Protection against Toxoplasma gondii cysts in pigs immunized with rROP2 plus Iscomatrix.

This study aimed to evaluate the humoral immune response in pigs immunized intranasally and intramuscularly with recombinant Toxoplasma gondii rROP2 protein in combination with the adjuvant Iscomatrix. Twelve mixed breed pigs divided into three groups (n=4) were used, G1 received recombinant ROP2 proteins (200 µg/dose) plus Iscomatrix, G2 received PBS plus Iscomatrix, and G3 as the control group. The intranasal (IN) and intramuscular (IM) routes were used. Animals were challenged orally with VEG strain oocysts and treated on day three after challenge. Fever, anorexia, and prostration were the clinical signs observed in all animals. All the G1 animals produced antibodies above the cut-off on the day of the challenge, while the G2 and G3 remained below the cut-off. Better partial protection against parasitemia and cyst tissue formation was observed in G1 than G3. The protection factors against tissue cyst formation were 40.0% and 6.1% for G1 and G2, respectively, compared to G3. In conclusion, there were not systemic antibody responses in pigs with IN immunization with rROP2+Iscomatrix; however, after IM immunization, those animals produced higher titers than animal controls. We associated these results with partial protection obtained against parasitemia and tissue cysts formation.

Neospora caninum DNA in feces of crab-eating fox (Cerdocyon thous - Linnaeus, 1776) from northeastern Brazil.

Neospora caninum is an obligate intracellular tissue cyst-forming coccidian parasite and it was first described in dogs. Despite the relevance of wild canids in the transmission chain of N. caninum, there are few studies in Brazil. The aim of the present study was to detect N. caninum DNA in feces of free-range and captive crab-eating fox (Cerdocyon thous) from different states of northeastern Brazil. Fecal samples of eighteen crab-eating foxes (fifteen individually and three pools) were collected in sterile containers and were kept cool at -20 °C until further processing. All fecal samples were subjected to DNA extraction. A nested PCR targeting the ITS-1 gene was performed for N. caninum. All the positive bands were extracted from the gel and purified. Forward and reverse strands were sequenced and the nucleotide sequences obtained were compared with N. caninum sequences deposited in Genbank. The alignment was edited and phylogenetic reconstruction was based on the ITS1 gene sequences. Thirteen stool samples were PCR-positive for N. caninum DNA. Nine out of thirteen positive samples showed similarity between 99%-100% for N. caninum in relation to the sequence U25044.1 stored at GenBank. The crab-eating fox could have an important role in the sylvatic cycle of Neospora caninum in Brazil. Experimental infections studies involving these wild canids may confirm if the crab-eating foxes are definitive hosts of N. caninum.

Co-Infection by Sarcoptes scabiei and Microsporum gypseum in Free-Ranging Crab-Eating Fox, Cerdocyon thous (Linnaeus, 1766)

ABSTRACT The aim of the present study was to describe the clinical manifestation, treatment and outcome of a case of co- infection by Sarcoptes scabiei and Microsporum gypseum in Cerdocyon thous (crab-eating fox) from Northeastern Brazil.

An outbreak of caprine toxoplasmosis - investigation and case report / Surto de toxoplasmose caprina - investigação e relato de caso

ABSTRACT: The 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.

RESUMO: O presente estudo teve como objetivo investigar um surto de aborto em um rebanho de cabras leiteiras no município de Arapoti, Paraná, Brasil. No início do surto, foram coletadas amostras de sangue de 33 cabras com sinais clínicos; mais tarde, de todo o rebanho caprino, dois gatos e dois cachorros. Foram obtidas amostras de leite das 78 cabras em lactação. Quatro amostras ambientais de solo e quatro de resíduos de comedouro também foram coletadas. O teste de imunofluorescência (IFI) foi utilizado para o sorodiagnóstico, a análise molecular foi conduzida por meio da reação em cadeia da polimerase (PCR), para isolamento do agente etiológico utilizou-se o bioensaio. Os resultados da IFI revelaram que 76,53% (137/179) das cabras, todos os cães e gatos eram soropositivos para Toxoplasma gondii. O bioensaio revelou uma amostra de camada leucocitária e duas de leite contaminadas com T. gondii viável. Na PCR, 11 amostras de sangue total, oito de leite, três resíduos alimentares e todas as amostras de solo foram positivas. Os resultados do presente estudo confirmaram um surto causado por contaminação 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, reforçando a necessidade de assistência técnica veterinária e boas práticas de manejo.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats.

The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 µg of rROP2 proteins plus 20 µg of Quil-A, G2 received 100 µg of BSA plus 20 µg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats / rROP2 de Toxoplasma gondii como potencial vacina contra a eliminação de oocistos em gatos domésticos

Abstract The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

Resumo O objetivo do presente estudo foi avaliar a eliminação de oocistos de Toxoplasma gondii em gatos imunizados pela via nasal com proteínas ROP2 de T. gondii. Doze gatos sem raça definida (Felis catus) foram divididos em três grupos experimentais G1, G2 e G3 (n = 4). Os animais do G1 receberam 100 μg de proteínas de rROP2 mais 20 μg de Quil-A, G2 recebeu 100 μg de albumina de soro bovino (BSA) junto com 20 μg de Quil-A, e o G3 recebeu apenas solução salina (grupo de controle). Todos os tratamentos foram realizados pela via intranasal nos dias 0, 21, 42 e 63. O desafio foi realizado em todos os grupos no dia 70 com aproximadamente 800 cistos de tecido da cepa ME-49 por via oral. Os animais de todos os grupos tiveram as suas fezes examinadas e o número de oocistos foi determinado durante 20 dias após o desafio. Os animais de G1 eliminaram menos oocistos (86,7%) do que os grupos controles. O ELISA foi utilizado para detectar IgG e IgA anti-rROP2, no entanto, não houve correlação entre o número de eliminhação de oocistos com os níveis de anticorpos IgG ou IgA. No presente trabalho, apesar da menor produção de oocistos no grupo imunizado (G1) em relação aos grupos controles (G2 e G3), não foi possível associar o uso de rROP2 pela via nasal com proteção contra eliminação de oocistos de T. gondii. Para o futuro, a utilização de outras proteínas recombinantes, ou mesmo vacina de DNA, em combinação com rROP2 poderia ser utilizada para tentar melhorar a eficácia deste tipo de vacina.

Application of different techniques to detect Toxoplasma gondii in slaughtered sheep for human consumption / Utilização de diferentes técnicas para detecção de Toxoplasma gondii em ovinos abatidos para consumo humano

Abstract The aim of this study was to investigate occurrence of Toxoplasma gondii in sheep slaughtered in the state of Alagoas, Brazil, by means of different diagnosis techniques. Serum samples and tissues from 100 slaughtered sheep were used. To detect antibodies, the indirect immunofluorescence antibody test (IFAT) was used, and tissues from seropositive animals (cut-off ≥1:64) were submitted to Polymerase Chain Reaction (PCR) and immunohistochemistry (IHC). To assess the concordance between the direct techniques, the kappa test was used. In the IFAT, it was observed that 14% (14/100) of the ovine samples were serum-positive. In the PCR, 21.43% (3/14) of the animals were positive and in IHC, it was observed that 7.14% (1/14) were positively stained for T. gondii in cerebral tissue. Histopathologically, the predominant finding was the presence of mononuclear cell infiltrate in the heart and a perivascular cuff in the cerebrum and cerebellum. The concordance between the direct diagnosis techniques was moderate (k=0.44). Thus, it is important to use different direct techniques in diagnosing toxoplasmosis in naturally infected sheep.

Resumo O objetivo deste estudo foi pesquisar a ocorrência de Toxoplasma gondii em ovinos abatidos no Estado de Alagoas, Brasil por meio de diferentes técnicas de diagnóstico. Foram utilizadas amostras de soros e tecidos de 100 ovinos abatidos. Para a pesquisa de anticorpos foi utilizada a Reação de Imunofluorescência Indireta (RIFI), e os tecidos dos animais soropositivos (ponto de corte ≥1:64) foram submetidos às técnicas de Reação de Cadeia da Polimerase (PCR) e Imunohistoquímica (IHQ). Para o estudo da concordância entre as técnicas diretas foi empregado o teste Kappa. Na RIFI, 14% (14/100) das amostras foram soro-positivas. Na PCR, 21,43% (3/14) dos animais foram positivos e, na IHC, 7,14% (1/14) apresentaram marcação positiva para T. gondii no tecido cerebral. Na histopatologia, o achado predominante foi o infiltrado celular mononuclear no coração e manguito perivascular no cérebro e cerebelo. A concordância entre as técnicas diretas de diagnóstico foi moderada (K= 0,44). Desse modo, é importante utilizar diferentes técnicas diretas no diagnóstico da toxoplasmose em ovinos naturalmente infectados.

Ocorrência de Cryptosporidium spp. em dois centros de treinamento de equinos de Curitiba, Paraná / Occurrence of Cryptosporidium spp. in two centers of training horses in Curitiba, Paraná

O presente trabalho investigou a ocorrência de parasitismo por Cryptoporidium spp. em equinos alojados em dois centros de treinamento de equinos localizados no municipio de Curitiba, Paraná. Foram examinados 108 cavalos, sendo 48 procedentes do Centro de treinamento 1 (CT1) e 60 do Centro de Treinamento 2 (CT2). As coletas de amostras de fezes foram realizadas no período de outubro de 2010 a janeiro de 2011. A metodologia utilizada para a confirmação da presença de oocistos de Cryptosporidium spp. foi a técnica de Ziehl-Neelsen modificada. A ocorrência encontrada foi de 18,52% para o total de animais examinados. Houve diferença estatística significativa (p > 0,05) quando comparadas as prevalências encontradas nos dois centros de treinamento, sendo no CT1 de 4,16% e no CT2 de 30%. Não houve associação entre a prevalência e a idade, o sexo e raça (p > 0,05).

This study investigated the occurrence of parasitism by Cryptosporidium spp. in horses housed in two training centers located in Curitiba, Paraná. A total of 108 horses were examined, 48 from the Training Center 1 (CT1) and 60 of the Training Centre 2 (CT2). Collection of stool specimens occurred from October 2010 to January 2011. The methodology used to confirm the presence of Cryptosporidium spp. was the Ziehl-Neelsen modified. The occurrence found was 18.52% for the total animals examined. There was a statistically significant difference (p > 0.05) comparing prevalence rates in the two training centers: 4.16% in CT1 and 30% in CT2. There was no association between prevalence and the age, sex and race (p > 0.05) of the horses.

Perfil epidemiológico de casos de surtos de doenças transmitidas poralimentos ocorridos no Paraná, Brasil / Epidemiological profile of foodborne diseases outbreaks occurred in Parana state, Brazil

A investigação dos surtos de Doenças Transmitidas por Alimentos (DTA) é desafiadora. Para a Vigilância em Saúde é fundamental a identificação de grupos e fatores de risco associados às DTA. O objetivo deste trabalho foi caracterizar o perfil epidemiológico dos surtos de DTA ocorridos nos 29 municípiosque compõe a 2ª Regional de Saúde, da Secretaria Estadual de Saúde do Paraná, Brasil, entre 2005-2008. Tabularam-se 472 laudos de alimentos do Laboratório Central do Estado do Paraná (LACEN-PR) e fichas notificadas no Sistema de Informação de Agravos de Notificação (SINAN) de casos suspeitos de DTA. O grupo mais acometido foi de indivíduos do sexo feminino com 50,51% dos casos. Indivíduos entre 20 a 49 anos de idade foram os mais frequentemente envolvidos com 49,50% das ocorrências. Restaurantes (27,65%) e residências dos indivíduos acometidos (36,17%) foram os locais de preparo econsumo mais frequentemente envolvidos. Manipulação/preparo inadequados dos alimentos (36,95%) eforma de consumo inadequada (23,90%) foram os fatores causais mais predominantes. A maior parte dasconfirmações dos surtos fundamentou-se em achados clínico-epidemiológicos (44,68%). Com relaçãoaos micro-organismos identificados nas amostras analisadas, os resultados apontaram predominância Escherichia coli (indicador sanitário), seguido pelo Bacillus cereus e Staphyloccus aureus. Os alimentos mais frequentemente envolvidos foram o leite (29,68%) e as frutas, verduras e legumes (24,21%). Com a pesquisa é possível orientar medidas preventivas e de controle de DTA na população pesquisada.

The investigation of outbreaks of Food Borne Diseases (FBD) is challenging. For the Health Surveillance is essential to identify groups and risk factors associated with the DTA. The aim of this study was to characterize the epidemiology of outbreaks of DTA occurred in 29 counties that comprise the 2nd Regional Health, the Department of Health of Paraná, Brazil, from 2005-2008. Tabulated to 472 reports of food the Central Laboratory of the State of Paraná (LACEN-PR) chips and notified the Information System for Notifiable Diseases (SINAN) of suspected cases of DTA. The group most affected were females with 50.51% of the cases. Individuals between 20 and 49 years of age were most often involved in 49.50% of cases. Restaurants (27.65%) of affected individuals and households (36.17%) were the preparation and consumption sites most frequently involved. Manipulation / inadequate preparation of food (36.95%) and inadequate form of consumption (23.90%) were the most prevalent causative factors.Most confirmations of outbreaks was based on clinical and epidemiological (44.68%). With respect to micro-organisms identified in the samples analyzed, the results showed predominance Escherichia coli(health indicator), followed by Bacillus cereus and Staphylococcus aureus. The foods most frequently involved were milk (29.68%) and fruits and vegetables (24.21%). With this study can guide preventive measures and control of DTA in the population studied.

Diagnosis and isolation of Toxoplasma gondii in horses from Brazilian slaughterhouses.

This study aimed to investigate anti-Toxoplasma gondii antibodies and to isolate the parasite from the brains of horses processed at slaughterhouses in Brazil. We collected brain and blood samples from 398 horses of various ages, from six Brazilian states. Serum samples were evaluated by indirect fluorescent antibody test (IFAT cut-off titre ≥ 1:64), and brains were submitted to mouse bioassay. Among the 398 horses, positivity for T. gondii was identified in 46 (11.6%) by IFAT and in 14 (3.5%) by mouse bioassay. In 12 of those 14 bioassays, mice were positive only by IFAT (cut-off titre ≥ 1:16), T. gondii being isolated in the remaining two. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 18S rDNA to differentiate among T. gondii, Neospora caninum, and Sarcocystis neurona, we found that two of the 14 brains were positive for T. gondii only. For genotyping of the two isolates and the PCR-positive brain, we performed PCR-RFLP based on 13 markers, and SAG2 all samples were Toxoplasma gondii type I. Collectively, IFAT of horse sera and mouse bioassay identified positivity in 60 (15%) of the samples. Our results show that some horses sent to slaughter in Brazil have been exposed to T. gondii.

Diagnosis and isolation of Toxoplasma gondii in horses from brazilian slaughterhouses / Diagnóstico e isolamento de Toxoplasma gondii em equídeos de frigoríficos brasileiros

This study aimed to investigate anti-Toxoplasma gondii antibodies and to isolate the parasite from the brains of horses processed at slaughterhouses in Brazil. We collected brain and blood samples from 398 horses of various ages, from six Brazilian states. Serum samples were evaluated by indirect fluorescent antibody test (IFAT cut-off titre ≥ 1:64), and brains were submitted to mouse bioassay. Among the 398 horses, positivity for T. gondii was identified in 46 (11.6%) by IFAT and in 14 (3.5%) by mouse bioassay. In 12 of those 14 bioassays, mice were positive only by IFAT (cut-off titre ≥ 1:16), T. gondii being isolated in the remaining two. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 18S rDNA to differentiate among T. gondii, Neospora caninum, and Sarcocystis neurona, we found that two of the 14 brains were positive for T. gondii only. For genotyping of the two isolates and the PCR-positive brain, we performed PCR-RFLP based on 13 markers, and SAG2 all samples were Toxoplasma gondii type I. Collectively, IFAT of horse sera and mouse bioassay identified positivity in 60 (15%) of the samples. Our results show that some horses sent to slaughter in Brazil have been exposed toT. gondii.

O objetivo do estudo foi investigar anticorpos anti-Toxoplasma gondii e isolar o parasita do cérebro de equídeos abatidos em matadouros-frigoríficos no Brasil. Colheram-se amostras de 398 cérebros e sangue de equídeos machos e fêmeas de idades variadas, provenientes de seis estados brasileiros. As amostras de soro foram avaliadas pelo teste de imunofluorescência indireta (IFI) para T. gondii (ponto de corte ≥ 64), e os fragmentos de cérebros foram submetidos ao bioensaio em camundongos. Por meio da IFI, 46 (11,6%) equídeos foram soropositivos. Pelo bioensaio em camundongos, 14 (3,5%) cérebros de equídeos testados foram positivos. Em doze dos bioensaios, os camundongos foram positivos somente pela IFI (ponto de corte ≥ 16) e T. gondii foi isolado nos outros dois bioensaios. A PCR-RFLP com base em 18S rDNA para diferenciar entre T. gondii, Neospora caninum, e Sarcocystis neurona foram feitas em todos os 14 cérebros e dois foram positivos apenas para T. gondii. De dois isolados positivos para T. gondii e do cérebro positivo à PCR em que realizou-se a PCR-RFLP, com base em 13 marcadores e SAG2, a genotipagem mostrou ser o T. gondii tipo I para todas as amostras. A IFI de soros de equídeos e do bioensaio em camundongos identificaram positividade em 60 (15%) amostras testadas. Os resultados mostram que alguns cavalos enviados para abate foram expostos ao T. gondii.