic uremic syndrome) an renal failure cases and was responsible for a considered number of deaths (44). The strain responsible for the outbreak, harbours besides the O104 antigen-associated gene wzx

O104

and the gene encoding the H4 flagellar antigen, fliCH4 several unusual properties. Firstly, its DNA content exists for 80% of the DNA of an enteroaggregative E. coli, which is likely more associated in human-to-human transmission and 20% of the genome consistent of a VTEC strain (Mellmann et al., 2011). The stain was positive for the shiga-toxin 2a gene (vtx2a) but negative for the LEE locus (intimine gene) and the enterohemolysin (ehxA). The E.coli strain was also multi-resistant to different antibiotics and in addition the strain was resistant to third generation cephalosporins, like ceftazidim (Taz), and expressed the ESBL phenotype.

From an initial epidemiological case-control study conducted in Hamburg, it was suggested that an association occurred between the disease and the consumption of raw tomatoes, cucumber and lettuce. Later, it became clear that contaminated seeds of sprouts were at the origin of the outbreak. At the time of the outbreak no methodology to detect this outbreak strain in vegetables existed and efforts from different laboratories were made to develop one. The Belgian National Reference Laboratory located at the Sci-entific Institute of Public Health (ISP/WIV), developed 2 methods. Both methods consisted of an enrichment step followed by isolation on Tryptone Bile X-glucuronic TBX medium after 6h and 24h of incubation and identification and confirmation of the typical blue colonies by real-time PCR (Bugarel et al, 2010 and Perelle et al., 2004). Both methods differ apart in the stage were this selective agent – Taz, was added: in the TBX for method 1 and in the enrichment broth for method 2. Detection of E. coli O104:H4 was also done directly on the enrichment broth by real-time PCR after 6h and 24h of incubation (Bugarel et al, 2010 and Perelle et al., 2004).

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Figure 1: Plating out of the enrichment broth on TBX after 6 h incubation at 37°C (cucumber) by using method 1 and 2 the blue colonies are E. coli O104:H4 the (outbreak strain)

Validation was performed on different kind of vegetables including tomatoes, lettuce, cucumber and sprouts. It became clear, from the validation, that for bacteriological detection, method 1 with enrichment in buffered peptone water followed by plating out on TBX supplemented with selective compound was the most sensitive. In this method, the background flora was more inhibited. Although for direct detection, with real time PCR on the enrichment broth added of selective supplement, the method 2 was better. During the validation, the use of an intern amplification control (IAC) in the real time PCR reaction was optimized. It became clear that inhibition occurred for vegetables: a 1/100 dilution was necessary to obtain a positive PCR signal for red lettuce. For 2011, no positive samples of E. coli O104:H4 were observed and for 2012 more than 3500 analyses have been planned by the FASFC.

plating out of the enrichment broth on TBX after 6 h incubation at 37°C (cucumber) by using method 1 and 2 the blue colonies are E. coli O104:H4 the (outbreak strain)

METHOD 2 METHOD 1

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Figure 2: The dissociation curve after real time PCR detection of E. coli O140:H4 in cucumber (24h of incubation by using method 2) the green curve is the typical melting curve after amplification of the wzw

O104 gene for the detection

of E. coli O104:H4; the red curve is the typical melting curve of the internal amplification control.

[email protected]

Figure 2: The dissociation curve after real time PCR detection of E. coli O140:H4 in cucumber (24h of incubation by using method 2)

The green curve is the typical melting curve after amplification of the wzwO104 gene for the detection of E. coli O104:H4.

The red curve is the typical melting curve of the internal amplification control.

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Emerging contaminants

Introduction

Humans are exposed to a cocktail of chemicals, of which several are persistent. Heavy metals, dioxins, polychloro-biphenyls (PCBs), poly aromatic hydrocarbons (PAHs) and pesticides have been studied for a long time, therefore possible sources and toxic effects are relatively well known. However, a large group of emerging contaminants exist, of which little is known about their possible exposure routes and possible toxic effects. Examples include plasticizers, flame retardants, photo initiators, preservatives, chemicals used in cosmetics and packaging, drug resi-dues and nanoparticles. In some cases the metabolites are more toxic than the original compound. Not all risks are of anthropogenic origin, mycotoxins, phytotoxins, phycotoxins and bacterial toxins are examples of biotoxins whose effects are not fully documented.

Emerging contaminants

A wide variety of contaminants are considered as ‘emerging’:• Personal healthcare products: triclosan, parabenes• Synthetic musks: used as fragrant in perfumes and in healthcare products (e.g. musk xylene, musk keton)• Chlorinated paraffins: flame retardants, plastic additives, caulk• Pesticides and herbicides• Bisphenol A: additives in plastic (packaging)• Phtalates: plasticizers (e.g. octylphenol, nonylphenol)• Nanoparticles: structures < 100 nm, used in food, medicine, construction and textile • Phytoestrogens: isoflavones, coumestanes• Brominated compounds: polybrominated diphenylethers (PBDEs) as flame retardants, insulation and plastics• Perfluorinated hydrocarbons: perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA)• Non halogenated compounds: carboxylic acid, formaldehyde • Pharmaceutics: painkillers, hormones, antibiotics, antidepressants

Most manmade chemicals can be found in areas with human activity and in numerous cases the source of con-tamination can be found. PFOS and PFOA have been found in the vicinity of fluoropolymer producing factories. The heat resistant and water and fat repellant properties of fluoropolymers are used in anti-stick cookware.

There are literally hundreds of sources of these emerging contaminants coming from consumer products that end up in the environment. Frequently used products like shampoo, sunscreen, pesticides, plastics, flame retardants and pharmaceuticals are possible exposure routes of these emerging contaminants besides industrial processes.

Household products do not only pose a problem through direct exposure via their intended use, but can also cause long term problems. Sludge from sewage treatment plants is sometimes used in agriculture and contami-nants seep from landfills and end up in the environment. The use of several compounds has been banned by the European Commission, but some of these are still widely used in rising economies like China and India. An impor-tant part of the production of these countries is exported to Europe and the U.S., so potentially harmful products are still available on the European market. In July 2007 pentachlorophenol was found in guar gum originating from India, which was destined for human consumption. Despite a worldwide ban against DDT, it is still used in India as a pesticide in agriculture.

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Government actions prove successful against point contamination from local industrial sources, but a diffuse pol-lution remains. The presence of these substances in the polar regions demonstrates that long distance transport is possible.

Toxicological effects

Some of these chemicals are endocrine disruptors and known carcinogens, others apparently have little impact after brief exposure. Unfortunately only a few studies exist where the effects of prolonged exposure have been studied. The greatest danger lies in the fact that exposure to several substances elicits effects that are not only additive but can also be synergistic. The impact of the combined effects of these substances may be even greater than the expected effect of each substance separately.

Substances such as triclosan and triclocarban which are used in personal care products such as soap, deodorant, toothpaste and mouthwash are endocrine disruptors. Genistein, a phytoestrogen occurring in soy, disrupts the endocrine system and is teratogenic. PBDEs from flame retardants also have an impact on the endocrine system, PBDEs in breast milk are associated with lower birth weight and cryptorchidism in newborn boys. Elevated levels of PBDEs have been correlated to low IQ. Nitromusks and polycyclic musks are synthetic fragrances that are widely used in cosmetics, detergents, washing powder, shampoo, soap and air fresheners. Musk xylene and musk ketone are endocrine disruptors and enhance genotoxicity of other substances such as aflatoxin B1 and some PAHs, and are potential carcinogens. Bisphenol A and phthalates, such as octyl-and nonylphenol have an anti-androgenic effect and can cause damage to the testis, they also disrupt the function of the thyroid gland and affect the sexual development of the fetus. Other emerging contaminants such as chlorinated paraffins cause inflammation and cancer. The effects of some substances such as nanoparticles and the new alternative flame retardants are not known, as for these relatively new contaminants little or no data is available.

Most of the research into the effects of contaminants is based on toxicity studies of a single molecule. When a substance elicits an effect, it is often the case that the investigated concentration is considerably higher than the concentration of a ‘natural’ exposure. Often no effect can be observed when investigating the effects of certain substances, by exposure to concentrations similar to those in the environment. Some are even believed not to be harmful. The general consensus is that drugs in the environment have no adverse effects given the very low con-centrations, especially when compared with therapeutic doses. In reality it appears that nothing is known about the impact of prolonged exposure to low concentrations of drugs, let alone to mixtures thereof.

Research suggests that mixtures of these emerging contaminants as occurring in the environment, may have a greater impact than can be expected from the cumulative effects of individual substances. This appears to be more pronounced with substances that affect the same biochemical process, but influence different points in this process. Virtually all studies on these compounds focus on the molecules in their original form. The degradation products or metabolites are rarely investigated and in most cases they are not even known. Metabolites of PAHs, e.g. dihydrodiol, quinone, diol epoxide, are more toxic than the parent molecule. Exposure to these substances oc-curs not only through the diet, but also by inhalation and in some cases via absorption through the skin.

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Legislation

Many of the substances listed here are not routinely monitored and often there is no legislation in place. Due to increasing knowledge from research and consumer awareness, some of these substances have recently been regulated. For example, the use of nonylphenol and nonylphenol ethoxylates in detergents is prohibited (Direc-tive 76/769/EEC) and bisphenol A may no longer be used in the production of infant feeding bottles since March 2011. Since 2004 (Directive 2004/28/EC) a risk analysis for possible persistence in the environment and harmful effects is required for all medicinal products for veterinary purposes,.

These substances are not systematically tracked, but given their occurrence in our world and their possible nega-tive impact on human health and the environment, they certainly deserve more attention.

Huig Vanderperren, [email protected]

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Emerging threats: new GMO and UGMS. Broeders, S. De Keersmaecker, N. RoosensWIV-ISP, Unit Platform Biotechnology and Molecular Biology (PBB)

A genetically modified organism (GMO) is an organism of non-human origin in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination (Commission Directive 2001/18/EC), i.e. by inserting a transgenic construct into the genome which will change the charac-teristics of the organism. To date, mainly plants have been genetically modified to be brought onto the food and feed market. This commercialisation is strictly regulated in the European Union by Commission Regulations EC/1829/2003 and EC/1830/2003, laying down the need for traceability and a labelling threshold to ensure con-sumers’ freedom of choice in eating GMO. These regulations imply that the biotech companies producing these GMO need to introduce a dossier for authorisation which includes the genetically modified (GM) testing material and a detection/quantification method for the GM event. The recommended technique hereto is real-time PCR and the method should be event-specific. This implies that it should identify unequivocally the specific GMO by targeting the junction sequence between the transgenic insert and the plant genome.

The completeness of the dossier is evaluated by the European Food Safety Authority (EFSA) and a risk assessment is performed in collaboration with the EU member states, while the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) checks the GM testing material and the performance of the submitted real-time PCR method. When all set criteria are fulfilled, the EU-RL organises a ring trial to validate the real-time PCR method with the support of the National Reference Laboratories (NRL) for GMO detection. The final dossier is published onto the EU-RL website (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm) and needs to be implemented in the GMO detection laboratories of the EU member states. This assures that the different enforcement laboratories use standardised and harmonised methods for GMO detection and allows achieving comparable analysis results between the different GMO detection laboratories in the EU member states.

At the moment, 23 single GM events and 19 stacked events are authorised under regulation EC/1829/2003. Another 10 single events and 4 stacked events fall under the new ‘Low Level Presence’ (LLP) regulation (Commis-sion Regulation EU/619/2011). The majority of these events show insect resistance or herbicide tolerance and standardised methods for identification and quantification are available as submitted by the biotech companies. European reports, however, state that the number and diversity of GMO (including animal GMO) being com-mercialised worldwide will increase significantly in the next 5 years (from 25 to 120 events, from 2 to 15 types of genes) (Stein & Rodriguez-Cerezo, 2009). These will include new types of genes to create new traits such as pathogen resistance, altered crop composition and abiotic stress tolerance,… Additionally, new crops, as for example eggplant, melon, papaya,… are being genetically modified. Finally, the GMO in food and feed currently commercialised have been developed by American and European biotech companies. These developers have a major interest in requesting authorisation for the introduction of their GM event on the European market as their products are meant for exportation. However, in 2015 more than half of the GMO will be developed in Asia by research institutions. As these GMO are only intended for local consumption (Stein & Rodriguez-Cerezo, 2009) no application for EU authorisation will be filed. Consequently, no information will be delivered and thus no EU vali-dated and harmonised identification methods will be available for these unauthorised GMO (UGM). Due to their possible accidental presence in food and feed products imported in the EU from Asia, the amount of UGM on the European market might increase steadily. In addition, the presence of these substances in food and feed samples will not have been evaluated for their risk on human health and the environment, and thus might be considered as a possible emerging threat.

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The growing number of authorised GMO and UGM, possibly present in food and feed samples, will have an influ-ence on the way enforcement laboratories operate. Identifying all the present authorised GMO events one by one is already not rational as it is extremely expensive and time consuming. In the near future, the increased number of events will make this way of analysing simply impossible. Moreover, no event-specific methods are available for the UGM and so they are undetectable with such kind of approach. Therefore, the WIV-ISP GMOlab (coordina-tor of the Belgian NRL for GMO detection) puts a major effort in the development and validation of an extensive number of real-time PCR screening methods. These methods are targeting species-specific sequences (Mbongolo Mbella et al., 2011), generic markers (Barbau-Piednoir et al., 2010) and GM-specific elements (Barbau-Piednoir et al., 2011). The unique combination of these screening methods together with a patented matrix-based decision support system, called CoSYPS (Van den Bulcke et al., 2010) allows narrowing down the number of GM events to be further identified.

Currently, the GMOlab is using 17 screening markers simultaneously under ISO 17025 accreditation for routine analysis of food and feed samples. The CoSYPS approach has several advantages. Firstly, the combination of the 17 screening markers allows covering all events currently authorised in the EU as well as those under the LLP regulation. This permits to drastically reduce the number of identifications to be done. Secondly, it is a modular tool which implies that at any moment new methods can be added when new events (both plant and animal) become authorised. Thirdly, the matrix-based approach can be implemented for other purposes than GMO detec-tion (e.g. pathogenic bacteria). Last, but not least, this system permits to screen also for UGM by using the present markers and including new ones. The pioneering work performed by the GMOlab in this field has recently been recognised at EU level especially in the frame of the lately adopted law on Chinese rice (Commission Decision 2011/884/EU). This decision states that a control for GMO needs to be carried out on rice and rice-containing sam-ples from China and hereto the analytical screening methods developed at WIV-ISP have been proposed.

Figure 1: Products in GMO analysis

Real-time PCR amplification curves: detection of presence of GM element

Real-time PCR dissociation curves: confirmation of specific amplicon

Products in GMO analysis Real-time PCR amplification curves: detection of presence of GM element Real-time PCR amplificatie curves: detectie van de aanwezigheid van een GM element Courbes d’amplification de la PCR en temps réel : détection d’un élément GM Real-time PCR dissociation curves: confirmation of specific amplicon

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The upcoming threat of the increasing amount of GMO and UGM in the European food and feed chain will thus intensify the tasks of GMO detection laboratories. Not only will they need to implement more identification methods (if available) but they will also have to focus on the use of an intensive screening procedure. Hereto, new screening markers need to be developed to continue covering all authorised GMO but also to more intensively screen for UGM. The project UGMMONITOR (financed by the FOD/SPF) will permit to develop new screening markers to target plant as well as animal UGM and to update the CoSYPS consequently.

At the moment, real-time PCR is the method of choice for the detection of GMO. The GMOlab, however, is also actively involved in the research of alternative analytical approaches to detect and identify GMO and UGM. The establishment of the BIOTECHlab (belonging to the same unit as the GMOlab) facilitates investigating the use of various molecular technologies for the analysis of genetic modifications. The in-house sequencing platform of the BIOTECHlab allows determining the nucleotide sequence amplified in between the primers used for screening. This additional source of data may be particularly useful to verify or determine the sequence of the GMO in case of ambiguous results. Other technologies, like DNA walking will be developed to determine unknown sequences reading from the anchored screening primers (UGMMONITOR project). Additional screening tools such as the multiplexing xMAP technology (commonly known as Luminex) enabling the simultaneous (up till 500) detection of different GMO (both authorised and unauthorised in the EU) will be explored.

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Figure 2a: Real-time PCR amplification curves: detection of presence of GM element

Figure 2b: Real-time PCR dissociation curves: confirmation of specific amplicon

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References

Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. Official Jour-nal of the European Union, L 106, p 1-38

Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on geneti-cally modified food and feed. Official Journal of the European Union, L 268, p 1-23

Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products pro-duced from genetically modified organisms and amending Directive 2001/18/EC. Official Journal of the European Union, L 268, p 24-28

Commission Regulation (EU) No 619/2011 of 24 June 2011 laying down the methods of sampling and analysis for the official control of feed as regards presence of genetically modified material for which an authorisation proce-dure is pending or the authorisation of which has expired. Official Journal of the European Union, L 166, p 9-15

Stein, A. & Rodriguez-Cerezo, E. (2009). The global pipeline of new GM crops. Implications of asynchroneous ap-proval for international trade. EU23846-EN.

Mbongolo Mbella, E. G., Lievens, A., Barbau-Piednoir, E., Sneyers, M., Leunda-Casi, A., Roosens, N. & Van den Bulcke, M. (2011). SYBR®Geen qPCR methods for detection of endogenous reference genes in commodity crops: a step ahead in combinatory screening of genetically modified crops in food and feed products. European Food Research

Technology, Vol. 232, pp. 485-196

Barbau-Piednoir, E., Lievens, A., Mbongolo-Mbella, G., Roosens, N., Sneyers, M., Leunda-Casi, A. & Van den Bulcke, M. (2010). SYBR®Green qPCR screening methods for the presence of “35S promoter” and “NOS terminator” elements in food and feed products. European Food Research Technology, Vol. 230, No. 3, pp. 383-393

Barbau-Piednoir, E., Lievens, A., Vandermassen, E., Mbongolo-Mbella, G., Leunda-Casi, A., Roosens, N., Sneyers, M. & Van den Bulcke, M. (2011). Four new SYBR®Green qPCR screening methods for the detection of Roundup Ready®, LibertyLink®, and CryIAb traits in genetically modified products. European Food Research Technology. Vol. 234, No. 1, pp. 13-23

Van den Bulcke, M., Lievens, A., Barbau-Piednoir, E., Mbongolo Mbella, G., Roosens, N., Sneyers, M. & Leunda Casi, A. (2010). A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants. Analytical Bioanalytical Chemistry, Vol. 396, No. 6, pp. 2113-2123

2011/884/EU: Commission Implementing Decision of 22 December 2011 on emergency measures regarding unauthorised genetically modified rice in rice products originating from China and repealing Decision 2008/289/EC. Official Journal of the European Union, L 343, p 140-148

[email protected] [email protected] [email protected]

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Tick-Borne Encephalitis Virus Seropositive Dog Detected in Belgium: Multi-species Screening of Sentinels for Public HealthSophie Roelandt1,2, Paul Heyman3, Marina De Filette4,5, Sirkka Vene6, Yves Van der Stede1,7, Ann Brigitte Caij8, Paul Tavernier9, Alexandre Dobly2, Hendrik De Bosschere10, Philip Vyt11, Carole Meersschaert12, and Stefan Roels2

1Unit for Coordination of Veterinary Diagnosis, Epidemiology and Risk Assessment (CVD-ERA), Operational Directo-rate of Interactions and Surveillance, Veterinary and Agrochemical Research Centre (VAR-CODA-CERVA), Brussels, Belgium ; 2Unit of Pathology & Prionology, Operational Directorate of Interactions and Surveillance, Veterinary and Agrochemical Research Centre, (VAR-CODA-CERVA), Brussels, Belgium ; 3Research and Reference Laboratory for Vector-Borne Diseases, Queen Astrid Military Hospital, Brussels, Belgium ; 4Department for Molecular Biomedi-cal Research, Flemish Institute for Biotechnology (VIB), Ghent, Belgium ; 5Laboratory of Gene Therapy, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, Belgium ; 6Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden ; 7Department of Immunology, Faculty of Veterinary Medicine, Univer-sity of Ghent, Merelbeke, Belgium ; 8Unit of Enzootic and (Re)emerging Viral Diseases (ENZOREM), Operational Directorate of Viral Diseases, Veterinary and Agrochemical, Research Centre (VAR-CODA-CERVA), Brussels, Belgium ; 9Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, Belgium ; 10Medisch Labo Bruyland, Kortrijk, Belgium ; 11Mediclab, Ghent, Belgium ; 12Laboratoire Dr. Jean Collard, Liège, Belgium.

Introduction (Roelandt et al., 2010)

Tick-borne encephalitis virus (TBEV) is an important emerging tick-borne viral infection of humans and dogs in Eu-rope. The Western TBEV subtype is transmitted by Ixodes ricinus ticks and occasionally by unpasteurized milk from domestic ruminants. Western TBEV is currently the most important arthropod-borne viral infection in humans in Europe, causing tick-borne encephalitis (TBE), a biphasic disease with fever and multifocal neurological signs, including meningo-encephalitis. All neurological patients need intensive care hospitalization and many develop permanent sequelae called ‘post-encephalitic syndrome’. TBE is an increasing public health risk in several Euro-pean countries and despite low mortality rates it results in a very high risk to society and health care. TBE is also emerging among Europe’s canine population and its distribution is steadily expanding over Western Europe in parallel with human TBE. Consequently, a higher number of canine TBE cases are likely to be diagnosed asawareness increases in the veterinary community. Although in 50% of dogs seroconversion occurs without any clinical signs, TBEV can cause pyrexia, lethargy, loss of appetite, and multifocal neurological signs. Most dogs develop a strong IgG immune response, which is detectable in cerebrospinal fluid and serum.

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The best diagnostic tests for TBEV are indirect IgG enzyme-linked immunosorbent assay (ELISA), hemagglutina-tion inhibition (HI), seroneutralization assay (SN), rapid fluorescent focus inhibition test (RFFIT), or immunoblotting tests. SN and RFFIT tests are considered to be highly specific confirmation/reference tests, whereas ELISA is more prone to cross-reactions and false positives. In Belgium, TBE is still considered an exotic disease and medical and veterinary TBEV surveillance is currently virtually nonexistent. Therefore, CODA-CERVA conducted a first serological screening of Belgian dogs for TBEV.

Canine Study (Roelandt et al., 2011)

A commercial ELISA test (Immunozym FSME/TBE IgG All Species-ELISA®, Progen Biotechnik, GmbH, Heidelberg, Germany) was adapted for the detection of TBEV-specific IgG antibodies in canine sera. Serum samples of Belgian dogs (Fig.1) were obtained from three diagnostic laboratories from Northern (n = 688) and Southern Belgium (n = 192) and were tested alongside positive and negative human and dog control samples. ELISA-positive and bor-derline samples were subjected to a TBEV RFFIT confirmation test, as well as SN and HI tests to rule out West Nile and Louping Ill viruses. One Flemish dog was confirmed TBEV seropositive by RFFIT.

The clinical history of the seropositive but asymptomatic dog included traveling close to (but not within) known endemic areas abroad, as well as tick exposure at home and insufficient tick prevention, but since these risk fac-tors did not seem to occur simultaneously in space and time and since the travelling occurred many years before the TBEV serological testing, this could not explain with certainty where and when TBEV infection was acquired. It was concluded that further surveillance would be necessary to determine whether this dog remains a single travel-related case or whether it represents an early warning of a possible future emergence of TBEV in Belgium.

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Figure1: Belgian canine samples (n = 880) were used in this study. Three colored pins represent the three partici-pating laboratories. One dot represents one dog. Purple triangles represent enzyme-linked immunosorbent assay (ELISA)-positive and borderline samples.

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Screening animal sentinels for TBEV (Roelandt et al., 2010)

It is known that the TBEV tick vector, I. ricinus, has more than 300 known natural hosts, including small and large mammals, birds and reptiles. Most of these hosts are subclinically infected with TBEV and become immune for life. Additionally, pets as well as farm animals and wildlife can be used as sentinels for TBEV detection, offering a practi-cal index for public health surveillance and risk assessment. Larger mammals such as wild boar, roe deer, dogs and domestic ruminants (cattle, sheep, and goats) have already proven to be good TBEV sentinels in European TBEV surveillance, since the prevalence in these hosts can be much higher than in the local tick or human populations.

Taking into account these epidemiological features and the findings from the canine study, it would be prudent to further validate and standardize an ELISA screening test for estimating prevalence of infection or exposure to TBEV in several species. Such a diagnostic test would be needed to enable a data-based risk analysis for TBE in Bel-gium. Continued serological screening of TBEV in dogs and other domestic and wild sentinels is advisable to gain more insight into the current situation and could contribute in a cost-effective way to a continuous public health epidemiosurveillance program for TBE(V). Finally, since a recent analysis showed that in Europe between 30% and 80% of human viral meningo-encephalitis cases remain unexplained, such a program should also include targeted clinical and risk-based surveillance in the human population.

Currently, a targeted multi-species screening and ELISA validation study is being conducted by a Belgian consor-tium. Serum samples of humans, as well as from domestic and wild species will be tested in ELISA and SN, and hereby species-specific seroprevalences as well as ELISA test accuracy will be evaluated.

Acknowledgments

The canine TBE study was funded by a grant from the Belgian Federal Government, Federal Public Service Depart-ment of Public Health, Safety of the Food Chain and Environment, as a part of the Wildsurv Project (contract RT 07/5). The multi-species study became possible through multidisciplinary cooperation of the consortium partners.

References

Roelandt S, Heyman P, Tavernier P, Roels S., 2010. Tick-borne encephalitis in Europe: review of an emerging zoono-sis. Vlaams Diergeneeskundig Tijdschrift 2010 (1), 23:31

Roelandt S, Heyman P, De Filette M, Vene S, Van der Stede Y, Caij AB, Tavernier P, Dobly A, De Bosschere H, Vyt P, Meersschaert C, Roels S, 2011. Tick-borne encephalitis virus seropositive dog detected in Belgium: Screening of the canine population as sentinels for public health. Vector-borne zoonotic diseases 2011; 11(10), 1371:1376. DOI: 10.1089/vbz.2011.0647. Epub. 2011 Sep 15. PMID: 21919722.

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Schmallenberg virus in BelgiumBrigitte Caij, Nick De Regge, Thierry van den BergCODA-CERVA (Veterinary and Agrochemical Research Centre)

On 18 November 2011 the Friedrich Loeffler Instituut (FLI) in Germany announced the discovery of a new Orthobunyavirus, the so-called « Schmallenberg virus », named after the German town where the virus was first identified, i.e. Schmallenberg, a picturesque health resort in the Sauerland region.

Orthobunyaviruses belong to the arbovirus group, a group of viruses that reproduce in blood-sucking vectors (mosquitoes, midges, ticks) before being transmitted to other hosts (birds, animals, humans) by these vectors. Arboviruses have a worldwide distribution and may cause considerable economic losses in livestock industry.

The emergence of an orthobunyavirus in our region was new. Until late last year they were found mainly in ruminants in Africa, Asia and Australia. They are transmitted by biting insects such as midges (Culicoides) and mosquitoes. As a rule, these viruses cause no or only mild clinical symptoms in adult animals but an infection of pregnant animals by lead to abortion or premature birth of offspring with malformations.

Foto © ITG

Photo 1 : A midget (the smaller of the two insects) and a mosquito

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The Akabane virus is the best known and best characterized virus of these orthobunyaviruses. It was first isolated in cattle with reproductive symptoms in Japan in the 1980s. The first genetic analyses showed that the Schmallen-berg virus is related to the Akabane virus although both viruses are not completely identical.

The Schmallenberg virus was identified in November 2011 after certain problems had been reported in August and September in dairy holdings in our neighbouring countries (Germany and The Netherlands). These problems included milk drop, watery diarrhea and fever and could not be accounted for by the presence of already known endemic and exotic viruses. The identification of the novel virus and the development of a detection test allowed to establish the presence of this novel virus in serum samples taken during the period of disease and thus confir-med the presence of the virus in these countries. In Belgium, the development of a diagnostic test for this virus made it possible to establish that the virus was already present in September in serum samples taken in two dairy holdings that encountered problems such as fever and a reduction in milk yield.

In addition to the clinical symptoms (milk drop, fever and diarrhea) found in adult animals in the summer, a se-cond series of clinical symptoms due to the Schmallenberg virus appeared late 2011. These symptoms were found in lambs and calves and are due to an infection during pregnancy that affects both the mother and the fetus. When pregnant animals contract Schallenberg they give birth to lambs and calves that are affected by congenital malformations of the limbs, the neck and the brain.

Photos 2 and 3 : deformed calves

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Several orthobunyaviruses being transmitted by mosquitoes and midges, it was expected that the Schmallenberg virus would also be transmitted by these vectors. Recently, the CODA-CERVA was able to establish the presence of the virus in the heads of midges caught early in the autumn of 2011 in Belgium, thus confirming this hypothesis. The activity of midges depends on the ambient temperature which reaches its lowest level in winter and gradu-ally increases as from early spring. We anxiously await the next vector season to find out if midges that carried the virus have survived winter and have transmitted the virus to the next generation through their eggs. We must also wait to find out if the significant spread of the virus in 2011 has conferred sufficient immunological protection to large numbers of sheep and cattle to limit the consequences of the incursion in the coming season.

Brigitte [email protected]

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EMERGING MARINE BIOTOXINS – report from the seminar 2012Mirjana Andjelkovic

Marine biotoxins (also called phycotoxins) are produced by certain phytoplankton species (diatoms and dino-flagellates) and can accumulate in various marine species such as fish, crabs or filter feeding bivalves (shellfish) such as mussels, oysters, scallops and clams. In shellfish, toxins mainly accumulate in the digestive glands with-out causing adverse effects on the shellfish itself. However, when substantial amounts of contaminated shellfish are consumed by humans this may cause severe intoxication. Approximately 60,000 human intoxications yearly with overall mortality of approximately 1.5% are related to toxins produced by algae (including freshwater cyano toxins) (1). This is both public health and economic problem. The necessary regular monitoring of specifically susceptible sites has a serious economic impact, moreover if the site gets closed during harvest, collection of that year as well as that of the following one can be hindered. In the case that toxic products make it to market result-ing in fatalities or illness, consumers need to be compensated.

Currently five groups of marine toxins are regulated in Europe: amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, okadaic acid and pectenotoxin group, azaspiracid group, and yessotoxin group (EC/853/2004). Another group, neurotic shellfish poisoning toxins (e.g. brevetoxin) is regulated in US, New Zealand and Australia. These toxins are regularly produced in the Gulf of Mexico, particularly in coastal regions of south-west Florida.

Regulated toxins have been for years detected by bioassays. Recently effort is made to accept HPLC and more specifically LCMS based detection methods as reference methods due to their increased international use and sensitivity. Their implementation in both, research and routine monitoring, led to detection of new marine toxins and toxin congeners. These are seen as emerging marine toxins. They are not on the radar of the European legisla-tion or in other regions of the world. Scientists and legislative authorities are aware of these toxins but do not have sufficient amount of information to:1. elucidate the activity of all unknown toxins;2. estimate the toxicity to humans;3. evaluate risk to humans.

The emerging group of marine biotoxins have been principally monitored in shellfish. Countries as France, Italy, Spain and Norway report mostly palytoxins (PlTX, ostreocin-D, ovatoxin-A, homopalytoxin, bishomopalytoxin, neopalytoxin, deopalytoxin and 42-hydroxypalytoxin) and cyclic imines [spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs)]. Toxin levels of some cyclic imines vary from 2–585 ng/g (2) to 11–7950 ng/g (3) in shellfish from France and Italy, respectively, whereas reported values for Spanish mussels were in the range of 13–20 ng/g (4). Putative palytoxin was for the first time detected in Italian waters at 1,350 ng for plankton pellet and 1,950 ng for butanol extract. It was therefore suggested that this toxin was the causative agent responsible for the Genoa 2005 outbreak of respiratory illness in people exposed to marine aerosols. There-fore some of these toxins are not only a concern of consumers but also may be seen as a public health problem related to environment. The cyclic imines group of toxins have been identified as nicotinic receptor blockers and are recognised as “fast acting toxins”. PlTX is one of the most toxic non-peptide substances known, showing remarkable biological activity even at very low concentration. This toxin and its analogs have become a global

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concern due to the poison’s effects on animals and especially on humans. On the other hand, there are more fre-quent reports on ciguatera fish poisoning including frequent fatalities. Ciguatera fish poisoning is caused by the consumption of coral reef fish contaminated by ciguatoxin and related toxins from dinoflagellates (microalgae) and cyanobacteria. This poisoning, which is a prevalent tropical and subtropical disease, has been long known but now increasing number of incidences makes it more worldwide problem. Likewise the increasing analytical pos-sibilities make it possible to respond the need to know and control it.

At the recent Seminar on emerging marine toxins in Vigo, Spain during two days the experts were discussing these groups of toxins. The European reference laboratory of marine toxins hosted the meeting at the new loca-tion which was also an official opening of the labs.

The lecturer session was opened with the talk of prof dr T. Yasumoto a leading figure in marine toxin research. His group in Tokio is currently collecting data and making a database of fish species related to ciguatera fish poison-ing in Pacific waters. They are extracting the toxins and working on the production of the reference material which would enable detection of the toxins. The material will be available even this year for a limited number of laboratories. The conclusions of prof Yasumoto are that ciguatoxins are region- and fish species specific but the in-traspecies variations were small. While some congeners like CTX1B type toxins are dominant in Okinawa (south of Japan), CTX3C type is more present Miyazaki (south-west part), and in fish from Marcus Island toxins of both types. Moreover the tendency of finding this toxins in northern part of Japan is rising. The lab is performing the chemical analysis (LC-MS/MS) of all the material with low sensitivity.

Seriousness of the ciguatera toxins in Canary Islands was further presented during round table sessions. Madeira Island (Portugal) was also pointed as a problematic spot for these toxins. Nevertheless, other countries also face the problem due to import or shipping of fish caught during sport fishing in Caribbean. Since 2004 there are some episodes of ciguatera toxins implicating big amberjack fish (Seriola sp) and affecting around 70 people. Several causative possibilities were listed. Pollution, climate change have been already brought in correlation with toxic algal blooms. Similarly, the Canary Islands authorities and scientists are relating the occurrence to climate change, inflow of balast water of incoming boats and ships, possibility of fish migration from Caribbean, or ap-pearance of an algae foreign to those waters (Gambierdiscus excentricus). Official control program of the region covers since 2010 these toxins and has introduced some new control strategies in fish. The samples are immedi-ately checked with immunoassay kit (Ciga-Check) and then referred to the lab for: mouse bioassay, cytotoxicity test and additionally confirmed by LC-MS/MS in the EURL-MB. The big disadvantage in the efforts is the detection method which is not sensitive enough. To improve its performance the standard should be developed and an in-terlaboratory validation study should be organised. Studies of these toxins were pointed out as being of common interest and topic for the future work in many European countries.

Following group of toxins were palytoxins on which prof. dr Aurelia Tubaro from University of Trieste gave some insights. She performs an extensive work on toxicity of palytoxins. Research with mice as well as cell cultures delivered the knowledge on the dose of these highly toxic compounds. It is noticed that already at 30 µg/kg dose functional impairment of skeletal and myocardial muscles can be seen. However prof Tubaro stressed that the knowledge on pharmacokinetics, repeated oral toxicity (basis for NOAEL) as well as assessment of cardiotoxicity of palytoxins but also ovatoxins is scarce. It is noteworthy to mention here the symptoms of the intoxication with those compounds. Experimental animals exerted the behaviour as: scratching, jumping, respiratory distress and paralysis; if the animal would recover it would usually lose weight.

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Palytoxins were mentioned several times during the seminar both due to their high toxicity, and allergic-like reaction that they may trigger in persons handling them in the lab or tourist and inhabitants around the area with algal bloom. There are hypotheses that some other components, like some volatile compounds released by microalgae (Ostreopsis) can be blamed for these reactions.

Northern European countries are also facing some toxins that were previously mainly recognised to appear in warmer waters. Norway is reporting since 2009 presence of spirolides produced by Alexandrium ostendfeldi and pinnatoxins produced by Vulcanodinium rugosum. The latter group of toxins is proven to have more congeners with two formed (PnTx F and G) in algae and the other metabolising out of this two via hydrolysis in shellfish. Pin-natoxins are found all over the coast of Norway in low levels but with only one “hot spot” where concentrations of the toxins may be higher. Dr Chris Miles (Norwegian Veterinary Institute) presented the actions and objectives that Norway has to overcome this problem. According to him a monitoring program for these toxins is in place already for some years. The shellfish and non-shellfish species are monitored (blue mussel, European oysters, scallops, cockles, horse mussel, sea snail, crabs, and sea worms) by SPATT discs. The authority is very interested in decod-ing the pattern of “hot spot” locations, to answer the question why are these toxins present all year around, and characterising the causative organism or whether a mixture of organisms could be responsible for this “subtropic occurrence” in northern waters.

Likewise, dr P. Hess (IFREMER) reported presence of pinnatoxins all year around in low concentrations (PnTx G around 100 mg/kg) in some specific areas of France. These are warm lagoons. The situation is monitored via Of-ficial Control Program and a scientific project in collaboration with an institute in New Zealand. Although there are no reports of human illness due to SPXs, GYMs, PnTXs or PtTXs episodes of toxicity, some cases involving non-specific symptoms such as gastric distress and tachycardia were recorded in individuals in Nova Scotia, Canada consuming shellfish during times when SPXs were known to be present. However these could not be definitively ascribed to SPXs since they were not consistent with the signs of toxicity shown in mice. These toxins are seen as very toxic with LD50 of 10 µg/g.

The cyclic imines may be analysed by bio-analytical and physicochemical methods to describe the active mecha-nism as explained by dr R.Araoz (National Research Centre, France). They act on nicotininc acetylcholine receptors which can be present in the brain. Decline, disruption or alterations of nicotininc cholinergic mechanisms con-tribute to dysfunctions such as epilepsy, schizophrenia, Parkinson’s disease, autism, dementia, Alzheimer’s disease and addiction. The neurotoxic doses of PnTx and SPX is comparable to those of paralytic shellfish poisoning. Since these toxins are not regulated and less known all information is necessary and functional methods would provide many answers. However to overcome lack of sensitivity of e.g. receptor binding assay these test should be coupled to MS.

The management of the emerging toxins may be explained as contemporary approach (dr J.Diogene, IRTA). The classical approach on toxins is based on the toxic episodes and experience that scientist got while the contem-porary approach lies on the understanding the source of toxins, vectors of intoxications, structure and risk assess-ment of new occurrences. Therefore we make difference between known and “classical” toxins that are regulated and those which are non-regulated; between local and foreign and those with identified source and unknown source.

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It can be concluded that there is no list of emerging toxins partially due to interpretation of emergent in the local context. However a lot was said about: palytoxins, ovatoxins, brevetoxins, cyclic imines (gymnodimins, pinnatox-ins, pteriatoxins and spirolides), azaspiracids and ciguatera toxins. What is necessary is the systematic identifica-tion of toxin vectors and the toxins. The evolution from the classical approach to the contemporary is enabled by technical advances and awareness when conducting monitoring program.

Finally, at the end of symposium and a meeting of a special working group formed on emerging toxins, partici-pants came to a conclusion that ciguatera toxins may be a topic of common efforts for validating a detection method in an international validation study. This work would include work on reference materials and their easier availability.

Mirjana Andjelkovic, WIV-ISP Brussels [email protected]

References

1. Lina Kantiani & Marta Llorca & Josep Sanchís & Marinella Farré & Damià Barceló. Emerging food contaminants: a review; Anal Bioanal Chem (2010). 398:2413–2427)2. Amzil Z, Sibat M, Royer F, Masson N, Abadie E. Report on the First Detection of Pectenotoxin-2, Spirolide-A and Their Derivatives in French Shellfish (2007) Marine Drugs 5:168–1793. Ciminiello P, Dell’Aversano C, Fattorusso E, Forino M, Tartaglione L, Boschetti L, Rubini S, Cangini M, Pigozzi S, Poletti R. Complex toxin profile of Mytilus galloprovincialis from the Adriatic sea revealed by LC–MS (2010) Toxicon 55:280–2884. González AV, Rodríguez-Velasco ML, Ben-Gigirey B, Botana LM. First evidence of spirolides in Spanish shellfish (2006) Toxicon 48:1068–1074

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The trainings for the approved laboratories organized by the FASFC in co-operation with the National Reference Laboratories are available on the website of the FASFC

(www.favv.be > Business Sectors > Laboratories > Trainings).

The schedule is updated regularly, it is therefore recommended to check the website from time to time.

Other interesting workshops and symposia are mentioned below.

Date Subject Place More information (website)

12-17.08.2012 58th ICoMSTInternational Congress of Meat Science and Tech-nology

Montréal, Canada http://www.biocircle-project.eu/media/6894/icomst_2012_bro-chure_belgique.pdf

26-31.08.2012 32nd International Symposium on Haloge-nated Persistent Organic Pollutants (POP’s) - DIOXIN 2012

Cairns, Australia http://www.dioxin2012.org/

11-13.09.2012 Fourth international con-ference on Feed Safety – Methods and Challenges

Beijing, P.R. China http://www.feedsafety.org/

17-18.09.2012 3rd International Fresenius ConferenceEndocrine Disruptors

Mainz, Germany http://www.akademie-fresenius.com/english/konferenz/output.php?thema=3&kurs=335

20-21.09.2012 17th Conference on Food Microbiology

Brussels, Belgium www.bsfm.be

30.09-3.10.2012 126th AOAC Annual Mee-ting & Exposition

Las Vegas, USA http://www.aoac.org

29-30.10.2012 5th International Frese-nius Conference“Food Allergens”

Mainz, Germany http://www.akademie-fresenius.com/english/konferenz/output.php?thema=3&kurs=294

4-8.11.2012 IDF World Dairy Summit Cape Town, South Africa

www.wds2012.com

Workshops & Symposia

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5-9.11.2012 7th Conference of The World Mycotoxin Forum® and XIIIth IUPAC Inter-national Symposium on Mycotoxins and Phycot-oxins

Rotterdam, the Netherlands

http://www.wmfmeetsiupac.org/html/welcome.aspThis unique combined event, WMF meets IUPAC, will build on the success of the previous conferences which were held separately all over the world.The aim of WMF meets IUPAC is to increase awareness of human and animal health risks due to natural toxicant contamination in agricultural commodities and seafood, and of potential risk management options, technologies and strategies for minimized contamination. The event will focus in particular on mycotoxins, phycotoxins and plant toxins.

14-16.11.2012 Chemical Reactions in Foods VII

Prague, Czech Replublic

http://www.crf2012.eu/

28.01-1.02.2013 Advanced Food Analysis Wageningen, The Netherlands

http://www.rafa2011.eu/pdf/Advanced_food_analysis_210911.pdf

7-10.05.2013 EuroFoodChem XVII Istanbul, Turkey http://www.arber.com.tr/eurofoodchemxvii.org/index.php/homewith state-of-the-art knowledge and applications in food chemis-try and complementary disciplines

25.08-28.08.2013 127th AOAC Annual Mee-ting & Exposition

Chicago, USA http://www.aoac.org

25-29.08.2013 24th International Conference of the World Association for the Ad-vancement of Veterinary Parasitology (WAAVP)

Perth Exhibition Centre, Perth, Western Australia

http://www.waavp.org/

28.10-1.11.2013 IDF World Dairy Summit Yokohama, Japan http://www.fil-idf.org/Public/SiteEventType.php?ID=23123

semi-annuel newsletter - n°8 july 2012 labinfo

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