universidade federal do paranÁ vivien midori

UNIVERSIDADE FEDERAL DO PARANÁ

VIVIEN MIDORI MORIKAWA

OCCURRENCE OF ZOONOSES AND INFECTIOUS DISEASES IN BARBARY

SHEEP (Ammotragus lervia) FROM THE CURITIBA ZOO

CURITIBA

2014

VIVIEN MIDORI MORIKAWA

OCCURRENCE OF ZOONOSES AND INFECTIOUS DISEASES IN BARBARY

SHEEP (Ammotragus lervia) FROM THE CURITIBA ZOO

Tese apresentada ao Programa de Pós-Graduação em

Ciências Veterinárias, Área de Concentração em

Sanidade Animal e Medicina Veterinária Preventiva,

Setor de Ciências Agrárias, Universidade Federal do

Paraná, como requisito parcial à obtenção do título de

Doutor em Ciências Veterinárias.

Orientador: Prof. Dr. Ivan Roque de Barros Filho

CURITIBA

2014

Aos meus queridos pais, Ida e Maçacatu,

por todo amor e apoio incondicional;

Ao Armando,

por me ajudar a ser uma pessoa melhor a cada dia.

Dedico.

AGRADECIMENTOS

Ao meu orientador, Prof. Dr. Ivan Roque de Barros Filho, por ter me aceito

como sua orientada e proporcionado a realização desta pesquisa.

Ao Prof. Dr. Alexander Welker Biondo, por toda orientação, apoio, incentivo e

amizade.

Ao Prof. Dr. Fabiano Montiani-Ferreira, pela orientação, ajuda e

encaminhamentos junto à Pós-Graduação.

Ao Prof. Dr. Rogério Ribas Lange por participar das coletas, sempre nos

auxiliando com seu vasto conhecimento na área.

Ao anestesista e amigo Rogério Robes por estar sempre à disposição e

gentilmente participar de todos os procedimentos.

À residente e amiga Cristina Kraemer Zimpel, pelo companheirismo e ajuda,

tanto nas coletas como na parte escrita.

À secretaria e amiga Maria José Maeda por todo auxílio com os documentos

da Pós-Graduação.

À toda equipe do Zoológico de Curitiba, em especial à Oneida, Marcelo,

Manoel, Carlos, Nancy e Tamandaré por toda ajuda durante a execução do estudo.

AGRADECIMENTO ESPECIAL à bióloga e amiga Dra. Tereza Cristina

Castellano Margarido, exemplo de profissional, por todo o incentivo, apoio e por me

ensinar tantas coisas.

Ao Instituto Biológico de São Paulo, em especial às pesquisadoras Maria do

Carmo Lara, Eliana Villalobos, Maristela Cardoso, Eliana Scarcelli, Alessandra

Nassar, Cristina Dib, Vanessa Castro, Lilia Paulin, Adriana Nogueira e Liria Okuda

por gentilmente terem me recebido no Instituto e proporcionado a realização dos testes.

À Luciana Gequelin, pelo auxílio com os testes diagnósticos.

Aos colegas Igor Paploski e Mariana Kikuti, por prontamente terem me

auxiliado com as análises estatísticas e a redação dos artigos.

Aos amigos Claudio Fontes e Pedro Sanches por toda ajuda durante o estudo e

apoio de sempre.

Às queridas amigas Dayana, Gilda, Sueli, Dirciane, Maysa e Haiuly, pelo

ombro amigo, convívio e apoio em todos os momentos.

Às amigas e veterinárias do coração Kelly e Audrey que torcem por mim e

comemoram comigo cada conquista.

Ao querido amigo Antonio, por estar sempre presente alegrando a minha vida.

À minha querida irmã Silvia, que mesmo estando longe sempre me apóia com

palavras doces e de conforto.

À Dona Clélia e Sr. Mario pelo carinho e cuidados comigo.

Aos meus pais, pelo amor, doação em prol dos meus estudos e da minha

felicidade.

Ao meu companheiro Armando, agradeço infinitamente por todo amor e pela

paciência nas horas difíceis. Obrigada por ser meu porto seguro.

RESUMO

Doenças infecciosas, particularmente as zoonoses, tem impactos significativos na

saúde humana e animal, e a sua fonte de infecção está cada vez mais relacionada aos

animais selvagens. O desenvolvimento de programas aplicáveis de vigilância das

doenças em seus reservatórios selvagens é essencial. Em zoológicos, a grande

concentração de animais proporciona um ambiente ideal para a transmissão de

doenças infecciosas entre as espécies animais. O risco de transmissão de zoonoses

para funcionários e visitantes também existe, de modo que é extremamente

necessário o monitoramento de potenciais reservatórios ou hospedeiros nestes locais.

O objetivo deste estudo foi avaliar a ocorrência de algumas doenças infecciosas

importantes em aoudads (Ammotragus lervia) do zoológico de Curitiba. Esta tese

está dividida em sete capítulos. O capítulo inicial apresenta uma introdução do

assunto, enfatizando a relevância e a finalidade da pesquisa. O segundo capítulo

apresenta uma revisão, expondo os dados atuais disponíveis na literatura sobre a

ocorrência de zoonoses e doenças infecciosas em aoudads. O terceiro capítulo é uma

pesquisa intitulada "Ocorrências de anticorpos anti-Toxoplasma gondii e anti-

Neospora caninum em aoudads no zoológico de Curitiba, sul do Brasil", publicada

na Revista Brasileira de Parasitologia Veterinária. O estudo revelou que o aoudad

está exposto a esses patógenos e, portanto, pode atuar como hospedeiro

intermediário, disseminando as doenças intra e inter espécies em áreas

compartilhadas. No quarto capítulo, a primeira infecção documentada de

Mycobacterium tuberculosis em aoudads é relatada. O quinto capítulo descreve um

inquérito sorológico para detecção de anticorpos anti-Leptospira spp. e anti-Brucella

spp. em aoudads, indicando que o animal está exposto às leptospiras e pode atuar

como um sentinela de exposição ambiental. A pesquisa relatada no sexto capítulo

revelou o isolamento de Campylobacter jejuni em amostras de fezes de aoudads,

alertando para o potencial risco zoonótico. E finalizando, o sétimo capítulo

apresenta um estudo sorológico para detecção do vírus da língua azul, artrite-

encefalite caprina e maedi-visna em amostras de sangue de aoudads, concluindo que

o animal está exposto ao vírus da língua azul. Em conclusão, aoudads estão expostos

a vários patógenos e podem desempenhar um papel importante no ciclo

epidemiológico de doenças infecciosas, como hospedeiros e sentinelas de exposição

ambiental. O desenvolvimento de programas adequados para um sistema de

vigilância de agentes infecciosos é extremamente necessário e está intimamente

integrado à vigilância da saúde pública, promovendo oportunidades para controlar

esses patógenos antes que possam afetar a saúde humana e animal.

Palavras-chave: Ammotragus lervia. Doenças infecciosas. Zoonoses. Zoológicos.

ABSTRACT

Infectious diseases, particularly zoonoses, have significant impacts on human and

animal health and are increasingly originating from wildlife. The development of

applicable programs for surveillance for the diseases in their wildlife reservoirs is

essential. In zoos, the large concentration of animals provides an optimal

environment for the transmission of infectious diseases among animal species. The

risk of zoonoses transmission to the staff and visitors also exists, so that the

monitoring of potential reservoirs or hosts in theses places is extremely necessary.

The aim of this study was to evaluate the occurrence of some important infectious

diseases in Barbary sheep at the Curitiba zoo. This thesis is divided into seven

chapters. The initial chapter provides an introduction of the subject, emphasizing the

relevance and the purpose of the research. The second chapter presents a review,

showing the current data available in the literature about the occurrence of zoonoses

and infectious diseases in Barbary sheep. The third chapter is a research entitled

“Occurrences of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in

Barbary sheep at Curitiba zoo, southern Brazil” published in the Brazilian Journal of

Veterinary Parasitology. The study revealed that Barbary sheep is exposed to these

pathogens and therefore may act as intermediate host, spreading toxoplasmosis and

neosporosis within and between species in shared areas. In the fourth chapter, the

first documented infection of Mycobacterium tuberculosis in Barbary sheep species

is reported. The fifth chapter describes a serological survey for detection of anti-

Leptospira spp. and anti-Brucella spp. antibodies in Barbary sheep, indicating that

the animal is exposed to leptospires and can act as a sentinel of environmental

exposure. The research reported in the sixth chapter revealed the isolation of

Campylobacter jejuni in fecal samples of Barbary sheep, warning to the potential

zoonotic risk. And lastly, the seventh chapter presents a serological study to detect

bluetongue, caprine arthritis-encephalitis and maedi-visna virus in blood samples of

Barbary sheep, concluding that the animal is exposed to the bluetongue virus. In

conclusion, Barbary sheep are exposed to several pathogens and may play an

important role in the epidemiological cycle of infectious diseases, as hosts and

sentinels of environmental exposure. The development of appropriate programs for a

surveillance system to infectious pathogens is sorely needed and closely integrated

to public health surveillance, providing opportunities to control such pathogens

before they can affect human and animal health.

Keywords: Ammotragus lervia. Infectious diseases. Zoonoses. Zoos.

LIST OF TABLES

TABLE 1 - Prevalence of Toxoplasma gondii and Neospora caninum infection

stratified by age, sex and housing location...................................... 42

TABLE 2 Prevalence of anti-Leptospira spp. antibodies stratified by age, sex

and housing location........................................................................ 61

TABLE 3 - Prevalence of antibodies against bluetongue virus by AGID and

ELISA tests stratified by age, sex and housing

location............................................................................................ 82

LIST OF ABBREVIATIONS AND SYMBOLS

UFPR Universidade Federal do Paraná

IUCN International Union for Conservation of Nature

USA United States of America

DNA Deoxyribonucleic Acid

PCR

ELISA

Polymerase Chain Reaction

Enzyme Linked Immunosorbent Assay

IFAT Indirect Immunofluorescence Antibody Test

IgG Immunoglobulin G

BT Bluetongue

OIE World Organisation for Animal Health

BTV Bluetongue Virus

IBGE Instituto Brasileiro de Geografia e Estatística

CDC Centers for Disease Control and Prevention

qPCR Real-Time Polymerase Chain Reaction

WHO World Health Organization

MAT Microscopic Agglutination Test

EFSA European Food Safety Authority

AGID Agar Gel Immunodiffusion

PANAFTOSA Centro Pan-Americano de Febre Aftosa

CAEV Caprine Arthritis-Encephalitis virus

spp. species

% percent °C degrees Celsius

m2 square meters

μm micrometer

L liter

mL milliliter

μL microliter

mg milligram

rpm rotations per minute

UI international unit

® trademark

= equal

/ divided

< less than

≥ greater than or equal

SUMMARY

1. INTRODUCTION ........................................................................................ 11

1.1. General purpose ........................................................................................... 14

1.2. Specific purposes ......................................................................................... 14

1.3. References ...................................................................................................

2. OCCURRENCE OF ZOONOSES AND INFECTIOUS DISEASES IN

BARBARY SHEEP (Ammotragus Lervia): A REVIEW…...………....…….

15

16

2.1. Introduction .................................................................................................

2.2. Development ...............................................................................................

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2.3. Conclusion ................................................................................................... 24

2.4. References ...................................................................................................

3. OCCURRENCES OF ANTI-Toxoplasma gondii AND ANTI-Neospora

caninum ANTIBODIES IN BARBARY SHEEP AT CURITIBA ZOO,

SOUTHERN BRAZIL .....................................................................................

3.1. Introduction .................................................................................................

3.2. Material and Methods ..................................................................................

3.3. Results and Discussion ................................................................................

3.4. References ...................................................................................................

4. FIRST REPORT OF Mycobacterium tuberculosis INFECTION IN

BARBARY SHEEP (Ammotragus lervia) AT THE CURITIBA ZOO,

SOUTHERN BRAZIL .....................................................................................

4.1. Introduction .................................................................................................



4.4. References ...................................................................................................

5. SEROLOGICAL SURVEY OF ANTI-Leptospira spp. ANTIBODIES

IN BARBARY SHEEP (Ammotragus lervia) AT THE CURITIBA ZOO,

SOUTHERN BRAZIL .....................................................................................

5.1. Introduction .................................................................................................



5.4. References ...................................................................................................

6. ISOLATION OF Campylobacter jejuni IN BARBARY SHEEP

(Ammotragus lervia) AT CURITIBA ZOO, SOUTHERN BRAZIL ...........

6.1. Introduction .................................................................................................



6.4. References ...................................................................................................

7. DETECTION OF ANTIBODIES AGAINST THE BLUETONGUE

VIRUS IN BARBARY SHEEP (Ammotragus lervia) AT CURITIBA

ZOO, SOUTHERN BRAZIL ..........................................................................

7.1. Introduction .................................................................................................



7.4. References ...................................................................................................

8. GENERAL CONCLUSIONS ......................................................................

9. SUPPLEMENTS ..........................................................................................

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1. INTRODUCTION

The knowledge of pathogens prevalence in wildlife of major infectious diseases

has a great importance for environmental protection strategies and public health

implications, considering its zoonotic potential.

Emerging infectious diseases have a major effect on human health and have

increased since the end of the 20th century; an estimated 75% of them are considered

zoonotic (Taylor, Latham e Woolhouse, 2001; Chomel, Belotto e Meslin, 2007).

Wild animals in particularly, are thought to be the source of more than 70% of all

emerging infections (Kuiken et al., 2005; Chomel, Belotto e Meslin, 2007) and these

wildlife pathogens can affect human health, agricultural production and also wildlife

conservation (Cleaveland, Laurenson e Taylor, 2001; Chomel, Belotto e Meslin,

2007).

Zoos are places that can be optimal environments for the transmission of

infectious diseases among animal species (Yesilbag, Alpay e Karakuzulu, 2011),

with a large concentration of animals sharing nearby areas, subjected to the same

environmental conditions, but with different behaviors and single characteristics of

each animal species. Nowadays, zoos have a staff of veterinarians, biologists and

other professionals that have been at the forefront of setting guidelines and standards

for appropriate care of wild animals (Schroeder, 1976; Fowler, 2006), however, the

biggest challenge for the staff is that the zoo consists of an inexhaustible source of

information that changes constantly, with new diseases and reemerging ones,

requiring technical approaches to each circumstance.

In zoos it is possible to lead researches that can bring benefits even to human

health, since the information obtained in zoo collections also allowed public health

12

veterinarians to solve problems such as tuberculosis in a collection of non-human

primates that acquired Mycobacterium tuberculosis (Fredrickson et al., 1971;

Fowler, 2006). Despite being an important source of data, zoos have been linked to

several zoonotic outbreaks, and more than 25 outbreaks of human infectious

diseases were associated with visitors to animal exhibits (Bender et al., 2004;

Chomel, Belotto e Meslin, 2007). Therefore, the introduction of preventive measures

and the monitoring to enable the exposure of healthy animals is vital. Lastly, there is

still the risk to keepers, which in their usual activities are in closer contact with the

animals and at constant risk of contracting infections, in case of animals being

carriers.

Although wildlife constitutes a large and often unknown reservoir and can

also be a source for reemergence of previously controlled zoonoses (Chomel,

Belotto e Meslin, 2007), little information exists regarding the role of the Barbary

sheep (Ammotragus lervia), as a pathogen reservoir. It is an African single species,

classified as "vulnerable" by the International Union for Conservation of Nature

(Nowak, 1999), which holds some primitive and unique features, and is grouped into

the suborder Ruminantia, subfamily of Caprinae, with the domesticated genera

Capra and Ovis (Mereu et al., 2008).

The purpose of this study is to evaluate the occurrence of some important

infectious diseases and zoonoses in Barbary sheep at the Curitiba zoo and, from this

data, establish preventive actions for animal and human health, with the

development of protocols to identify potential animal reservoirs or hosts, adoption of

management measures to enable quick diagnosis, and therefore prevent the

occurrence of infectious diseases and interspecies transmission, in the complex

environment of the zoo.

13

First of all, a review research is reported in this thesis, addressing the

theoretical basis for current data available in the literature. The research describes

the occurrence of some major infectious diseases and their importance in wildlife,

particularly in Barbary sheep.

After that, a research paper relates the occurrences of anti-Toxoplasma

gondii and anti-Neospora caninum antibodies in Barbary sheep. All 17 healthy

captive-born Barbary sheep housed at Curitiba zoo were included in the study and

the aliquots were sent to the Biological Institute of São Paulo, Center for Animal

Health Research and Development, for serological tests to be performed. This paper

was published in the Brazilian Journal of Veterinary Parasitology (supplement 2).

Then, the first report of Mycobacterium tuberculosis infection in Barbary

sheep at the Curitiba zoo is described. Using molecular techniques, Real-Time

Polymerase Chain Reaction, M. tuberculosis was detected of the lung tissue sample

in the Central Laboratory of the Paraná State.

And lastly, the three upcoming articles relate the prevalence of three

important agentes of infectious diseases in Barbary sheep: Leptospira spp.,

Campylobacter jejuni and bluetongue virus, indicating exposure to these pathogens.

14

1.1. GENERAL PURPOSE

Evaluate the occurrence of some important infectious diseases and zoonoses in

Barbary sheep at the Curitiba zoo.

1.2. SPECIFIC PURPOSES

Perform serological diagnosis of Toxoplasma gondii and Neospora caninum.

Perform molecular techniques to detect Mycobacterium tuberculosis.

Evaluate the prevalence of anti-Leptospira spp. antibodies.

Evaluate the prevalence of anti-Brucella spp. antibodies.

Evaluate the occurrence of Campylobacter spp. in fecal samples by bacteriological

procedure for isolation and biochemical identification.

Perform serological diagnosis of bluetongue, caprine arthritis-encephalitis and

maedi-visna.

15

1.3. REFERENCES

References organized and formatted using the Endnote software version X4 and

ABNT style.

BENDER, J. B. et al. Reports of zoonotic disease outbreaks associated with animal

exhibits and availability of recommendations for preventing zoonotic disease

transmission from animals to people in such settings. J Am Vet Med Assoc, v. 224, n.

7, p. 1105-9, 2004.

CHOMEL, B. B.; BELOTTO, A.; MESLIN, F. X. Wildlife, exotic pets, and emerging

zoonoses. Emerg Infect Dis, v. 13, n. 1, p. 6-11, 2007.

CLEAVELAND, S.; LAURENSON, M. K.; TAYLOR, L. H. Diseases of humans and

their domestic mammals: pathogen characteristics, host range and the risk of

emergence. Philos Trans R Soc Lond B Biol Sci, v. 356, n. 1411, p. 991-9, 2001.

FOWLER, M. E. Historical perspective of zoo and wildlife medicine. J Vet Med

Educ, v. 33, n. 3, p. 326-30, 2006.

FREDRICKSON, L. E. et al. An epizootic of tuberculosis in a municipal zoo: a public

health problem. J Am Vet Med Assoc, v. 159, n. 11, p. 1474-6, 1971.

KUIKEN, T. et al. Public health. Pathogen surveillance in animals. Science, v. 309, n.

5741, p. 1680-1, 2005.

MEREU, P. et al. Complete nucleotide mtDNA sequence of Barbary sheep

(Ammotragus lervia). DNA Seq, v. 19, n. 3, p. 241-5, 2008.

NOWAK, R. M. Walker's mammals of the world. 6th ed. Baltimore: Johns Hopkins

Univesity Press, 1999.

SCHROEDER, C. R. Zoo medicine: yesterday and today. J Am Vet Med Assoc, v.

169, n. 1, p. 61-9, 1976.

TAYLOR, L. H.; LATHAM, S. M.; WOOLHOUSE, M. E. Risk factors for human

disease emergence. Philos Trans R Soc Lond B Biol Sci, v. 356, n. 1411, p. 983-9,

2001.

YESILBAG, K.; ALPAY, G.; KARAKUZULU, H. A serologic survey of viral

infections in captive ungulates in Turkish zoos. J Zoo Wildl Med, v. 42, n. 1, p. 44-8,

2011.

16

OCCURRENCES OF ZOONOSES AND INFECTIOUS DISEASES IN

BARBARY SHEEP (Ammotragus Lervia): A REVIEW

ABSTRACT

Wild animals are thought to be the source of more than 70% of all emerging infectious

diseases and some species in particular have the potential to act as host and reservoir.

The aim of this research was describe the occurrence of some major infectious diseases

and their importance in wildlife, particularly in Barbary sheep. This review

demonstrated that Barbary sheep has been part of the epidemiological cycle of

infectious diseases, including zoonoses, from relevance to both human and animal

health.

Keywords: Emerging diseases, zoonoses, Barbary sheep.

RESUMO

Os animais selvagens são considerados fonte de infecção para mais de 70% das doenças

infecciosas emergentes, e algumas espécies em particular têm o potencial para atuar

como hospedeiro e reservatório. O objetivo desta pesquisa foi descrever a ocorrência de

algumas doenças infecciosas relevantes e sua importância na fauna silvestre,

especialmente para o aoudad. Este revisão demonstrou que o aoudad tem participado do

ciclo epidemiológico de doenças infecciosas, incluindo zoonoses, de relevância tanto

para a saúde humana quanto animal.

Palavras-chave: Doenças emergentes, zoonoses, aoudad.

17

2.1. INTRODUCTION

The occurrence of emerging and reemerging infectious diseases, affecting

human and animals, have increased since the end of the 20th century and it is estimated

that 75% are zoonotic (Taylor, Latham e Woolhouse, 2001; Chomel, Belotto e Meslin,

2007). The recent concern about emerging diseases and zoonoses is the interface that

has been established between livestock and wildlife (Cunningham, 2005). In some

situations, due to environmental changes, the pathogens can infect multiple host species,

become self-sustaining in the new host and lead to outbreaks in humans, livestock and

wildlife (Woolhouse, 2002). A well-known example is bovine tuberculosis, which is

primarily a disease of domestic cattle and goats, but can affect many other domestic and

wild species, as well as humans (Gortázar et al., 2007).

Particularly wild animals are thought to be the source of more than 70% of all

emerging infectious diseases (Kuiken et al., 2005) and zoos provide optimal

environments for the transmission of infectious diseases among animal species

(Yesilbag, Alpay e Karakuzulu, 2011). The possibility of diseases transmission from

wild animals to humans has important public health implications and the knowledge of

the prevalence of different pathogens in wildlife species is one of the most important

strategies for human and environmental protection (Artois, 1993).

Several prevalence studies have been conducted covering occurrence of

infectious diseases in wildlife, however in most situations the role in the disease cycle is

still unclear. According to some authors, some wild species in particular, such as the

Barbary sheep (Ammotragus lervia), an African ungulate, have the potential to act as

host and reservoir for important infectious diseases (Candela et al., 2009). Thus, the aim

of this research was to perform a review of Barbary sheep and the occurrence of the

18

most important zoonoses and infectious diseases that may be involved with this species.

2.2. DEVELOPMENT

Ammotragus lervia is an ungulate mammal with unique and primitive

characteristics, classified as "vulnerable" species (VU A2cd) by the IUCN (International

Union for Conservation of Nature). It originates from the rocky and arid countries of

Africa and was successfully introduced in the USA in the mid-twentieth century and

Spain, with the purpose of sport hunting (Gray e Simpson, 1980; Nowak e Paradiso,

1991). Common names for the species are Barbary sheep, aoudad, arui, Barbarian,

Saharan mouflon among others (Gray e Simpson, 1980). As physical characteristics, the

head and body length ranges between 1,30 m and 1,65 m and the weight ranges between

40 kg to 55 kg for females, and 100 kg to 145 kg in males. The pelage is generally

brownish red, with whitish areas inside the ears, chin and near the ventral region. The

Barbary sheep has a mane in the ventral region consisting of long, soft hair under the

throat, chest and upper part of the front legs. The horns of males are very heavy,

wrinkled and length reach measuring 840 mm. Females have prominent horns also

(Nowak e Paradiso, 1991). Reproductive activity occurs throughout the year, although

mating occurs more widely from September to November, and births from March to

May. The gestation period varies from 154 to161 days and sexual maturity is reached at

about 18 months (Gray e Simpson, 1980).

Barbary sheep is grouped into the suborder Ruminantia, subfamily Caprinae,

which includes the domesticated genera Capra and Ovis (Nowak, 1999). Previous

researches have shown that Barbary sheep is a single species with some characteristics

19

that make it particularly interesting. In 2007, the complete sequence of the

mitochondrial DNA genome of the species was obtained by PCR (Polymerase Chain

Reaction) (Mereu et al., 2008). The genomic organization was corresponding to other

mammalian mitochondrial genomes and the molecule length is formed by 16 530 base

pairs, and has similar features to the sheep and goat genome. As a final result, it was

observed that Ammotragus, Capra and Ovis depart from a common ancestor, but there

is a closer relationship between Ammotragus and Capra (Mereu et al., 2008).

According to some authors, there is a potential risk of Barbary sheep act as host,

and often reservoir of important infectious diseases. It is also consider the importance of

co-infection as a relevant factor in epidemiological studies, especially in wild

populations, which the occurrence of multiple infections is common (Candela et al.,

2009). Next, will be described the occurrence of some major infectious diseases and

their importance in wildlife, particularly in Barbary sheep.

The toxoplasmosis is a disease caused by intracellular protozoan Toxoplasma

gondii, whose definitive hosts are domestic and wild felines (Kikuchi et al., 2004) and

is widely prevalent in humans and other animals worldwide, especially in goats causing

abortion or neonatal mortalities (Dubey e Beattie, 1988). T. gondii infection not only

results in significant reproductive and economic losses, but also has implications for

public health, since consumption of infected meat or milk can facilitate zoonotic

transmission (Faria et al., 2007).

T. gondii infections in zoos are very important once many animals die in

captivity by clinical toxoplasmosis and also due to the risk of exposure to visitors,

especially children and elderly. Although most human and animals infections are

asymptomatic, toxoplasmosis can cause serious implications in children infected

congenitally and in people with low immunity. In addition, a significant risk factor is

20

the animal exhibition in open enclosures, which corroborates to the hypothesis that

animals in captivity are predominantly exposed to the oocysts ingestion and sequential

contamination (De Camps, Dubey e Saville, 2008).

In 2006, a study conducted in Spain reported for the first time seroprevalence

data of T. gondii in Barbary sheep. Serum samples were collected from several species

of wild ruminants, in six regions of the country and tested by MAT (Modified

Agglutination Test) at dilutions of 1:25, 1:50 and 1:500. The cut-off appointed was

equal or greater than 1:25. The Barbary sheep seroprevalence was 10.0% and associated

to some risk factors such as the presence of cats, weather conditions, animal species

involved and serological tests. The results indicated a wide distribution of T. gondii

among wild animals in Spain and suggested that the consumption of raw or

undercooked meat, as well as the handling of carcasses, can be important sources of

people infection (Gauss et al., 2006).

Neospora caninum is an apicomplex protozoan similar to T. gondii. Neosporosis

is the cause of many neuromuscular disorders, paralysis and death in dogs (Barber et al.,

1997) and a major cause of abortion in cattle worldwide, causing huge economic losses

and occasionally clinical signs in horses, goats, sheep and deer (Dubey, 2003). Due to

its worldwide distribution, N. caninum was suspected to have a cosmopolitan

carnivorous as definitive host. In 1998, domestic dogs were confirmed as definitive

hosts from the liberation of oocysts in feces after consuming tissues of mice infected

with N. caninum (Mcallister et al., 1998).

The first diagnosis of neosporosis in wild animals was post mortem in mule deer

(Odocoileus hemionus columbianus) in California (Woods et al., 1994). In 2007, the

first case of N. caninum in Barbary sheep was reported in Spain. ELISA (Enzyme

Linked Immunosorbent Assay) and IFAT (Indirect Immunofluorescence Antibody Test)

21

were performed as confirmatory tests, detecting the presence of IgG. Antibody titers

found were 1:200 (cut-off 1:50) and the prevalence in Barbary sheep was 7.7%

(Almeria et al., 2007). The spectrum of wild herbivores that act as intermediate hosts

have increased gradually, including many species of ruminants, and other wild

carnivores (raccoons and red foxes) that are likely involved in the sylvatic cycle of the

parasite. Thus, the possibilities for research involving neosporosis in wildlife are

numerous and should eventually explain the current questions (Gondim, 2006),

emphasizing that the recognition of a wild cycle is another complicating factor to

control the disease (Rosypal e Lindsay, 2005).

Leptospirosis is a worldwide zoonosis, caused by spirochetes bacteria of the

genus Leptospira (Greene et al., 2006). It is an infectious, contagious widespread

disease that affects humans, domestic animals and wildlife, assuming considerable

importance as an economic and public health problem (Faine et al., 1999). The rat,

Rattus norvegicus, is the most important reservoir (Farrington e Sulzer, 1982).

Leptospirosis may cause a range of clinical manifestations since a mild infection,

characterized by nonspecific symptoms, to Weil's disease, which is the most severe

form causing icterus, bleeding and acute kidney injury (Andrade, De Francesco Daher e

Seguro, 2008).

Although a previous survey reported no detectable titers for leptospirosis in

Barbary sheep (Hampy, Pence e Simpson, 1979), leptospirosis incidence in wildlife is

usual and constitutes an important epidemiological factor to the maintenance and spread

of the disease to human and animals. Zoos can potentially be a source of infection for

human leptospirosis (Mortimer, 2005) and constitute a serious public health problem. In

captivity, the sharing areas and the consequent interaction between pathogen and host

may expose animals from different ecologic areas to a common environment and to a

22

different serovars not found in their native habitat (Luna-Alvarez et al., 1996; Ullmann,

Hoffmann, et al., 2012).

Another important zoonosis studied was brucellosis, a disease caused by

Brucella genus bacteria, involved with reproductive problems outbreaks, including

abortion and infertility in several animal species worldwide (Cutler, Whatmore e

Commander, 2005). Brucella melitensis, followed by Brucella abortus and Brucella

suis are the main species involved in human infection, and therefore, are the main target

of eradication campaigns (Munoz et al., 2010). The human disease is quite varied with

nonspecific clinical signs, may be similar to mild to severe flu or can develop

complications affecting the nervous, skeletal muscle and cardiac systems (Galinska e

Zagorski, 2013).

Studies have shown that some wildlife species can act as asymptomatic hosts for

Brucella spp. representing contamination sources to the environment, animals and

human (Thorne, 2001; Godfroid, Nielsen e Saegerman, 2010). Although wild ruminants

have been suggested to hold brucellosis, previous studies revealed no antibody

responses to Brucella spp. infections in Barbary sheep (Hampy, Pence e Simpson, 1979;

Candela et al., 2009; Munoz et al., 2010). However, the risk of infection exists and can

be high in overabundant wildlife situations, in contact with infected livestock (Gortázar

et al., 2007).

Not least, tuberculosis disease was also included in this research. It is one of the

most important diseases in the world and annually more than nine million human cases

are reported (Who, 2012). Mycobaterium tuberculosis is considered primarily a human

pathogen and has been reported in domestic and wildlife species living in close contact

with humans (Michalak et al., 1998; Montali, Mikota e Cheng, 2001).

23

The role of Barbary sheep as a pathogenic reservoir to humans remains to be

fully established, but previous studies have described that they can be host or reservoir

and spreading factor of M. tuberculosis complex, showing antibodies prevalence against

Mycobacterium bovis in Barbary sheep, from a free-ranging (49.5%) and captive (8.0%)

populations in Spain (Candela et al., 2009). Furthermore, the existence of wildlife

tuberculosis reservoirs is the major limiting factor for the disease control in livestock

(Phillips et al., 2003).

Finally, an important viral disease was studied. The bluetongue (BT) is an

infectious and noncontagious disease according to the World Organisation for Animal

Health - OIE (Oie, 2014), caused by the bluetongue virus (BTV). It is a disease that

affects domestic and wild ruminants, transmitted by some species from the genus

Culicoides midges (Yesilbag, Alpay e Karakuzulu, 2011) and the economic and sanitary

impact of BT involves more than direct losses in affected herds with reduced milk yield,

infertility and abortion, but also rigorous restrictions on international trade of animals

and their products (Legisa et al., 2013; Oie, 2014).

The role of wild animals in the BT epidemiology is still unclear, even though the

susceptibility of wild ruminants as potential reservoirs is known and raises the

possibility that BTV could spread among captive ruminants housed in zoos (Vilar et al.,

2011). Serologic evidence of BT has been previously documented in a Barbary sheep in

USA, detected by Official Modified Compliment Fixation test, in which antibody titers

higher than 1:10 were considered positive (Hampy, Pence e Simpson, 1979).

24

2.3. CONCLUSION

This research revealed that Barbary sheep has been part of the epidemiological

cycle of infectious diseases, including zoonoses, from relevance to both human and

animal health. Since 1979, studies in Barbary sheep have detected serological evidences

of important diseases, which are worldwide spread in nowadays and have established an

interesting interface between livestock and wildlife. The monitoring of emerging

diseases in their wildlife reservoirs is needed for a better understand of the diseases

cycles, to recognize and identify all animals involved and establish preventive measures

of public health surveillance and species conservation.

25

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29

OCCURRENCES OF ANTI-Toxoplasma gondii AND ANTI-Neospora caninum

ANTIBODIES IN BARBARY SHEEP AT CURITIBA ZOO, SOUTHERN

BRAZIL

ABSTRACT

Barbary sheep (Ammotragus lervia) have the potential to act as hosts of important

infectious diseases, particularly zoonoses. Blood samples from 17 Barbary sheep at the

Curitiba zoo were collected to evaluate occurrences of anti-Toxoplasma gondii and anti-

Neospora caninum antibodies, tested using the Indirect Immunofluorescence Antibody

test (IFAT). Anti-T. gondii and anti-N. caninum antibodies were detected in 4/17

(23.5%) and 4/17 (23.5%) samples, respectively. The present study has shown that

Barbary sheep at Curitiba zoo were exposed to T. gondii and N. caninum and therefore

may act as intermediate hosts, spreading toxoplasmosis and neosporosis within and

between species in shared areas.

Keywords: Ammotragus lervia, Toxoplasma gondii, Neospora caninum, zoonoses,

zoos.

RESUMO

Aoudads (Ammotragus lervia) tem o potencial para atuar como hospedeiros de

importantes doenças infecciosas, em particular as zoonoses. Amostras de sangue de 17

aoudads do zoológico de Curitiba foram coletadas para avaliar ocorrências de

anticorpos anti-Toxoplasma gondii e anti-Neospora caninum, testados pela Reação de

Imunofluorescência Indireta (RIFI). Anticorpos anti-T. gondii e anti-N. caninum foram

detectados em 4/17 (23,5%) e 4/17 (23,5%) das amostras dos aoudads, respectivamente.

O presente estudo revelou que os aoudads no zoológico de Curitiba estão expostos a T.

gondii e N. caninum e, portanto, podem atuar como hospedeiros intermediários e fatores

de difusão da toxoplasmose e da neosporose intra e interespécies em áreas

compartilhadas.

Palavras-chave: Ammotragus lervia, Toxoplasma gondii, Neospora caninum,

zoonoses, zoológicos.

30

3.1. INTRODUCTION

Wildlife may represent a large and often unknown reservoir system that can

potentially become a source for emerging or reemerging diseases, particularly zoonoses,

which were previously absent or under control (Chomel, Belotto e Meslin, 2007).

Although the role of wild animals in harboring and transmitting infectious agents has

been reported in relation to several species, endangered species may be difficult to

assess and therefore their role remains to be fully established. In addition, infectious

diseases may also present risks to successful maintenance of wild animals in captivity

(Yesilbag, Alpay e Karakuzulu, 2011).

Translocation or introduction of wild species, particularly exotic ones, which is a

regular practice among zoos, may present serious risks to the resident population, since

new pathogens can be introduced to a hypothetically controlled environment. In such

scenarios, zoos may provide an optimal environment for infectious disease transmission

because of the greater animal density and sharing of space between different animal

species (Yesilbag, Alpay e Karakuzulu, 2011).

According to some authors, some species in particular, such as the Barbary

sheep (Ammotragus lervia), have the potential to act as hosts and reservoirs for

important infectious diseases (Candela et al., 2009). The Barbary sheep or aoudad was

successfully introduced into the United States in the early to mid-twentieth century for

the purposes of sport hunting and now is classified as "vulnerable" (VU A2cd) by the

IUCN (International Union for Conservation of Nature) (Nowak, 1999). It is a singular

African species that has some unique primitive features and is grouped into the suborder

Ruminantia, subfamily of Caprinae, which includes the domesticated genera Capra and

Ovis. A closer phylogenetic connection between the genera Ammotragus and Capra was

31

recently observed (Mereu et al., 2008).

In zoos, Toxoplasma gondii infection is important because of the risk of visitor

exposure and because animals in captivity may die from clinical toxoplasmosis (De

Camps, Dubey e Saville, 2008). Regarding neosporosis, which is one of the major

causes of abortion in several domestic species (Dubey, 2003), the wild cycle may

present a complicating factor with regard to disease control (Rosypal e Lindsay, 2005).

Although toxoplasmosis and neosporosis are important diseases worldwide, the

exposure of Barbary sheep to these protozoan pathogens has been scarcely reported to

date. Therefore, this study aimed to evaluate occurrences of anti-Toxoplasma gondii and

anti-Neospora caninum antibodies in Barbary sheep at Curitiba zoo, state of Paraná,

southern Brazil.

3.2. MATERIAL AND METHODS

Curitiba zoo, which was established in 1982, is now among the largest Brazilian

zoos with approximately 2300 specimens, several of which were born in captivity,

distributed across an area of 530 square meters (Javorouski e Biscaia, 2012). Curitiba

(25°25'47"S, 49°16'19"W) is the capital of the state of Paraná, in southern Brazil, and is

the eighth most populous city in Brazil, with an estimated population of 1,751,907

inhabitants (Ibge, 2010).

A total of 17 healthy captive-born Barbary sheep, of both genders and different

ages, were included in the present study. Blood samples were taken by means of

physical restraint without chemical sedation, following a 24-hour fasting period. Out of

the total sample, five of these sheep were kept on display in a public exhibition and 12

32

were kept in an isolation area. Each individual was identified by means of an ear tag

number and color, and the individual information gathered included date of birth,

gender and housing location. Blood samples (20 mL) were collected intravenously and

placed in tubes without anti-coagulant and kept at room temperature (25°C) until visible

clot retraction. The material was then centrifuged at 3000 rpm for 5 minutes and the

serum was separated and kept at -20°C until testing.

The aliquots were sent to the Biological Institute of São Paulo, Center for

Animal Health Research and Development, for serological tests to be performed. The

serum samples were tested for anti-Toxoplasma gondii antibodies by means of the

Indirect Immunofluorescence Antibody Test (IFAT). Each serum sample was tested at a

range of dilutions, using a cut-off of 1:64 (Camargo, 1974). In addition, individual

serum samples were tested for the presence of anti-Neospora caninum antibodies by

means of IFAT (Dubey et al., 1988), on slides for optical microscopy (PGC-Scientifics,

catalogue number 60-5453-46) sensitized with tachyzoites of N. caninum. Each of these

serum samples was tested at a range of dilutions, using a cut-off of 1:50. The specimens

were examined under a fluorescence microscope with 40x objective.

Laboratory data were entered into an Excel spreadsheet. Contingency tables for

associations were created in Epi Info 7 (Cdc, 2013) and then analyzed in OpenEpi

(Dean, Sullivan e Soe, 2013). Prevalence ratios and 95% confidence intervals were

calculated, and Fisher’s exact test was used to compare proportions between variables at

a 5% significance level.

33

3.3. RESULTS AND DISCUSSION

Anti-Toxoplasma gondii and anti-Neospora caninum antibodies were detected,

respectively, in 4/17 (23.5%) and 4/17 (23.5%) of the sampled Barbary sheep. Only

2/17 (11.7%) of them showed antibodies simultaneously for T. gondii and N. caninum.

The prevalences of T. gondii and N. caninum infection were evaluated stratified

by age, sex and place where the animals were housed (public exhibition or isolation

area) (Table 1). The mean age of the sampled animals was 4.5 years, with a standard

deviation of 2.7 years. Thus, a cut-off of 5 years was chosen to dichotomize the age

variable, i.e. forming one group of animals aged less than 5 years and another of

animals aged greater than or equal to 5 years.

The prevalence of T. gondii infection in animals aged less than 5 years was 2/10

(20.0%) and it was 2/7 (28.6%) in animals aged greater than or equal to 5 years, which

corresponded to a prevalence ratio of 1.4 (95% CI: 0.3 – 7.9), i.e. higher in the group of

animals aged greater than or equal to 5 years, although this difference was not

statistically significant (Fisher p-value = 0.99). The same values were obtained when N.

caninum infection and the animals’ ages were evaluated.

Regarding gender, the T. gondii prevalence in females was 1/7 (14.3%) and it

was 3/10 (30.0%) in males, making the prevalence in males 2.1 times (95% CI: 0.3 –

16.3) greater in males than in females, but this difference was not statistically

significant (Fisher p-value = 0.88). The N. caninum prevalence was 0/7 (0%) in females

and 4/10 (40.0%) in males. Since the prevalence in one group was zero, it was not

possible to calculate the prevalence ratio.

The T. gondii prevalence among the animals housed in the isolation area was

1/12 (8.3%) and among the animals on public display it was 3/5 (60.0%). The

34

prevalence of T. gondii in the animals on public display was 7.2 times (95% CI: 0.9 –

53.6) greater than in the animals kept in isolation. This difference was not statistically

significant, but was close to the significance threshold (Fisher p-value = 0.10). For N.

caninum, the prevalence among animals housed in the isolation area was 2/12 (16.7%)

and among animals on public display it was 2/5 (40.0%). The prevalence was 2.4 times

(95% CI: 0.5 – 12.6) greater in the animals on public display than in the animals in the

isolation area, even though this difference was not statistically significant (Fisher p-

value = 0.38).

The present study has revealed occurrences of two important infectious agents in

Barbary sheep, which are a singular wild species that shares a common ancestor with

domestic species in the genera Capra and Ovis (Mereu et al., 2008). Emerging and

reemerging infectious diseases may have a major effect and impact on human and

animal health, and produce economic losses. Animals, particularly of wild species, are

thought to be reservoirs for more than 70% of all emerging infections (Kuiken et al.,

2005; Chomel, Belotto e Meslin, 2007). Pet zoos, where children are allowed to

approach and feed captive wild and domestic animals, have been linked to several

zoonotic outbreaks. Not surprisingly, over 25 outbreaks of human infectious diseases

associated with visitors to animal exhibits were identified from 1990 to 2000 (Bender et

al., 2004; Chomel, Belotto e Meslin, 2007).

Antibodies against T. gondii were at higher levels in the current report than in

the first previous report, and were present in 4/17 (23.5%) versus 1/10 (10.0%) of the

Barbary sheep, respectively (Gauss et al., 2006). The differences may be due to

different geographical locations, which were reported in the earlier study to be the most

important factor affecting infection prevalence. Similar results of 6/25 (24.0%) for

captive Barbary sheep were found more recently, while the prevalence among free-

35

range Barbary sheep was 1/67 (1.5%) (Candela et al., 2009).

Since Curitiba zoo is located in a humid subtropical climate with an average

annual temperature of 16 °C, the relatively higher prevalence may be associated with

the local weather (Ibge, 2010). Our findings corroborate previous studies that have

shown that there may be higher prevalence of toxoplasmosis infection in areas that are

more shaded and have relatively higher humidity than in areas with lower shade and

rain and higher evaporation and desiccation (Smith e Frenkel, 1995; Gauss et al., 2006).

Although age, sex and housing location were not statistically significant, the Barbary

sheep on public display were 7.2 times more likely to have T. gondii infection than were

those housed in the isolation area. Hence, captive animals on public display in open

enclosures may be at higher risk of infection, as previously observed (De Camps, Dubey

e Saville, 2008). Since the prevalence of T. gondii infection also depends on the

presence of felids (Gauss et al., 2006), our study may provide a warning with regard to

nearby wild cat enclosures and circulation of domestic felids in zoo areas.

Neospora caninum co-infection was also investigated because previous studies

reported multiple infections in animal populations (Petney e Andrews, 1998; Candela et

al., 2009). Although neosporosis was first reported in wild animals in a dead black-

tailed deer (Woods et al., 1994), and antibodies have recently been reported in Barbary

sheep (Almeria et al., 2007) and several other free-range and captive wild animals, the

role of wild animals in the N. caninum life cycle remains unclear (Dubey, 2003;

Almeria et al., 2007). Although antibodies against N. caninum in the present study

occurred in 4/17 animals (23.5%), i.e. more frequently than in a previous survey, which

found them in 1/13 Barbary sheep (7.7%), no significant differences regarding sex or

age were observed in either study (Almeria et al., 2007). Both studies used the IFAT,

with 1:50 as the cut-off (Cheadle, Lindsay e Blagburn, 1999; Ortuno, Castella e

36

Almeria, 2002).

T. gondii and N. caninum occurrences in Barbary sheep can be compared with

occurrences in domestic goats, which are genetically related to each other (Mereu et al.,

2008). Our findings may indicate, as observed in domestic goats, that males and females

are equally exposed to the risk of infection (Faria et al., 2007). However, other factors

such as geographical area and environmental exposure may impede adequate

comparison, as observed among domestic goats in northeastern Brazil, which presented

higher occurrence of T. gondii 75/306 (24.5%) and lower occurrence of N. caninum

10/306 (3.3%) (Faria et al., 2007). Similar rates among domestic goats were also

observed in another study in southeastern Brazil, which showed higher occurrence of T.

gondii 113/394 (28.7%) and lower occurrence of N. caninum 25/394 (6.4%) (Figliuolo

et al., 2004). These differences may be explained by the level of exposure and climatic

factors, thus reflecting the abundance of viable parasitic stages in the environment and

influencing the overall prevalence (Faria et al., 2007).

Captive wild species, particularly carnivores kept in zoos, may share

environments and act as sources of T. gondii and N. caninum infection for herbivores

such as Barbary sheep or other related susceptible wild species. A recent study

conducted among wild captive carnivores kept in zoos in the Brazilian states of São

Paulo and Mato Grosso, and in Brasilia, showed that antibodies to T. gondii and N.

caninum were present in 102/161 (63.4%) and 70/161 (50.3%) wild cats, respectively,

using IFAT. In addition, 49/98 (50.5%) and 40/98 (41.2%) wild canids were

seropositive for T. gondii and N. caninum antigens, respectively, using IFAT (Andre et

al., 2010). Relatively lower prevalence was found in wild canids in zoos and sanctuaries

in the states of Paraná, Santa Catarina and Rio de Janeiro and in Brasilia, which found

prevalences of 20/50 (40.0%) for T. gondii and 18/50 (36.0%) for N. caninum (Mattos

37

et al., 2008). More recently, seropositivity for T. gondii was observed in 38/57 samples

(66.7%) samples from Neotropical felids kept at the Bela Vista Biological Sanctuary,

Itaipu Binacional hydroelectric development, southern Brazil (Ullmann et al., 2010),

which was similar to the prevalence of 26/37 (64.4%) that was found in an early study

on captive exotic wild felids in 12 different zoos (Silva et al., 2001). Overall, these

results suggest that despite efforts to control such infections in zoo facilities,

particularly among wild-caught carnivore specimens, animals may be exposed to

infectious agents due to other environmental sources.

Although T. gondii seroprevalence may be associated with several risk factors,

particularly the presence of domestic cats, a comprehensive study conducted on 865

neotropical felids in 78 cities in 20 Brazilian states showed that the presence of

domestic cats roaming in zoos was not an independent risk factor for toxoplasmosis

(Ramos Silva et al., 2007). Moreover, the seroprevalence of T. gondii and N. caninum

in roaming and/or feral cats has not been documented to date in Brazilian zoos, and

further studies should be conducted in order to fully establish the role of domestic cats

as potential sources of infection among captive wild animals.

Finally, reports of wild species that could serve as intermediate hosts for N.

caninum have been gradually increasing. These species include Barbary sheep, many

other ruminant species and possibly rhinoceroses, thus providing new insights into

current questions regarding the N. caninum cycle in wildlife (Gondim, 2006).

In conclusion, this study has shown that Barbary sheep in Curitiba zoo, Paraná,

southern Brazil, are exposed to T. gondii and N. caninum and may act as intermediate

hosts for spreading toxoplasmosis and neosporosis. Barbary sheep may play an

important role in the protozoan cycle as a sentinel for measuring parasite dissemination

in zoos and for checking whether circulating pathogens are present. In addition, wildlife

38

staff should be aware of the inherent risks involved in introducing and providing shelter

for such animal species. A better understanding of pathogen occurrence in wildlife

species, particularly zoonotic pathogens, may serve as the basis for future

environmental protection and public health strategies.

39

3.4. REFERENCES


ABNT style.

ALMERIA, S. et al. Seroprevalence of Neospora caninum in non-carnivorous wildlife

from Spain. Vet Parasitol, v. 143, n. 1, p. 21-8, 2007.

ANDRE, M. R. et al. Antibodies to Toxoplasma gondii and Neospora caninum in

captive neotropical and exotic wild canids and felids. J Parasitol, v. 96, n. 5, p. 1007-9,

2010.

BENDER, J. B. et al. Reports of zoonotic disease outbreaks associated with animal

exhibits and availability of recommendations for preventing zoonotic disease

transmission from animals to people in such settings. J Am Vet Med Assoc, v. 224, n.

7, p. 1105-9, 2004.

CAMARGO, M. E. Introdução às técnicas de imunofluorescência. Rev Bras Patol

Clín, v. 10, n. 4, p. 143-171, 1974.



Dis, v. 28, n. 5, p. 481-9, 2009.

CDC. Atlanta, 2013. Available from: < http://wwwn.cdc.gov/epiinfo/7/ >. Cited 2013

Jul 03.

CHEADLE, M. A.; LINDSAY, D. S.; BLAGBURN, B. L. Prevalence of antibodies to

Neospora caninum in dogs. Vet Parasitol, v. 85, n. 4, p. 325-30, 1999.





94, n. 3, p. 648-53, 2008.

DEAN, A. G.; SULLIVAN, K. M.; SOE, M. M. OpenEpi: Open Source Epidemiologic

Statistics for Public Health, Version. 2013. Available from: <

http://www.OpenEpi.com , updated 2013 Apr 06 >. Cited 2013 Jul 03.

DUBEY, J. P. Review of Neospora caninum and neosporosis in animals. Korean J

Parasitol, v. 41, n. 1, p. 1-16, 2003.

http://wwwn.cdc.gov/epiinfo/7/

http://www.openepi.com/

40

DUBEY, J. P. et al. Newly recognized fatal protozoan disease of dogs. J Am Vet Med

Assoc, v. 192, n. 9, p. 1269-85, 1988.

FARIA, E. B. et al. Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum

antibodies in goats slaughtered in the public slaughterhouse of Patos city, Paraiba State,

Northeast region of Brazil. Vet Parasitol, v. 149, n. 1-2, p. 126-9, 2007.

FIGLIUOLO, L. P. C. et al. Prevalence of anti-Toxoplasma gondii and anti-Neospora

caninum antibodies in goat from São Paulo State, Brazil. Small Rum Res, v. 55, n. 1-3,

p. 29-32, 2004.

GAUSS, C. B. et al. Prevalence of Toxoplasma gondii antibodies in red deer (Cervus

elaphus) and other wild ruminants from Spain. Vet Parasitol, v. 136, n. 3-4, p. 193-200,

2006.

GONDIM, L. F. Neospora caninum in wildlife. Trends Parasitol, v. 22, n. 6, p. 247-52,

2006.

IBGE. Population estimates for Curitiba. 2010. Available from: <

http://www.ibge.gov.br/cidadesat/painel/painel.php?codmun=410690 >. Cited 2014 Oct

30.

JAVOROUSKI, M. L.; BISCAIA, S. A. Zoológico Municipal de Curitiba 30 anos.

Curitiba: 2012.


5741, p. 1680-1, 2005.

MATTOS, B. C. et al. [Seroprevalence of antibodies anti-Neospora caninum and anti-

Toxoplasma gondii in captive wild canids]. Rev Bras Parasitol Vet, v. 17 Suppl 1, p.

267-72, 2008.





ORTUNO, A.; CASTELLA, J.; ALMERIA, S. Seroprevalence of antibodies to

Neospora caninum in dogs from Spain. J Parasitol, v. 88, n. 6, p. 1263-6, 2002.

PETNEY, T. N.; ANDREWS, R. H. Multiparasite communities in animals and humans:

frequency, structure and pathogenic significance. Int J Parasitol, v. 28, n. 3, p. 377-93,

1998.

http://www.ibge.gov.br/cidadesat/painel/painel.php?codmun=410690

41

RAMOS SILVA, J. C. et al. Risk factors associated with sero-positivity to Toxoplasma

gondii in captive neotropical felids from Brazil. Prev Vet Med, v. 78, n. 3-4, p. 286-95,

2007.

ROSYPAL, A. C.; LINDSAY, D. S. The sylvatic cycle of Neospora caninum: where do

we go from here? Trends Parasitol, v. 21, n. 10, p. 439-40, 2005.

SILVA, J. C. et al. Seroprevalence of Toxoplasma gondii in captive neotropical felids

from Brazil. Vet Parasitol, v. 102, n. 3, p. 217-24, 2001.

SMITH, D. D.; FRENKEL, J. K. Prevalence of antibodies to Toxoplasma gondii in wild

mammals of Missouri and east central Kansas: biologic and ecologic considerations of

transmission. J Wildl Dis, v. 31, n. 1, p. 15-21, 1995.

ULLMANN, L. S. et al. Serological survey of Toxoplasma gondii in captive

Neotropical felids from Southern Brazil. Vet Parasitol, v. 172, n. 1-2, p. 144-6, 2010.

WOODS, L. W. et al. Systemic neosporosis in a California black-tailed deer

(Odocoileus hemionus columbianus). J Vet Diagn Invest, v. 6, n. 4, p. 508-10, 1994.



2011.

42

Table 1 - Prevalence of Toxoplasma gondii and Neospora caninum infection stratified by age, sex and housing location.

Characteristic Toxoplasma gondii Neospora caninum

n/N % PR (95% CI)* p-value n/N % PR (95% CI) p-value

Age

<5 years 2/10 20.0 - -

2/10 20.0 - -

≥5 years 2/7 28.6 1.4 (0.3 - 7.9) 0.99

2/7 28.6 1.4 (0.3 - 7.9) 0.99

Sex

Female 1/7 14.3 - -

0/7 0.0 - -

Male 3/10 30.0 2.1 (0.3 - 16.3) 0.88

4/10 40.0 NA** 0.18

Place

Isolation area 1/12 8.3 - -

2/12 16.7 - -

Public display 3/5 60.0 7.2 (0.9 - 53.6) 0.10 2/5 40.0 2.4 (0.5 - 12.6) 0.38

* Prevalence ratio /confidence interval

** Not applicable

n = Total number of seropositive animals

N = Total number of sampled animal

43

FIRST REPORT OF Mycobacterium tuberculosis INFECTION IN BARBARY

SHEEP (Ammotragus lervia) AT THE CURITIBA ZOO, SOUTHERN BRAZIL

ABSTRACT

Tuberculosis is one of the most important world diseases caused by Mycobacterium

tuberculosis complex infecting both humans and animals. A sudden death of a Barbary

sheep in Curitiba zoo, and presence of multifocal nodules in lungs at necropsy aroused

the tuberculosis suspicious. Real-Time Polymerase Chain Reaction (qPCR) from organs

and fluid was performed. qPCR detected M. tuberculosis of a lung sample. This

research reports for the first time M. tuberculosis infection in Barbary sheep, which may

provide a better understanding for species conservation, captivity transmission,

reservoir potential and public health impact to Zoo personal and visitors.

Keywords: Ammotragus lervia, Mycobacterium tuberculosis, zoos.

RESUMO

A tuberculose é uma das doenças mundiais mais importantes causada pelo complexo

Mycobacterium tuberculosis e pode infectar pessoas e animais. A morte repentina de

um aoudad no Zoológico de Curitiba, e a presença de nódulos multifocais no pulmão à

necropsia, levantou a suspeita de tuberculose. Foi realizada a Reação em Cadeia da

Polimerase em Tempo Real (qPCR) de fragmentos de órgãos e fluido. A qPCR detectou

a presença de M. tuberculosis nas amostras de pulmão. Este estudo relata pela primeira

vez a infecção por M. tuberculosis em aoudad, e pode prover um melhor entendimento

sobre conservação de espécies, transmissão em cativeiro, potencial reservatório e

impacto na saúde pública de visitantes e funcionários dos zoológicos.

Palavras-chave: Ammotragus lervia, Mycobacterium tuberculosis, zoológicos.

44

4.1. INTRODUCTION

The introduction of wild species, considered a common practice in zoos, may

characterize significant risks to the local population, since new pathogens can be

introduced to a in theory, controlled environment (Yesilbag, Alpay e Karakuzulu,

2011). Wildlife may represent a massive and regularly unknown reservoir system that

can potentially become a font to emerging or reemerging diseases, particularly zoonoses

(Chomel, Belotto e Meslin, 2007).

Tuberculosis is one of the most important diseases in the world and annually

more than nine million human cases are reported (Who, 2012). This infection has been

reported in several mammals, including wild captive animals, and a common finding in

these cases is the exposure to domestic animals or humans that have the disease

(Kaneene et al., 2010). Mycobaterium tuberculosis is considered primarily a human

pathogen. It has been reported in domestic and wildlife species living in close contact

with humans (Michalak et al., 1998; Montali, Mikota e Cheng, 2001) and also in free-

ranging wildlife (Alexander et al., 2002). Additionally, animals can be also a potential

source of M. tuberculosis infection for humans.

Some species, such as the Barbary sheep (Ammotragus lervia) have the potential

to act as reservoirs for important infectious diseases (Candela et al., 2009). It is an

African species with some unique primitive features, grouped into suborder Ruminantia,

subfamily Caprinae, which includes the domesticated genera Ovis and Capra. A closer

phylogenetic connection between the genera Ammotragus and Capra was recently

observed (Mereu et al., 2008).

The role of Barbary sheep as a pathogenic reservoir to humans remains to be

fully established, but previous studies have described that they can be host or reservoir

45

and spreading factor of M. tuberculosis complex, and have shown high prevalence of

antibodies against M. bovis and M. avium ssp. paratuberculosis (Candela et al., 2009;

Munster et al., 2013). The agent investigation in captive animals is very important, once

humans can be infected and develop the disease, even without close contact with

infected animals (Akkerman et al., 2014). Besides that, Barbary sheep is mostly

involved with co-infections (Candela et al., 2009) and more research about infectious

agents can indicate the real health status of the zoo, also warning about the risks to all

the staff and visitors.

The present study reported the first (to the best of the author’s knowledge)

documented infection of M. tuberculosis in Barbary sheep at Curitiba zoo, southern

Brazil.


Curitiba zoo, which was established in 1982, is among the largest Brazilian zoos

with approximately 2300 animals of 300 species, several of which were born in

captivity, distributed across 530 square meters area (Javorouski e Biscaia, 2012).

Curitiba (25°25'47"S, 49°16'19"W) is the capital of Paraná state, in southern Brazil, and

is the eighth most populous city in Brazil, with an estimated population of 1,751,907

inhabitants in 2010 (Ibge, 2010).

In April 2013, an apparently healthy eleven year-old male captive-born Barbary

sheep died without any specific clinical sign. The animal was kept in an isolation area

with another four healthy males. Postmortem examination revealed multifocal yellowish

white nodules in lungs and enlarged mediastinal lymph nodes with multiple nodules.

46

Additionally, it was found a significant volume of free sero-sanguineous fluid in the

thoracic cavity. Multiple organs fragments were fixed in formaldehyde 10% and sent to

histological exam. It was observed pronounced diffuse neutrophilic inflammatory

infiltrate within alveoli and bronchioles, associated with multifocal necrosis areas and

marked autolysis. Morphological diagnosis was suppurative bronchopneumonia

associated with necrosis areas, and it was not possible to completely rule out the

development of an infectious process, such as tubercles. Lungs, lymph nodes, nodules,

liver fragments and fluid were sent to Central Laboratory of the Paraná State. DNA

extraction and purification were performed using the automated platform Nuclisens

easyMAG (bioMérieux, Boxtel, Netherlands). During the extraction procedure, 20 µl of

plasmid is added to each specimen in order to provide the target for the inhibition

control system. Using molecular techniques, Real-Time Polymerase Chain Reaction

(qPCR) (MTB Q - PCR Alert Kit, Nanogen Advanced Diagnostics®), M. tuberculosis

was detected of the lung tissue sample. qPCR was used to detect the IS6110 region,

which is specific for M. tuberculosis complex (Eisenach et al., 1990). The ABI Prism

7500 (Applied Biosystems, Foster, CA, USA) was used as defined by the

manufacturer´s instructions.


M. tuberculosis was detected of the lung tissue sample. The importance of this

pathogen occurrence in zoos or animal parks is growing and is emphasized by the

difficulty of replacing some rare and endangered species, economic losses and risks to

public health (Thoen et al., 2009). Furthermore, sometimes there is a reluctance to

47

publicize findings due to public relations impacts and loss of revenue (Kaneene et al.,

2010). The present study has revealed occurrence of M. tuberculosis in Barbary sheep,

an exotic species, however this zoonotic agent has already infected tapirs in the same

Brazilian zoo in 2006, the first report of RD Rio M. tuberculosis infecting wildlife

(Murakami et al., 2012). Not surprising, animals particularly of wild species, are

thought to be reservoirs for more than 70% of all emerging infections (Kuiken et al.,

2005; Chomel, Belotto e Meslin, 2007), despite the efforts to control such infections in

zoo facilities, through the adoption of preventive measures like restricted access to the

employees, use of personal protective equipment, exclusive material of each enclosure

and quarantine.

Reports of zoos wild species that could serve as host or reservoir for M.

tuberculosis have been greatly increased. Some of these species include bonobos

(Akkerman et al., 2014), chimpanzees, Asian elephants (Charlesworth et al., 2013),

Brazilian tapir (Michel et al., 2003), antelopes, capuchin, vervet monkeys (Michel et al.,

2013), Rocky Mountain goats, black rhinoceros (Oh et al., 2002). It is important to

emphasize that clinical signs are only rarely apparent in wild animals (Kaneene et al.,

2010), which makes diagnosis more difficult. Additionally, treatment of infected

wildlife has been limited to animals in captivity and is not recommended for animals

that cannot be monitored.

Considering that the infected captive-born Barbary sheep was kept in an non-

visiting and isolation area, the probable infection source and zoonotic transmission may

have been the animal keepers and veterinary staff, playing as carriers of M. tuberculosis

from other zoo areas. All keepers and veterinary staff are regularly submitted to

occupational periodic exams, which include infectious diseases tests, and there is no

report of anyone positive for tuberculosis in the data collected.

48

In conclusion, the present study has shown that Barbary sheep in the Curitiba

zoo, Paraná, southern Brazil, may also be infected by M. tuberculosis. This occurrence

may provide a better understanding for species conservation, captivity transmission,

reservoir potential and public health impact to Zoo personal and visitors.

49

4.4. REFERENCES


ABNT style.

AKKERMAN, O. W. et al. Infection of great apes and a zoo keeper with the same

Mycobacterium tuberculosis spoligotype. Med Microbiol Immunol, v. 203, n. 2, p.

141-4, 2014.

ALEXANDER, K. A. et al. Mycobacterium tuberculosis: an emerging disease of free-

ranging wildlife. Emerg Infect Dis, v. 8, n. 6, p. 598-601, 2002.



Dis, v. 28, n. 5, p. 481-9, 2009.

CHARLESWORTH, K. E. et al. Bug Breakfast in the Bulletin: Diagnosis, investigation

and management of tuberculosis at an Australian zoo. N S W Public Health Bull, v. 24,

n. 1, p. 49, 2013.



EISENACH, K. D. et al. Polymerase chain reaction amplification of a repetitive DNA

sequence specific for Mycobacterium tuberculosis. J Infect Dis, v. 161, n. 5, p. 977-81,

1990.



30.


Curitiba: 2012.

KANEENE, J. B. et al. Tuberculosis in wild animals. Int J Tuberc Lung Dis, v. 14, n.

12, p. 1508-12, 2010.


5741, p. 1680-1, 2005.




50

MICHALAK, K. et al. Mycobacterium tuberculosis infection as a zoonotic disease:

transmission between humans and elephants. Emerg Infect Dis, v. 4, n. 2, p. 283-7,

1998.

MICHEL, A. L. et al. Mycobacterium tuberculosis at the human/wildlife interface in a

high TB burden country. Transbound Emerg Dis, v. 60 Suppl 1, p. 46-52, 2013.

MICHEL, A. L. et al. Mycobacterium tuberculosis infections in eight species at the

National Zoological Gardens of South Africa, 1991-2001. J Zoo Wildl Med, v. 34, n. 4,

p. 364-70, 2003.

MONTALI, R. J.; MIKOTA, S. K.; CHENG, L. I. Mycobacterium tuberculosis in zoo

and wildlife species. Rev Sci Tech, v. 20, n. 1, p. 291-303, 2001.

MUNSTER, P. et al. Distribution of Mycobacterium avium ssp. paratuberculosis in a

German zoological garden determined by IS900 semi-nested and quantitative real-time

PCR. Vet Microbiol, v. 163, n. 1-2, p. 116-23, 2013.

MURAKAMI, P. S. et al. Detection of RD(Rio) strain of Mycobacterium tuberculosis

in tapirs (Tapirus terrestris) from a zoo in Brazil. J Zoo Wildl Med, v. 43, n. 4, p. 872-

5, 2012.

OH, P. et al. Human exposure following Mycobacterium tuberculosis infection of

multiple animal species in a Metropolitan Zoo. Emerg Infect Dis, v. 8, n. 11, p. 1290-3,

2002.

THOEN, C. O. et al. Tuberculosis: a re-emerging disease in animals and humans. Vet

Ital, v. 45, n. 1, p. 135-81, 2009.

WHO. World Health Organization report: Global tuberculosis report 2012., 2012.

Available from: < http://www.who.int/tb/publications/global_report/en/ >. Cited 2014

Oct 20.



2011.

http://www.who.int/tb/publications/global_report/en/

51

SEROLOGICAL SURVEY OF ANTI-Leptospira spp. ANTIBODIES IN

BARBARY SHEEP (Ammotragus lervia) AT THE CURITIBA ZOO, SOUTHERN

BRAZIL

ABSTRACT

Leptospirosis is a worldwide zoonosis, affecting humans, domestic and wild animals.

The present study aimed to evaluate prevalence of anti-Leptospira spp. antibodies in

Barbary sheep at the Curitiba zoo. Microscopic Agglutination Test (MAT) was

performed using 17 serogroups. Antibodies against Leptospira spp. were observed in

23.53% samples and Icterohaemorrhagiae was the only prevalent serogroup. The

presence of anti-Leptospira antibodies in Barbary sheep indicate exposure to

leptospires, thus monitoring and preventive measures are necessary in zoo’s captive

animals, since they can act as sentinels of environmental exposure in an area with high

movement of persons.

Key words: Leptospirosis, Ammotragus lervia, zoo.

RESUMO

A leptospirose é uma zoonose mundial que afeta seres humanos, animais domésticos e

selvagens. O presente estudo objetivou avaliar a prevalência de anticorpos anti-

Leptospira spp. em aoudads do zoológico de Curitiba. Foi realizado o teste de

Soroaglutinação Microscópica (SAM) utilizando 17 sorogrupos. Anticorpos contra

Leptospira spp. foram observados em 23,53% das amostras de aoudads e

Icterohaemorrhagiae foi o único sorogrupo prevalente. A presença de anticorpos em

aoudads indica exposição a leptospiras portanto monitoramento e medidas preventivas

são necessários em animais confinados em zoológicos, uma vez eles podem atuar como

sentinelas de exposição ambiental em uma área com alta circulação de pessoas.

Palavras-chave: Leptospirose, Ammotragus lervia, zoológico.

52

5.1. INTRODUCTION

Leptospirosis is an important worldwide zoonosis transmitted mainly by rats,

Rattus norvegicus, in urban areas (Adler e De La Pena Moctezuma, 2010). It is an acute

infectious disease, which affects humans, domestic and wild animals, causing economic

losses in livestock and of great importance in public health (Faine et al., 1999).

In developing countries, a higher incidence of the disease is related to high

temperatures and rainfall, during specific periods of the year (Tassinari et al., 2008).

Around 10,000 cases of human leptospirosis are reported in Brazil every year (Mcbride

et al., 2005) and case-fatality rates range from 10.0 to 15.0% (Ko et al., 1999) in

patients with classical clinical manifestation (also known as Weil’s syndrome), but can

be as high as 74.0% in patients that develops severe pulmonary hemorrhagic syndrome

(Gouveia et al., 2008). Curitiba has reported an average of 135 human leptospirosis

cases per year in the past five years (Sinan, 2014) and is among the cities with the

highest human mortality rates from leptospirosis in Paraná state (Sms, 2011).

Wildlife may act as reservoirs to specific serogroups of Leptospira and the

exposure to captive wild animals at zoos can be an important source of zoonotic

infection (Chomel, Belotto e Meslin, 2007). Free-ranging wild animals are exposed to a

huge variety of Leptospira serogroups and usually exhibit anti-Leptospira antibody

titers to serogroups native of its areas. However, in captivity, the common environment

of a wide range of potential reservoirs and hosts may result in exposure to a variety of

serogroups (Luna-Alvarez et al., 1996; Ullmann, Hoffmann, et al., 2012).

Some species, such as the Barbary sheep (Ammotragus lervia) have the potential

to act as hosts, and probably reservoirs for important infectious diseases (Candela et al.,

2009). Previous surveys showed evidence of infection or exposure to pathogens as

53

Mycobacterium bovis, Mycobacterium paratuberculosis, Salmonella spp., Toxoplasma

gondii and Neospora caninum in Barbary sheep (Candela et al., 2009; Morikawa et al.,

2014). Serological monitoring of pathogen exposure in zoo captive animals may help

understanding the susceptibility of these species to infectious agents as well as a guide

to preventive measures considering the high circulation of human visitors. Thus, the

present study aimed to evaluate prevalence of anti-Leptospira spp. antibodies in Barbary

sheep at the Curitiba zoo.


Curitiba zoo is among the largest Brazilian zoos with approximately 2300

animals of 300 species, distributed across 530 square meters area (Javorouski e Biscaia,

2012). All 17 captive-born Barbary sheep, otherwise healthy animals, from Curitiba zoo

were included in the present study. Each animal was uniquely identified and individual

information such as age, sex and housing location were collected. Blood samples were

obtained by jugular venipuncture. Serum was obtained by centrifugation at 3000 rpm

for 5 minutes and stored at -20°C until testing.

Microscopic Agglutination Test (MAT) using a cut-off titer of 100 was

performed (Faine et al., 1999). Seventeen serogroups were tested: Australis (serovars

Australis and Bratislava), Autumnalis (serovars Autumnalis and Butembo), Ballum

(serovar Castellonis), Bataviae (serovar Bataviae), Canicola (serovar Canicola),

Celledoni (serovar Whitcombi), Cynopteri (serovar Cynopteri), Djasiman (serovar

Sentot), Grippotyphosa (serovar Grippotyphosa), Hebdomadis (serovar Hebdomadis),

Icterohaemorrhagiae (serovars Copenhageni and Icterohaemorrhagiae), Javanica

54

(serovar Javanica), Panama (serovar Panama), Pomona (serovar Pomona), Pyrogenes

(serovar Pyrogenes), Sejroe (serovars Hardjo and Wolffi) and Shermani (serovars

Shermani and Tarassovi).

Samples serum were also tested to brucellosis by screening with the Buffered

Acidified Antigen or Rose Bengal Test, and positive samples were confirmed by the

Complement Fixation Test (Alton, Jones e Angus, 1988).

Both laboratory and epidemiological data were entered into an Excel

spreadsheet. Contingency tables for associations were created in Epi Info 7 (Cdc, 2013)

and analyzed in OpenEpi (Dean, Sullivan e Soe, 2013). Prevalence ratios and 95%

confidence intervals were calculated, and Fisher’s exact test was used to compare

proportions between variables at a 5% significance level.


Antibodies against Leptospira spp. were observed in 23.53% (4/17) Barbary

sheep sampled. Prevalence of serologic evidence of exposure to pathogen was stratified

by age, sex and housing condition (public display or isolation area) (Table 2). The mean

age of the sampled animals was 5 years, with a standard deviation of 2.7 years. The

prevalence of anti-Leptospira spp. antibodies in animals aged ≥ 5 years was 0.5 (95%

CI: 0.1 - 3.7) times the observed in animals aged <5 years (p-value = 0.60). None of the

female animals were seropositive (0/7) whereas 40% of males (4/10) presented anti-

Leptospira antibodies. Seroprevalence among the animals housed on public display was

7.2 times higher than those in the isolation area (Prevalence ratio: 7.2; 95% CI: 0.97 –

53.6; p-value = 0.05). All four seropositive animals presented antibodies titer ranging

55

from 100 to 400 for serogroup Icterohaemorrhagiae, and all sera were negative for

brucellosis.

The present study has identified the seroprevalence of 23.53% (4/17) of anti-

Leptospira spp. antibodies in Barbary sheep at the Curitiba zoo. Similar prevalence has

been described in previous studies with captive wild animals in different Brazilian

cities, from zoos and research centers. Several animals species were studied, such as

lion tamarins in Rio de Janeiro (Lilenbaum et al., 2005), New World monkeys in

Salvador (Pinna et al., 2012), llama, crab-eating fox, maned wolf, coatimundi, zebra,

spider monkey (Lilenbaum et al., 2002), yagouaroundi, margay, mountain lion and

jaguar (Lilenbaum et al., 2004) in Rio de Janeiro zoo, and captive neotropical felids in

Foz do Iguaçu (Guerra Neto et al., 2004). Prevalence in these studies ranged from

5.68% to 56.80% and Icterohaemorrhagiae was one of the predominant serovars (Corrêa

et al., 2004; Guerra Neto et al., 2004; Esteves et al., 2005; Lilenbaum et al., 2005;

Pimentel et al., 2009; Pinna et al., 2012; Ullmann, Neto, et al., 2012). Captivity in zoos

may allow contact of wild captive animals to free-ranging wildlife and synanthropic

animals such as rodents, raccoons, opossums and stray dogs due to close proximity of

both urban and sylvatic environments. These animals can be considered reservoirs or

hosts of leptospires (Faine et al., 1999). Particularities of environmental settings where

urban zoos are located can explain the predominance of Icterohaemorrhagiae serogroup

in this study. This serogroup is commonly reported in urban areas of Latin America

affecting humans and animal, carried by Rattus norvegicus (Ko et al., 1999).

The absence of Brucella spp. antibodies was frequently in previous studies with

Barbary sheep (Candela et al., 2009; Munoz et al., 2010), other wild ungulates (e.g.

Capra i. ibex) (Giacometti et al., 1995) and Cervus elaphus (Rhyan et al., 1997),

suggesting these animals are probably not hosts for these pathogens, or a effective

56

health controlling at the zoo.

Curitiba zoo is located in Iguaçu Municipal Park, an area with humid subtropical

climate with an average annual temperature of 16 °C (Ibge, 2010), which gives

favorable conditions to the survival of leptospires in the environment, for several

months (Faine e Stallman, 1982; Langston e Heuter, 2003). Previous studies were

conducted in an adjacent area to the Curitiba zoo, also located in Iguaçu Municipal

Park, called Vila Pantanal, a riverside slum community where human leptospirosis is

considered endemic, where Leptospira spp. seroprevalence was established for owned

dogs and cart horses. The first study showed Leptospira spp. antibody prevalences of

9.2% and 16.0% in sampled dogs, Canicola and Icterohaemorrhagiae as the

predominant serogroups (Morikawa, 2010). An even higher prevalence of antibodies

against Leptospira spp. was observed in cart horses (75.8%), also with

Icterohaemorrhagiae as the most frequent serogroup (80.8%) (Finger et al., 2014). In

this area, poor sanitary conditions and infrastructure, proximity to garbage dump sites,

open sewers and flooding were associated to seroprevalence of leptospirosis (Bier et al.,

2012; Finger et al., 2014). Since Vila Pantanal and Curitiba zoo are both located in

Iguaçu Municipal Park and Iguaçu River go through them, it may be considered that

animals from both places are submitted to the same environmental conditions and risk

factors. Thus, it is possible to expect the occurrence of same serogroup

Icterohaemorrhagiae.

Presence of rats at the Curitiba zoo is a critical problem. Chemical control is

only applied in specific situations. Efforts are focused on preventing and diminishing

conditions for the establishment of sinantropic animals, which can be challenging due to

the management of resident captive animals. Zoo staff is periodically trained to avoid

leaving waste on the ground, keeping disposal containers closed and feeders properly

57

cleaned, with an emphasis on the usage of personal protective equipment, especially in

area prone to flooding inside the zoo.

Due to a small number of animals tested, this study did not have enough

statistical power to detect factors associated with higher prevalence of anti-Leptospira

spp. antibodies in Barbary sheep. Still, it is noteworthy that out of all animals from this

species kept at the zoo, a higher prevalence was found in younger, male and housed in

public display animals. Further studies are necessary to a better understand of these

findings. A borderline significant P value for the association of public housing display

and higher seroprevalence highlights a worrisome situation, where visitors may also be

exposed to higher environmental exposure to this pathogen. Exhibiting captive animals

on public display in open enclosures may be at higher risk of infection, as previously

observed in a seroepidemiology survey of infectious diseases in zoos (De Camps,

Dubey e Saville, 2008).

Although leptospirosis does not appear to be a significant clinical issue for

Barbary sheep at the Curitiba zoo and seropositive animals presented low titers, a

23.53% overall prevalence may indicate environmental exposure to leptospires. This is

particularly important since animals in public display housing had higher

seroprevalence, raising concerns regarding environmental exposures to visitors. This

issue is being address with continuous training to the zoo staff. Serological monitoring

of captive wild animals in zoos may provide a better understanding of health hazards

that ultimately will aid in species conservation by comprehending reservoir potential,

public health impact and on guiding specific preventive measures.

58

5.4. REFERENCES


ABNT style.

ADLER, B.; DE LA PENA MOCTEZUMA, A. Leptospira and leptospirosis. Vet

Microbiol, v. 140, n. 3-4, p. 287-96, 2010.

ALTON, G. G.; JONES, L. M.; ANGUS, R. D. Techniques for the brucellosis

laboratory. Paris: Intitut National de la Recherche Agronomique, 1988. 545p.

BIER, D. et al. Spatial Distribution of Seropositive Dogs to Leptospira spp. and

Evaluation of Leptospirosis Risk Factors Using a Decision Tree. Acta Scientiae

Veterinariae, v. 40, n. 3, p. 1054, 2012.



Dis, v. 28, n. 5, p. 481-9, 2009.


Jul 03.



CORRÊA, S. H. R. et al. Epidemiologia da Leptospirose em animais silvestres na

Fundação Parque Zoológico de São Paulo. Braz J Vet Res Anim Sci, v. 41, p. 189-193,

2004.



94, n. 3, p. 648-53, 2008.




ESTEVES, F. M. et al. Detecção de anticorpos para Leptospira spp. em animais e

funcionários do Zoológico Municipal de Uberaba, MG. Arq Inst Biol, v. 72, n. 3, p.

283-288, 2005.

FAINE, S. et al. Leptospira and Leptospirosis. Melbourne: MedSci, 1999. 272.

FAINE, S.; STALLMAN, N. D. Amended Descriptions of the Genus Leptospira

Noguchi 1917 and the Species L. interrogans (Stimson 1907) Wenyon 1926 an L.



59

biflexa (Wolbach and Binger 1914) Noguchi 1918. International Journal of

Systematic Bacteriology, v. 32, n. 4, p. 461-463, 1982.

FINGER, M. A. et al. Serological and molecular survey of Leptospira spp. among cart

horses from an endemic area of human leptospirosis in Curitiba, southern Brazil Rev

Inst Med Trop Sao Paulo, v. 56, n. 6, p. 473-6, 2014.

GIACOMETTI, M. et al. [Seroepidemiologic investigations in the Alpine ibex (Capra i.

ibex) of Piz Albris in the canton of Grigioni (Switzerland)]. Schweiz Arch Tierheilkd,

v. 137, n. 12, p. 537-42, 1995.

GOUVEIA, E. L. et al. Leptospirosis-associated severe pulmonary hemorrhagic

syndrome, Salvador, Brazil. Emerg Infect Dis, v. 14, n. 3, p. 505-8, 2008.

GUERRA NETO, G. et al. Ocorrência de anticorpos contra Leptospira spp. em felídeos

neotropicais pertencentes ao criadouro de animais silvestres da Itaipu Binacional e ao

Zoológico Municipal Bosque Guarani, Foz do iguaçu, Estado do Paraná. ARS

Veterinaria, v. 20, n. 1, p. 075-080, 2004.



30.


Curitiba: 2012.

KO, A. I. et al. Urban epidemic of severe leptospirosis in Brazil. Salvador

Leptospirosis Study Group. Lancet, v. 354, n. 9181, p. 820-5, 1999.

LANGSTON, C. E.; HEUTER, K. J. Leptospirosis. A re-emerging zoonotic disease.

Vet Clin North Am Small Anim Pract, v. 33, n. 4, p. 791-807, 2003.

LILENBAUM, W. et al. Leptospiral antibodies in wild felines from Rio de Janeiro

Zoo, Brazil. Vet J, v. 168, n. 2, p. 191-3, 2004.

LILENBAUM, W. et al. Leptospirosis antibodies in mammals from Rio de Janeiro

Zoo, Brazil. Res Vet Sci, v. 73, n. 3, p. 319-21, 2002.

LILENBAUM, W. et al. Leptospiral antibodies in captive lion tamarins

(Leontopithecus sp) in Brazil. Vet J, v. 169, n. 3, p. 462-4, 2005.

LUNA-ALVAREZ, M. A. et al. Investigación serológica de leptospirosis en fauna

silvestre mantenida en cautiverio en el zoológico de Chapultepec de la ciudad de

México. Vet Méx, v. 27, n. 3, p. 229-234, 1996.


60

MCBRIDE, A. J. et al. Leptospirosis. Curr Opin Infect Dis, v. 18, n. 5, p. 376-86,

2005.

MORIKAWA, V. M. Estudo sorológico da infecção por Leptospira spp. em uma

area de ocupação irregular e alto risco para a doença em cães em Curitiba, PR.

2010. Disssertação. Universidade Federal do Paraná, Curitiba.

MORIKAWA, V. M. et al. Occurrences of anti-Toxoplasma gondii and anti-Neospora

caninum antibodies in Barbary sheep at Curitiba zoo, southern Brazil. Rev Bras

Parasitol Vet, v. 23, n. 2, p. 255-9, 2014.

MUNOZ, P. M. et al. Spatial distribution and risk factors of Brucellosis in Iberian wild

ungulates. BMC Infect Dis, v. 10, p. 46, 2010.

PIMENTEL, J. S. et al. Inquérito sorológico para toxoplasmose e leptospirose em

mamíferos selvagens neotropicais do Zoológico de Aracaju, Sergipe. Pesq Vet Bras, v.

29, n. 12, p. 1009-1014, 2009.

PINNA, M. H. et al. Detection of anti-Leptospira antibodies in captive nonhuman

primates from Salvador, Brazil. Am J Primatol, v. 74, n. 1, p. 8-11, 2012.

RHYAN, J. C. et al. Survey of free-ranging elk from Wyoming and Montana for

selected pathogens. J Wildl Dis, v. 33, n. 2, p. 290-8, Apr 1997.

SINAN. Brasil. Ministério da Saúde. Leptospirose: Casos confirmados notificados no

Sistema de Informação de Agravos de Notificação. 2014. Available from: <

http://dtr2004.saude.gov.br/sinanweb/tabnet/tabnet?sinannet/lepto/bases/leptobrnet.def.

>. Cited 2014 Nov 6.

SMS. Secretaria Municipal de Saúde de Curitiba, Boletim epidemiológico.

Leptospirose: situação atual em Curitiba. . v. 1, p. 2-3, 2011.

TASSINARI, W. S. et al. Detection and modelling of case clusters for urban

leptospirosis. Trop Med Int Health, v. 13, n. 4, p. 503-12, 2008.

ULLMANN, L. S. et al. Serologic survey for Leptospira spp. in captive neotropical

felids in Foz do Iguacu, Paraná, Brazil. J Zoo Wildl Med, v. 43, n. 2, p. 223-8, 2012.

ULLMANN, L. S. et al. Epidemiology of leptospirosis at Sorocaba Zoo, São Paulo

state, Southeastern Brazil. Pesq Vet Bras, v. 32, n. 11, p. 1174-1178, 2012.

http://dtr2004.saude.gov.br/sinanweb/tabnet/tabnet?sinannet/lepto/bases/leptobrnet.def

61

Table 2. Prevalence of anti-Leptospira spp. antibodies stratified by age, sex and housing

location.

Characteristic Anti-Leptospira spp. antibodies

n/N % PR (CI 95%)** p-value

Age

<5 years 3/10 30.0 - -

≥5 years 1/7 14.3 0.5 (0.1 - 3.7) 0.60

Sex

Female 0/7 0.0 - -

Male 4/10 40.0 NA* 0.10

Housing

Isolation 1/12 8.3 - -

Public display 3/5 60.0 7.2 (0.97 - 53.6) 0.05

** Prevalence ratio (95% confidence interval)

* Not applicable

n = Total number of seropositive animals

N = Total number of sampled animals

62

ISOLATION OF Campylobacter jejuni IN BARBARY SHEEP (Ammotragus lervia)

AT CURITIBA ZOO, SOUTHERN BRAZIL

ABSTRACT

Campylobacter jejuni is the major cause of human campylobacteriosis and a broad

range of animal acts as hosts and reservoirs, however little is known about the

epidemiology of the disease in wild animal populations. Barbary sheep fecal samples

were collected and subjected to bacteriological procedure for isolation and biochemical

identification of Campylobacter spp. A total of 5.8% (1/17) Barbary sheep was positive

for C. jejuni. Barbary sheep may be infected by C. jejuni and might represent a potential

zoonotic risk to humans, especially to the keepers, veterinarians and visitors.

Keywords: Campylobacter jejuni, Barbary sheep, zoos.

RESUMO

Campylobacter jejuni é a maior causa de campilobacteriose e tem uma ampla gama de

hospedeiros e reservatórios animais, porém pouco se conhece sobre a sua epidemiologia

em populações de animais selvagens. Amostras de fezes de aoudads foram coletadas e

submetidas a procedimentos bacteriológicos para isolamento e identificação bioquímica

de Campylobacter spp. Um total de 5,8% (1/17) dos aoudads foi positive para C. jejuni.

Em conclusão, aoudads podem se infectar com C. jejuni representando um potencial

risco zoonótico para os seres humanos, em especial tratadores, veterinários e visitantes.

Palavras-chave: Campylobacter jejuni, aoudads, zoológicos.

63

6.1. INTRODUCTION

One characteristic of many emerging diseases is to have an animal as a host or

reservoir (Cleaveland, Laurenson e Taylor, 2001). About 60% of infectious diseases and

75% of emerging diseases worldwide are zoonotic (Taylor, Latham e Woolhouse,

2001), and identification of pathogen’s sources and transmission routes is required to

guide disease control measures (Batz et al., 2005).

Campylobacter spp. is an important causative agent of acute bacterial enteritis

worldwide (Aarestrup et al., 1997; Engberg et al., 2000), frequently found in nature and

domestic animals (Blaser e Reller, 1981). Epidemiological data have shown that

ingestion of contaminated food or water and direct contact with infected animals are the

most important sources of human disease (Blaser e Reller, 1981).

Campylobacter jejuni is the major cause of human campylobacteriosis and

accounts for approximately 90% of cases (Gillespie et al., 2002). The disease is

characterized by a self-limiting abdominal discomfort, diarrhea and fever, during around

7 days. In rare cases, it can trigger Guillain-Barre syndrome, where severe paralysis and

neurological disorders develops (Altekruse et al., 1999; Moore et al., 2005). Although it

is classified as the third most common foodborne disease in the European Union (Efsa,

2013), transmission routes have not been entirely clarified (Misawa et al., 2000; Aquino

et al., 2010). C. jejuni has a broad range of hosts and reservoirs, including a variety of

mammalian, avian, and other animals (Mccarthy et al., 2007). Studies have suggested

that Campylobacter jejuni infection is common in zoo animals (Luechtefeld, Cambre e

Wang, 1981; Gifford, Shane e Smith, 1985; Misawa et al., 2000).

Barbary sheep (Ammotragus lervia) have the potential to act as hosts and

reservoirs for important infectious diseases (Candela et al., 2009) and this study aimed

64

to evaluate the prevalence of Campylobacter spp. in Barbary sheep from the Curitiba

zoo, to verify their potential role as a reservoir of this pathogen.


All 17 healthy captive-born Barbary sheep from Curitiba zoo were investigated

for the presence of C. jejuni. Animals were both males (58.8%) and females (41.2%%),

from 1 to 11 years of age and kept in either isolation area or public display. Fecal

samples were collected using swabs and placed in tubes with specific means of transport

and maintained under refrigeration between 2°C and 8°C.

Samples were subjected to bacteriological procedure for isolation and

biochemical identification of Campylobacter spp. and subsequent selection of C. jejuni

strains (Castro, Genovez e Scarcelli, 1997; Hunt, Abeyta e Tran, 2001). The suspension

was made in physiological saline and 2 mL was filtered through cellulose ester

membrane (Millipore), with a pore 0,65 μm, using a plastic support (swinex; Millipore)

and sterile syringes. Aliquots of 100 μL of the filtrate were plated on brucella agar

medium (Difco) supplemented with 10% defibrinated sheep blood (ABS). Aliquots of

100 μL (without filtration) were also plated on selective medium (ABS-ATB)

consisting of brucella blood agar supplemented with antibiotic mixture (Dufty, 1967)

consisting of polymyxin B (1,000 UI/L), cycloheximide (20 mg/L), novobiocin

(5mg/L), and bacitracin (15.000UI/L). The plates were incubated at 37°C under

microaerophilic atmosphere (5% CO2), for 48 hours. After that, suspected colonies

were identified by presumptive methods: Gram staining, observation of mobility in dark

65

field microscope and oxidase test. Once characterized Campylobacter genus, hippurate

hydrolysis biochemical test, specific to C. jejuni, was performed.

To characterize the strain of C. jejuni obtained from the animals, we performed

PCR for detection of the hip gene, which encodes the enzyme hipuricase, specific of C.

jejuni species. DNA was extracted from suspensions of isolates of Campylobacter spp.

using a commercial kit (Bacterial Genomic PREP Mini Spin Prep - GE Healthcare),

according to manufacturer's specifications.


A single (5.8%) Barbary sheep was positive for C. jejuni. The positive Barbary

sheep was a one year-old female, kept in an isolation area, with no public access. The

animal was healthy and did not show any clinical signs, suggesting a subclinical

infection. As most animals housed at isolation area, reason for such was to avoid

overcrowding of public display housings.

Positivity for C. jejuni in this study corroborate with previous researches in

which C. jejuni has been isolated of a wide variety of captive wild animals, mostly

healthy, in zoos around the world (Fernandez, Rocha e Trabulsi, 1987; Adesiyun,

Caesar e Inder, 1998; Misawa et al., 2000; Hoar et al., 2007; Stirling et al., 2008; De

Haan et al., 2013; Tel, Bozkaya e Keskin, 2013).

None of the Barbary sheep housed on public display of the zoo were positive. In

Curitiba zoo, as the majority of traditional zoos, visitors have limited access to the

animals. Their housings are surrounded mainly by fences and railings, for health and

safety reasons, reducing consequently the risk of zoonotic transmission of pathogens to

66

visitors.

Since C. jejuni can spread widely through multiple environments such as soil

and natural waters, as a consequence of fecal contamination (Studer, Luthy e Hubner,

1999; Jones, 2001; French et al., 2005), assessment of all animals in the zoo would be

necessary and justified due to proximity of animal enclosures and sharing common

source of drinking water. Natural water sources from the zoo such as lakes should also

be examined, since previous studies reported the presence C. jejuni in water supply

(Stirling et al., 2008).

In scenarios such as zoos, where there is a large concentration of animals,

attention is needed to the conditions that can provide environmental stressors and the

opportunity for horizontal transmission of infectious agents such as overcrowding, lack

of safety on diet preparation, inadequate conditions of temperature and humidity of

housing areas and the frequent presence of humans (Matsusaki et al., 1986).

As campylobacteriosis is considered a foodborne disease, basic food safety

measures on diet preparation should be taken to prevent the infection. In the Curitiba

zoo, all food is prepared following specific rules of safety, hygiene and quality. The

food comes from registered companies with certified to perform their activities and is

stored in a specific room for foodstuffs. There is a single central sector where all food is

prepared. The food distribution is done in a box truck, to each individual animal

enclosure by the keepers. Understanding diseases that may affect wildlife are vital

aspects of zoo management programs. The opportunities of contact between keepers and

diseases carried by zoo animals are an important point of action to avoid the infection of

zoo keepers. Zoos should include on its management programs details on how to

proceed in situations where opportunities of infection occur.

We here report that C. jejuni is also found in Barbary sheep born and kept at

67

zoos. This might represent a risk, especially to the keepers and veterinarians from the

zoo, but possibly to visitors depending on the possibilities of contact in each zoo. The

prevention of campylobacteriosis should focus on following basic rules of food safety

and continuous veterinary surveillance of pathogens in all animals in the zoo, with

constant monitoring of animals, water and food sources and environmental conditions.

Guidance must be given to the public to respect physical barriers and avoid feeding the

animals, preventing human to animal transmission of foodborne pathogens.

68

6.4. REFERENCES


ABNT style.

AARESTRUP, F. M. et al. Antimicrobial susceptibility patterns of thermophilic

Campylobacter spp. from humans, pigs, cattle, and broilers in Denmark. Antimicrob

Agents Chemother, v. 41, n. 10, p. 2244-50, 1997.

ADESIYUN, A. A.; CAESAR, K.; INDER, L. Prevalence of Salmonella and

Campylobacter species in animals at Emperor Valley Zoo, Trinidad. J Zoo Wildl Med,

v. 29, n. 2, p. 237-9, 1998.

ALTEKRUSE, S. F. et al. Campylobacter jejuni - an emerging foodborne pathogen.

Emerg Infect Dis, v. 5, n. 1, p. 28-35, 1999.

AQUINO, M. H. et al. Diversity of Campylobacter jejuni and Campylobacter coli

genotypes from human and animal sources from Rio de Janeiro, Brazil. Res Vet Sci, v.

88, n. 2, p. 214-7, 2010.

BATZ, M. B. et al. Attributing illness to food. Emerg Infect Dis, v. 11, n. 7, p. 993-9,

2005.

BLASER, M. J.; RELLER, L. B. Campylobacter enteritis. N Engl J Med, v. 305, n. 24,

p. 1444-52, 1981.



Dis, v. 28, n. 5, p. 481-9, 2009.

CASTRO, A. G. M.; GENOVEZ, M. E.; SCARCELLI, E. Monitoramento de

Campylobacter spp ao longo da linha de abate de frangos de corte. Arq Inst Biol, v. 64,

p. 21-26, 1997.

CLEAVELAND, S.; LAURENSON, M. K.; TAYLOR, L. H. Diseases of humans and

their domestic mammals: pathogen characteristics, host range and the risk of

emergence. Philos Trans R Soc Lond B Biol Sci, v. 356, n. 1411, p. 991-9, 2001.

DE HAAN, C. P. et al. Multilocus sequence types of environmental Campylobacter

jejuni isolates and their similarities to those of human, poultry and bovine C. jejuni

isolates. Zoonoses Public Health, v. 60, n. 2, p. 125-33, 2013.

DUFTY, J. H. Diagnosis of vibriosis in the bull. Aust Vet J, v. 43, n. 10, p. 433-7,

1967.

69

EFSA. European Food Safety Authority. The European Union summary report on

trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011.

EFSA J, v. 11, p. 3129, 2013.

ENGBERG, J. et al. Prevalence of Campylobacter, Arcobacter, Helicobacter, and

Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation

methods for Campylobacters. J Clin Microbiol, v. 38, n. 1, p. 286-91, 2000.

FERNANDEZ, H.; ROCHA, M. B.; TRABULSI, L. R. Ocorrência de Campylobacter

jejuni em animais do zoológico. Rev Facul Med Vet Zootec Univ Sao Paulo, v. 24, n.

2, p. 239-241, 1987.

FRENCH, N. et al. Spatial epidemiology and natural population structure of

Campylobacter jejuni colonizing a farmland ecosystem. Environ Microbiol, v. 7, n. 8,

p. 1116-26, 2005.

GIFFORD, D. H.; SHANE, S. M.; SMITH, R. E. Prevalence of Campylobacter jejuni in

felidae in Baton Rouge, Louisiana. Int J Zoonoses, v. 12, n. 1, p. 67-73, 1985.

GILLESPIE, I. A. et al. A case-case comparison of Campylobacter coli and

Campylobacter jejuni infection: a tool for generating hypotheses. Emerg Infect Dis, v.

8, n. 9, p. 937-42, 2002.

HOAR, B. M. et al. Evaluation of the enteric microflora of captive whooping cranes

(Grus americana) and sandhill cranes (Grus canadensis). Zoo Biol, v. 26, n. 2, p. 141-

53, 2007.

HUNT, J. M.; ABEYTA, C.; TRAN, T. Campylobacter. Bacteriological analytical

manual. 2001. Available from: < http://www.cfsan.fda.gov/~ebam/bam-7.html >.

Cited 2014 Nov 05.

JONES, K. Campylobacters in water, sewage and the environment. Symp Ser Soc Appl

Microbiol, n. 30, p. 68S-79S, 2001.

LUECHTEFELD, N. W.; CAMBRE, R. C.; WANG, W. L. Isolation of Campylobacter

fetus subsp jejuni from zoo animals. J Am Vet Med Assoc, v. 179, n. 11, p. 1119-22,

1981.

MATSUSAKI, S. et al. Prevalence of Campylobacter jejuni and Campylobacter coli

among wild and domestic animals in Yamaguchi Prefecture. Microbiol Immunol, v.

30, n. 12, p. 1317-22, 1986.

MCCARTHY, N. D. et al. Host-associated genetic import in Campylobacter jejuni.

Emerg Infect Dis, v. 13, n. 2, p. 267-72, 2007.

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MISAWA, N. et al. Isolation of Campylobacter species from zoo animals and

polymerase chain reaction-based random amplified polymorphism DNA analysis. Vet

Microbiol, v. 71, n. 1-2, p. 59-68, 2000.

MOORE, J. E. et al. Campylobacter. Vet Res, v. 36, n. 3, p. 351-82, 2005.

STIRLING, J. et al. Prevalence of gastrointestinal bacterial pathogens in a population

of zoo animals. Zoonoses Public Health, v. 55, n. 3, p. 166-72, 2008.

STUDER, E.; LUTHY, J.; HUBNER, P. Study of the presence of Campylobacter jejuni

and C. coli in sand samples from four Swiss chicken farms. Res Microbiol, v. 150, n. 3,

p. 213-9, 1999.



2001.

TEL, O. Y.; BOZKAYA, F.; KESKIN, O. Salmonella, Campylobacter, and

Chlamydophila in bald ibis (Geronticus eremita) feces in Turkey. J Zoo Wildl Med, v.

44, n. 1, p. 21-6, 2013.

71

DETECTION OF ANTIBODIES AGAINST THE BLUETONGUE VIRUS IN

BARBARY SHEEP (Ammotragus lervia) AT CURITIBA ZOO, SOUTHERN

BRAZIL

ABSTRACT

The bluetongue (BT) is an infectious and noncontagious disease that affects domestic

and wild ruminants, transmitted by some species from the genus Culicoides midges.

Clinical disease is often seen in sheep and the role of wild animals in the BT cycle is

still unclear. Serum samples were tested by the Agar Gel Immunodiffusion (AGID) and

Enzyme Linked Immunosorbent Assay (ELISA). Antibodies were observed in 35.3%

(6/17) Barbary sheep by AGID test and in 41.2% (7/17) by ELISA test. Our findings

demonstrated the occurrence of antibodies against BTV in Barbary sheep, which

indicate that these animals have been exposed and have responded immunologically to

that pathogen.

Keywords: Bluetongue, Barbary sheep, zoos.

RESUMO

A língua azul é uma doença infecciosa e não contagiosa que acomete ruminantes

domésticos e silvestres, transmitida por algumas espécies de mosquitos do gênero

Culicoides. A doença clínica é frequente em ovinos e o papel dos animais silvestres no

ciclo do vírus ainda não está claro. Amostras de soro foram testadas pela Imunodifusão

em Gel de Ágar (IDGA) e Ensaio de Imunoadsorção Enzimática (ELISA). Anticorpos

foram observados em 35,3% (6/17) pela IDGA e em 41,2% (7/17) pelo ELISA. Nossos

resultados demonstraram a ocorrência de anticorpos contra o vírus da língua azul em

aoudads, indicando que esses animais tem sido expostos e respondem

imunologicamente a este patógeno.

Palavras-chave: Língua azul, aoudad, zoológicos.

72

7.1. INTRODUCTION

The bluetongue (BT) is an infectious and noncontagious disease according to the

World Organisation for Animal Health - OIE (Oie, 2014), caused by the bluetongue

virus (BTV), classified in the Orbivirus genus in the family Reoviridae (Mertens et al.,

2005) with at least 26 serotypes reported worldwide to date (Maan et al., 2012).

It is a disease that affects domestic and wild ruminants, transmitted by some

species from the genus Culicoides midges, and considered to be endemic in some

regions of the Middle East, Africa, Australia and America (Yesilbag, Alpay e

Karakuzulu, 2011). The economic and sanitary impact of BT involves more than direct

losses in affected herds with reduced milk yield, infertility and abortion, but also

rigorous restrictions on international trade of animals and their products (Legisa et al.,

2013; Oie, 2014).

Much of Brazil composes the epidemic zone for BT because of this favorable

climatic conditions to vector development (Gibbs e Greiner, 1994). The first report was

in 1978, when it was described antibodies against BTV in sheep and cattle, in São Paulo

state (Silva, 1978). Clinical disease is often seen in sheep and outbreaks with deaths

have occurred in Brazilian states such as Paraná, Rio Grande do Sul and Rio de Janeiro

(Clavijo et al., 2002; Antoniassi et al., 2010; Balaro et al., 2014). Cattle and goats

usually show a subclinical disease (Maclachlan et al., 2009), although acute infections

may occur when the virus emerges in some populations in areas where it is not

generally encountered (Coetzee et al., 2012).

Overall, the role of wild animals in the BT epidemiology is still unclear, even

though the susceptibility of wild ruminants as potential reservoirs is known and raises

the possibility that BTV could spread among captive ruminants housed in zoos (Vilar et

73

al., 2011).

Barbary sheep is an African ruminant, included with genera Capra and Ovis in

the subfamily Caprinae (Mereu et al., 2008). According to some authors, this singular

species have the potential to act as host for important infectious diseases (Candela et al.,

2009) and this study aimed to evaluate the detection of antibodies against the BTV in

Barbary sheep at Curitiba Zoo, southern Brazil.


A total of 17 healthy captive-born Barbary sheep, of both genders and different

ages, were sampled. Each individual was identified by means of an ear tag number and

color, and the individual information gathered included date of birth, gender and

housing location. By means of physical restraint without chemical sedation, blood

samples were collected intravenously and placed in tubes without anti-coagulant. The

material was then centrifuged at 3000 rpm for 5 minutes and the serum was separated

and kept at -20°C until testing. The aliquots were sent to the Biological Institute of São

Paulo, Center for Animal Health Research and Development, for serological tests to be

performed.

Serum samples were tested by the Agar Gel Immunodiffusion (AGID) and

ELISA (Enzyme Linked Immunosorbent Assay), provided by the PANAFTOSA,

performed according to the manufacturer's protocol. All procedures were used following

the OIE recommendations (Oie, 2014). Serum samples were also tested to caprine

arthritis-encephalitis and maedi-visna virus, by the AGID.

74

Laboratory data were inserted into an Excel spreadsheet. Contingency tables for

associations were created in Epi Info 7 (Cdc, 2013) and then analyzed in OpenEpi

(Dean, Sullivan e Soe, 2013). Prevalence ratios and 95% confidence intervals were

calculated, and Fisher’s exact test was used to compare proportions between variables at

a 5% significance level.


All sera were negative to antibodies against caprine arthritis-encephalitis and

maedi-visna virus.

Antibodies against BTV were observed in 35.3% (6/17) Barbary sheep by AGID

test and in 41.2% (7/17) by ELISA test. The prevalence rates of antibodies against

bluetongue virus were evaluated and stratified by age, sex and housing location (public

display or isolation area) (Table 3). The mean age of the sampled animals was 4.5 years,

with a standard deviation of 2.7 years. Thus, a cut-off of 5 years was chosen to

dichotomize the age variable, forming one group of animals aged less than 5 years and

another of animals aged greater than or equal to 5 years.

Using AGID test, the prevalence of BTV in animals aged less than 5 years was

2/10 (20.0%) and 4/7 (57.1%) in animals aged greater than or equal to 5 years, which

corresponded to a prevalence ratio of 2.9 (0.7 - 11.5), higher in the group of animals

aged greater than 5 years, although this difference was not statistically significant

(Fisher p-value = 0.16). The prevalence was 5/7 (71.4%) in females and 1/10 (10.0%) in

males, which corresponded to a prevalence ratio of 0.1 (0.0 – 0.9) and Fisher p-value =

0.03. This difference was statistically significant, so the prevalence in females is higher

75

than in males. The prevalence was 0/5 (0%) in the group of animals housed on public

display and 6/12 (50.0%) in the group of animals housed in the isolation area. Since the

prevalence in one group was zero, it was not possible to calculate the prevalence ratio.

Using ELISA test, the prevalence of BTV in animals aged less than 5 years was

3/10 (30.0%) and 4/7 (57.1%) in animals aged greater than or equal to 5 years, which

corresponded to a prevalence ratio of 1.9 (0.6 – 6.0), higher in the group of animals

aged greater than 5 years, although this difference was not statistically significant

(Fisher p-value = 0.35). The prevalence was 5/7 (71.4%) in females and 2/10 (20.0%) in

males, which corresponded to a prevalence ratio of 0.3 (0.1 – 1.1). This difference was

not statistically significant, but was close to the significance threshold (Fisher p-value =

0.06). The prevalence was 0/5 (0%) in the group of animals housed on public display

and 7/12 (58.3%) in the group of animals housed in the isolation area. Since the

prevalence in one group was zero, it was not possible to calculate the prevalence ratio.

In the present study, two tests were performed to detect antibodies against BTV

and prevalence rates were 35.3% (6/17) by AGID test and 41.2% (7/17) by ELISA test.

Both tests have been used in serological surveys. The AGID test is widely used because

it is simple to perform and the antigens are relatively easy to produce, but is no longer

considered sufficiently accurate for use in the support of international trade. The major

disadvantage of the AGID test is the lack of specificity in differentiating BTV

antibodies to another Orbivirus species, like virus of the epizootic hemorrhagic disease.

Nowadays, ELISA is recommended for BTV antibodies detection. Similar prevalence

rate was found in a previous study with Barbary sheep conducted in USA, where 50%

(6/12) were reactors (Hampy, Pence e Simpson, 1979). In another study realized in

Turkish zoos, none of Barbary sheep was reactor (Yesilbag, Alpay e Karakuzulu, 2011).

Although age and housing location were not statistically significant on both

76

tests, the prevalence in females is statistically higher than in males. Previous researches

usually do not stratify the findings regarding to sex (Lager, 2004; Nogueira et al., 2009;

Dorneles et al., 2012) but in some outbreaks females were most affected (Antoniassi et

al., 2010; Balaro et al., 2014).

Several studies were conducted in zoos worldwide, once these places can be

optimal environments for the transmission of infectious diseases among wild animal

species (Yesilbag, Alpay e Karakuzulu, 2011). Previous survey in 49 zoos in the

northern Europe in 2006/2007 related BT outbreaks (Sanderson, 2010). It is interesting

to point that no clinical signs were reported in any of their African ruminant species and

since BT is native-born in Africa, many of these species are likely to develop a elevated

level of resistance to the disease (Vilar et al., 2011). Accordingly, as Barbary sheep is

also an African ruminant, the present study tried to evaluate how is the participation of

Barbary sheep in the cycle of BTV, although the dynamics of BTV in African ruminants

is not well known (Vilar et al., 2011). Moreover, serological studies from 30 species of

zoos due for importation from Africa into the USA, exposed that 13 species had

antibodies to BTV (House, Groocock e Campbell, 1982), warning to the potential risk

of introducing BTV in a controlled environment as zoo, through animals imported

(Vilar et al., 2011).

The participation of wild ruminants is increasingly in the BT epidemiology and

has been described in further studies, showing prevalence rates more frequently in red

deer (Garcia-Bocanegra et al., 2011; Corbiere et al., 2012). Thus, a complex multi-host

and multi-pathogen of the disease involving 26 different serotypes has recently

emerged, showing two cycles, a wild cycle driven by deer of subfamily Cervinae and a

domestic cycle driven by sheep, cattle and others, which are possibly linked through

Culicoides vectors of many species (Ruiz-Fons et al., 2014). Researches in Brazil

77

involving wildlife found prevalence of 39.0% in free-living collared peccaries (Pecari

tajacu) (Gerber et al., 2012) and 15.6% and 2.8% in lowland tapir (Tapirus terrestris)

from Atlantic Forest and Pantanal, respectively (Medici, Mangini e Fernandes-Santos,

2014).

BT occurrence in Barbary sheep can be compared with occurrences in domestic

sheep which are genetically related to each other (Mereu et al., 2008). In Brazil,

prevalence rates were described in Rio Grande do Sul state (0.16%), lower when

compared to other regions of Brazil, probably attributed to climatic conditions, less

favorable to the propagation vector (Costa et al., 2006), in Mato Grosso do Sul state

(10.9%) (Tomich et al., 2009), in Distrito Federal (52.3%) (Dorneles et al., 2012) and in

São Paulo state (74.1 % and 65.0%) (Nogueira et al., 2009), indicating the virus is

largely distributed in sheep herds in Brazil. The occurrence found in serological tests

without characteristic clinical signs of the disease may indicate that BT is spread

silently in the country.

The BTV distribution is restricted to areas where climatic conditions favor

increased vector population and activity (Wittmann e Baylis, 2000). Curitiba is located

in a humid subtropical climate with an average annual temperature of 16 °C (Ibge,

2010). In 2001, an outbreak of sheep was reported in Curitiba experiencing a severe and

acute disease, where 128 from a flock of 130 sheep were euthanized. Serological

evidence of the presence of BTV was investigated and seroconversion was detected in

70.0% of the animals on the farm (Clavijo et al., 2002), which indicates the circulation

of the vector and hence the disease occurrence.

Although infection is asymptomatic in cattle, they are considered important

reservoirs and amplifying hosts to the BTV because of the long viremic phase, that

usually occurs in the absence of clinical signs, with high viral titers during this period

78

(Barratt-Boyes e Maclachlan, 1994)(Ward, Carpenter e Osburn, 1994). The presence of

cattle housing in the zoo and cattle herds next to the zoo warn of this risk and a

preventive measure would be to test all cattle for BTV.

Our findings demonstrated the occurrence of antibodies against BTV in Barbary

sheep, which indicate that these animals have been exposed and have responded

immunologically to that agent. The absence of clinical manifestations of disease may be

due to differences in susceptibility among races or differences in pathogenicity among

BTV serotypes (Purse et al., 2005) or may indicate that Barbary sheep can act as a BT

host or reservoir. In Brazil the knowledge about the BT epidemiology and their

occurrence in different species, mostly in wild species, still needs further studies. This

research emphasized the need for serological and entomological surveillance systems

particularly into and next to the zoo, with an effective contingency plan in case of

infection is detected.

79

7.4. REFERENCES


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ANTONIASSI, N. A. B. et al. Alterações clínicas e patológicas em ovinos infectados

naturalmente pelo vírus da língua azul no Rio Grande do Sul. Pesq Vet Bras, v. 30, n.

12, p. 1010-1016, 2010.

BALARO, M. F. et al. Outbreak of Bluetongue virus serotype 4 in dairy sheep in Rio

de Janeiro, Brazil. J Vet Diagn Invest, v. 26, n. 4, p. 567-570, 2014.

BARRATT-BOYES, S. M.; MACLACHLAN, N. J. Dynamics of viral spread in

bluetongue virus infected calves. Vet Microbiol, v. 40, n. 3-4, p. 361-71, 1994.



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CLAVIJO, A. et al. Isolation of bluetongue virus serotype 12 from an outbreak of the

disease in South America. Vet Rec, v. 151, n. 10, p. 301-2, 2002.

COETZEE, P. et al. Bluetongue: a historical and epidemiological perspective with the

emphasis on South Africa. Virol J, v. 9, p. 198, 2012.

CORBIERE, F. et al. Bluetongue virus serotype 1 in wild ruminants, France, 2008-10.

J Wildl Dis, v. 48, n. 4, p. 1047-51, 2012.

COSTA, J. R. R. et al. Prevalência de anticorpos contra o vírus da língua azul em

bovinos e ovinos do sudoeste e sudeste do Rio Grande do Sul. Arq Bras Med Vet

Zootec, v. 58, n. 2, p. 273-275, 2006.




DORNELES, E. M. S. et al. Prevalence of bluetongue virus antibodies in sheep

fromDistrito Federal, Brazil. Semina Ci Agrárias, v. 33, n. 4, p. 1521-1524, 2012.

GARCIA-BOCANEGRA, I. et al. Role of wild ruminants in the epidemiology of

bluetongue virus serotypes 1, 4 and 8 in Spain. Vet Res, v. 42, p. 88, 2011.



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GERBER, P. F. et al. Orbivirus infections in collared peccaries (Tayassu tajacu) in

southeastern Brazil. J Wildl Dis, v. 48, n. 1, p. 230-2, 2012.

GIBBS, E. P.; GREINER, E. C. The epidemiology of bluetongue. Comp Immunol

Microbiol Infect Dis, v. 17, n. 3-4, p. 207-20, 1994.

HAMPY, B.; PENCE, D. B.; SIMPSON, C. D. Serological studies on sympatric

Barbary sheep and mule deer from Palo Duro Canyon, Texas. J Wildl Dis, v. 15, n. 3,

p. 443-6, 1979.

HOUSE, J. A.; GROOCOCK, C. M.; CAMPBELL, C. H. Antibodies to bluetongue

viruses in animals imported into United States zoological gardens. Can J Comp Med,

v. 46, n. 2, p. 154-9, 1982.



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LAGER, I. A. Bluetongue virus in South America: overview of viruses, vectors,

surveillance and unique features. Vet Ital, v. 40, n. 3, p. 89-93, 2004.

LEGISA, D. et al. Phylogenetic analysis of bluetongue virus serotype 4 field isolates

from Argentina. J Gen Virol, v. 94, n. Pt 3, p. 652-62, 2013.

MAAN, N. S. et al. Identification and differentiation of the twenty six bluetongue virus

serotypes by RT-PCR amplification of the serotype-specific genome segment 2. PLoS

One, v. 7, n. 2, p. e32601, 2012.

MACLACHLAN, N. J. et al. The pathology and pathogenesis of bluetongue. J Comp

Pathol, v. 141, n. 1, p. 1-16, 2009.

MEDICI, E. P.; MANGINI, P. R.; FERNANDES-SANTOS, R. C. Health assessment of

wild lowland tapir (tapirus terrestris) populations in the atlantic forest and pantanal

biomes, Brazil (1996-2012). J Wildl Dis, v. 50, n. 4, p. 817-28, 2014.



MERTENS, P. P. C. et al. Orbivirus, Reoviridae. In: FAUQUET, C. M.;MAYO, M. A.,

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NOGUEIRA, A. H. C. et al. Detecção de anticorpos contra o vírus da língua azul na

região de Araçatuba, São Paulo, Brasil. Cienc Anim Bras, v. 10, n. 4, p. 1271-1276,

2009.

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Bluetongue virus. 2014.

PURSE, B. V. et al. Climate change and the recent emergence of bluetongue in Europe.

Nat Rev Microbiol, v. 3, n. 2, p. 171-81, 2005.

RUIZ-FONS, F. et al. The role of wildlife in bluetongue virus maintenance in Europe:

lessons learned after the natural infection in Spain. Virus Res, v. 182, p. 50-8, 2014.

SANDERSON, S. Bluetongue in non-domestic ruminants: experiences gained in EAZA

zoos during the 2007 & 2008 BTV8 and BTV1 epizootics. In: (Ed.). Transmissible

Diseases Handbook: Ed J. Kaandorp. European Association of Zoo and Wildlife

Veterinarians (EAZWV), 2010. p.1-10.

SILVA, F. J. F. Estudos de ocorrência da língua azul em São Paulo: Comissão de

Estudos do Ministério da Agricultura. Portaria Ministerial n.150 (relatório) 1978.

TOMICH, R. G. P. et al. Sorologia para o vírus da língua azul em bovinos de corte,

ovinos e veados campeiros no Pantanal sul-mato-grossense. Arq Bras Med Vet

Zootec, v. 61, n. 5, p. 1222-1226, 2009.

VILAR, M. J. et al. Culicoides vectors of bluetongue virus in Chester Zoo. Vet Rec, v.

168, n. 9, p. 242, 2011.

WARD, M. P.; CARPENTER, T. E.; OSBURN, B. I. Host factors affecting

seroprevalence of bluetongue virus infections of cattle. Am J Vet Res, v. 55, n. 7, p.

916-20, 1994.

WITTMANN, E. J.; BAYLIS, M. Climate change: effects on culicoides--transmitted

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2011.

82

Table 3. Prevalence of antibodies against bluetongue virus by AGID and ELISA tests stratified by age, sex and housing location.

Characteristic Bluetongue virus (AGID) Bluetongue virus (ELISA)

n/N % PR (CI 95%) p-value * n/N % PR (CI 95%) p-value *

Age

<5 < 5 years

2/10 20.0 - -

3/10 30.0 - -

≥ 5 years

4/7 57.1 2.9 (0.7 - 11.5) 0.16

4/7 57.1 1.9 (0.6 - 6.0) 0.35

Sex

Female

5/7 71.4 - -

5/7 71.4 - -

Male

1/10 10.0 0.1 (0.0 - 0.9) 0.03

2/10 20.0 0.3 (0.1 - 1.1) 0.06

Housing

Isolation

6/12 50.0 - -

7/12 58.3 - -

Public display 0/5 0.0 NA** 0.10 0/5 0.0 NA** 0.04

* 2-sided Fisher's exact Test

** Not Applicable

n = Seropositive animals

N = Sampled animals

83

8. GENERAL CONCLUSIONS

With the growing understanding of the significant importance of wildlife in the

epidemiological cycle of infectious diseases, as a reservoir or host, there is an increased

recognition of the need to establish an animal surveillance system, especially for

zoonotic pathogens. A better understanding of pathogen occurrence in wildlife species

improves the basis for environmental protection and public health strategies.

In this thesis, the Barbary sheep was used as a model to investigate the

occurrence of important infectious diseases and zoonoses in the Curitiba zoo. The data

revealed that Barbary sheep are exposed to several pathogens such as Toxoplasma

gondii, Neospora caninum, Leptospira spp., bluetongue virus, and therefore may play

an important role in the epidemiological cycle as hosts and sentinels of environmental

exposure, measuring the spreading and circulation of these pathogens into the zoo.

Relative to Mycobacterium tuberculosis and Campylobacter jejuni isolation, the number

of similar cases reported in captive animals in zoos has been greatly increased, and

often neglected. Since humans can be infected and develop these disease, control

strategies must be deployed urgently.

Development of appropriate programs for surveillance system to infectious

pathogens is closely integrated to public health surveillance and provides opportunities

to control such pathogens before they can affect human and animal health, environment,

food supply and economics. The actions should be focused on the recognition of the

pathogen presence and the understanding of its epidemiology, to prevention and control.

Identification of the main risk factors and which animals are potential reservoirs is also

important.

Every zoo must have a specific program to control infectious diseases and

84

zoonoses, according to its environment and requirements. Basically it should include

routine actions to diseases prevention and a strategy plan in case of outbreaks. Also, it is

important to note about the growing need for disease surveillance in free-ranging

wildlife. Lastly, to reduce risk for zoonoses, the public should be aware of the inherent

risks involved in wildlife, so that a program of continuing education is essential in the

zoo.

85

SUPPLEMENTS

SUPPLEMENT 1 – Animal Use Ethics Committee (Certificate) ........................... 86

SUPPLEMENT 2 – Published article: “Occurrences of anti-Toxoplasma gondii and

anti-Neospora caninum antibodies in Barbary sheep at Curitiba zoo, southern

Brazil” ...................................................................................................................... 87

SUPPLEMENT 3 – Biological Institute - Result (Toxoplasma gondii) .................. 92

SUPPLEMENT 4 – Biological Institute - Result (Neospora caninum) .................. 93

SUPPLEMENT 5 – Central Laboratory of the Paraná State – Result (Mycobacterium

tuberculosis) ………………………………………………………………………. 94

SUPPLEMENT 6 – Biological Institute – Result (Leptospira spp.) ....................... 95

SUPPLEMENT 7 – Biological Institute – Result (Campylobacter jejuni).............. 96

SUPPLEMENT 8 – Biological Institute – Result (bluetongue virus) ..................... 98

SUPPLEMENT 9 – Curitiba zoo map ................................................................... 100

86

SUPPLEMENT 1 – Animal Use Ethics Committee (Certificate)

87

SUPPLEMENT 2 – Published article: “Occurrences of anti-Toxoplasma gondii and

anti-Neospora caninum antibodies in Barbary sheep at Curitiba zoo, southern

Brazil”

92

SUPPLEMENT 3 – Biological Institute – Result (Toxoplasma gondii)

93

SUPPLEMENT 4 - Biological Institute – Result (Neospora caninum)

94

SUPPLEMENT 5 – Central Laboratory of the Paraná State – Result (Mycobacterium

tuberculosis)

95

SUPPLEMENT 6 – Biological Institute – Result (Leptospira spp.)

96

SUPPLEMENT 7 – Biological Institute – Result (Campylobacter jejuni)

98

SUPPLEMENT 8 – Biological Institute – Result (bluetongue virus)

SECRETARIA DE AGRICULTURA E ABASTECIMENTO AGÊNCIA PAULISTA DOS AGRONEGÓCIOS

INSTITUTO BIOLÓGICO

LVB REG LPA 07 REF. LVB POP LPA 040

REVISÃO: 01 PÁGINA: 1 DE 1

EMISSÃO: 10/08/12 LABORATÓRIO DE VIROSES DE BOVÍDEOS RESULTADO DE ANÁLISE

TRIAGEM Nº: 0939/13 Data de entrada: 16/05/2013 Horário: 08:20

Proprietário: ZOOLOGICO DE CURITIBA – MATERIAL DE PESQUISA

Município/Estado: CURITIBA / PR

Requisitante: PROF. IVAN BARROS (UFPR)

Espécie animal: AOUDADS

Data da colheita: 13/05/2013

Laboratório registrado no CRMV-SP sob o número 00879/J Responsável Técnica Edviges Maristela Pituco CRMV-SP 4770

Av. Conselheiro Rodrigues Alves, 1252 – Vila Mariana – Cep: 04014-002 São Paulo/SP Fone/Fax: (11) 5087-1786 / 5087-1765 e-mail: [email protected]

1-410 13766 NÃO REAGENTE IMUNODIFUSÃO

Fabricante do kit: Panaftosa Não reagente: Não evidenciado linha de

identidade entre amostra teste e o antígeno. Ensaio realizado na temperatura 22 a 25°C

2-195 13767 NÃO REAGENTE

3-O4 13768 NÃO REAGENTE


5-501 13770 REAGENTE

6-31 13771 REAGENTE


8-42 13773 REAGENTE



11-408 13776 REAGENTE







EXAME: LINGUA AZUL – ANTICORPO MATERIAL: SORO

IDENTIFICAÇÃO DO ANIMAL N°LVB13 RESULTADO MÉTODO

LIBERADO POR: Liria Hiromi Okuda-CRMV-SP 8572

DATA: 25/06/2013 ASS.

99

SECRETARIA DE AGRICULTURA E ABASTECIMENTO AGÊNCIA PAULISTA DOS AGRONEGÓCIOS

INSTITUTO BIOLÓGICO

LVB REG LPA 07 REF. LVB POP LPA 040

REVISÃO: 01 PÁGINA: 1 DE 1

EMISSÃO: 10/08/12 LABORATÓRIO DE VIROSES DE BOVÍDEOS RESULTADO DE ANÁLISE

TRIAGEM Nº: 0939/13 Data de entrada: 16/05/2013 Horário: 08:20

Proprietário: ZOOLOGICO DE CURITIBA – MATERIAL DE PESQUISA

Município/Estado: CURITIBA / PR

Requisitante: PROF. IVAN BARROS (UFPR)

Espécie animal: AOUDADS

Data da colheita: 13/05/2013

Laboratório registrado no CRMV-SP sob o número 00879/J Responsável Técnica Edviges Maristela Pituco CRMV-SP 4770

Av. Conselheiro Rodrigues Alves, 1252 – Vila Mariana – Cep: 04014-002 São Paulo/SP Fone/Fax: (11) 5087-1786 / 5087-1765 e-mail: [email protected]

EXAME: LINGUA AZUL – ANTICORPO MATERIAL: SORO

IDENTIFICAÇÃO DO ANIMAL N°LVB13 RESULTADO MÉTODO


ELISA Kit: PANAFTOSA


3-O4 13768 NÃO REAGENTE



6-31 13771 REAGENTE


8-42 13773 REAGENTE



11-408 13776 REAGENTE


13-304 13778 REAGENTE





LIBERADO POR: Liria Hiromi Okuda-CRMV-SP 8572

DATA: 25/06/2013 ASS.

100

SUPPLEMENT 9 – Curitiba zoo map

Barbary sheep enclosure

universidade federal do paranÁ vivien midori

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