Made available in DSpace on 2016-04-28T20:16:21Z (GMT). No. of bitstreams: 1 2006-Gil Vicente Oliveira da Silva.pdf: 2177431 bytes, checksum: 46c0c2291cd76db4f34ccb7eeaa34c10 (MD5) Previous issue date: 2006-04-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The equines piroplasmosis are diseases that may cause debilitating progress or fatal episodies, its etiologic agents are the species Theileria equi and Babesia caballi. The maintenance of these species in nature, as well as the widespread ability, are limitants factor to genetic improvement of equines herds in view of the internationals restrictions referring the accomited animals transit with these patogens. In Brazil, in economic terms these patogens have a great importance because the national erd is stimated in 5,800,000 of animals, that dropped with the asinines, arrive in 8,300,000 of animals. The eluciding of links to the natural disease history, overall the ones that propitiate the perpetuation of it in our herds, becomes urgent and necessary. Note that some species have a sight convivence with equines, among it is the Canis familiaris. With purpose to evaluate the possibility of C. familiaris to be involved on epizootiologic natural chain of T. equi, we used two boarding: experimental and epizootiologic. For the first, using material from seventeen dogs and puppies that were divided in three groups: G1 Puppies; G2 Dogs; G3 immunossuprimitted dogs and puppies, both ixodids-free since birth until end of experimental evaluations referring the possibility to C. Familiaris to be a able host to T. equi. to evaluate the occurrence of T. equi in these animals, were used some diagnostic tools, as follow: parasitologic aspect blood smears in immersion optical microscopy (IOM); sorology: using research of antibodies against T. equi, with immunofluorescence indirect test (IFT); and molecular using four differents protocols of polimerase chain reaction (PCR), in follow material: blood with EDTA; fragment of tissues; Boophilus microplus nymphs and Rhipicephalus sanguineus in adult stage. In the second moment, evaluated, for three times, twelve dogs from enzootic stability area to T. equi with a sight convivence with horses since birth occasions, using for it IMO and IFT, forming G4 group. The results were negatives in all observations. Concluding that the T. equi specie did not show the ability to establishment and development in C. Familiaris; and that specie do not involved in epizootiologic natural chain of T. equi, in municipality of Seropedica, State of Rio de Janeiro, Brazil. / As piroplasmoses eq?inas s?o doen?as que variam de progress?o debilitante a fatal, tendo como agentes causais ?s esp?cies Theileria equi e Babesia caballi. A manuten??o dessas esp?cies na natureza, bem como a habilidade de dispers?o das mesmas, s?o fatores limitantes quanto ao melhoramento gen?tico dos plant?is eq?inos tendo em vista as restri??es internacionais referentes ao tr?nsito de animais acometidos com estes pat?genos. No Brasil, em termos econ?micos estas parasitoses assumem grande vulto tendo em vista que o plantel nacional de eq?inos ? estimado em 5.800.000 de cabe?as que somado aos asininos chega-se ao total de 8.300.000. A elucida??o dos elos referentes ? hist?ria natural da doen?a, sobretudo os que propiciam a perpetua??o da mesma em nossos plant?is, torna-se urgente e necess?rio. Nota-se que algumas esp?cies mant?m um conv?vio estreito com eq??deos dentre as quais a Canis familiaris. Com o prop?sito de avaliar a possibilidade da esp?cie C. familiaris estar envolvida na cadeia epizootiol?gica da esp?cie T. equi, utilizamos duas abordagens: experimental e epizootiol?gica. Para a primeira, utilizando materiais procedentes de dezessete c?es adultos e filhotes os quais foram divididos em tr?s grupos: G1 C?es filhotes; G2 C?es adultos; G3 C?es adultos e filhotes imunossuprimidos, mantidos livres de ixod?deos, desde a ocasi?o do nascimento at? o t?rmino das avalia??es experimentais referentes ? possibilidade da esp?cie C. familiaris ser hospedeira vi?vel de T. equi. Para avaliar o estabelecimento da T. equi nestes animais, foram utilizadas para isto as seguintes ferramentas diagn?sticas: aspecto parasitol?gico - esfrega?os sangu?neos ? microscopia ?ptica de imers?o; sorol?gico - por meio de pesquisa de anticorpos anti-T. equi, utilizando a rea??o de imunofluoresc?ncia indireta (RIFI); e molecular, utilizando quatro diferentes protocolos de rea??o de polimeriza??o em cadeia (PCR), nos seguintes materiais: sangue total com EDTA; fragmentos de diversos ?rg?os; ninfas de Boophilus microplus e adultos de Rhipicephalus sanguineus. No segundo momento, Avaliou-se, por tr?s vezes, doze c?es provenientes de ?rea de estabilidade enzo?tica para T. equi com estreita conviv?ncia com cavalos desde a ocasi?o de seus nascimentos, utilizou-se para isto a MOI e RIFI, formando o grupo G4. Os resultados foram negativos em todas as observa??es. Conclui-se que a esp?cie T. equin?o se mostrou h?bil quanto ao estabelecimento e desenvolvimento em C. familiaris, e que esta esp?cie n?o est? envolvida na cadeia epizootiol?gica natural da T. equi, no Munic?pio de Serop?dica, Estado do Rio de Janeiro, Brasil.

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.