In an attempt to develop quantitative real-time PCR (qPCR) assays for the detection of equine piroplasms, sequence heterogeneity in the V4 hypervariable region of the 18S ribosomal RNA (rRNA) gene sequences within both Theileria equi and Babesia caballi from South Africa was discovered. A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was therefore carried out using horse and zebra samples from different geographical locations around South Africa. We evaluated the ability of a recently developed T. equi-specific qPCR assay in detecting all T. equi 18S rRNA variants identified in South Africa. We further present the first report on the development and application of a TaqMan minor groove binder (MGB™) qPCR assay, targeting the 18S rRNA gene, for the detection of B. caballi infections in equine blood samples. Despite the ability of the 18S rRNA T. equi- and B. caballi-specific qPCR assays to detect all known 18S rRNA gene sequence variants thus far identified in South Africa, the existence of as yet undetected variants in the field cannot be overlooked. Other qPCR assays targeting alternative genes could be developed which, used in conjunction with the 18S rRNA qPCR assays, may provide better confirmation of test results. A T. equi-specific qPCR assay targeting the equi merozoite antigen gene (ema-1) was recently developed for the detection of T. equi parasites in the midgut of Rhipicephalus (Boophilus) microplus nymphs. This assay was not able to detect T. equi in all South African samples that were confirmed positive by other molecular and serological assays. Sequence characterization of the ema-1 gene from South African isolates revealed the existence of variation in the regions where the qPCR primers and probes had been designed. Based on these observations, a conserved region of the ema-1 gene was selected and targeted in the development of an ema-1-specific TaqMan MGB™ qPCR assay, which was shown to have a higher sensitivity than the previously reported ema-1 qPCR assay. The rhoptry-associated protein (rap-1) gene from South African B. caballi isolates was also characterized following the failure of a B. caballi-specific competitive-inhibition enzyme-linked immunosorbent assay (cELISA) to detect B. caballi antibody in the sera of infected horses from South Africa. The genome walking PCR technique was used to amplify the complete rap-1 gene sequence from two South African B. caballi isolates. Significant heterogeneity in the rap-1 gene sequences and in the predicted amino acid sequences was found. Marked amino acid sequence differences in the carboxy-terminal region, and therefore the probable absence of the monoclonal antibody binding site, explains the failure of the cELISA to detect antibody to B. caballi in sera of infected horses in South Africa. This is the first comprehensive molecular study of the parasites that cause equine piroplasmosis in South Africa. Our results add further to the existing knowledge of piroplasmosis worldwide and will be invaluable in the development of further molecular or serological diagnostic assays. / Thesis (PhD)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Com o objetivo de avaliar os equinos alojados no Jóquei Clube de São Paulo, Brasil, quanto a presença de anticorpos contra Theileria equi e Babesia caballi, foram testadas 180 amostras de soro sanguíneo por meio da técnica de C-ELISA (Competitive Enzyme-Linked Immunosorbent Assay), metodologia atualmente recomendada pela OIE (Organização Internacional de Epizootíases) por ter alta sensibilidade e especificidade. A frequência de animais com sorologia positiva para Theileria equi foi de 6,66% (12/180), para Babesia caballi foi de 22,3% (40/180) e para infecção concomitante foi de 6,66% (12/180). Os resultados sorológicos obtidos por este estudo revelam que 35,5% (64/180) dos animais possuem anticorpos contra a babesiose equina sendo que a maioria dos animais acometidos tem dois e três anos de idade e portanto estão há menos tempo no hipódromo. Fatores como a ausência de carrapatos vetores, o uso de terapias babesicidas repetidas e o longo tempo de permanência dos animais no Jóquei após o tratamento, favorecem a diminuição dos títulos de anticorpos sem que ocorra reinfecção. Esses fatores podem justificar o menor número de animais com sorologia positiva para a doença nos cavalos com idade acima de quatro anos. Considerando-se esses resultados sugere-se que os animais sejam avaliados sorologicamente ao ingressarem no Jóquei Clube de São Paulo para que o uso de medicamentos contra a doença seja feito de forma adequada e para que os sinais clínicos compatíveis com babesiose equina em animais sorologicamente negativos sejam melhor avaliados e considerados em diagnósticos diferenciais. / In order to evaluate the presence of antibodies against Theileria equi and Babesia caballi in horses kept at the Jockey Club in São Paulo city, Brazil, a total of 180 samples of blood serum was tested using the Competitive Enzyme-Linked Immunosorbent Assay (C-ELISA test). This methodology has been recommended by the International Organization of Epizooties (IOE) due to its high sensitivity and specificity. The frequency of seropositive animals for Theileria equi, Babesia caballi and for both was 6.66% (12/180), 22.3% (40/180) and 6.66% (12/180), respectively. Serological results showed that 35.5% of the animals (64/180) had antibodies against equine piroplasmosis; they were from two to three years old and were at the Jockey Club for a shorter period of time. Factors such as absence of thick vectors, repeated therapy using babesicidal drugs and the long period of time that the animals stayed in the Jockey Club after treatment favoured the lowering of antibody titers with no reinfection. These factors might be responsible for the fewer number of animals with positive serology for the disease in horses over four years of age. Based on these findings, animals should be serologically evaluated at the time of entrance into the Jockey Club so that the use of drugs against the disease be performed properly and clinical signs suggestive of equine babesiosis in serologically negative animals be better evaluated and considered for differential diagnosis.

Com o objetivo de avaliar os equinos alojados no Jóquei Clube de São Paulo, Brasil, quanto a presença de anticorpos contra Theileria equi e Babesia caballi, foram testadas 180 amostras de soro sanguíneo por meio da técnica de C-ELISA (Competitive Enzyme-Linked Immunosorbent Assay), metodologia atualmente recomendada pela OIE (Organização Internacional de Epizootíases) por ter alta sensibilidade e especificidade. A frequência de animais com sorologia positiva para Theileria equi foi de 6,66% (12/180), para Babesia caballi foi de 22,3% (40/180) e para infecção concomitante foi de 6,66% (12/180). Os resultados sorológicos obtidos por este estudo revelam que 35,5% (64/180) dos animais possuem anticorpos contra a babesiose equina sendo que a maioria dos animais acometidos tem dois e três anos de idade e portanto estão há menos tempo no hipódromo. Fatores como a ausência de carrapatos vetores, o uso de terapias babesicidas repetidas e o longo tempo de permanência dos animais no Jóquei após o tratamento, favorecem a diminuição dos títulos de anticorpos sem que ocorra reinfecção. Esses fatores podem justificar o menor número de animais com sorologia positiva para a doença nos cavalos com idade acima de quatro anos. Considerando-se esses resultados sugere-se que os animais sejam avaliados sorologicamente ao ingressarem no Jóquei Clube de São Paulo para que o uso de medicamentos contra a doença seja feito de forma adequada e para que os sinais clínicos compatíveis com babesiose equina em animais sorologicamente negativos sejam melhor avaliados e considerados em diagnósticos diferenciais. / In order to evaluate the presence of antibodies against Theileria equi and Babesia caballi in horses kept at the Jockey Club in São Paulo city, Brazil, a total of 180 samples of blood serum was tested using the Competitive Enzyme-Linked Immunosorbent Assay (C-ELISA test). This methodology has been recommended by the International Organization of Epizooties (IOE) due to its high sensitivity and specificity. The frequency of seropositive animals for Theileria equi, Babesia caballi and for both was 6.66% (12/180), 22.3% (40/180) and 6.66% (12/180), respectively. Serological results showed that 35.5% of the animals (64/180) had antibodies against equine piroplasmosis; they were from two to three years old and were at the Jockey Club for a shorter period of time. Factors such as absence of thick vectors, repeated therapy using babesicidal drugs and the long period of time that the animals stayed in the Jockey Club after treatment favoured the lowering of antibody titers with no reinfection. These factors might be responsible for the fewer number of animals with positive serology for the disease in horses over four years of age. Based on these findings, animals should be serologically evaluated at the time of entrance into the Jockey Club so that the use of drugs against the disease be performed properly and clinical signs suggestive of equine babesiosis in serologically negative animals be better evaluated and considered for differential diagnosis.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-05-03T14:32:38Z No. of bitstreams: 1 Neurisvan Ramos Guerra.pdf: 2637002 bytes, checksum: 451efc2cbd7dd76dda9b010b8b0189ba (MD5) / Made available in DSpace on 2018-05-03T14:32:39Z (GMT). No. of bitstreams: 1 Neurisvan Ramos Guerra.pdf: 2637002 bytes, checksum: 451efc2cbd7dd76dda9b010b8b0189ba (MD5) Previous issue date: 2017-02-20 / The equid industry in Brazil occupies a prominent position worldwide, with about eight million equids. Diseases caused by protozoa such as Babesia caballi, Theileria equi and Neospora spp. as well as parasites that cause zoonotic protozooses such as Leishmania spp. and Toxoplasma gondii represent one of the main obstacles in the development of the sector. Therefore, this study aims to detect infection by Leishmania spp., Babesia caballi, Theileria equi, Toxoplasma gondii and Neospora spp. and their respective risk factors in equidae created with different management forms. To perform the tests, 400 samples of whole blood and serum from clinically healthy equines, including horses, mules and donkeys from 41 rural properties in the state of Pernambuco were analyzed. In order to detect Leishmania spp., direct parasitological and Polymerase Chain Reaction (PCR) tests were performed. Concerning the presence of infection by Babesia caballi and Theileria equi, direct parasitological tests and enzyme-linked immunosorbent assay (ELISA) were used for anti-Babesia caballi and anti-Theileria equi immunoglobulins detection. For the determination of seroprevalences of toxoplasmosis and neosporosis, modified agglutination (MAT) tests were used to identify anti-T. gondii IgG antibodies and anti-Neospora spp. All samples were negative for Leishmania spp. in the tests, suggesting that equidae do not participate in the epidemiological chain of leishmaniasis in the studied areas. The prevalence of anti-Babesia caballi and anti-Theileria equi antibodies of 4.3% (17/400; CI: 2.6-6.9) and the presence of B. caballi and T. equi in the serological tests revealed a prevalence of 10.8% (43/400; CI: 8.0 - 14.3), respectively, and co-infection was detected in 1% (4/400) of the animals. These data allow the characterization of areas of enzootic instability in the sites surveyed. Anti-T. gondii IgG antibodies were detected in 12.5% (50/400) of the animals analyzed. When evaluating the risk factors for T. gondii infection, only the mesoregion factor (p = 0.029) was associated with infection, particularly Zona da Mata (OR = 3). The results reveal the presence of the parasite in the studied area, which may represent a link in the transmission chain of toxoplasmosis. Seropositivity for Neospora spp. was 5.7% (23/400) and the variables age, breeding type and region presented statistical significance. In relation to age, it was observed that animals older than 11 years presented 11.8 times more chances of being serum-reactive wjhen compared with young animals (<2,5) and the prevalence found shows that the parasite is dispersed in the areas studied and that the variables age, breeding type and region are the most important risk factors for the occurrence of infection in equidae, and should be considered in the prevention of the disease. Considering the results found in the present study, the diagnosis of the various diseases present in the State of Pernambuco, when performed at an early stage, allows the application of preventive and control measures, contributing significantly to animal health and public health. / A equideocultura do Brasil ocupa posição de destaque mundial, com cerca de oito milhões de cabeças. Doenças causadas por protozoários como Babesia caballi, Theileria equi e Neospora spp. além de parasitos que causam protozooses zoonóticas a exemplo de Leishmania spp. e Toxoplasma gondii representam um dos principais entraves no desenvolvimento do setor. Diante disso, esse estudo tem como objetivo determinar as prevalências e fatores de risco associados às infecções por Leishmania spp., Babesia caballi (Nuttall & Strickland, 1910), Theileria equi (Mehlhorn & Schein, 1998), Toxoplasma gondii (Nicolle & Manceaux, 1909) e Neospora spp. em equídeos submetidos a diferentes regimes de criação. Para realização dos exames, foram analisadas 400 amostras de sangue total e soro de equídeos clinicamente saudáveis, incluindo equinos (387/400), muares (9/400) e asininos (4/400) provenientes de 41 propriedades rurais do estado de Pernambuco. Com a finalidade da detecção de Leishmania spp., foram realizados os exames parasitológicos diretos e Reação em cadeia da Polimerase (PCR). No intuito de averiguar a presença de infecção por Babesia caballi e Theileria equi foram utilizados os exames parasitológico direto e Ensaio de Imunoadsorção Enzimática (ELISA), para detecção de imunoglobulinas anti-Babesia caballi e anti-Theileria equi. Para determinação das soroprevalências da toxoplasmose e neosporose foram utilizados os testes de aglutinação modificado (MAT) para identificação de anticorpos IgG anti-T. gondii e IgG anti-Neospora spp. Todas as amostras resultaram negativas para Leishmania spp. nos testes, o que sugere que os equídeos não participam da cadeia epidemiológica das leishmanioses nas áreas estudadas. Quanto à presença de B. caballi e T. equi, os testes sorológicos revelaram uma prevalência de anticorpos anti-Babesia caballi e anti-Theileria equi de 4.3% (17/400; I.C. 2,6 – 6.9%) e 10,8% (43/400; I.C. 8.0 – 14.3), respectivamente e foi detectada co-infecção em 1% (4/400) dos animais. Tais dados permitem caracterizar como áreas de instabilidade enzoótica os locais pesquisados. Anticorpos IgG anti-T. gondii foram detectados em 12,5% (50/400) dos animais analisados. Quando avaliados os fatores de risco para infecção por T. gondii, apenas o fator mesorregião (p=0,029) apresentou associação com a infecção, particularmente Zona da Mata (OR=3). Os resultados revelam a presença do parasito na área estudada, o que pode representar um elo na cadeia de transmissão da toxoplasmose. A soropositividade para Neospora spp. total foi de 5,7% (23/400) e as variáveis idade, tipo de criação e região apresentaram significância estatística. Em relação à idade, observou-se que animais acima de 11 anos apresentaram 11,8 vezes de chances a mais de serem sororreagentes quando comparados com os animais jovens (<2,5) e a prevalência encontrada demonstra que o parasito está disperso nas áreas estudadas e que as variáveis idade, tipo de criação e região são fatores de riscos mais importantes para ocorrência da infecção em equídeos, devendo ser considerados na prevenção da doença. Considerando os resultados encontrados no presente estudo, o diagnóstico das diversas doenças presentes no estado de Pernambuco, quando realizado de forma precoce, possibilita a aplicação de medidas preventivas e de controle, contribuindo significativamente com a sanidade animal e saúde pública.