Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-03-13T14:24:56Z No. of bitstreams: 1 PGCA14MA204.pdf: 193153 bytes, checksum: 775a55b56bd27e5412d5e2e42810041c (MD5) / Made available in DSpace on 2018-03-13T14:24:56Z (GMT). No. of bitstreams: 1 PGCA14MA204.pdf: 193153 bytes, checksum: 775a55b56bd27e5412d5e2e42810041c (MD5) Previous issue date: 2014-02-11 / PROMOP / CAPES / Anaplasma marginale (THEILER, 1910), Babesia bovis (BABES, 1888) and B. bigemina (SMITH & KILBORN, 1893) are obligatory intraerythrocytic microorganisms that are responsible for high mortality and morbidity rates in several regions of Brazil. The goal of this project was to investigate the infections dynamics and the pathogens prevalence in cattle located in the mountain region of Santa Catarina, Brazil. For this purpose, 257 blood samples from animals with age between four months and eleven years were collected from March 2012 to July 2013, in sixteen cities of the region. The samples were grouped in five classes according to age, Group A were compounds of the animals with six months or less; Group B animals from 7 to 12 months, Group C animals from 13 to 24 months, Group D animals from 25 to 36 months and Group E animals with more than 36 months. Blood samples were submitted to hematological analysis, conducted at Laboratório Clínico Veterinário – Centro de Ciências Agroveterinárias (CAV), and pathogen investigation was examined by Multiplex-PCR, in Laboratório de Bioquímica de Hemoparasitas e Vetores – LABHEV (CAV). Erythrogram and WBC were performed on electronic counter (CELM CC-530), and differential leukocyte counts by cytological analysis in optical microscope. The DNA extraction was performed by the phenol-chloroform method. Using the Multiplex-PCR technique, we were able to detect Anaplasma marginale prevalence in about 27.24%, B. bovis in 29.57% and B. bigemina in 16.73% of the animals. Statistical analysis showed correlation between the presence of the microorganisms and the animals groups age. The hematological modifications most significant were found on WBC, where leukocytosis was founded in the overall average of animals. However, infections were delivered in healthy animals, which shows sub-clinical infections in animals studies. The study area was characterized in enzootic instability, since the prevalence of microorganisms occurred in less than 75% of the population, situation that requires care in vector control and parasites to be conducive to outbreaks of disease / Anaplasma marginale (THEILER, 1910), Babesia bovis (BABES, 1888) e B. bigemina (SMITH & KILBORN, 1893) são microorganismos intra-eritrocitários obrigatórios responsáveis por causar prejuízos ligados a alta morbidade e mortalidade em várias regiões do Brasil. Com intuito de avaliar a dinâmica de infecção e prevalência desses parasitas em bovinos da região serrana de Santa Catarina, 257 amostras de sangue de animais com idade entre três meses e onze anos, foram coletadas entre março de 2012 e julho de 2013, em dezesseis municípios da região. As amostras foram divididas em cinco classes de acordo com a idade, onde o grupo (A) foi composto de animais de 4 a 6 meses, o grupo (B) animais entre 7 e 12 meses, o grupo (C) de 13 a 24 meses, o grupo (D) de 25 e 36 meses e no grupo (E) com mais de 36 meses. As amostras foram utilizadas para análises hematológicas, processadas no Laboratório Clínico Veterinário do Centro de Ciências Agroveterinárias (CAV) e para pesquisa dos agentes parasitários pela Multiplex-PCR, no Laboratório de Bioquímica de Hemoparasitas e Vetores - LABHEV. Eritrograma e leucograma foram realizados em contador eletrônico (CELM CC-530) e contagem diferencial de leucócitos por análise citológica em microscopia óptica. A extração de DNA foi realizada pelo método de fenol e clorofórmio. Com o uso da técnica Multiplex-PCR, foram detectadas prevalência de A. marginale em 27,24% dos bovinos, B. bovis em 29,57% e B. bigemina em 16,73% dos animais. Através da análise estatística verificou-se associação entre presença dos microorganismos e as classes etárias. A alteração hematológica mais relevante encontrada foi na porção leucocitária, onde a leucocitose foi observada na média geral dos animais amostrados. Contudo, as infecções foram apresentadas em animais saudáveis, o que demonstra infecções sub-clínicas nos bovinos estudados. A região estudada foi caracterizada em estado de instabilidade enzoótica, já que a prevalência dos microrganismos se deu em menos de 75% da população estudada, situação que requer cuidados no controle de vetor e parasitos por ser propícia à ocorrência de surtos da doença

Orientadora: Rosangela Zacarias Machado / Banca: Jose Mauricio Barbante Duarte / Banca: Natalino Hajime Yoshinari / Resumo: Poucos relatos têm sido feitos sobre o diagnóstico da erliquiose e babesiose em felinos domésticos e selvagens brasileiros, os quais são baseados diretamente pela presença de mórulas em leucócitos e piroplasmas em eritrócitos, e indiretamente pela detecção de anticorpos anti-Ehrlichia canis. O presente estudo teve como objetivo realizar a detecção molecular de E. canis e B. canis e a presença de anticorpos da classe IgG contra esses hemoparasitas em amostras de sangue e soro, respectivamente. Neste utilizamos 72 felídeos selvagens brasileiros mantidos em cativeiro em algumas instituições e zoológicos. Pela Reação de Imunofluorescência Indireta (RIFI), dezoito (25%) e cinqüenta e três (73,6%) dos 72 animais amostrados foram sororeagentes frente aos antígenos de E. canis e B. canis, respectivamente. Na PCR para E. canis, onze (15,3%) dos 72 animais amostrados foram positivos. Os amplicons foram confirmados por seqüenciamento e o DNA de E. canis encontrado mostrou grande similaridade genética com amostras de E. canis isoladas no Brasil, México, Portugal, Grécia e Taiwan, com 98% de similaridade. Nenhuma das amostras foi positiva na PCR para B. canis. Destaca-se a importância e a primeira detecção molecular de E. canis e presença de anticorpos anti-E. canis e anti-B. canis em felídeos selvagens brasileiros mantidos em cativeiro. / Abstract: Few are the reports that have been carried out on ehrlichiosis and babesiosis diagnostic in Brazilian domestic and wild felids, which are based directly on the presence of morulae in leucocytes and piroplasms in erythrocytes, and indirectly by detection of antibodies against E. canis. The aim of this study was to detect molecularly E. canis and B. canis and the presence of anti-E. canis and B. canis IgG antibodies in the blood and sera samples, respectively, from 72 Brazilian wild captive felids maintained in some instituitions and zoos. Eighteen (25.0%) and fifty-three (73.6%) out of 72 animals were seroreagent for E. canis and B. canis antigen, respectively, by IFA (Indirect Immunofluorescent Assay). Eleven (15.3%) of the 72 samples were positive for nPCR E. canis. The amplicons were confirmed by sequencing and the E. canis DNA found appeared to be closely related to E. canis samples from Brazil, Mexico, Portugal, Greece and Taiwan with 98% percent identity. None of the 72 samples were positive for B. canis by PCR. This is the first molecular detection of E. canis and presence of seroreactivity for both B. canis and E. canis in Brazilian wild captive felids. / Mestre

Com o objetivo de estudar a fauna parasitária de Cerdocyon thous e Pseudalopex gymnocercus foram estimadas a prevalência, a intensidade média de infecção e abundância de helmintos gastrintestinais e artrópodes nesses hospedeiros. Foram capturados 40 canídeos silvestres na Região Sul do Estado do Rio Grande do Sul, nos municípios de Pedro Osório e Pelotas. Os animais foram capturados usando-se armadilha tipo Live Trap. Logo após a captura, os animais eram sedados, momento em que se coletava sangue e buscava-se coletar artrópodes ectoparasitos mediante exame minucioso. Em seguida os animais eram transportados para o Laboratório de Parasitologia do Departamento de Microbiologia e Parasitologia do Instituto de Biologia da UFPel, onde eram sacrificados usando-se pentobarbital sódico. Após a necropsia, os compartimentos do trato digestório, respiratório e renal foram isolados e examinados na busca de parasitos. O crânio dos animais foi usado para identificação taxonômica dos canídeos, dos quais 45% eram C. thous e 55% P. gymnocercus, podendo-se inferir que ambos canídeos são encontrados habitando a região fisiográfica desse trabalho. A totalidade dos animais (100%) apresentou-se parasitado. Amblyomma aureolatum é o carrapato com maior prevalência em P. gymnocercus (23,7%) e C. thous (61,11%) na região sul do Rio Grande do Sul. Nos nematódeos, os parasitos mais freqüentes foram: Ancylostoma caninum (C. thous 22,22%e P. gymnocercus 45,45%), Molineus felineus (C. thous 5,56% e P. gymnocercus 9,90%), Strongyloides spp. (C. thous 16,67% e P. gymnocercus 22,73%), Trichuris spp (C. thous 11,11% e P. gymnocercus 13,64%), e Capillaria hepática (C. thous 5,56% e P. gymnocercus 13,64%). Nos trematódeos, os parasitos encontrados foram: Alaria alata (C. thous 50,00% e P. gymnocercus 36,36%) e Athesmia heterolecithodes (C. thous 5,56%). Nos cestódeos, os parasitos encontrados foram: Spirometra spp (C. thous 61,11% e P. gymnocercus 54,55%) e Diphillobotriidae (C. thous 77,78% e P. gymnocercus 81,82%). Nos Acanthocephala os parasitos encontrados foram: Centrorhynchus sp. (C. thous 5,56%). Nos protozoários, os 7 parasitos encontrados foram: Babesia spp (P. gymnocercus 13,64%), diagnosticada por exame direto. / With the objective of studying parasitic fauna of Cerdocyon thous and Pseudalopex gymnocercus the prevalence, mean intensity of infection and abundance of gastrintestinal helminths and arthropods were observed in those hosts. 40 wild canids were captured in the South Area of the State of Rio Grande do Sul, in the counties of Pedro Osório and Pelotas. The animals were captured being used trap type “Live Trap”. Therefore after the capture, the animals were anesthesied and in that moment, the blood was collected and the presence or not of ectoparasites was observed. Therefore the animals were transported for the Laboratory of Parasitology, Department of Microbiology and Parasitology, Institute of Biology, UFPel, where they were sacrificed being used sodic pentobarbitol. After this, the portions of the intestinal and respiratory tract and kidney were isolated and examined in the search of parasites. The skull of the animals was used for the taxonomic identification of the hosts. C. thous (45%) and P. gymnocercus (55%) where observed. All hosts (100%) were parazited by some parasite type. Two species of Amblyomma were identified: A. aureolatum and A. tigrinum. Among the nematoda, the most frequent parasites were: Ancylostoma caninum (C. thous 22,22%e P. gymnocercus 45,45%), Molineus felineus (C. thous 5,56% e P. gymnocercus 9,90%), Strongyloides spp. (C. thous 16,67% e P. gymnocercus 22,73%), Trichuris spp (C. thous 11,11% and P. gymnocercus 13,64%), and Capillaria hepática (C. thous 5,56% and P. gymnocercus 13,64%). Among the trematoda, were found Alaria alata (C. thous 50,00% and P. gymnocercus 36,36%) and Athesmia heterolecithodes (C. thous 5,56%). Among the cestoda, the found parasites were: Spirometra spp. and Diphillobotriidae. Acanthocephala the found parasites were: Centrorhynchus sp. and among the protozoa, the found parasites were: Babesia spp (P. gymnocercus 13,64%), diagnosed by direct exam.

Com o objetivo de estudar a fauna parasitária de Cerdocyon thous e Pseudalopex gymnocercus foram estimadas a prevalência, a intensidade média de infecção e abundância de helmintos gastrintestinais e artrópodes nesses hospedeiros. Foram capturados 40 canídeos silvestres na Região Sul do Estado do Rio Grande do Sul, nos municípios de Pedro Osório e Pelotas. Os animais foram capturados usando-se armadilha tipo Live Trap. Logo após a captura, os animais eram sedados, momento em que se coletava sangue e buscava-se coletar artrópodes ectoparasitos mediante exame minucioso. Em seguida os animais eram transportados para o Laboratório de Parasitologia do Departamento de Microbiologia e Parasitologia do Instituto de Biologia da UFPel, onde eram sacrificados usando-se pentobarbital sódico. Após a necropsia, os compartimentos do trato digestório, respiratório e renal foram isolados e examinados na busca de parasitos. O crânio dos animais foi usado para identificação taxonômica dos canídeos, dos quais 45% eram C. thous e 55% P. gymnocercus, podendo-se inferir que ambos canídeos são encontrados habitando a região fisiográfica desse trabalho. A totalidade dos animais (100%) apresentou-se parasitado. Amblyomma aureolatum é o carrapato com maior prevalência em P. gymnocercus (23,7%) e C. thous (61,11%) na região sul do Rio Grande do Sul. Nos nematódeos, os parasitos mais freqüentes foram: Ancylostoma caninum (C. thous 22,22%e P. gymnocercus 45,45%), Molineus felineus (C. thous 5,56% e P. gymnocercus 9,90%), Strongyloides spp. (C. thous 16,67% e P. gymnocercus 22,73%), Trichuris spp (C. thous 11,11% e P. gymnocercus 13,64%), e Capillaria hepática (C. thous 5,56% e P. gymnocercus 13,64%). Nos trematódeos, os parasitos encontrados foram: Alaria alata (C. thous 50,00% e P. gymnocercus 36,36%) e Athesmia heterolecithodes (C. thous 5,56%). Nos cestódeos, os parasitos encontrados foram: Spirometra spp (C. thous 61,11% e P. gymnocercus 54,55%) e Diphillobotriidae (C. thous 77,78% e P. gymnocercus 81,82%). Nos Acanthocephala os parasitos encontrados foram: Centrorhynchus sp. (C. thous 5,56%). Nos protozoários, os 7 parasitos encontrados foram: Babesia spp (P. gymnocercus 13,64%), diagnosticada por exame direto. / With the objective of studying parasitic fauna of Cerdocyon thous and Pseudalopex gymnocercus the prevalence, mean intensity of infection and abundance of gastrintestinal helminths and arthropods were observed in those hosts. 40 wild canids were captured in the South Area of the State of Rio Grande do Sul, in the counties of Pedro Osório and Pelotas. The animals were captured being used trap type “Live Trap”. Therefore after the capture, the animals were anesthesied and in that moment, the blood was collected and the presence or not of ectoparasites was observed. Therefore the animals were transported for the Laboratory of Parasitology, Department of Microbiology and Parasitology, Institute of Biology, UFPel, where they were sacrificed being used sodic pentobarbitol. After this, the portions of the intestinal and respiratory tract and kidney were isolated and examined in the search of parasites. The skull of the animals was used for the taxonomic identification of the hosts. C. thous (45%) and P. gymnocercus (55%) where observed. All hosts (100%) were parazited by some parasite type. Two species of Amblyomma were identified: A. aureolatum and A. tigrinum. Among the nematoda, the most frequent parasites were: Ancylostoma caninum (C. thous 22,22%e P. gymnocercus 45,45%), Molineus felineus (C. thous 5,56% e P. gymnocercus 9,90%), Strongyloides spp. (C. thous 16,67% e P. gymnocercus 22,73%), Trichuris spp (C. thous 11,11% and P. gymnocercus 13,64%), and Capillaria hepática (C. thous 5,56% and P. gymnocercus 13,64%). Among the trematoda, were found Alaria alata (C. thous 50,00% and P. gymnocercus 36,36%) and Athesmia heterolecithodes (C. thous 5,56%). Among the cestoda, the found parasites were: Spirometra spp. and Diphillobotriidae. Acanthocephala the found parasites were: Centrorhynchus sp. and among the protozoa, the found parasites were: Babesia spp (P. gymnocercus 13,64%), diagnosed by direct exam.

Para determinação da ocorrência de transmissão transplacentária da Theileria equi em neonatos eqüinos foram avaliados 50 potros da raça Puro Sangue Lusitano, machos e fêmeas, bem como suas respectivas mães, logo após o parto. Foram colhidas amostras de sangue total, tanto das mães como dos neonatos, entre as primeiras cinco horas pós parto para pesquisa de Theileria equi e Babesia caballi através da técnica de RT-PCR. Utilizou-se o kit de detecção baseado no fluofóro intercalante de DNA SYBERgreen. Um total de 46% das éguas apresentaram resultado positivo para Theileira equi e 54% se mostraram negativas, enquanto que 66% dos potros apresentaram resultados positivos e 34% negativos, sendo que 73,9% dos potros positivos nasceram de mães também positivas. Já para Babesia caballi, 16% das éguas foram positivas e 84% negativas, assim como 2% dos potros foram positivos e 98% negativos. O teste de RT-PCR é bastante sensível e específico, mas pode resultar em falso negativo, apesar de ser eficaz na detecção da Theileria equi e Babesia caballi nos eqüinos. Estes dados permitem concluir que existe a possibilidade de transmissão transplacentária de Theileria equi. / The occurrence of transplacentary transmission of Theileria equi in horses was determined by evaluating 50 young male and female horses of the breed Lusitano Horses as well as their respective mothers. Colts and fillies were evaluated as soon as they were born. Total blood samples were collected from both mother and offspring within the first five hours right after the parturition to analyse Theileria equi and Babesia caballi through the RT-PCR technique. It was used the kit of detection based on DNA SYBERgreen. This study showed us that 46% of the female horses had positive results for Theileira equi and 54% negative results while 66% of the male horses had positive results and 34% of them, negative ones. Moreover, 73.9% of the positive young horses also had their mothers positive. However, for Babesia caballi 16% of the female horses had positive results and 84% negative ones while 2% of the male horses had positive results and 98%, negative ones. The RT-PCR test is very sensitive and specific but it can occur false-negative results although it is efficient in detecting Theileria equi and Babesia caballi in horses. In conclusion, the data show us that there is a possibility of transplacentary transmission of Theileria equi.

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.

La babésiose canine est une maladie parfois mortelle due a des protozoaires des genres Babesia et Theileria transmis par des tiques dont Rhipicephalus sanguineus. Malgré la présence soutenue de R. sanguineus dans le Sud de la France, son rôle dans la transmission de la maladie dans cette région reste incertain. D'autre part, le statut taxonomique de R. sanguineus reste confus et mérite d'être clarifié. Les objectifs de ce travail ont été de caractériser (i) les tiques R. sanguineus s.l. parasitant les chiens dans différentes régions du Sud de la France, (ii) les espèces des genres Babesia/Theileria potentiellement transmises par ces tiques et (iii) l'impact des variations génétiques et/ou des co-infections sur la compétence vectorielle. Au total 140 sangs de chien et 242 tiques R. sanguineus s.l. ont été récoltées et analysées entre 2010 et 2012 par PCR, PCR-RFLP et séquençage. De l'ADN de B. vogeli et B. canis a été détecté dans 13,6% et 12,9% des chiens et dans 10,7% et 1,7% des tiques respectivement avec, parfois, des prévalences significativement différentes entre les régions. Un seul chien a été détecté porteur d'ADN de T. annae. Des comparaisons phylogénétiques et phylogéographiques de séquences de R. sanguineus (ADNr-mt 12S et 16S) et B. vogeli (ARNr 18S) à l'échelle mondiale ont confirmé l'affiliation des tiques de France aux R. sanguineus des régions « tempérées » et suggèrent l'existence d'une coévolution entre le protozoaire et son vecteur. Ce travail apporte de nouvelles connaissances sur la babésiose canine en France et supporte l'existence d'au moins deux populations au sein du groupe sanguineus pouvant présenter des compétences vectorielles différentes / Study on Rhipicephalus sanguineus sensu lato ticks and their potential role in the transmission of canine babesiosis in France: prevalence of parasite infections, vector genetic diversity and epidemiology. Canine babesiosis is a potentially fatal disease caused by protozoa of the genera Babesia and Theileria transmitted by ticks including Rhipicephalus sanguineus. Although R. sanguineus is a major tick species in southern France, its role in the transmission of the disease in this area remain unknown. Moreover, the taxonomic status of R. sanguineus is controversial and needs to be clarified. The aims of this work were to characterize (i) the ticks R. sanguineus s.l. that parasitize dogs in different areas of southern France, (ii) the Babesia/Theileria species potentially transmitted by these ticks in this area and (iii) the impact of genetic variations and/or co-infections on vector competence. A total of 140 dog bloods and 588 R. sanguineus s.l. were collected from 2010 to 2012 from which all dogs bloods and 242 ticks were screened using specific PCR, PCRRFLP and sequencing. B. vogeli and B. canis DNA were evidenced in 13.6% and 12.9% of dogs and in 10.7% and 1.7% of R. sanguineus ticks respectively with sometimes significant prevalence differences between areas. A single case of T. annae infection was detected in a dog. Phylogenetic and phylogeographic analyzes performed at a global scale on 12S and 16S mt-rDNA for ticks and 18S rDNA for B. vogeli confirmed the affiliation of R. sanguineus from France with “temperate” species and suggested the existence of a coevolution process between the pathogen and its vector. This work provides new information on the epidemiology of canine babesiosis in France and supports the existence of at least two populations in the R. sanguineus group in the world with possible different vector competences

Babesia canis est un hémoparasite du phylum des Apicomplexes transmis par la morsure de tique et responsable de la piroplasmose canine en Europe. Dans la perspective de développer un vaccin recombinant, nous avons réalisé deux études visant à mieux comprendre les interactions hôte/parasite au cours du cycle érythrocytaire. Nous avons étudié d'une part le rôle des antigènes parasitaires solubles (APS) dans le déclenchement des signes cliniques et d'autre part celui de l'antigène Bc28.2 codé par la famille multigénique Bc28 dans les mécanismes d'échappement à l'hôte.Les APS de B. canis induisent une réponse protectrice anti-maladie chez les chiens vaccinés. Toutefois, leur rôle exact dans la pathogénèse reste à définir. Contrairement à ce qui est décrit dans la pathogenèse à B. bovis, nos analyses réfutent l'hypothèse qu'ils pourraient agir sur le système kallicréine-kinine plasmatique. Par contre elles suggèrent, pour la première fois chez le genre Babesia, un rôle direct des APS dans le déclenchement précoce de la réponse inflammatoire observée au cours de la pathogenèse.De part leur fonction essentielle dans la survie parasitaire, les antigènes localisés à la surface de l'érythrocyte (rôle dans l'agglutination des hématies parasitées) ou des mérozoïtes (rôle dans l'invasion) sont de bons candidats vaccins. Cependant et probablement dans une perspective d'échappement à l'hôte, ils sont codés par des familles multigéniques. Chez B. canis, la famille multigénique Bc28 contient le gène Bc28.1 qui code pour un antigène à ancrage GPI préalablement caractérisé à la surface du mérozoïte. Nous montrons qu'un autre gène, désigné Bc28.2 (plusieurs copies dans le génome) contient 2 cadres de lecture chevauchant et serait capable d'exprimer des antigènes polymorphes de 28 kDa et 50 kDa par un mécanisme de recodage traductionnel +1. De façon originale, ces protéines seraient localisées non pas à la surface des mérozoïtes mais à la surface des hématies parasitées. / Babesia canis is an apicomplexan haemoparasite transmitted by tick bite and responsible of canine babesiosis in Europe. Understanding host/parasite relationships during the erythrocytic cycle is crucial for further development of a recombinant vaccine. In that way, the role of soluble parasite antigens (SPA) in the onset of clinical signs and the role of Bc28.2 antigen (encoded by the B. canis Bc28 multigene family) in host evading process were investigated.B. canis-derived SPA induce a protective anti-disease immunity in vaccinated dogs but their precise role during the pathogenesis remains unknown. In contrast to B. bovis, our analysis disproved the hypothesis that B. canis SPA could act on the plasma kallikrein-kinin system. However they strongly suggest, for the first time in the genus Babesia, a direct role of these SPA in the onset of the inflammatory response which is early observed during pathogenesis. Because of their essential function in the parasite survival, antigens located on the surface of infected erythrocyte (role in agglutination of erythrocytes) or on the surface of merozoites (role in the invasion) are good vaccine candidates.However, and probably for host evading, they are encoded by multigene families. In B. canis, the Bc28 multigene family contains the Bc28.1 gene that encodes for a GPI-anchored antigen previously characterized on the merozoite surface. We demonstrated here, that another gene designated Bc28.2, is multicopy and composed of two overlapping open reading frames (Orf1 and Orf2). It allows, though +1 programmed ribosomal frameshift, expression of polymorphic antigens of 28 kDa and 50 kDa. Unexpectedly, these proteins seem localized on the surface of parasitized erythrocytes, suggesting they play a crucial function in evading host through agglutination process of infected erythrocytes.

Made available in DSpace on 2015-03-26T13:47:01Z (GMT). No. of bitstreams: 1 texto completo.pdf: 424006 bytes, checksum: e70b787e481758ae2e9bc9b63c6cb946 (MD5) Previous issue date: 2006-12-14 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / It was evaluated the immunology response given by the SBbo23290 and SBm7462 synthetic peptides to the control of Babesia bovis and Riphicephalus (Boophilus) microplus, inoculated by subcutaneous route, individually and simultaneously (Group I) and in a cocktail of both peptides (Group II). Each inoculation was done with intervals of 30 days. In the 30th and 34th days after the 3th inoculation, it was applied in each animal, 4000 larvaes of R. (B.) microplus, free of blood parasites and also, it was inoculated a sample of Babesia bovis UFV1 9th passage with a concentration of 1x106 babesias/mL, respectively. According to the results, the animals inoculated with the Group II obtained greater clinic response after challenging with both parasites. Besides, these animals showed high levels of total IgG specific to the SBbo23290, beginning one week after 1st inoculation, with titles of 1,06 ± 0,2486. The titles with high affinity of total IgG to SBm7462 was presented from the 8th week, with levels of 1,75 ± 0,1849 and 1,13 ± 0,2819 to Groups I and II, respectively. Both Group showed high levels of IgG1 over IgG2 isotopes with high affinity to SBbo23290. The Group I showed titles of total IgG and IgG1 with high affinity to SBm7462 and low efficiency (EF) to control the ticks, different of the obtained in the Group II, where it was found high titles of total IgG, low levels of IgG1 and EF of 46,80 %. These results suggest that, intraclonal competition was present after presentation by APCs (DC or WC1.1+) to CD4+ and lymphocytes B in the germinal centers, with major affinity by specific clones to SBbo23290 over SBm7462. It is possible that these changes were determined by antigen solubility. / Avaliou-se a resposta imunológica conferida pelos peptídeos SBbo23290 e SBm7462 para o controle de Babesia bovis e Riphicephalus (Boophilus) microplus, ao serem aplicados simultaneamente na apresentação individual (Group I) e em associação (Group II). Foram realizadas três aplicações em cada grupo, via subcutânea, com intervalos de 30 dias. O desafio foi feito 30º e 34º dias após a 3ª inoculação, colocando-se 4000 larvas de R. (B.) microplus, cepa livre de hemoparasitas, e inoculando-se a cepa de B. bovis UFV1 9ª passagem numa concentração de 1x106 parasitas/mL, respectivamente. De acordo com os resultados obtidos, os animais do Grupo II, apresentaram uma melhor resposta clínica frente ao desafio, acompanhado de altos níveis de IgG total específicos para o peptídeo SBbo23290, a partir da 1ª semana após a inoculação, com títulos de 1,06 ± 0,2486. Os títulos de IgG total específicos para SBm7462 estiveram presentes a partir da 8ª semana com níveis de 1,75 ± 0,1849 e 1,13 ± 0,2819 para Grupo I e II, respectivamente. Ambos os tratamentos apresentaram maiores níveis de isotipos IgG1 sobre IgG2 específicos para SBbo23290. O Grupo I, apresentou títulos de IgG total e IgG1 específicos para SBm7462 e baixa eficácia (EF) no controle dos carrapatos, diferente do obtido no Grupo II, onde encontrou-se altos títulos de IgG total, níveis baixos do IgG1 e uma EF de 46,80 %. Sugere-se que estes resultados foram determinados por competição intraclonal em populações após a apresentação por APCs (DC ou WC1.1+) a CD4+ e linfócitos B nos centros germinais, com maior afinidade por clones específicos para SBbo23290 sobre SBm7462, talvez determinado por solubilidade antigênica.

Made available in DSpace on 2015-03-26T13:47:27Z (GMT). No. of bitstreams: 1 texto completo.pdf: 923002 bytes, checksum: d038ed7630080e45d27557c298896b35 (MD5) Previous issue date: 2006-12-15 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Sixteen bovines belonged to Bos taurus taurus species, serumly and parasitically negatives to hemoparasites, were divided in four groups of inoculation: Control groups, Saponine group, Treatment group I and Treatment group II. The following study had the objective to evaluate and measure the cellular immune response and the protection given to bovines, through simultaneous vaccination of synthetics immunogenics SBbo 23290 in opposite sides (Treatment I) and through vaccination of the same immunogenics SBm 7462 + SB 23290 in the same dose (Treatment II). All animals were inoculated three times, through subcutaneous, with intermissions of thirty days. The challenges were done thirty and thirty-four days after third inoculation, placing 2000 grubs of Ripicephalus Boophilus miroplus, out of hemoparasites, and lately inoculating the strain of Babesia bovis UFV1 ninth passing, in the concentration of 1 x 106 Babesias/ml, respectively. The results show that, along the study, animals vaccinated in two forms related obtained an alteration of the sub population of lymphocytes present in the peripheral blood. Among the population of lymphocytes studied, a bigger increase for the sub population of cells B CD21+ was seen. Through the study of kinetic production of interleukin 4, was valued the activation of blood peripheral mononuclear cells (PBMC) cultivated after vaccination of animals and beforehand stimulated together with synthetic peptides SBbo 23290 e SBm 7462. The quantification of IL-4 production showed low levels for Treatment group I, Treatment group II and Saponine group. / Dezesseis bovinos pertencentes a espécie Bos taurus taurus, negativos sorologicamente e parasitologicamente à hemoparasitas foram divididos em quatro grupos de inoculação: grupo Controle, grupo adjuvante Saponina, grupo Tratamento I e grupo com Tratamento II. O presente estudo teve como objetivo avaliar a resposta imune celular e a proteção conferida aos bovinos, pela vacinação simultânea com os imunógenos sintéticos SBm 7462 e SBbo 23290 em lados opostos (Tratamento I) e pela vacinação dos mesmos imunógenos SBm 7462 + SBbo 23290 na mesma dose (Tratamento II). Todos os animais foram inoculados três vezes, via subcutânea, com intervalos de trinta dias. O desafio foi feito 30 e 34 dias após a terceira inoculação, colocando-se 2000 larvas de Ripicephalus Boophilus microplus, livres de hemoparasitas, e posteriormente inoculando a cepa de Babesia bovis UFV1 - 9ª passagem, na concentração de 1x 106 Babesias/mL, respectivamente. Os resultados demonstraram que, ao longo do estudo, os animais vacinados das duas formas desenvolveram uma modificação das sub-populações de linfócitos presentes no sangue periférico. Dentre as populações de linfócitos estudadas houve um maior aumento para as sub-populações de células B CD21+. Por meio de um estudo da cinética de produção de Interleucina-4 avaliou-se a ativação de células sanguíneas mononucleares periféricas (PBMC) cultivadas após a vacinação dos animais e previamente estimuladas juntamente com os peptídeos sintéticos SBbo 23290 e SBm 7462 .Os resultados da produção de IL-4 apresentaram baixos níveis para os grupos Tratamento I, Tramento II e Saponina.