Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia (SRS), an economically devastating rickettsial disease of farmed salmonids. SRS responds poorly to antibiotic treatment and no effective vaccine is available for its control. A molecular biology approach was used to characterize and identify antigens of P. salmonis that would be suitable to use as a recombinant subunit vaccine to aid in the control of SRS. A system for routine and reliable growth of P. salmonis was established using a chinook salmon (Oncorhynchus tshawytscha) embryo cell line. A purification protocol to separate P. salmonis from host cell material was devised using a combination of differential and Percoll density gradient centrifugation. Purified P. salmonis was used to generate polyclonal rabbit antisera. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis confirmed that P. salmonis was effectively separated from host cell debris and that immunoreactive antigens identified by rabbit antisera were surface associated. Rabbit anti- P. salmonis sera recognized the lipooligosaccharide component of bacterial lipopolysaccharide, and 7 protein antigens with relative mobilities of 27, 24, and 16 kDa and 4 migrating between 50–80 kDa. P. salmonis lipopolysaccharide was observed to be predominantly low m.w., but less abundant high m.w. species containing O-antigen were present. Genomic DNA was isolated from purified P. salmonis and used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal anti-P. salmonis serum was used to identify immunoreactive clones. A lambda clone encoding an immunoreactive 17 kDa outer surface protein (OspA) of P. salmonis was identified. The 4,983 by insert contained a high molar percentage of adenine and thymine, encoded four intact ORF's, and represented the first non-ribosomal DNA sequence data from P. salmonis. OspA is modified as a bacterial lipoprotein in Escherichia coli and is most closely homologous to a rickettsial 17 kDa surface lipoprotein previously only observed within the genus Rickettsia. A codon optimized version of ospA was constructed and the lipoprotein nature of OspA was determined to be a limiting factor in its production in E. coli. High level production of immunoreactive OspA targeted to inclusion bodies was achieved in E. coli by combining OspA with an N-terminal fusion protein. The OspA fusion was recognized by convalescent salmon sera thereby identifying OspA as an excellent candidate for a recombinant vaccine against P. salmonis. Vaccine preparations using P. salmonis bacterins were found to elicit variable immune responses in coho salmon (Oncorhynchus kisutch) that resulted in either protection or immunosuppression of vaccinates which varied with antigen dosage. Recombinantly produced OspA elicited an astonishing level of protection in vaccinated coho salmon with a relative percent survival (RPS) as high as 59%. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein which are universally immunogenic in mammalian immune systems were incorporated into an OspA fusion protein. Addition of the TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a 3-fold increase in the number of coho salmon protected (83% RPS). These results represent an effective monovalent recombinant subunit vaccine for the rickettsial pathogen, P. salmonis. / Graduate

Graduation date: 1997

A serosurvey was conducted to determine rickettsial infection rates in dogs from two distinct areas in Indiana. Sera were collected from dogs and tested for the presence of antibodies to R. rickettsii, R. montana, R. rhipicephali, and R. bellii using the micro-immunofluoresence test. Results from this study indicate an association between canine and human rickettsial infections. Dogs in southwestern Indiana were found to have significantly higher rickettsial infection rather than those in east central Indiana. Human RMSF cases have also been reported more frequently from southern Indiana.All rickettsial species were detected at some level, with many dogs reacting to more than one antigen Evidence suggests that R. montana is the predominant rickettsial species in Indiana. In addition, indicative of a more suitable tick habitat, dogs sampled from rural areas were seropositive more frequently than the urban/suburban dogs. This study suggests that dogs are exposed to the same tick population as humans and can serve as indicators of the presence of rickettsial agents. Indiana residents should be aware of the potential for RMSF transmission throughout the state. / Department of Physiology and Health Science

Human monocytic ehrlichiosis (HME) is a tick-borne infectious disease caused by the bacterium Ehrlichia chaffeensis and transmitted by the ixodid tick Amblyomma americanum. The first confirmed case of HME in Indiana occurred in 1994. Since then, there have been an additional 17 confirmed cases reported from 11 counties.A total of 498 A. americanum and 25 Dermacentor variabilis ticks were collected from counties in southern Indiana during May and June 1998, pooled, and examined for the presence of E. chaffeensis using nested PCR with primers HE 1 and HE3, which are specific for the 16S rRNA gene of E. chaffeensis. Ten pools of adult A. americanum specimens tested positive for E. chaffeensis DNA. This represented a minimum infection rate (MIR) of 3.82%. None of the A. americanum nymphs or adult D. variabilis ticks tested positive.In addition, 325 white-tailed deer blood samples from Indiana and 327 from Ohio were collected during November, 1998 and tested for the presence of E. chaffeensisreactive antibodies using an indirect immunofluoescence assay (IFA). Evidence of such antibodies was found in deer killed in six Indiana counties where infection rates ranged from 43% - 64% and four Ohio counties where infection rates ranged from 4% - 25%.The results from this study support the view that the distribution of E. chaffeensis closely follows that of A. americanum in the North Central United States. This is the first report of E. chaffeensis-reactive antibodies in white-tailed deer from Ohio. / Department of Biology

Potomac Horse Fever (PHF) is a disease of great concern to many horse owners in the Potomac River area of Maryland and Virginia. It is caused by a rickettsia, Ehrlichia risticii. The involvement of an arthropod vector has been suspected because of the seasonal epidemiology of the disease. This research was an attempt to identify and evaluate potential arthropod vectors. A seasonal activity study of biting arthropods attacking horses in endemic areas of Maryland and Virginia identified five potential vectors: (1) Simulium jenningsi (Diptera: Simuliidae), (2) Stomoxys calcitrans (Diptera: Muscidae), (3) Culicoides obsoletus (Diptera: Ceratopogonidae), (4) C. variipennis, and (5) Dermacentor variabilis (Acari: Ixodidae). These five arthropod species were given status as potential vectors because they were collected feeding on horses just prior to and throughout the PHF season. Simulium jenningsi and D. variabilis have the closest seasonal association with the occurrence of PHF as presented in this study. D. variabilis was determined to have the greatest potential due to its reported association with other rickettsial diseases. A series of laboratory and field studies were designed to examine the potential role of D. variabilis in the transmission of E. risticii. We first attempted to transmit E. risticii by feeding adult D. variabilis collected from an endemic farm on susceptible horses. Other laboratory studies included mouse to horse and mouse to mouse transmission attempts using ticks fed on mice inoculated with E. risticii. A serological survey of 105 trapped field rodents (host of immature D. variabilis) on endemic farms in Maryland showed all specimens collected to be negative for PHF antibodies. These studies and others gave no indication of D. variabilis's involvement in the transmission of the disease in nature. The other species mentioned above were not examined. / Ph. D.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The Brazilian spotted fever is an infectious disease transmitted by ticks to humans. The occurrence of Rickettsia rickettsii agent has been reported in Brazil since 1920 and is considered the main bacteria involved in Brazilian Spotted Fever. Since 2000, four other current species have been identified in the country, as follows: Rickettsia riphicephali, Rickettsia amblyommi, Rickettsia parkeri and Rickettsia felis. R. parkeri was first isolated in Amblyomma maculatum on the Gulf Coast of the United States in 1937, but until 2004 was considered as non-pathogenic agent, when was the first recognized case of spotted fever in humans caused by this species. Recently a new human rickettsial infection was reported to cause disease in São Paulo, being called Rickettsia parkeri Strain Atlantic Forest. This study aimed to detect and identify proteins with potential to stimulate the immune system of the host of this new strain described. Therefore, we performed total protein extraction R. parkeri Strain Atlantic Forest from infected VERO cell samples. The extracted proteins were fractionated by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE). The proteins were transferred to nitrocellulose membranes by semi-dry electrotransfer system and subjected to Western blot. Subsequently, the membrane was incubated with domestic rabbit hyperimmune serum against R. parkeri Strain Atlantic Forest (primary antibodies) followed by incubation with anti-rabbit IgG peroxidase-conjugated antibody (secondary antibody) to detect the primary antibodies bound to the proteins . Obtaining the hyperimmune rabbit serum was performed by experimental infection of R. parkeri Strain Atlantic Forest in domestic rabbit, intraperitoneally. After incubation, the disclosure of immunoreactive proteins was performed using a chromogenic substrate. 2 immunoreactants were detected proteins with more than 78 kDa (200 e 130 kDa) and 5 with less than 78 kDa. By comparing existing proteomic maps and the molecular weight of these proteins showed that, it is suggested that rOmpA (200 kDa) and rOmpB (130 kDa) are among the proteins detected and the remaining proteins found are members of the family of surface antigens cell (Sca - Surface cell antigen). However, one can not say that these proteins represent only reactive immunity to R. parkeri Strain Atlantic Forest, or that are homologous to other species. However, studies using the Modern Liquid Chromatography technique associated with mass spectrometry (LC/MS/MS) must be conducted for protein characterization. Preliminary results obtained in this study allowed further elucidation of protein profile of this agent, providing subsidies for the development of highly sensitive and specific diagnoses, as well as studies allow for the realization of a vaccine, as an alternative for the control and prevention of Brazilian Spotted Fever. / A Febre Maculosa Brasileira é uma doença infecciosa, transmitida por carrapatos ao homem. A ocorrência do agente Rickettsia rickettsii tem sido relatada no Brasil desde 1920, sendo considerada a principal bactéria envolvida na Febre Maculosa Brasileira. Desde 2000, outras quatros espécies circulantes foram identificadas no país, sendo: Rickettsia riphicephali, Rickettsia amblyommi, Rickettsia parkeri e Rickettsia felis. R. parkeri foi isolada pela primeira vez em Amblyomma maculatum, na Costa do Golfo dos Estados Unidos da América em 1937, porém até 2004 era considerada como agente não patogênico, quando então houve o primeiro caso reconhecido de Febre Maculosa em humanos ocasionado por essa espécie. Uma nova riquetsiose humana foi descrita como causadora da doença no Estado de São Paulo, sendo denominada de Rickettsia parkeri cepa Mata Atlântica. O presente trabalho teve como objetivo detectar e identificar proteínas com potencial de estimular o sistema imune do hospedeiro desta nova cepa descrita. Para tanto, foi realizado a extração proteica total de R. parkeri cepa Mata Atlântica a partir de amostras de células VERO infectadas. As proteínas extraídas foram fracionadas por eletroforese em gel de poliacrilamida na presença de dodecil-sulfato de sódio (SDS-PAGE). Estas proteínas foram transferidas para membranas de nitrocelulose através do sistema semi-seco de eletrotransferência e submetidas à técnica de Western blot. Posteriormente, a membrana foi incubada com soro hiperimune de coelho doméstico contra R. parkeri cepa Mata Atlântica (anticorpos primários), seguido de incubação com anticorpo anti-IgG de coelho conjugado com peroxidase (anticorpo secundário), para detectar os anticorpos primários ligados às proteínas. A obtenção do soro de coelho hiperimune foi realizada através da infecção experimental de R. parkeri cepa Mata Atlântica em um coelho doméstico, via intraperitoneal. A revelação das proteínas imunorreativas foi efetuada através de substrato cromogênico. Foram detectadas 2 proteínas imunorreagentes com mais de 78 kDa (200 e 130 kDa ) e 5 com menos de 78 kDa. Através da comparação de mapas proteômicos existentes e pelo peso molecular que estas proteínas apresentaram, sugere-se que rOmpA (200 kDa) e rOmpB (130 kDa) estejam entre as proteínas detectadas e as demais proteínas encontradas, sejam membros da família de antígenos de superfície celular (Sca Surface cella ntigen). No entanto, não se pode afirmar que estas proteínas apresentam imunidade reativa única para R. parkeri cepa Mata Atlântica, ou se são homólogas a outras espécies de riquetsias. Contudo, estudos empregando a técnica de Cromatografia Líquida Moderna associada a Espectrometria de Massas (LC/MS/MS) deverão ser conduzidos para a caracterização proteica. Os resultados preliminares obtidos neste estudo permitiram maior elucidação do perfil proteico deste agente, fornecendo assim subsídios para o desenvolvimento de diagnósticos altamente sensíveis e específicos, bem como permitir estudos para a realização de uma vacina, como alternativa para o controle e prevenção da Febre Maculosa Brasileira.