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Felines are definitive hosts of Toxoplasma gondii and can shed oocysts in their feces, contaminating the environment. Sporulated oocysts are highly resistant to the environment and have higher infectivity, which are attributed to many toxoplasmosis outbreaks. The aim of the present study was to evaluate a quantitative polymerase chain reaction (qPCR) technique for the detection of T. gondii oocysts shed by cats. Twelve cats from a previous vaccine experiment were challenged orally with 600 cysts of the TgDoveBr8 strain on day 72. Fecal samples were collected daily using the centrifugal flotation technique, with microscopic examination (Sheather technique) and qPCR for 20 days after the challenge. Cats from all groups shed oocysts in their feces. Five negative cats in the Sheather were positive according to qPCR on the 3rd day post-inoculation (dpi). Oocysts were detected on the 4th dpi using the Sheather; however, there was no statistical difference between the two methods (p=0.1116). In addition, there was no statistically significant difference in oocyst shedding between the groups according to the Sheather technique (p=0.6534) and qPCR (p=0.9670). In conclusion, these results demonstrate that qPCR can be used as an alternative to the Sheather to detect and quantify T. gondii oocysts. Resumo Felinos são hospedeiros definitivos do Toxoplasma gondii e podem eliminar oocistos nas fezes, contaminando o meio ambiente. Oocistos esporulados são altamente resistentes ao meio ambiente com elevada infectividade, sendo atribuído a muitos surtos de toxoplasmose. O objetivo do estudo foi avaliar a reação em cadeia da polimerase quantitativa (qPCR) para a detecção de oocistos de T. gondii eliminados por gatos. Doze gatos de um experimento prévio de vacina foram desafiados por via oral com 600 cistos da cepa TgDoveBr8 no dia 72. Amostras fecais foram coletadas diariamente pela técnica de centrifugo-flutuação seguida de exame microscópico (técnica de Sheather) e qPCR por 20 dias após desafio. Gatos de todos os grupos eliminam oocistos nas fezes. Cinco gatos negativos na técnica Sheather foram positivos de acordo com a qPCR no 3º dia pós-inoculação (dpi). Oocistos foram detectados no 4º dpi no Sheather; entretanto, não houve diferença estatística entre os dois métodos (p=0,1116). Não houve diferença estatisticamente significativa na eliminação de oocistos entre os grupos de acordo com a técnica de Sheather (p = 0,6534) e qPCR (p = 0,9670). Em conclusão, esses resultados demonstram que qPCR pode ser usada como uma alternativa ao Sheather para detectar e quantificar oocistos de T. gondii.

Although leptospirosis has been considered a major concern in urban areas, no study to date has spatially and simultaneously compared both owner and dog serology in households of major cities. Accordingly, the aim of the present study was to assess the seroprevalence of Leptospira antibodies, evaluate associated risk factors and conduct spatial analyses in 565 randomly selected households, which included 597 dog owners and 729 dogs in Londrina, Southern Brazil. Seropositivity by MAT were detected in in 11/597 (1.84%) owners and in 155/729 (21.26%) dogs. The risk factors were evaluated with logistic regression analysis and spatial factors and case distribution were evaluated with kernel density analyses. The sera of 14/155 (9.03%) dogs reacted for more than one serovar with the same titer. Canicola was the most frequent serogroup, detected in 3/11 (27.27%) owners and 76/155 (49.03%) dogs. The highest titer among the owners was 1:3,200 and was detected in the same household with a titer of 1:800 in the dog. Simultaneous owner-dog seropositivity was found in 7/565 (1.23%) households, with three reacted against serogroup Canicola. Positive owners were detected in 4/565 (0.70%) households and positive dogs were detected in 141/565 (24.95%) households. The associated risks of infection for dogs were different from those associated with infection in owners. Risk analyses for Canicola also identified specific factors of infection. Regardless of owner and dog cases were not statistically clustered, the kernel map has shown dog positivity occurrence in the same hot locations and near positive owners. The dependent variable analysis and logit model suggested a greater likelihood of peri-domiciliary contact with Leptospira. In conclusion, exposure to Leptospira infection was significantly higher in dogs than in their owners and human cases spatially overlapped dog cases, implicating dogs as potential environmental sentinels for this disease. In addition, the associated risk may vary according to serogroup, and the observed simultaneous Canicola seropositivity of owner and dog has suggested intradomicile-transmitted infection.

Neospora caninum is an obligate intracellular protozoan parasite that infects domestic and wild animals. Canids are considered to be definitive hosts since they may shed oocysts into the environment through their feces. The disease is recognized as one of the major causes of bovine abortion worldwide, leading to important economic losses in the dairy and beef cattle industries. Previous studies have reported N. caninum infection in different species of birds; infection in birds has been associated with increased seroprevalence and reproductive problems in dairy cattle. Although the role of birds in the epidemiological cycle of neosporosis is unknown, birds are exposed to infection because they feed on the ground and could thus contribute to parasite dissemination. This review is focused on the current state of knowledge of neosporosis in birds.

Toxoplasma gondii is an intracellular parasite that can infect all warm-blooded animals including humans. Recent studies showed that T. gondii strains from South America are genetically diverse. The present work aimed to determine T. gondii prevalence in free-ranging chicken in northwest Parana state in Brazil by two serological tests, to isolate the parasites from seropositive chickens and to genotype the isolates. Antibodies to T. gondii in 386 serum samples from 24 farms were investigated by immunofluorescence antibody assay (IFA) and modified agglutination test (MAT). Samples having titers ≥ 16 were considered positive for both tests. Among the 386 serum samples, 102 (26.4%) were positive for IFA, 64 (16.6%) were positive for MAT, 47 (12.2%) were positive in both tests, and 119 (30.8%) were positive in at least one of the two tests. Brain and pool of heart, lung, and liver from the 119 seropositive chickens were used for mouse bioassay to isolate the parasites. Thirty eight (31.9%) of these seropositive chickens were considered positives in mouse bioassay and 18 isolates were obtained. The isolates were characterized by 10 PCR-RFLP genetic markers including SAG1, SAG2 (5'-3'SAG2, alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Results of genotyping were compared with the genotypes in ToxoDB database. It revealed ten genotypes, including ToxoDB PCR-RFLP genotypes #6 (n = 2), #19 (n = 1), #21 (n = 2), #111 (n = 2), #152 (n = 1), and #175 (n = 1) and four new types not described before. Our results confirmed a high genetic diversity of this parasite in southern Brazil and also showed that the use of two serological tests in combination can improve the chance of T. gondii isolation. More studies should be taken to determine the zoonotic potential of chickens in the transmission of T. gondii.

Neosporosis is an infectious disease caused by Neospora caninum, a protozoan parasite that has worldwide distribution and is responsible for enormous economic losses in cattle. Birds are considered a good bioindicator of environmental contamination, since they feed on the ground, being exposed to N. caninum oocysts. The aim of this study was to determine the occurrence of antibodies against N. caninum and to verify the presence of parasite DNA in brain from free-ranging eared doves (Zenaida auriculata) from Southern Brazil. For this purpose, blood and brain samples were collected from 249 doves for ELISA and PCR analysis respectively. The prevalence of N. caninum antibodies in doves was 31.72% (79/249) and detection of parasite DNA was not observed in none of birds. This is the first report of antibodies against N. caninum in doves Z. auriculata, what show us that these birds had previously contact with the parasite but since no N. caninum DNA was detected, more studies should be performed to elucidate the real importance of doves in the epidemiologic cycle of the N. caninum.

The aim of the present study was to investigate the occurrence of N. caninum associated with abortions of dairy cattle from Santa Catarina state, southern Brazil by using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and polymerase chain reaction (PCR). Blood from dairy cows that aborted along with intrathoracic fluid and tissue samples (brain, heart, liver, and lung) from their fetuses were collected and used for serology; PCR, histopathological, and immunohistochemistry (IHC) evaluations were also conducted. Twenty-one cows (51.2%) out of 41, and eight fetuses (26.7%) out of 30 were ELISA (HerdCheck, IDEXX) positive for N. caninum. Dams > 36 months of age had a higher risk of being serum positive than younger animals. PCR and IHC revealed that 38.8% (14/36) and 25.0% (9/36) of the fetuses were positive for N. caninum, respectively for each of the tests. Seropositive cows had a higher frequency of fetuses that were also positive by either intrathoracic fluid, PCR, or IHC. In summary, the present study observed a high frequency of N. caninum in abortions from dairy cows from southern Brazil, with a higher N. caninum prevalence found in cows that were older than 36 months. In addition, serology, PCR, and IHC should be used all together for better diagnosis of neosporosis in cattle. Resumo O objetivo deste estudo foi avaliar a ocorrência de N. caninum associado a abortamentos em vacas de leite do estado de Santa Catarina, sul do Brasil pelo uso das técnicas de ELISA (HerdCheck, IDEXX), reação em cadeia pela polimerase (PCR) e imunohistoquímica (IHC). O sangue das vacas leiteiras que abortaram, bem como, o líquido intratorácico e amostras de tecidos (cérebro, coração, fígado e pulmão) de seus fetos foram coletados e usados para sorologia, PCR (Np21+ e Np6+), e IHC. Vinte e uma vacas (51,2%) de um total de 41, bem como, oito fetos (26,7%) de um total de 30 foram positivos no ELISA (IDEXX) para N. caninum. As vacas > 36 meses de idade tiveram um maior risco de serem soropositivas do que os animais mais jovens. PCR e IHC revelaram que 38,8% e 25,0% dos fetos foram positivos para N. caninum, respectivamente para cada um dos testes. As vacas soropositivas tiveram uma maior frequência de fetos que também foram positivos no fluído intratorácico, na PCR ou na IHC. Em resumo, o presente estudo observou uma alta frequência de N. caninum em abortos de vacas leiteiras na região estudada, com maior prevalência de N. caninum em vacas com mais de 36 meses de idade. Além disso, sorologia, PCR e IHC deveriam ser utilizadas conjuntamente para melhor diagnóstico de neosporose em bovinos.

The present study aimed to isolate and genotype strains of T. gondii from pigs slaughtered for human consumption in South Brazil. Blood and tissues (heart, diaphragm, liver, tongue, and masseter) from 400 animals were collected at two slaughterhouses. Sera were obtained, and antibodies against T. gondii were detected by both indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT). The tissues of animals that tested positive in MAT, IFAT, or both (cut-off ≥ 64) were bioassayed. Twenty-six (6.5%) of the 400 animals were positive by serology. A total of 18 (69.2%) out of those 26 were positive in the mouse bioassay. The isolates were characterized by using 10 PCR-RFLP genetic markers. Fourteen isolates were fully genotyped, and four isolates were genotyped using nine of the 10 markers. All isolates belonged to ToxoDB PCR-RFLP genotype #206. The present study reports on genotype #206 in pigs for the first time, and it confirms the atypical nature of the Brazilian T. gondii isolates. Additionally, even with low levels of antibodies detected in pig herds, pork presents a T. gondii infection risk for humans.

The objective of this study was to determine factors associated with vegetable contamination with zoonotic protozoan. Samples of water, soil and vegetables were collected from July/2014 to May/2016, totaling 83 samples, 21 properties of Londrina region, Paraná, Brazil. DNA amplification of Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis in the samples was conducted using polymerase chain reaction (PCR). The PCR results were positive for T. gondii in 12.9% (8/62), Cryptosporidium spp. in 11.3% (7/62) and G. intestinalis in 25.8% (16/62) of the samples. DNA sequencing identified C. parvum in five samples and G. intestinalis Assemblage E in three. The statistical associations demonstrated greater probability of positive samples for T. gondii and for at least one of the three protozoa when the source of irrigation water was the river; a greater chance of positive samples for Cryptosporidium spp. when deer were present on the property; and a smaller chance of positive samples for at least one of the three etiologic agents when soil was supplemented with limestone. The results expose some critical contamination points, providing support for training farmers on good management practices during the production process. Resumo O trabalho teve como objetivo determinar os fatores associados à contaminação de vegetais por protozoários zoonóticos. Amostras de água, solo e vegetais foram coletadas de julho/2014 a maio/2016, totalizando 83 amostras de 21 propriedades da região de Londrina, Paraná, Brasil. A amplificação de fragmentos de DNA de T. gondii, Cryptosporidium spp. e Giardia intestinalis foi realizada por meio da reação em cadeia da polimerase (PCR). Os resultados da PCR foram positivos para T. gondii em 12,9% (8/62), Cryptosporidium spp. em 11,3% (7/62) e G. intestinalis. em 25,8% (16/62) das amostras. O sequenciamento de DNA identificou C. parvum em cinco amostras e G. intestinalis, Assemblage E em três amostras. As associações estatísticas evidenciaram maior probabilidade de amostras serem positivas para T. gondii ou para pelo menos um dos três protozoários quando a fonte de água de irrigação era o rio; uma maior chance de amostras positivas para Cryptosporidium spp. quando havia cervos na propriedade; e uma menor chance das amostras serem positivas para pelo menos um dos três agentes etiológicos quando o solo era suplementado com calcário. Os resultados expõem alguns pontos críticos de contaminação, fornecendo suporte para capacitar os agricultores em boas práticas de gestão durante o processo de produção.

EnglishThe present study aimed to investigate an abortion outbreak in a dairy goat herd in the municipality of Arapoti, Parana, Brazil. At the beginning of the outbreak, blood samples were collected from 33 goats with clinical signs; later, of the whole goat herd, two cats and two dogs. Milk samples were collected from 78 lactating goats. Four environmental soil samples and four samples of feed residue from goat feeders were collected too. Immunofluorescence antibody test (IFA) was used for serodiagnosis, the molecular analysis was conducted by means of the polymerase chain reaction (PCR), for the isolation of the etiological agent the bioassay was used. The results of the IFA revealed that 76.53% (137/179) of the goats, two dogs and two cats were seropositive for Toxoplasma gondii. Bioassay revealed one buffy coat and two milk sample having viable T. gondii. In the PCR, 11 whole blood samples, eight milk, three feeder troughs, and all soil samples were positive. The findings of the present study confirmed an outbreak caused by environmental contamination (of soil and feed) with T. gondii oocysts that could have been shed by kittens that lived on the farm and had access to the stock of goat food, facilitating this contamination, which reinforces the need for veterinary assistance and good management practices on farms. Key words: toxoplasma; goats. IFA; PCR; bioassay. portuguesO presente estudo teve como objetivo investigar um surto de aborto em um rebanho de cabras leiteiras no municipio de Arapoti, Parana, Brasil. No inicio do surto, foram coletadas amostras de sangue de 33 cabras com sinais clinicos; mais tarde, de todo o rebanho caprino, dois gatos e dois cachorros. Foram obtidas amostras de leite das 78 cabras em lactacao. Quatro amostras ambientais de solo e quatro de residuos de comedouro tambem foram coletadas. O teste de imunofluorescencia (IFI) foi utilizado para o sorodiagnostico, a analise molecular foi conduzida por meio da reacao em cadeia da polimerase (PCR), para isolamento do agente etiologico utilizou-se o bioensaio. Os resultados da IFI revelaram que 76,53% (137/179) das cabras, todos os caes e gatos eram soropositivos para Toxoplasma gondii. O bioensaio revelou uma amostra de camada leucocitaria e duas de leite contaminadas com T. gondii viavel. Na PCR, 11 amostras de sangue total, oito de leite, tres residuos alimentares e todas as amostras de solo foram positivas. Os resultados do presente estudo confirmaram um surto causado por contaminacao ambiental (de solo e alimentos) com oocistos de T. gondii que, provavelmente, foram eliminados por gatos que permaneceram na fazenda e tinham acesso ao estoque de alimento dos caprinos, reforcando a necessidade de assistencia tecnica veterinaria e boas praticas de manejo. Palavras-chave: toxoplasma; cabras. IFI; PCR; bioensaio.

Made available in DSpace on 2015-11-03T15:29:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-10-01. Added 1 bitstream(s) on 2015-11-04T10:14:47Z : No. of bitstreams: 1 S1984-29612014000400443.pdf: 604594 bytes, checksum: f2dd2f1f3e36bfd53ab7d2708b790958 (MD5) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Pombos silvestres (Zenaida auriculata), comuns em áreas urbanas, rurais e selvagens em muitas regiões do Brasil, são frequentemente predados por gatos domésticos. Sendo assim, os isolados de T. gondii obtidos de pombos podem refletir uma maior diversidade ambiental do que os outros hospedeiros. O objetivo do presente estudo foi avaliar a soroprevalência, isolar e genotipar T. gondii de Z. auriculata. Amostras de soro e tecido foram coletadas de 206 pombos para o teste de aglutinação modificado (MAT) e o bioensaio em camundongos. A prevalência de anticorpos contra T. gondii em pombos foi 22,3% (46/206), com títulos variando de 16 a 4096, e T. gondii foi isolado de 12 pombos. Cinco genótipos foram detectados por PCR-RFLP, incluindo os genótipos ToxoDB #1, #6, #17, #65 e um genótipo não descrito anteriormente (ToxoDB#182). Esse é o primeiro relato de isolamento de T. gondii de Z. auriculata. Este estudo também confirmou a diversidade dos isolados de T. gondii e a presença de tipo clonal II (ToxoDB #1) no Brasil. Eared doves (Zenaida auriculata), which are common in urban, rural and wild areas in many regions of Brazil, are frequently prey for domestic cats. Therefore Toxoplasma gondii isolates obtained from doves may reflect greater environmental diversity than those from other hosts. The aim of the present study was to evaluate T. gondii seroprevalence, isolate and genotype strains from Z. auriculata. Serum and tissue samples were collected from 206 doves for use in the modified agglutination test (MAT) and mouse bioassay. The prevalence of T. gondii antibodies in the doves was 22.3% (46/206), with titers ranging from 16 to 4096, and T. gondii strains were isolated from 12 of these doves. Five genotypes were detected by means of PCR-RFLP, including ToxoDB genotypes #1, #6, #17 and #65, and one genotype that had not previously been described (ToxoDB#182). This was the first report on isolation of T. gondii from Z. auriculata. This study confirmed the genetic diversity of T. gondii isolates and the existence of clonal type II (ToxoDB genotype #1) in Brazil. Univ Estadual Londrina, Dept Vet Prevent Med, Lab Protozool, BR-86051990 Londrina, PR, Brazil Univ Tennessee, Dept Microbiol, Knoxville, TN 37996 USA Univ Estadual Paulista, Sch Agr &Vet Sci, Dept Vet Pathol, UNESP, Jaboticabal, SP, Brazil Univ Estadual Paulista, Sch Agr &Vet Sci, Dept Vet Pathol, UNESP, Jaboticabal, SP, Brazil CAPES: 1345/2011 CAPES: 10259/12-0 FAPESP: 2010/01597-2