A Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) is morphologically similar and genetically identical, based on SSU rRNA gene comparisons, to two agents responsible for human babesiosis in North America and is closely related to the European parasite, Babesia divergens. The ribosomal RNA (rRNA) internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S rRNA genes of Babesia isolates were sequenced and analyzed. The rRNA ITS region sequences of three isolates, one each from Kentucky, Massachusetts and Great Britain, considered Babesia divergens-like organisms, were compared to two Babesia microti isolates, two Babesia odocoilei isolates and a well defined Babesia divergens isolate. The two B. divergenslike isolates from North America shared identical rRNA ITS1-5.8S-ITS2 region sequences, and the clones of these isolates clustered into one clade in three phylogenetic analyses, suggesting that these isolates are conspecific. In vitro comparison of host erythrocyte specificity between the rabbit Babesia sp. and B. divergens was employed to discriminate between the two organisms and to determine the usefulness of in vitro techniques for Babesia sp. characterization. In vitro growth of the rabbit Babesia sp. was supported in human and cottontail rabbit erythrocytes, but not in bovine cells. Babesia divergens in vitro growth was supported in human and bovine erythrocytes, but not in cottontail rabbit cells. Morphological characteristics and size differences also distinguished the two parasites from one another. The erythrocyte specificity and parasite size differences reported in this study agree with previous in vivo results and validate the use of in vitro methods for characterization of Babesia species.

Babesia species have a worldwide distribution, affecting a wide range of mammalian hosts. The major route of transmission is inoculation by an infected Ixodid tick. Babesia species of major economic concern are those that cause bovine and equine babesiosis. Historically, bovine Babesia species, Babesia bovis and Babesia bigemina caused significant economic losses in the United States in the 1860’s, as thousands of cattle died. Also, outbreaks of equine babesiosis, caused by Babesia equi or Babesia caballi, have occurred in the United States resulting in the death of some horses and millions of dollars in losses. A constant risk of reinfection with bovine and equine Babesia species exists, as stray and smuggled animals from Mexico, where bovine babesiosis is endemic, may carry infected ticks as they cross the border, and, thousands of horses from B. equiand B. caballi-endemic regions are imported through Florida every year. Vaccines have been developed for a number of Babesia species, none of which result in sterile immunity. The live attenuated vaccine is the most commonly used vaccine against Babesia species. However, the basis for the vaccine is to maintain a carrier state in order to prevent disease. Other vaccine designs have been developed to invoke protection without a carrier state but have been unsuccessful. It has been shown that the cysteine protease is important in the life cycle of a number of parasitic organisms, making it a good target for vaccine development. The vaccine design for this study incorporated the cysteine protease of Babesia microti. Babesia microti naturally infects Peromyscus leucopus (white-footed mouse) and is the major cause of human babesiosis in the United States. Using B. microti in the vaccine design allowed for the use of a mouse model to determine whether the cysteine protease of other economically important Babesia species may make a good vaccine target. The vaccine design incorporated a prime-boost strategy, priming with DNA encoding the cysteine protease and boosting two times with either DNA encoding the cysteine protease or cysteine protease peptide, followed by parasite challenge. Analysis of daily percent parasitemias, packed cell volume, and seroconversion of all groups revealed that a protective immune response against B. microti was not elicited by this vaccine strategy.

Ph.D. / Bovine babesiosis, or redwater, is at present known to be caused by Babesia bigemina and Babesia bovis in the Republic of South Africa. Until recently, however, the only information on the natural transmission of these parasites in the country was based on observations made during the early part of this century and information on the developmental cycle of the parasites in their vectors was superficial or nonexistent ...

Babesiosis is caused by a genus of tick-transmitted apicomplexan parasites with considerable economic, medical, and veterinary impact. Bovine babesiosis is an important impediment to livestock production throughout the world. Limited options are available for control of this widespread protozoal disease. This study evaluated the protective effect of DNA vaccines incorporating Babesia cysteine proteases and Apical Membrane Antigen-1 separately and in combination. The Helios Gene Gun System was used to vaccinate BALB/c mice with plasmid DNA constructs encoding different B. microti proteins (pBmCP1, pBmAMA1 or a combination of pBmCP1 and pBmAMA1). An analysis of the parasitemia post-challenge supports the hypothesis that pBmCP1 and pBmAMA1 induce protective effects against the progression of the parasite. However, the combination of the two constructs given simultaneously has no marked effect on parasite progression. Furthermore, the data obtained from the packed cell volumes of the mice indicates that only BmCP1 is able to reduce this effect of clinical disease with any level of significance. Babesia bovis constructs containing Cysteine Protease-2 and Apical Membrane Antigen-1 were created and sequence verified for use in future vaccination studies. The results seen using the mouse model of Babesiosis may provide applicable information for the design of vaccines against other Babesia spp., particularly for B. bovis.

Universiẗat, Diss., 2004--München.

Submitted by TALITA BARBAN BILHASSI null (talitabarban@yahoo.com.br) on 2016-04-26T17:24:25Z No. of bitstreams: 1 TESE TALITA BARBAN BILHASSI.pdf: 3215997 bytes, checksum: 07aede5d76602ed82d16385cc21986cf (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-28T18:47:32Z (GMT) No. of bitstreams: 1 bilhassi_tb_dr_jabo.pdf: 3215997 bytes, checksum: 07aede5d76602ed82d16385cc21986cf (MD5) / Made available in DSpace on 2016-04-28T18:47:32Z (GMT). No. of bitstreams: 1 bilhassi_tb_dr_jabo.pdf: 3215997 bytes, checksum: 07aede5d76602ed82d16385cc21986cf (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Entre as principais causas de perdas produtivas em bovinos criados nos trópicos está a infestação pelo carrapato Rhipicephalus (Boophilus) microplus e, consequentemente, dos hemoparasitas transmitidos por ele. A resistência dos zebuínos e de animais cruzados com raças taurinas à infestação por esse ácaro é amplamente conhecida. Entretanto, no que se refere à suscetibilidade às babesioses bovinas, existem evidências de que o grupo genético também pode interferir na resistência, seguindo o mesmo padrão observado para o carrapato vetor, com os taurinos apresentando maior sensibilidade. Assim, este estudo teve por objetivo avaliar a parasitemia por Babesia bovis e Babesia bigemina em 50 novilhas da raça Canchim ( Charolês + Zebu) naturalmente infestadas pelo R. (B.) microplus nas quatro estações do ano durante 24 meses, além de caracterizar o perfil de citocinas que podem estar associados ao fenótipo de resistência e suscetibilidade aos hemoparasitas do gênero Babesia spp. Foram realizadas contagens de fêmeas adultas de carrapatos com tamanho igual ou superior a 4,5 mm de diâmetro, presentes no lado esquerdo de cada bovino. As amostras de DNA extraídas foram submetidas à amplificação por meio da Reação em Cadeia da Polimerase Quantitativa em Tempo Real (qPCR), utilizando iniciadores que flanqueiam fragmentos dos genes mitocondriais do citocromo b (mt-cyt B), específicos para B. bovis e B. bigemina. O RNA extraído do sangue, foi usado para sintetizar o DNA complementar (cDNA) para análise de expressão dos genes do IFN- , TNF- , IL-10 e IL-12B por meio da quantificação relativa (RTqPCR). Foram observadas diferenças significativas (P<0,05) entre os meses das avaliações para a contagem de carrapatos. Entretanto, não houve efeito significativo (P>0,05) nas colheitas realizadas entre novembro de 2013 a janeiro de 2014 e entre os meses janeiro e fevereiro de 2015. A frequência da parasitemia no rebanho foi de 98%. Dentre as amostras de DNA que puderam ser quantificadas pela qPCR, 98% e 95,4% foram positivas para B. bovis e B. bigemina, respectivamente. Com relação ao número de cópias (NC) dos fragmentos dos genes mt-cyt B específicos para B. bovis e B. bigemina foram observados efeitos significativos (P<0,05) para ambas as espécies e interação das mesmas com as colheitas realizadas nas diferentes estações do ano. A análise do nível de expressão de mRNA do IFN- , TNF- e IL-12B revelou que houve um efeito siginificativo (P<0,05) da interação entre animais dos extremos de resistência/suscetibilidade e estações do ano, exceto para IL-10. Conclui-se que, a qPCR apresenta alta sensibilidade e especificidade para o diagnóstico das babesioses bovinas em amostras de sangue e que a oscilação na carga parasitária nas diferentes estações do ano pode estar associada com o perfil de expressão de citocinas apresentada. / Among the main causes of production losses in cattle is in the tropics infestation by Rhipicephalus (Boophilus) microplus, and consequently the hemoparasites transmitted by it. The resistance of zebu and crossbred with European breeds to infestation by this mite is widely known. However, as regards susceptibility to bovine babesiosis, there is evidence that genetic group can also interfere in resistance following the same pattern observed in the tick vector, with the taurine presenting greater sensitivity. This study aimed to evaluate parasitaemia by Babesia bovis and Babesia bigemina in 50 heifers Canchim (⅝ Charolais + ⅜ Zebu) naturally infested by R. (B.) microplus in four seasons for 24 months, and characterize the profile of cytokines that may be associated with phenotype resistance and susceptibility by gender hemoparasites Babesia spp. Adult female ticks counts with size equal to or greater than 4.5 mm in diameter, present in the left side of each calf were performed. The extracted DNA samples were subjected to amplification by Reaction Polymerase Chain Quantitative Real Time (qPCR), using primers flanking fragments of mitochondrial gene cytochrome B (mt-cyt B) specific for B. bovis and B. bigemina. The RNA extracted from the blood was used to synthesize complementary DNA (cDNA) for expression analysis of genes IFN-γ, TNF-α, IL-10 and IL-12B by relative quantification (RT-qPCR). Significant differences were observed (P <0.05) between the months of reviews for the tick count. However, there was no significant effect (P>0.05) in samples taken between november 2013 and january 2014 and between the months january and february 2015. The frequency of parasitaemia in the herd was 98%. Among the samples of DNA that could be quantified by qPCR, 98% and 95.4% were positive for B. bovis and B. bigemina, respectively. Regarding the number of copies (NC) of fragments of genes mt-cyt B, specific to B. bovis and B. bigemina significant effects were observed (P<0.05) for both species and interaction between those harvests in different seasons. Analysis of the mRNA expression level of IFN-γ, TNF-α and IL-12B showed that there was a significant effect (P<0.05) interaction between the animal extreme resistance/susceptibility and seasons, except for IL-10. It is concluded that the qPCR has high sensitivity and specificity for the diagnosis of bovine babesiosis in blood samples and that the fluctuation in the parasitic load in the different seasons of the year may be associated with the profile of cytokine expression presented by herd animals Canchim during the experimental period. / FAPESP: 2013/16246-9

No description available.

The aim of this study was to determine the serologic and molecular occurrence of Rickettsia spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. in ticks and domestic and wild animals from two conservation units in the city of Natal, Rio Grande do Norte State, Brazil. The collection period was between October 2012 and August 2013. Serum samples were tested against Rickettsia spp. antigens and Ehrlichia canis by Indirect fluorescent antibody test. Tissue samples and ticks were processed for molecular detection of the pathogens. Twenty-seven marsupials and four rodents were captured, and up to three animals of each species were euthanized. In addition, serum samples from 155 domestic animals: 53 cats living inside the units, 29 dogs domiciled around the areas and 73 dgos of the Zoonosis Control Center of the City (ZCC). Twenty dogs from ZCC were also euthanized and samples of spleen were obtained. Antibodies to at least one of the Rickettsia species tested were detected in six Didelphis albiventris and in one Rattus rattus; 17% (17/102) of the dogs presented antibodies to E. canis and 13% (20/155) of all tested domestic animals (dogs and cats) were seropositive for Rickettsia spp. antigens. Three species of ticks (Amblyomma auricularium, Ixodes loricatus and Ornithodoros mimon) were collected and one A. auricularium was positive for Rickettsia amblyommii by PCR. Two D. albiventris spleen samples amplified PCR products for Ehrlichia spp. Spleen samples from three D. albiventris and spleen and lung sample from one Necromys lasiurus were positive for Babesia spp. by PCR test. Among the 20 spleen samples from dogs subjected to molecular analysis, eight were positive by PCR for E. canis and two for H. canis / O objetivo deste estudo foi determinar a ocorrência sorológica e molecular de Rickettsia , Ehrlichia , Hepatozoon e Babesia em carrapatos e mamíferos silvestres e domésticos, provenientes de duas unidades de conservação ambiental (UC) na cidade de Natal, Rio Grande do Norte, Brasil. O período de coleta foi de outubro de 2012 a agosto de 2013. Os soros foram testados contra antígenos de Rickettsia spp. e Ehrlichia canis através da reação de imunofluorescência indireta. Amostras de tecido e carrapatos foram processadas para a detecção molecular dos patogenos. Foram capturados 27 marsupiais e quatro roedores para coleta de sangue, destes foram eutanásiados ate três animais de cada espécie e coletadas amostras de baço e pulmão. Paralelo, amostras de soro de 155 animais domésticos: 53 gatos que viviam nas UCs, 29 cães domiciliados no entorno das areas e 73 cães do Centro de Controle de Zoonoses do município, dos quais 20 tiveram amostras de baço coletadas. Foram detectados anticorpos para, pelo menos, uma das espécies de Rickettsia testadas em seis Didelphis albiventris e em um Rattus rattus , e 17 % (17/102) dos cães apresentaram anticorpos anti- E. canis e 13% (20/155) de todos os animais domésticos (cães e gatos) foram soropositivos para antígenos de Rickettsia spp. Três espécies de carrapatos (Amblyomma auricularium , Ixodes loricatus e Ornithodoros mimon ) foram coletadas e um A. auricularium foi positivo para R. amblyommii pela PCR. Duas amostras de baço de D. albiventris amplificaram produtos de PCR para Ehrlichia spp. e amostras de baço de três D. albiventris e baço e pulmão de um Necromys lasiurus foram positivas para Babesia spp. pela PCR. Entre as 20 amostras de baço de cão submetidas a análises moleculares, oito foram positivas na PCR para E. canis e duas para H. canis

Thesis (MSc (Veterinary Tropical Diseases, Veterinary Science))--University of Pretoria, 2008. / Includes bibliographical references. Also available in print format.

The aim of this study was to determine the serologic and molecular occurrence of Rickettsia spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. in ticks and domestic and wild animals from two conservation units in the city of Natal, Rio Grande do Norte State, Brazil. The collection period was between October 2012 and August 2013. Serum samples were tested against Rickettsia spp. antigens and Ehrlichia canis by Indirect fluorescent antibody test. Tissue samples and ticks were processed for molecular detection of the pathogens. Twenty-seven marsupials and four rodents were captured, and up to three animals of each species were euthanized. In addition, serum samples from 155 domestic animals: 53 cats living inside the units, 29 dogs domiciled around the areas and 73 dgos of the Zoonosis Control Center of the City (ZCC). Twenty dogs from ZCC were also euthanized and samples of spleen were obtained. Antibodies to at least one of the Rickettsia species tested were detected in six Didelphis albiventris and in one Rattus rattus; 17% (17/102) of the dogs presented antibodies to E. canis and 13% (20/155) of all tested domestic animals (dogs and cats) were seropositive for Rickettsia spp. antigens. Three species of ticks (Amblyomma auricularium, Ixodes loricatus and Ornithodoros mimon) were collected and one A. auricularium was positive for Rickettsia amblyommii by PCR. Two D. albiventris spleen samples amplified PCR products for Ehrlichia spp. Spleen samples from three D. albiventris and spleen and lung sample from one Necromys lasiurus were positive for Babesia spp. by PCR test. Among the 20 spleen samples from dogs subjected to molecular analysis, eight were positive by PCR for E. canis and two for H. canis / O objetivo deste estudo foi determinar a ocorrência sorológica e molecular de Rickettsia , Ehrlichia , Hepatozoon e Babesia em carrapatos e mamíferos silvestres e domésticos, provenientes de duas unidades de conservação ambiental (UC) na cidade de Natal, Rio Grande do Norte, Brasil. O período de coleta foi de outubro de 2012 a agosto de 2013. Os soros foram testados contra antígenos de Rickettsia spp. e Ehrlichia canis através da reação de imunofluorescência indireta. Amostras de tecido e carrapatos foram processadas para a detecção molecular dos patogenos. Foram capturados 27 marsupiais e quatro roedores para coleta de sangue, destes foram eutanásiados ate três animais de cada espécie e coletadas amostras de baço e pulmão. Paralelo, amostras de soro de 155 animais domésticos: 53 gatos que viviam nas UCs, 29 cães domiciliados no entorno das areas e 73 cães do Centro de Controle de Zoonoses do município, dos quais 20 tiveram amostras de baço coletadas. Foram detectados anticorpos para, pelo menos, uma das espécies de Rickettsia testadas em seis Didelphis albiventris e em um Rattus rattus , e 17 % (17/102) dos cães apresentaram anticorpos anti- E. canis e 13% (20/155) de todos os animais domésticos (cães e gatos) foram soropositivos para antígenos de Rickettsia spp. Três espécies de carrapatos (Amblyomma auricularium , Ixodes loricatus e Ornithodoros mimon ) foram coletadas e um A. auricularium foi positivo para R. amblyommii pela PCR. Duas amostras de baço de D. albiventris amplificaram produtos de PCR para Ehrlichia spp. e amostras de baço de três D. albiventris e baço e pulmão de um Necromys lasiurus foram positivas para Babesia spp. pela PCR. Entre as 20 amostras de baço de cão submetidas a análises moleculares, oito foram positivas na PCR para E. canis e duas para H. canis