Occurrence of anti-Toxoplasma gondii, Neospora caninum and Leptospira spp. antibodies in opossums (Didelphis spp.) in São Paulo State, Brazil / Ocorrência de anticorpos anti-Toxoplasma gondii, anti-Neospora caninum e antiLeptospira spp. em gambás (Didelphis spp.) no estado de São Paulo, Brasil

Opossum (Didelphis spp.) is an omnivorous marsupial native to the Americas that shows synanthropic behavior in urban areas. Despite its proximity to domestic animals and humans, knowledge of its participation in the epidemiology of some zoonotic agents is substantial. This study aimed to determine the presence of antibodies against Toxoplasma gondii, Neospora spp. and Leptospira spp. in blood samples collected from opossums in 18 municipalities in the state of São Paulo, Brazil, between 2003 and 2008. Blood samples from 343 opossums: Didelphis aurita (n = 256) and Didelphis albiventris (n = 87) were obtained. These were tested to detect antibodies against T. gondii, using the modified agglutination test (MAT-Toto; cutoff ≥ 25); Neospora spp., using the indirect fluorescent antibody test (IFAT; cutoff ≥ 25); and Leptospira spp., using the microscopic agglutination test (MAT-Lepto; cutoff ≥ 100). Frequency of anti-T. gondii, Neospora spp. and Leptospira spp. antibodies were in 22.7%, 1.5% and 3.5%, respectively. The serogroups-serovars of Leptospira spp. presenting positive MAT-Lepto reactions were: AutumnalisButembo; Mini-Mini; Ballum-Castellonis; Icterohaemorrhagiae-Icterohaemorrhagiae; Icterohaemorrhagiae-Copenhageni and Grippotyphosa-Grippotyphosa or Bananal. This study demonstrated that these zoonotic agents are circulating in opossum populations in the state of São Paulo. Therefore, investigations regarding the role of marsupials in the epidemiology of each of these diseases should be conducted, especially to understand the behavior of these animals as zoonosis maintenance hosts.(AU)

O gambá (Didelphis spp.) é um marsupial onívoro nativo das Américas que apresenta comportamento sinantrópico em áreas urbanas. Apesar da sua proximidade com animais domésticos e o homem, o conhecimento da sua participação na epidemiologia de alguns agentes zoonóticos é fundamental. Este estudo objetivou determinar a presença de anticorpos contra Toxoplasma gondii, Neospora spp. e Leptospira spp. em amostras de sangue colhidas de gambás de 18 municípios do estado de São Paulo, Brasil, entre 2003 e 2008. Foram obtidas amostras sanguíneas de 343 gambás: Didelphis aurita (n = 256) e Didelphis albiventris (n = 87). As amostras foram testadas para detecção de anticorpos contra T. gondii, utilizando o teste de aglutinação modificado (TAM-Toxo; ponto de corte ≥ 25); Neospora spp., utilizando a reação de imunofluorescência indireta (RIFI; ponto de corte ≥ 25); e Leptospira spp., utilizando a soroaglutinação microscópica (SAM-Lepto; ponto de corte ≥ 100). As frequências de anticorpos contra T. gondii, Neospora spp. e Leptospira spp. foram 22,7%, 1,5% e 3,5%, respectivamente. Os sorogrupossorovares de Leptospira spp. que apresentaram soropositividade foram: Autumnalis-Butembo; Mini-Mini; Ballum-Castellonis; Icterohaemorrhagiae-Icterohaemorrhagiae; Icterohaemorrhagiae-Copenhageni e Grippotyphosa-Grippotyphosa ou Bananal. Esse estudo demonstrou que esses agentes estão circulando em populações de gambás no estado de São Paulo. Desta forma, investigações que visam determinar o papel dos marsupiais na epidemiologia de cada doença devem ser conduzidas, especialmente visando o entendimento do comportamento desses animais como hospedeiros dessas zoonoses.(AU)

Serologic survey of infectious diseases in populations of maned wolf (Chrysocyon brachyurus) and crab-eating fox (Cerdocyon thous) from Aguas Emendadas Ecological Station, Brazil.

Domestic dogs are reservoirs for many infectious diseases and may represent a potential source of infection for wild canid populations. A serologic investigation of antibodies to Toxoplasma gondii, Neospora caninum, Brucella abortus, and Leptospira spp. was conducted on three maned wolves (Chrysocyon brachyurus) and seven crab-eating foxes (Cerdocyon thous), all free-living, at the Aguas Emendadas Ecological Station (ESECAE), Federal District, Brazil, between February and October 2006. Out of the 10 samples analyzed, eight (80%) were seropositive for T. gondii: 3/3 (100%) of the maned wolves and 5/7 (71.4%) of the crab-eating foxes. None of the animals presented anti-N. caninum, B. abortus, and Leptospira spp. antibodies. This study demonstrated that the wild canid populations at ESECAE presented high exposure to T. gondii and indicated that there is high environmental contamination at the Station, which can be attributed to its proximity to urban zones, the presence of domestic cats in the study area, or the existence of other wild infected felines.

Pathogens of wild maned wolves (Chrysocyon brachyurus) in Brazil.

The maned wolf, Chrysocyon brachyurus, is an endangered Neotropical canid that survives at low population densities. Diseases are a potential threat for its conservation but to date have been poorly studied. We performed clinical evaluations and investigated the presence of infectious diseases through serology and coprologic tests on maned wolves from Galheiro Natural Private Reserve, Perdizes City, Minas Gerais State, southeastern Brazil. Fifteen wolves were captured between 2003 and 2008. We found high prevalences of antibody to canine distemper virus (CDV; 13/14), canine parvovirus (CPV; 4/14), canine adenovirus type 2 (13/14), canine coronavirus (5/11), canine parainfluenza virus (5/5), and Toxoplasma gondii (6/8), along with Ancylostomidae eggs in all feces samples. Antibodies against Leishmania sp. were found in one of 10 maned wolves, and all samples were negative for Neospora caninum. Evidence of high exposure to these viral agents was also observed in unvaccinated domestic dogs from neighboring farms. High prevalence of viral agents and parasites such as CDV, CPV, and Ancylostomidae indicates that this population faces considerable risk of outbreaks and chronic debilitating parasites. This is the first report of exposure to canine parainfluenza virus in Neotropical free-ranging wild canids. Our findings highlight that canine pathogens pose a serious hazard to the viability of maned wolves and other wild carnivore populations in the area and emphasize the need for monitoring and protecting wildlife health in remaining fragments of the Cerrado biome.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul / Diagnóstico sorológico e molecular e bioensaio em camundongo para detecção de Toxoplasma gondii em galinhas de criação livre do Pantanal do Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Os objetivos deste estudo foram investigar a ocorrência de Toxoplasma gondii e comparar os resultados obtidos no Teste de Aglutinação Modificada (MAT), Reação em Cadeia pela Polimerase (PCR) e o bioensaio em camundongos. Para tanto, 40 galinhas de criação livre de oito fazendas em áreas limítrofes ao Pantanal da Nhecolândia, Mato Grosso do Sul, foram eutanasiadas e amostras de sangue, o cérebro e o coração foram coletados. A frequência de anticorpos anti-T. gondii encontrada nas galinhas foi de 67,5% (27 amostras), considerando como ponto de corte a diluição 1:5. Entre as amostras analisadas, 7 (25,9%) foram positivas na diluição 1:5, 3 (11,1%) em 1:10, 2 (7,4%) em 1:20, 3 (11,1%) em 1:320, 1 ( 3,7%) em 1:640, 3 (11,1%) em 1:1.280, 2 (7,4%) em 1:2.560, 4 (14,8%) em 1:5.120 e 2 (7,4%) em 1:10.240. A partir da mistura de amostras de tecidos (cérebro e coração) das galinhas analisadas, 16 (40%) apresentaram bandas eletroforéticas compatíveis com T. gondii por PCR (gene B1). Na comparação das técnicas, revelou-se 59,26% de positividade na PCR entre os animais soropositivos no MAT (ponto de corte 1:5). Dos 141 camundongos inoculados, seis (4,44%) morreram de toxoplasmose aguda entre 15 e 23 dias após a inoculação. Os camundongos que sobreviveram foram sacrificados 74 dias após a inoculação, sendo encontrados 28 cistos nos cérebros de 10 grupos distintos. Das galinhas soropositivas, foram realizados 27 bioensaios e obtidos 11 (40,7%) isolados. Um maior número de isolamentos ocorreu em camundongos que foram inoculados com tecidos de galinhas que tinham altos títulos de anticorpos anti-T. gondii. Infecção crônica em camundongos foi observada em nove grupos (33,3%) de cinco propriedades diferentes. Entre os camundongos que sobreviveram, 25,6% foram positivos para T. gondii no MAT (1:25). Dos camundongos positivos na PCR, 87,5% também foram positivos no MAT. Já entre os camundongos PCR negativos 5,2% foram positivos para T. gondii no MAT. Concluiu-se através deste estudo que a ocorrência de infecção pelo T. gondii nas propriedades rurais estudadas foi alta, que a PCR direcionada ao gene B1, não confirma a viabilidade do parasita, porém pode ser utilizada como método de triagem para a seleção de galinhas infectadas por T. gondii, que os animais com título maior que 10 devem ser priorizados para a seleção de animais para bioensaio, pois para eles, as chances de isolamento do parasita são maiores e que a soroconversão em camundongos infectados experimentalmente não é um bom indicador de isolamento do agente.

Genotyping of Toxoplasma gondii isolates from free range chickens in the Pantanal area of Brazil.

The aim of this paper was to genetically characterize Toxoplasma gondii isolates from free range chickens in regions of Brazilian territory in the state of Mato Grosso do Sul (MS) where T. gondii strains have never been studied. In total, T. gondii isolates from 22 free range chickens were included in this study. Fifty chickens from Eldorado, thirty from Rio Verde and ten from Aquidauana were sampled between January and April 2007. In relation to the genetic diversity of T. gondii isolates from chickens in MS, the magnitude of the diversity in the isolates sampled in this study was comparable to the overall diversity in a composite data set. These 22 isolates in MS revealed 11 genotypes, whereas the 321 isolates ever genotyped in Brazil have revealed 95 genotypes. The values of Simpson's Diversity Index for the whole population of T. gondii isolates in Brazil, the whole population of T. gondii isolates from chickens in Brazil and the population surveyed in this study were 0.97, 0.95 and 0.90, respectively. Seven of the 11 genotypes revealed from chicken isolates from MS are newly described genotypes and six of them each have a single isolate. In conclusion, the results obtained from isolates in MS corroborate previous studies on T. gondii isolates in Brazil, thus confirming their diversity and atypicality. Nonetheless, the applicability of PCR-RFLP markers for epidemiological inferences remains controversial.

Serologic survey for selected infectious diseases in free-ranging Brazilian tapirs (Tapirus terrestris) in the cerrado of central Brazil.

From September 2000 to January 2002, a serologic survey was conducted in a population of free-ranging Brazilian tapirs (Tapirus terrestris) inhabiting Emas National Park and surrounding areas in Goiás state, central Brazil, as part of an ecologic study. Ten tapirs were immobilized with a tiletamine-zolazepam combination, and blood samples were collected. All sera were negative for Leptospira spp., Brucella abortus, and equine infectious anemia; and one of 10 animals was positive for Toxoplasma gondii. This report represents the first serologic survey for selected infectious diseases in a free-ranging population of Brazilians tapirs in central Brazil.

Genetic diversity among Toxoplasma gondii isolates of small ruminants from Brazil: novel genotypes revealed.

Recent studies indicated that Toxoplasma gondii isolates of many domestic animal hosts from Brazil are genetically and biologically different from those in USA and Europe. Despite of high pathogenicity of this parasite to small ruminants, the epidemiology and genetic diversity of T. gondii in these animals are not well understood in Brazil. In this study, a total of 28 T. gondii samples (16 isolates from sheep in São Paulo state, and 12 isolates from goats in the states of São Paulo and Rio Grande do Norte) were genotyped using genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified from these T. gondii isolates. Eight isolates (4 from sheep and 4 from goats) were grouped into the common clonal type BrI lineage. One sheep isolate was grouped to the type BrIII lineage. Five isolates grouped to three previously identified genotypes in Brazil, and 13 isolates grouped to six novel genotypes. Mixed genotype was found in one isolate from goat in São Paulo. No classical clonal Type I, II or III isolates were found, confirming previous reports that these clonal lineages are rare in Brazil. The allele types at the CS3 locus are strongly linked to mouse virulence of the parasite. The results of this study indicate that even though a large number of T. gondii genotypes have been identified from a variety of animal hosts in Brazil, high percentage of new genotypes are continuously identified from different animal species, suggesting extremely high diversity of T. gondii in the population.

Genetic diversity among capybara (Hydrochaeris hydrochaeris) isolates of Toxoplasma gondii from Brazil.

Recent studies indicate that Toxoplasma gondii isolates of many domestic hosts from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. However, little is known about genetics of T. gondii isolates from wild mammals in Brazil. In this study, genotypes of 36 T. gondii isolates from capybaras (Hydrochaeris hydrochaeris) from six counties in São Paulo state, Brazil, were determined. Sixteen genotypes were identified using 11 genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. No classical clonal Type I and Type II isolates were found, confirming other findings that these lineages are rare in Brazil. Eight of these 36 isolates were grouped into the common clonal lineages in Brazil, previously designed as Types BrI, BrII and BrIII. Seven of the 16 genotypes were reported for the first time in this study. Three of the 36 isolates showed mixed infections. Analysis of mortality rates in infected mice indicated that Type BrI is highly virulent, Type BrII is intermediately virulent and Type BrIII is non-virulent, which is in agreement with previous report. The allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. These genotyping results support previous findings that the T. gondii population is highly diverse in Brazil.

Isolation of Toxoplasma gondii from capybaras (Hydrochaeris hydrochaeris) from São Paulo State, Brazil.

The capybara (Hydrochaeris hydrochaeris) is a large rodent used for human consumption in certain areas of South America. In the present study, viable Toxoplasma gondii was isolated for the first time from this host. Antibodies to T. gondii were assayed in the sera of 64 capybaras from 6 counties of São Paulo State, Brazil, using the modified agglutination test (MAT, > or =1:25 dilution) and the indirect fluorescent antibody test (IFAT, > or =1:16 dilution), and antibodies were found in 48 (75%) by MAT, and 49 (76.6%) by IFAT. Samples of brain, heart, and tongue of 40 seropositive capybaras were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenates of 36 capybaras, and the isolates were designated TgCyBrl-36. Most isolates were lethal to mice; 17 of the 36 isolates killed 100% of infected mice, 11 isolates caused mortality in 25-90% of infected mice, and 8 isolates were nonpathogenic to mice. Results indicate that asymptomatic capybaras can harbor mouse-virulent T. gondii, and hence they can serve as a source of infection for humans.

[Risk factors and prevalence of antibodies anti-Neospora caninum in urban and rural dogs from Rio Grande do Sul, Brazil]. / Fatores de risco e prevalência de anticorpos anti-Neospora caninum em cães urbanos e rurais do Rio Grande do Sul, Brasil.

Serum samples from 339 dogs from rural (beef and dairy farms) and urban areas of Municipality of Pelotas, RS, were tested by indirect fluorescent antibody test to verify the presence of antibodies anti-Neospora caninum. Data from dogs took part of an epidemiologic questionnaire to identify possible risk factors associated with the seropositivity. Antibodies anti-N. caninum were found 15.6% (53/339) of serum samples, with 5.5% (6/109) of positive dogs from urban area and 20.4% (47/230) from rural area. The analysis multivariate of the risk factors in showed that rural dogs have 3.5 times more chance of contact with the parasite than urban dogs. Dogs with more than 3-years old have a greater risk (OD = 4.1) to be positive for N. caninum than dogs with less than 3-years. Rural dogs from beef farms presented greater risk (OD = 2.8) to be positive when compared with dogs from dairy farms. Seropositivity rate increased 2.2 times in farms which the carcasses of dead animals and aborted fetus were not appropriately removed. N. caninum is present in the southern region of the Rio Grande do Sul, infecting urban and rural dogs.

Prevalence of Neospora caninum antibodies in cattle from Santarém, Pará, Brazil.

Prevalence of anti-Neospora caninum antibodies was measured in serum samples randomly collected from dairy (40 cows from four farms) and beef cattle (120 animals from 12 farms) from the municipality of Santarém, Pará State, Brazil, calculated by using the Win Episcope 2.0 statistical program. The presence of anti-N. caninum antibodies was determined by indirect immunofluorescence-antibody test with a cut-off value of 1:100. We found that 13 farms (81.25%) showed infection rates above 10%, which indicates widespread distribution of N. caninum in the region. The frequency per animal was 19%. No difference was observed between the prevalence values in dairy and beef animals or between farms, which was probably due to the small number of dairy farms examined. The results confirm, for the first time, the presence of anti-N. caninum antibodies in cattle from Pará State and the necessity to further investigate the epidemiology of N. caninum in the Amazon region.

First isolation and molecular characterization of Toxoplasma gondii from finishing pigs from São Paulo State, Brazil.

Toxoplasma gondii infection is widely prevalent in humans in Brazil. Among the food animals, pigs are considered the most important meat source of T. gondii for infection in humans. In the present study, we report the first isolation of viable T. gondii from finishing pigs in Brazil. Antibodies to T. gondii were found in 49 (17%) of 286 pigs prior slaughter using the modified agglutination test (MAT) at a serum dilution of 1:25. Attempts were made to isolate T. gondii from 28 seropositive pigs. Samples of heart, brain, and tongue from each pig were pooled, digested in acid pepsin, and bioassayed in five mice per pig. Viable T. gondii was isolated from seven pigs; all isolates were lethal for mice. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR revealed that two isolates were Type I and five were Type III. The results indicate that phenotypically and genetically T. gondii isolates from pigs from Brazil are distinct from isolates of T. gondii from pigs in the USA.

Ocorrência de parasitos gastrintestinais em fezes de gatos das cidades de Säo Paulo e Guarulhos / Occurrence of gastrointestinal parasites in feces of cats from the cities of Säo Paulo and Guarulhos

Amostras de fezes foram colhidas de 138 gatos, de idade, sexo e raça variadas, capturados nas ruas das cidades de Säo Paulo e Guarulhos, para determinaçäo da presença de parasitos gastrintestinais. Os animais encontravam-se alojados individualmente no Centro de Controle de Zoonoses dos Municípios de Säo Paulo (107 gatos) e Guarulhos (31gatos). As fezes foram colhidas individualmente e processadas pela técnica de flutuaçäo em soluçäo de sacarose (d=1,203g/cm³). Dentre os protozoários o agente mais freqüente foi o Cystoisospora felis, em 36 gatos (26,09 por cento), seguido pelo Cystoisospora rivolta, em 34 (24,64 por cento), pelo Cryptosporidium parvum, em dois (1,45 por cento) e pelo Sarcocystis spp. em um animal (0,72 por cento). Dentre os helmintos, Toxocara cati foi o de maior ocorrência, com 39 gatos positivos (28,26 por cento), seguido pelo Ancylostoma spp., com 12 gatos positivos (8,70 por cento) e pelo Platynosomun fastosum em dois gatos (1,45 por cento). A presença de proglotes de Dipylidium caninum foi observada em duas amostras, quando da colheita nas gaiolas, dado este certamente subestimado, uma vez que o encontro de cápsula ovígera em fezes é bastante raro, sendo o diagnóstico feito pela observaçäo de proglotes nas fezes frescas. Os parasitos estavam presentes em infecçöes múltiplas em 25 animais (18,12 por cento) sendo as ocorrências mais comuns T.cati com Cystoisospora spp. e T.cati com Ancylostoma spp., ambas com 7,97 por cento de ocorrência (11 amostras)