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

Made available in DSpace on 2014-06-11T19:23:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-27Bitstream added on 2014-06-13T20:11:33Z : No. of bitstreams: 1 rondelli_mch_me_jabo.pdf: 1021961 bytes, checksum: 86f21b8998af136aaf5ef78e0fdec164 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A erliquiose em cães pode conduzir o animal ao óbito, tanto por alterações hematológicas quanto clínicas, que se explicam pelo caráter catabólico multifatorial da doença. O comprometimento adrenocortical pode ser esperado em virtude dos desafios impostos ao sistema imune, gerados pelas infecções e inflamações. Este estudo visou avaliar a ocorrência de alterações hormonais relacionadas às glândulas adrenais, em cães infectados naturalmente por Ehrlichia canis (n=21), positivos na identificação de anticorpos anti-E.canis (Dot-ELISA) e DNA da bactéria (nPCR). Para tanto, foram pesquisadas as concentrações séricas de cortisol, sulfato de dehidroepiandrosterona (DHEA-S) e proteínas de fase aguda (proteína C reativa e haptoglobina) pré e pósestimulação com o hormônio adrenocorticotrópico (ACTH), e comparadas com os valores obtidos de cães saudáveis (n=10). Os resultados demonstraram resposta semelhante dos cães doentes e saudáveis ao teste de estimulação com ACTH, no que concerne ao cortisol. Isto sugere que cães com erliquiose de ocorrência natural mantêm a capacidade de secretar cortisol adequadamente diante o estímulo do ACTH. As concentrações de DHEA-S foram significativamente mais elevadas nos cães doentes, nos momentos pré e pós-ACTH, em relação aos saudáveis, o que ilustra o estresse do organismo na doença em estudo. Não foram observadas diferenças significativas entre os momentos dentro de cada grupo. Aparentemente, a estimulação com ACTH não interferiu na concentração de DHEA-S dosado uma hora após sua administração, sugerindo que a resposta ao estímulo hormonal não foi detectada. Da mesma forma, a estimulação com ACTH não interferiu na concentração das proteínas de fase aguda, embora se apresentassem significativamente... / Ehrlichiosis in dogs may lead to death both for hematological and for clinical alterations that might be explained for its multifactorial catabolic character. The adrenocortical disruption may be expected during the course of the disease due to challenges on the immune system, generated by infection and inflammation. This study aimed to evaluate the occurrence of hormonal disturbances associated to the adrenal glands in dogs naturally infected by Ehrlichia canis (n=21), positive at the identification of anti-E. canis antibodies and of the bacterial DNA (nPCR). For this purpose, serum concentration of cortisol, dehydroepiandrosterone sulfate (DHEA-S) and acute phase proteins (C reactive protein and haptoglobin) pre and post-stimulation with adrenocorticotropic hormone (ACTH) were assessed and compared to values obtained from healthy dogs (n=10). The results have shown similar cortisol response to the ACTH stimulation test between sick and healthy dogs. It suggests that dogs with ehrlichiosis of natural occurrence maintain the capacity of secreting cortisol properly before the ACTH stimulation. DHEA-S concentrations were significantly increased in sick dogs at pre and post-ACTH moments, in relation to healthy dogs, which illustrates the organic stress in the studied disease. Significant differences between both moments were not observed in the groups. Apparently, ACTH stimulation has not interfered in DHEA-S concentration, assessed one hour after its administration, suggesting that the response to the hormonal stimulation was not detected. The same way, ACTH stimulation has not interfered in the acute phase concentrations, although they were significantly increased in sick dogs, which reinforces the role of an inflammatory process in the course of the disease. The results indicate... (Complete abstract click electronic access below)

Degree Not Listed / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Anaplasmataceae family rickettsial bacteria are mostly vector-transmitted pathogens causing important diseases in several vertebrates, including humans, canines, and ruminants. Ehrlichia chaffeensis, a tick-transmitted intraphagosomal rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis (HME). Little is known about how this and other related rickettsial organisms are able to reside and replicate within an acidified phagosome environment. Similarly, it is unclear how the infectious form of the bacterium maintains pH homeostasis in the extracellular milieu where the pH is about 7.35-7.45, before its infection to a naïve host cell. Sodium/cation: proton antiporters are integral membrane proteins reported from a wide range of species. They exchange sodium or other monovalent cations against protons across a plasma membrane in maintaining the cytoplasmic pH of a cell. We recently described a mutation within the Ech_0379 gene of E. chaffeensis that is predicted to encode for a Na+/H+ antiporter protein. The mutation caused the attenuated growth of the organism in vertebrate hosts, resulting in a reduced level of the bacterial presence in the circulation. In this study, we evaluated several antiporter protein genes of E. chaffeensis. Its genome contains 10 coding sequences encoding for polypeptides which may form at least six functional proteins. To define their function, a sodium sensitive Escherichia coli strain having a mutation in two of its three antiporter protein genes (EP432) is used to carry out the functional complementation assay with E. chaffeensis genes from their respective promoters. The EP432 strain has a growth defect during its replication in the presence of NaCl that can be restored with functional complementation. All six E. chaffeensis genes could complement the growth defect of EP432 under acidic pH, while Ech_0379 and Ech_0179 also complemented at basic pH. Ech_0179 complemented at neutral pH as well. The complementation of all genes at neutral and basic pHs, except Ech_0179 and Ech_0379, made EP432 E. coli strain be more sensitive to the presence of 200 mM NaCl. The channeling activity is verified independently by constructing a proteoliposome in vitro with the recombinant protein Ech_0379. The recombinant protein showed antiporter activity at all three pHs in the presence of 100 or 200 mM NaCl when assessed using the recombinant proteoliposome. This research is the first description of antiporter proteins of E. chaffeensis.

In the search for a better vaccine against Ehrlichia ruminantium infection in ruminants, four E. ruminantium open reading frames (ORFs) derived from the Welgevonden isolate were tested using either DNA vaccination or DNA primemodified viral or DNA prime-recombinant protein boost strategies. Both the DNA vaccination and the DNA prime.recombinant protein boost strategy provided complete protection against E. ruminantium Welgevonden needle challenge, while the DNA prime.modified viral boost strategy only provided 90 % protection. The DNA prime.recombinant protein boost strategy also coincided with elevated cellular immunology as was evident from increased IFN-ã production. Furthermore, we could show that the 1H12 DNA vaccine could induce protection against heterologous needle challenge when animals were immunised with the Welgevonden-derived 1H12 ORFs and challenged with selected E. ruminantium stocks. Unfortunately the DNA only and the DNA prime.recombinant protein boost strategy were not protective in the field. Therefore, our results suggest that there is a vast difference between needle challenge and natural tick infestation and that E. ruminantium organisms transmitted by ticks have the ability to evade the protective immunity induced by immunization with the four 1H12 ORFs. / Thesis (PhD)--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

Four 1H12 E. ruminantium open reading frames cloned into the pCMViUBs mammalian expression vector and used as a recombinant DNA vaccine against heartwater repeatedly provided complete protection in sheep (using a cocktail or the individual ORFs) against a laboratory needle challenge while 1/5 of sheep were protected after a natural tick challenge. The lack of protection under natural field conditions could be attributed to the delivery strategy used and therefore there is a need to investigate other delivery methods. Polymeric microparticles based on PLGA polymers have been used extensively to target the delivery of vaccine to antigen presenting cells, play a role in the induction of cellular immunity and can be used as a single dose vaccine mimicking prime/boost vaccination. In this study, the four 1H12 pCMViUBs_ORFs and their respective recombinant proteins were either encapsulated into or adsorbed onto microparticles using a modified double emulsion solvent evaporation technique. The particles were formulated to release DNA on day zero and day 21 and recombinant proteins on day 42 thus mimicking a two times DNA prime/recombinant protein-boost immunization strategy. Encapsulation did not have any detrimental effects on the stability of the recombinant proteins as determined by gel electrophoresis and western blotting. The in vitro incubation of microparticles in either a Float-A-Lyzer® dialyzer or an eppendorf tube showed the potential of microparticles to be used as a vaccine because of their release profiles that mimics a heterologous prime/boost immunization strategy. Microparticles formulated using polymers with low glycolide ratios released 80% of the encapsulated proteins within the first week of in vitro incubation with most of the proteins released on day 1. Microparticles formulated using polymers with 50:50 monomer ratios released the recombinant proteins during week 1 and 3 of in vitro incubation. These microparticles did not release any protein in week 2 (day 7-14). Microparticles with 0.5% cetyltrimethylammonium bromide (CTAB) on their surfaces adsorbed DNA and released more than 40% of DNA on day 1 with 100% release by day 14. RG502H microparticles formed with PVA as the internal phase viscosity enhancer released intact DNA only from day 12 to day 21. A cocktail of these microparticles could therefore be used as an autobooster vaccine thus reducing the need for repeated immunizations needed to obtain protective immunity. Potential scientific publication Tshikhudo, N.P., Pretorius, A., Putterill, J., and van Kleef, M. 2009, “Biodegradable microparticles as a single dose delivery system for Ehrlichia ruminantium vaccines”, Journal of Controlled Release, (draft manuscript). Publication of results in conference proceedings / abstracts NanoAfrica 2009: Biodegradable microspheres as a single dose delivery system for Ehrlichia ruminantium vaccines: N. Tshikhudo, A. Pretorius, J. Putterill and M. van Kleef. / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Ehrlichia ruminantium is an obligate intracellular pathogen and, as such, cell mediated immunity plays a key role in the control of bacterial replication and subsequent protection against heartwater in ruminants. IFN-γ has been shown to be a potent inhibitor of E. ruminantium growth in endothelial cells in vitro. Thus, identification of antigens that preferentially activate T cells to proliferate and secrete IFN-γ needs to be done so that they can be evaluated as vaccine candidates. Previously, a cocktail of four open reading frames (ORFs) that induce 100% protection after needle challenge have been identified. However, only 20% protection was obtained after tick challenge in the field, when administered as a DNA vaccine in sheep. Because only limited protection was obtained during a field vaccine trial, our research focused on the identification of additional ORFs as a mean to improve the efficacy of this vaccine. Because secreted proteins are reported to be major targets in a specific immune response we hypothesize that they may be potential heartwater vaccine candidates. Five ORFs (Erum5000, Erum5010, Erum7760, Erum8060 and Erum8610) encoding secreted E. ruminantium proteins were selected from the Welgevonden stock genome sequence using bioinformatics tools. The ORFs were cloned into a pET 102/D-TOPO® vector. The corresponding recombinant (r) proteins were expressed in a bacterial expression system and the expression was confirmed by immunoblots using anti-His antibodies and sheep sera. Proteins were purified by immobilized metal ion affinity chromatography and resulted in a yield of 200 μg-2000 μg per 100 ml and 1L cultures respectively. Four of the five recombinant proteins (rErum5000, rErum7760, rErum8060 and rErum8610) could be expressed. Recombinant proteins were assayed to determine whether they induce recall cellular immune responses in vitro. The peripheral blood mononuclear cells used in the assays were obtained from a naïve and four heartwater immune sheep. Significant proliferative responses (p ≤ 0.01) were evident for 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610). IFN-γ production was determined using an ELISPOT assay and 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610) induced IFN-γ production. Each recombinant protein had its own optimum concentration for inducing immune responses and the responses differed between animals. In addition, real-time PCR was used to measure IFN-γ and IL-4 gene expression by antigen stimulated immune PBMC. The real-time PCR results correlated with the ELISPOT assay results. Based on the results obtained, it can be concluded that a Th1 type immune response was elicited. Thus these proteins that induced proliferation and IFN-γ production may be important in protection against heartwater and will be tested in future vaccine studies. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / Unrestricted

Human Monocytic Ehrlichiosis (HME) is an emerging disease first described in 1987 and is transmitted by the bite of Amblyomma americanum. Over the past 10 years, the CDC has documented increasing ehrlichiosis case reports nationwide. Our study site is a golf-oriented retirement community located in the Cumberland Plateau of Tennessee. In 1993, four men at the study site had symptoms consistent with HME which prompted a CDC outbreak investigation and led community managers to mitigate ticks feeding on deer. The objectives of this study were to measure the efficacy of current tick mitigation attempts, to determine the level of infection and composition of tick-borne disease in the study area, and to assess which wildlife species are potentially acting as reservoirs for disease. Ticks were sampled in the community at eight sites of ‘4-poster’ acaricide applicator utilization and at seven untreated sites. Close to the ‘4-poster’ devices, larval, nymphal, and adult tick abundances were reduced by 90%, 68% and 49% respectively (larval p<0.001, nymphal p<0.001, adult p=0.005) relative to the untreated areas. We extracted DNA from A. americanum ticks collected at the treatment and non-treatment sites and tested for Ehrlichia spp. infections. Of 253 adult and nymphal A. americanum tested, we found 1.2% to be positive for Ehrlichia chaffeensis, 4.7% positive for Ehrlichia ewingii, and 1.6% positive for Panola Mountain Ehrlichia; in combination this prevalence is similar to that reported in other Ehrlichia-endemic areas of the eastern U.S.. We also performed blood meal analysis on DNA from A. americanum ticks and the results suggest that the most significant reservoir hosts for Ehrlichia spp. are white-tailed deer, turkeys, grey squirrels, and Passeriformes. We conclude that while the ‘4-poster’ acaricide applicators reduce the number of ticks close to treatment, at the density at which they are currently being used (8 applicators per 52.6 km2, average distance between applicators = 6.6km) they will have no large-scale effect on the community’s tick population. In order to accomplish area-wide reduction of A.americanum and Ehrlichia spp. in this locale, community managers should develop an integrated management strategy that utilizes other techniques in addition to ‘4-poster’ devices.

The rickettsial pathogen Ehrlichia ruminantium, transmitted by ticks of the genus Amblyomma, causes heartwater, an economically important, often fatal disease of domestic and wild ruminants in sub-Saharan Africa and in the Caribbean. The studies described in this thesis have contributed to understanding several aspects of heartwater. First, a real-time PCR method was developed in order to study the kinetics of infection with E. ruminantium in the mammalian host. The assay was validated for specificity and sensitivity and was used to estimate numbers of the organisms in the blood of infected sheep. However, organisms were only detected during the clinical phase of infection, indicating that the way in which it was applied did not provide sufficient sensitivity to follow the early stages of infection. This PCR assay was then used, together with transcription and proteomic analyses, to investigate differential gene expression of E. ruminantium in the arthropod and mammalian hosts, in order to identify genes that may allow the organisms to successfully adapt to different environments. These studies used in vitro tick and mammalian cell culture systems, as well as tissues from infected A. variegatum ticks, and initially focused on the map1 multigene family. Although transcripts for most of the map1 paralogs were detected in organisms grown in vitro, in both mammalian and tick cells, only transcripts from map1 and map1-1 were detected in infected ticks. Moreover, map1-1 transcripts were more abundant in midguts than in salivary glands whereas map1 transcripts were most abundant in salivary glands and were expressed at higher levels following several days of tick feeding on a mammalian host. Because of the quantities of material required, proteomic analysis was only possible using in vitro-cultured organisms. Comparison of proteins encoded by the map1 cluster in E. ruminantium grown in tick or bovine endothelial cell cultures, using 2D gels and MALDI-TOF analysis, revealed that different proteins predominated in the corresponding spots in 2D gels from the different cultures; products of the map1-1 gene were abundant in tick cells, while products of map1 were abundant in endothelial cells. The detection of higher levels of map1 transcripts in salivary glands than in midguts of infected ticks, together with the presence of abundant MAP1 protein in organisms grown in mammalian but not in tick cell lines, suggest that expression of this protein may be associated with infectivity for mammalian cells. In contrast, map1-1 transcripts were abundant both in midguts of infected ticks and in tick cell lines, and the protein was expressed at high levels in infected tick cell cultures. Since both of these stages have low infectivity for sheep, these results suggest that the MAP1-1 protein may play an important role within the vector, possibly associated with colonisation and replication of E. ruminantium in the tick midgut. Collectively these findings suggest that this multigene family is involved in functions of biological relevance in different stages of the life cycle of E. ruminantium. Lastly the suppression subtractive hybridisation (SSH) technique was applied to RNA extracted from E. ruminantium-infected endothelial and tick cell cultures in an attempt to sample a large portion of the E. ruminantium genome for differentially expressed genes; although not resulting in identification of any differentially transcribed genes in the present study, this method was shown to work in principle.

La Ehrlichiosis es una enfermedad zoonótica emergente transmitida a los humanos a través de la picadura de garrapatas infectadas, de gran importancia en países tropicales y sub-tropicales. En el presente estudio se determinó la seropositividad contra Ehrlichia chaffeensis en propietarios de perros con antecedentes de ehrlichiosis mediante la prueba de inmunofluorescencia indirecta (IFI) y su asociación con el sexo, edad, exposición a garrapatas y nivel de contacto con los perros mediante la prueba de Chi cuadrado. Se evaluaron 95 personas sin distinción de sexo, edad o condición socio económica cuyos perros tenían historia de ehrlichiosis reciente, los cuales llenaron un cuestionario con datos clínicos y epidemiológicos de importancia. El estudio se realizo entre Enero del 2009 y Diciembre del 2010. Los resultados del total de personas consideradas en el presente estudio indicaron que el 31.6% (30/95) presentaron anticuerpos contra Ehrlichia chaffeensis. Las variables edad y exposición a garrapatas resultaron estadísticamente significantes (p<0.05), frente a la seropositividad contra Ehrlichia chaffeensis. De los pacientes seropositivos, el 80% son personas menores de 40 años (24/30), mientras que el 20% son personas de 40 años a más (6/30). Asimismo, de los pacientes seropositivos el 93.3% estuvieron expuestos a garrapatas (28/30) mientras que el 6.7% no estuvieron expuestos (2/30). Estos hallazgos confirman la exposición a Ehrlichia chaffeensis en propietarios de perros con antecedentes de ehrlichiosis en Lima Metropolitana, evidenciando asociación de los resultados con los factores de riesgo evaluados, tales como edad y exposición a garrapatas.

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.