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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…...………....…….
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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 .................................................................................................
4.2. Material and Methods ..................................................................................
4.3. Results and Discussion ................................................................................
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 .................................................................................................
5.2. Material and Methods ..................................................................................
5.3. Results and Discussion ................................................................................
5.4. References ...................................................................................................
6. ISOLATION OF Campylobacter jejuni IN BARBARY SHEEP
(Ammotragus lervia) AT CURITIBA ZOO, SOUTHERN BRAZIL ...........
6.1. Introduction .................................................................................................
6.2. Material and Methods ..................................................................................
6.3. Results and Discussion ................................................................................
6.4. References ...................................................................................................
7. DETECTION OF ANTIBODIES AGAINST THE BLUETONGUE
VIRUS IN BARBARY SHEEP (Ammotragus lervia) AT CURITIBA
ZOO, SOUTHERN BRAZIL ..........................................................................
7.1. Introduction .................................................................................................
7.2. Material and Methods ..................................................................................
7.3. Results and Discussion ................................................................................
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
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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.
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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.
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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.
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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.
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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.
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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
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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
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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
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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
References organized and formatted using the Endnote software version X4 and
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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.
CANDELA, M. G. et al. Coinfection is an important factor in epidemiological studies:
the first serosurvey of the aoudad (Ammotragus lervia). Eur J Clin Microbiol Infect
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.
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.
DE CAMPS, S.; DUBEY, J. P.; SAVILLE, W. J. Seroepidemiology of Toxoplasma
gondii in zoo animals in selected zoos in the midwestern United States. J Parasitol, v.
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.
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.
KUIKEN, T. et al. Public health. Pathogen surveillance in animals. Science, v. 309, n.
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.
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.
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.
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.
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.
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.
4.2. MATERIAL AND METHODS
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.
4.3. RESULTS AND DISCUSSION
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
References organized and formatted using the Endnote software version X4 and
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.
CANDELA, M. G. et al. Coinfection is an important factor in epidemiological studies:
the first serosurvey of the aoudad (Ammotragus lervia). Eur J Clin Microbiol Infect
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.
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.
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.
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.
KANEENE, J. B. et al. Tuberculosis in wild animals. Int J Tuberc Lung Dis, v. 14, n.
12, p. 1508-12, 2010.
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.
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.
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.
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.
5.2. MATERIAL AND METHODS
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.
5.3. RESULTS AND DISCUSSION
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
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BIER, D. et al. Spatial Distribution of Seropositive Dogs to Leptospira spp. and
Evaluation of Leptospirosis Risk Factors Using a Decision Tree. Acta Scientiae
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CANDELA, M. G. et al. Coinfection is an important factor in epidemiological studies:
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CDC. Atlanta, 2013. Available from: < http://wwwn.cdc.gov/epiinfo/7/ >. Cited 2013
Jul 03.
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.
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.
DE CAMPS, S.; DUBEY, J. P.; SAVILLE, W. J. Seroepidemiology of Toxoplasma
gondii in zoo animals in selected zoos in the midwestern United States. J Parasitol, v.
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.
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.
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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,
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GOUVEIA, E. L. et al. Leptospirosis-associated severe pulmonary hemorrhagic
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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.
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http://www.ibge.gov.br/cidadesat/painel/painel.php?codmun=410690 >. Cited 2014 Oct
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JAVOROUSKI, M. L.; BISCAIA, S. A. Zoológico Municipal de Curitiba 30 anos.
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LILENBAUM, W. et al. Leptospiral antibodies in wild felines from Rio de Janeiro
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LILENBAUM, W. et al. Leptospirosis antibodies in mammals from Rio de Janeiro
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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.
6.2. MATERIAL AND METHODS
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.
6.3. RESULTS AND DISCUSSION
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
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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.
7.2. MATERIAL AND METHODS
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.
7.3. RESULTS AND DISCUSSION
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
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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”
88
89
90
91
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)
97
98
SUPPLEMENT 8 – Biological Institute – Result (bluetongue virus)
SECRETARIA DE AGRICULTURA E ABASTECIMENTO AGÊNCIA PAULISTA DOS AGRONEGÓCIOS
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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
4-404 13769 NÃO REAGENTE
5-501 13770 REAGENTE
6-31 13771 REAGENTE
7-849 13772 NÃO REAGENTE
8-42 13773 REAGENTE
9-307 13774 NÃO REAGENTE
10-45 13775 NÃO REAGENTE
11-408 13776 REAGENTE
12-301 13777 NÃO REAGENTE
13-304 13778 NÃO REAGENTE
14-25 13779 REAGENTE
15-407 13780 NÃO REAGENTE
16-302 13781 NÃO REAGENTE
17-23 13782 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
1-410 13766 NÃO REAGENTE
ELISA Kit: PANAFTOSA
2-195 13767 NÃO REAGENTE
3-O4 13768 NÃO REAGENTE
4-404 13769 NÃO REAGENTE
5-501 13770 REAGENTE
6-31 13771 REAGENTE
7-849 13772 NÃO REAGENTE
8-42 13773 REAGENTE
9-307 13774 NÃO REAGENTE
10-45 13775 NÃO REAGENTE
11-408 13776 REAGENTE
12-301 13777 NÃO REAGENTE
13-304 13778 REAGENTE
14-25 13779 REAGENTE
15-407 13780 NÃO REAGENTE
16-302 13781 NÃO REAGENTE
17-23 13782 REAGENTE
LIBERADO POR: Liria Hiromi Okuda-CRMV-SP 8572
DATA: 25/06/2013 ASS.
100
SUPPLEMENT 9 – Curitiba zoo map
Barbary sheep enclosure