No description available.

Zugl.: Berlin, Freie Univ., Diss., 2009

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Current bioinformatic methods such as molecular phylogenetics and phylogenomics provide us with good insight to symbiont evolution. Though modern science evolves rapidly, accelerates speed of acquiring novel discoveries and improves their quality, there is still endless row of questions waiting to be answered. This thesis focuses on origins of symbiosis between insects and Enterobacteria, and the mechanisms promoting association of bacteria with arthropods. The main emphasis is put on the secondary symbionts of the genus Sodalis (Enterobacteriaceae) and the pathogenic Anaplasma phagocytophilum (Anaplasmataceae) that seems to be undergoing first steps to become hereditary mutualist.

O patógeno intracelular Anaplasma marginale (Rickettsiales: Anaplasmataceae) é endêmico em muitas regiões tropicais e subtropicais do mundo. A infecção do bovino com A. marginale causa anaplasmose bovina, devido à replicação do microrganismo nos eritrócitos do hospedeiro, sendo responsável por consideráveis perdas econômicas à criação de gado. A transmissão de A. marginale para bovinos ocorre biologicamente por carrapatos ou mecanicamente por insetos hematófagos e instrumentos contaminados com sangue infectado. Uma cepa de A. centrale, naturalmente atenuada tem sido usada extensivamente para controle da anaplasmose bovina em diversas regiões do mundo. A vacina de A. centrale é produzida em bovinos esplenectomizados oriundos de áreas livres de carrapato, todavia uma contaminação acidental por A. marginale é um risco durante o processo de produção. Por este motivo, a produção da vacina deve ser avaliada, para garantir uma amostra de A. centrale livre de contaminação. Durante a fase aguda da doença, o diagnóstico é realizado pela observação de A. marginale por esfregaço sanguíneo, porém durante a fase crônica, este método não tem a sensibilidade necessária para detectar animais que portam níveis baixos de parasitemia. Sendo, o método de detecção molecular o mais indicado para detectar tanto anaplasmose aguda quanto crônica e diferenciar entre A. marginale e A. centrale. Deste modo, no presente trabalho um ensaio de PCR foi padronizado para detectar e diferenciar A. marginale de A. centrale em amostras de sangue bovino. Foram utilizados primers para o gene msp4 de Anaplasma sp. para a amplificação do material obtido a partir de uma extração de DNA de sangue congelado de bovinos experimentalmente infectados com A. centrale e A. marginale. A PCR se mostrou específica, sem amplificar o DNA de outros hemoparasitas. O teste se mostrou sensível para detectar a quantidade 0,25 pg para o DNA de A. centrale e 0,125 pg para detectar o DNA de A. marginale, mostrando-se útil para detectar animais à campo persistentemente infectados com A. marginale e vacinados com A. centrale, os quais foram indetectáveis pela microscopia óptica. Em resumo, a PCR é um teste mais sensível e específico que a microscopia óptica para detectar e diferenciar as espécies de A. centrale e A. marginale, mostrando-se útil para auxiliar no controle de qualidade durante a produção da vacina. / The intracellular pathogen Anaplasma marginale (Rickttsiales: Anaplasmataceae), is endemic in many tropical and subtropical regions of the world. Infection of cattle with A. marginale causes bovine anaplasmosis, due to replication of the microrganism inside the erythrocytes being responsible for considerable economic losses to livestock. The transmission of A. marginale to cattle occurs biologically by ticks or mechanically by blood sucking insects and instruments contaminated with infected blood. A strain of A. centrale, naturally attenuated, has been used extensively to control bovine anaplasmosis in various regions of the world. The vaccine with A. centrale is produced in splenectomized cattle from tick-free areas, however accidental contamination by A. marginale represents a risk during the production process. Therefore, production of the vaccine should be evaluated to ensure a sample of A. centrale free of contamination. During the acute phase of the disease, diagnosis is made by observing A. marginale by blood smear, but during the chronic phase, this method is not as sensitivity as required to detect animals that carry low levels of parasitemia. Being, the molecular detection method more appropriate for detecting both acute and chronic anaplasmosis and differentiate between A. centrale and A. marginale. Thus, in this study a PCR assay was standardized to detect and differentiate A. centrale and A. marginale in samples of bovine blood. Primers for msp4 gene of Anaplasma sp. were used for amplification of material obtained from a DNA extraction of frozen blood from bovines experimentally infected with A. centrale and A. marginale. The PCR showed to be specific, do not amplifying the DNA of other hemoparasites. The test was sensitive to detect the amount of 0.25 pg of DNA from A. centrale and 0.125 pg of DNA from A. marginale, proving to be useful for detecting field animals persistently infected with A. marginale and vaccinated with A. centrale, which were undetectable by optical microscopy. Briefly, PCR is a more sensitive and specific test than the optical microscopy analysis to detect and differentiate the species A. centrale and A. marginale showing to be useful to assist the quality control during the vaccine production.

O patógeno intracelular Anaplasma marginale (Rickettsiales: Anaplasmataceae) é endêmico em muitas regiões tropicais e subtropicais do mundo. A infecção do bovino com A. marginale causa anaplasmose bovina, devido à replicação do microrganismo nos eritrócitos do hospedeiro, sendo responsável por consideráveis perdas econômicas à criação de gado. A transmissão de A. marginale para bovinos ocorre biologicamente por carrapatos ou mecanicamente por insetos hematófagos e instrumentos contaminados com sangue infectado. Uma cepa de A. centrale, naturalmente atenuada tem sido usada extensivamente para controle da anaplasmose bovina em diversas regiões do mundo. A vacina de A. centrale é produzida em bovinos esplenectomizados oriundos de áreas livres de carrapato, todavia uma contaminação acidental por A. marginale é um risco durante o processo de produção. Por este motivo, a produção da vacina deve ser avaliada, para garantir uma amostra de A. centrale livre de contaminação. Durante a fase aguda da doença, o diagnóstico é realizado pela observação de A. marginale por esfregaço sanguíneo, porém durante a fase crônica, este método não tem a sensibilidade necessária para detectar animais que portam níveis baixos de parasitemia. Sendo, o método de detecção molecular o mais indicado para detectar tanto anaplasmose aguda quanto crônica e diferenciar entre A. marginale e A. centrale. Deste modo, no presente trabalho um ensaio de PCR foi padronizado para detectar e diferenciar A. marginale de A. centrale em amostras de sangue bovino. Foram utilizados primers para o gene msp4 de Anaplasma sp. para a amplificação do material obtido a partir de uma extração de DNA de sangue congelado de bovinos experimentalmente infectados com A. centrale e A. marginale. A PCR se mostrou específica, sem amplificar o DNA de outros hemoparasitas. O teste se mostrou sensível para detectar a quantidade 0,25 pg para o DNA de A. centrale e 0,125 pg para detectar o DNA de A. marginale, mostrando-se útil para detectar animais à campo persistentemente infectados com A. marginale e vacinados com A. centrale, os quais foram indetectáveis pela microscopia óptica. Em resumo, a PCR é um teste mais sensível e específico que a microscopia óptica para detectar e diferenciar as espécies de A. centrale e A. marginale, mostrando-se útil para auxiliar no controle de qualidade durante a produção da vacina. / The intracellular pathogen Anaplasma marginale (Rickttsiales: Anaplasmataceae), is endemic in many tropical and subtropical regions of the world. Infection of cattle with A. marginale causes bovine anaplasmosis, due to replication of the microrganism inside the erythrocytes being responsible for considerable economic losses to livestock. The transmission of A. marginale to cattle occurs biologically by ticks or mechanically by blood sucking insects and instruments contaminated with infected blood. A strain of A. centrale, naturally attenuated, has been used extensively to control bovine anaplasmosis in various regions of the world. The vaccine with A. centrale is produced in splenectomized cattle from tick-free areas, however accidental contamination by A. marginale represents a risk during the production process. Therefore, production of the vaccine should be evaluated to ensure a sample of A. centrale free of contamination. During the acute phase of the disease, diagnosis is made by observing A. marginale by blood smear, but during the chronic phase, this method is not as sensitivity as required to detect animals that carry low levels of parasitemia. Being, the molecular detection method more appropriate for detecting both acute and chronic anaplasmosis and differentiate between A. centrale and A. marginale. Thus, in this study a PCR assay was standardized to detect and differentiate A. centrale and A. marginale in samples of bovine blood. Primers for msp4 gene of Anaplasma sp. were used for amplification of material obtained from a DNA extraction of frozen blood from bovines experimentally infected with A. centrale and A. marginale. The PCR showed to be specific, do not amplifying the DNA of other hemoparasites. The test was sensitive to detect the amount of 0.25 pg of DNA from A. centrale and 0.125 pg of DNA from A. marginale, proving to be useful for detecting field animals persistently infected with A. marginale and vaccinated with A. centrale, which were undetectable by optical microscopy. Briefly, PCR is a more sensitive and specific test than the optical microscopy analysis to detect and differentiate the species A. centrale and A. marginale showing to be useful to assist the quality control during the vaccine production.

O patógeno intracelular Anaplasma marginale (Rickettsiales: Anaplasmataceae) é endêmico em muitas regiões tropicais e subtropicais do mundo. A infecção do bovino com A. marginale causa anaplasmose bovina, devido à replicação do microrganismo nos eritrócitos do hospedeiro, sendo responsável por consideráveis perdas econômicas à criação de gado. A transmissão de A. marginale para bovinos ocorre biologicamente por carrapatos ou mecanicamente por insetos hematófagos e instrumentos contaminados com sangue infectado. Uma cepa de A. centrale, naturalmente atenuada tem sido usada extensivamente para controle da anaplasmose bovina em diversas regiões do mundo. A vacina de A. centrale é produzida em bovinos esplenectomizados oriundos de áreas livres de carrapato, todavia uma contaminação acidental por A. marginale é um risco durante o processo de produção. Por este motivo, a produção da vacina deve ser avaliada, para garantir uma amostra de A. centrale livre de contaminação. Durante a fase aguda da doença, o diagnóstico é realizado pela observação de A. marginale por esfregaço sanguíneo, porém durante a fase crônica, este método não tem a sensibilidade necessária para detectar animais que portam níveis baixos de parasitemia. Sendo, o método de detecção molecular o mais indicado para detectar tanto anaplasmose aguda quanto crônica e diferenciar entre A. marginale e A. centrale. Deste modo, no presente trabalho um ensaio de PCR foi padronizado para detectar e diferenciar A. marginale de A. centrale em amostras de sangue bovino. Foram utilizados primers para o gene msp4 de Anaplasma sp. para a amplificação do material obtido a partir de uma extração de DNA de sangue congelado de bovinos experimentalmente infectados com A. centrale e A. marginale. A PCR se mostrou específica, sem amplificar o DNA de outros hemoparasitas. O teste se mostrou sensível para detectar a quantidade 0,25 pg para o DNA de A. centrale e 0,125 pg para detectar o DNA de A. marginale, mostrando-se útil para detectar animais à campo persistentemente infectados com A. marginale e vacinados com A. centrale, os quais foram indetectáveis pela microscopia óptica. Em resumo, a PCR é um teste mais sensível e específico que a microscopia óptica para detectar e diferenciar as espécies de A. centrale e A. marginale, mostrando-se útil para auxiliar no controle de qualidade durante a produção da vacina. / The intracellular pathogen Anaplasma marginale (Rickttsiales: Anaplasmataceae), is endemic in many tropical and subtropical regions of the world. Infection of cattle with A. marginale causes bovine anaplasmosis, due to replication of the microrganism inside the erythrocytes being responsible for considerable economic losses to livestock. The transmission of A. marginale to cattle occurs biologically by ticks or mechanically by blood sucking insects and instruments contaminated with infected blood. A strain of A. centrale, naturally attenuated, has been used extensively to control bovine anaplasmosis in various regions of the world. The vaccine with A. centrale is produced in splenectomized cattle from tick-free areas, however accidental contamination by A. marginale represents a risk during the production process. Therefore, production of the vaccine should be evaluated to ensure a sample of A. centrale free of contamination. During the acute phase of the disease, diagnosis is made by observing A. marginale by blood smear, but during the chronic phase, this method is not as sensitivity as required to detect animals that carry low levels of parasitemia. Being, the molecular detection method more appropriate for detecting both acute and chronic anaplasmosis and differentiate between A. centrale and A. marginale. Thus, in this study a PCR assay was standardized to detect and differentiate A. centrale and A. marginale in samples of bovine blood. Primers for msp4 gene of Anaplasma sp. were used for amplification of material obtained from a DNA extraction of frozen blood from bovines experimentally infected with A. centrale and A. marginale. The PCR showed to be specific, do not amplifying the DNA of other hemoparasites. The test was sensitive to detect the amount of 0.25 pg of DNA from A. centrale and 0.125 pg of DNA from A. marginale, proving to be useful for detecting field animals persistently infected with A. marginale and vaccinated with A. centrale, which were undetectable by optical microscopy. Briefly, PCR is a more sensitive and specific test than the optical microscopy analysis to detect and differentiate the species A. centrale and A. marginale showing to be useful to assist the quality control during the vaccine production.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Melinda J. Wilkerson / Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant zoonotic pathogens of dogs and humans worldwide. In tropical regions such as Grenada, West Indies, dogs represent a major reservoir for E. canis and A. platys, and they are often co-infected. The purpose of this study was to develop a serologic, multiplex bead-based assay to detect species-specific exposures to E. canis, A. platys, and E. chaffeensis in dogs for purposes of surveillance and public health. Peptides from specific outer membrane proteins of P30 for E. canis, OMP1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads and assays were optimized using the multiplex Luminex xMAP® platform. In experimentally infected dogs, the multiplex assay successfully detected antibodies for E. canis and E. chaffeensis, but not A. platys. In the Grenadian population (n=104), the multiplex assay and the in-house ELISA, the SNAP® 4Dx®, detected A. platys antibodies as well as Ehrlichia spp.. Multiplex assay results were found to have “good” and “very good” agreement with the ELISA and IFA for E. canis antibody-positive dogs (K value of 0.73 and 0.84 respectively), while ELISA and IFA had “very good” agreement with each other (K value of 0.85). A. platys multiplex results had only “poor” agreement with ELISA and IFA (K value of -0.02 and 0.01, respectively), while the ELISA and IFA tests had “moderate” agreement with each other (K value of 0.5). These tests showed the prevalence of exposure to E. canis to be comparable with previous studies (38% in 2014), but a doubling of exposure to A. platys determined by IFA and 4Dx® from 9% in 2006, to 20% in 2014. Bayesian modeling (performed on E. canis data only) suggested conditional independence between the IFA, 4Dx®, and MAG tests using consensus priors calculated from literature, and that the bead-assay had comparable sensitivity and specificity to the IFA and ELISA tests. In conclusion, the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays; however, more work needs to be done to assess performance in populations of dogs with exposures to multiple species of Ehrlichia. Further, the reasons for low seroreactivity to A. platys need to be further investigated.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 10

Canine tick-borne diseases are an emerging problem within Australia and throughout the world. This thesis investigates Babesia gibsoni and Anaplasma platys infections in dogs in Australia and also explores the evolutionary relationships and taxonomy of the canine piroplasm species and the members of the order Piroplasmida. A nested PCR-RFLP assay was developed for the detection and differentiation of the canine piroplasm species and was found to have a high detection limit, capable of detecting a 2.7 x 10-7 % parasitaemia or the equivalent of 1.2 molecules of target DNA. Detection of piroplasm DNA applied to Whatman FTA“ classic cards using nested-PCR was found to have a lower detection limit than when using DNA extracted from whole blood but higher than IsoCode‘ Stix or QIAamp extraction from filter paper based techniques. The nested PCR-RFLP assay was further evaluated for the detection of B. gibsoni infection in dogs being exported from Australia to New Zealand and compared to the current screening methods, the Immunofluorescent Antibody Test (IFAT) and microscopy. Of 235 dogs screened, 11 were IFAT positive, 1 was microscopy positive and 3 were PCR positive for B. gibsoni, highlighting the discordance that exists between various detection techniques. Replacing microscopic examination of blood smears with PCR-RFLP is suggested for screening dogs entering New Zealand, in addition to revising the current IFAT cut-off titre to minimize false positive results. The first case of B. gibsoni in New South Wales is also reported. A study was also conducted to further investigate the recent discovery of B. gibsoni in Australia and the association of this infection with American Pit Bull Terriers in an epidemiological study. Both American Pit Bull Terriers (n = 100) and other dog breeds (n = 51) were screened for B. gibsoni using IFAT and PCR-RFLP. A questionnaire was also completed by each dog owner regarding thethe husbandry and habits these dogs. Fourteen dogs were positive for B. gibsoni using IFAT and/or PCR-RFLP and all were American Pit Bull Terriers. Dogs that were male and/or were bitten by or were biters of other American Pit Bull Terriers were statistically more likely to be B. gibsoni positive, thus suggesting that blood-to-blood transmission may contribute to the spread of this disease. Experimental B. gibsoni infections were established in vivo to investigate the efficacy of combined atovaquone and azithromycin therapy and to determine the detection limits of PCR, IFAT and microscopy during various stages of infection. While atovaquone and azithromycin produced a reduction in circulating parasite levels, it did not cause total eradication, and possible drug resistance also developed in one dog. PCR was found to be most useful in detecting early and acute stage infections, while IFAT was most useful during chronic and acute infections. Microscopy is suggested to be only useful for detecting acute stage infections. This study also describes the detection of B. gibsoni in tissue samples during chronic infection for the first time, suggesting possible sequestration of this parasite. Anaplasma platys has also only recently been reported in Australia and the distribution, molecular-charcterisation, pathogenesis, co-infection with Babesia canis vogeli and treatment of infection with doxycycline were investigated. For the first time, A. platys is reported in Western Australia, Queensland and Victoria, with each isolate found to be genetically identical on the basis of the 16S rRNA gene. No correlation could be established between A. platys infection and the development of clinical signs or pathogenesis and definitive treatment using doxycycline could not be determined. Isolates of canine piroplasms from various geographical locations worldwide (n = 46), including Australia were characterised on the basis of multiple gene loci to explore the distribution, genetic variation and possible phylogeographical relationships of these species. Separate genotypes of B. canis vogeli, B. canis canis and B. gibsoni are suggested and may be correlated to different geographical origins. Characterization of B. canis vogeli, B. canis canis and B. canis rossi on the basis of the HSP 70 gene and B. gibsoni on the basis of the ITS 1, 5.8S rRNA gene and ITS 2 is described for the first time. Elevation of each of the B. canis subspecies, with the exclusion of B. canis presentii, to separate species is also proposed. The current paraphyly and taxonomic confusion associated with the members of the order Piroplasmida led to a review of the phylogenetic and taxonomic status of this group of organisms. Phylogenetic relationships are determined using 18S rRNA gene, 5.8S rRNA gene, HSP 70 gene and combined loci analyses. Rearrangement of the Piroplasmida into three families, including the new family Piroplasmiidae is proposed, in addition to the establishment of two new genera, the Piroplasma (Patton, 1895) and the Achromaticus (Dionisi, 1899). Other proposed schemes of classification and the limitations of phenotypic characteristics in taxonomic classification within the Piroplasmida are also discussed.