Neospora caninum is an apicomplexan parasite that causes neuromuscular paralysis in dogs and abortions in cattle. N. caninum is responsible for losses of several million dollars to the dairy and beef industries in several parts of the world. The key players in the host immune response to N. caninum include CD4+ T cells, the Th1 cytokines IL-12, Interferon gamma and IgG2a isotype antibodies. There are currently no chemotherapeutic agents that are effective against adult cattle neosporosis. A commercially available, inactivated vaccine induces the undesirable Th2 type of immunity against N. caninum. Therefore, two approaches towards vaccine development against N. caninum that were designed to induce potent cell mediated immunity have been explored in this dissertation. The first approach consisted of the development of a bivalent recombinant vaccine for both brucellosis and neosporosis, while the second approach involved gamma irradiation of N. caninum tachyzoites for use as an attenuated vaccine against N. caninum. Since N. caninum research has been conducted with several strains of mice and the different strains of mice vary in their susceptibility to infection with N. caninum, there is a need to develop a standard lab animal model for N. caninum. A gerbil and a C57BL/6 mouse model for N. caninum vaccine testing have been developed. It was found that the LD50 of N. caninum tachyzoites in gerbils was 9.3 x105 tachyzoites per gerbil delivered intra-peritoneally, (i.p) while for C57BL/6 mice the LD50 was 1.5 x107 tachyzoites per mouse delivered i.p. Vertical transmission rates in C57BL/6 mice infected with N. caninum tachyzoites during mid-gestation were determined and found to be in the range of 96-100%. Putative protective antigens of N. caninum that included MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in B. abortus strain RB51 to create recombinant vaccine strains. C57BL/6 mice were vaccinated with either the recombinant strains or the irradiated tachyzoites. Antigen specific IgG2a and IgG1 responses and high levels of interferon gamma and IL-10 were induced by vaccination. Mice vaccinated with irradiated tachyzoites, RB51-MIC1 and RB51-GRA6 were completely protected against lethal challenge, while the mice vaccinated with RB51-SRS2, RB51-GRA2 and RB51-MIC3 were partially protected. To determine the efficacy of the vaccines in preventing vertical transmission of N. caninum, mice were vaccinated and bred after administration of a booster dose four weeks after the primary vaccination. Antigen specific IgG1 and IgG2a and significant levels of IFN-ã and IL-10 were detected in vaccinated, pregnant mice. Pregnant mice were challenged with 5 x 106 N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups three weeks after birth and examined for the presence of N. caninum by a semi-nested PCR. Protection against vertical transmission elicited by the RB51-GRA6, RB51-MIC3, irradiated tachyzoite, RB51-GRA2, RB51-MIC1 and RB51-SRS2 vaccinated groups were 43%, 38%, 34%, 34%, 18%, and 7% respectively. Since not all the antigens that were highly protective against acute disease were not very effective in preventing vertical transmission, the role of the selected antigens in preventing acute disease and vertical transmission appear to differ. Only GRA6 was found to be effective in protecting against an acute lethal challenge as well as preventing vertical transmission 43% of the time. In summary, two animal models for the testing of N. caninum vaccines were developed. N. caninum protective antigens were successfully expressed in B. abortus strain RB51. The irradiated tachyzoite and recombinant RB51-Neospora vaccines were highly effective in protecting against acute neosporosis and partially protective against vertical transmission. Therefore, both these approaches show great promise as practical and effective means to achieve the goal of successful prophylaxis against N. caninum induced abortions and reduce the chances of vertical transmission. / Ph. D.

Brucella abortus is a facultative intracellular bacterial pathogen, which causes abortion in cattle and undulant fever in human. B. abortus strain RB51 (Strain RB51) is the official vaccine for bovine brucellosis in the USA. B. abortus strain RB51 can be used as a vector for the over-expression of its own (homologous) as well as heterologous protective antigens. The immune system can detect these heterologous antigens and produce a response. Expressing a protein in different bacterial compartments has been shown to affect its accessibility to the immune system and the way the antigen is processed by antigen presenting cells. In order to determine if the immune response is affected by the localization of the antigen, green fluorescent protein (GFP) was expressed at three different locations in B. abortus strain RB51, outer-membrane (OM), periplasmic space (PS) and in the cytoplasmic region (CR) of B. abortus strain RB51. This localization was obtained by transforming strain RB51 with plasmids pBBg18sGFP and pBBgSsGFP, in which the 18 kDa Brucella lipoprotein and the Brucella Cu/Zn SOD protein signal sequences were added to the GFP sequence to cause OM and PS expression respectively. No signal sequences were added to the plasmid pBBgGFP for CR only expression. Expression and localization of GFP in the different compartments in recombinant B. abortus strain RB51 were confirmed by electron microscopy and antibody absorption experiments. Groups of 5 female BALB/c mice each were injected and boosted with three recombinant strains and appropriate controls. Mice were bled and their anti-GFP antibody production was assessed. None of the immunized mice produced specific antibodies against GFP, probably due to the low expression of the heterologous antigen observed in this study by strain RB51 observed in this study. It will be necessary to produce new recombinants which are able to express higher amounts of GFP to answer if localization of heterologous antigen within the recombinant RB51 affects the level of a specific immune response. / Master of Science

Aging is accompanied by dysregulated immune function resulting in increased susceptibility of the elderly to diseases caused by microbial pathogens. There exists a multitude of data suggesting decreased resistance of the elderly to a variety of intracellular pathogens but there is no data relating the effect of aging on the immune response against Brucella. To elucidate the mechanism of immune dysregulation in old, old and young DBA/2 and BALB/c mice were infected with wild-type B. abortus strain 2308. The old and young mice were also vaccinated with vaccine B. abortus strain RB51 over-expressing Cu-Zn superoxide dismutase (SOD) and then challenged with B. abortus strain 2308 to determine the effect of vaccination in old vs. young mice. Specific IgG1 and IgG2a response to Brucella antigens were also evaluated to determine the effect of aging on Th-specificity of the immune response against Brucella infection. The immune response in aged vs. young mice was further assessed using RT-PCR and cytokine antibody array to determine the type of T-helper response. The experimental results indicate that all mice, regardless of age, survived infection ranging from doses of 2 x 104 to - 2 x 108 CFU. Though the older DBA/2 mice had a higher organism burden after 1 week of infection, these mice cleared Brucella infection more efficiently (5 weeks post-infection) than young mice. Vaccination with strain RB51 over-expressing SOD provided significant protection in young DBA/2, young BALB/c and old BALB/c mice but not in old DBA/2 mice after strain 2308 challenge. The results also suggest that old mice produced a different magnitude of IgG1 and IgG2a response to bacterioferritin and SOD of Brucella. The data suggests that both Th17 as well as Th1 responses were accentuated in old mice as compared to young mice following infection with Brucella. How the Th17 and Th1 branches of immune system work together enabling old mice to clear Brucella better than young mice warrants future investigation. / Master of Science

A brucelose bovina é uma das zoonoses mais difundidas no mundo e provoca prejuízos econômicos decorrentes de problemas reprodutivos. Diversos investimentos e esforços têm sido feitos para preveni-la em várias partes do mundo. Em destaque no cenário da pesquisa mundial sobre brucelose, está a busca por uma vacina que seja inócua aos humanos e animais, garanta uma boa imunidade e, principalmente não interfira nos testes de diagnóstico. Desde a criação da primeira vacina contra brucelose, diversas outras foram desenvolvidas, no entanto, poucas foram aceitas e usadas em larga escala. As perspectivas para o futuro das vacinas contra brucelose, demostram ser tão variadas quanto o seu passado. Nesse contexto, o objetivo desse trabalho é fazer uma reflexão sobre as vacinas contra brucelose considerando as principais vacinas desenvolvidas no passado, as utilizadas na atualidade e as perspectivas para a vacinologia contra brucelose. / Bovine brucellosis is one of the most widespread zoonoses in the world and it causes economical losses due to reproductive problems. Several investiments and efforts have been done to prevent bovine brucellosis in many parts of the world. The search for a vaccine which is harmless to both human and animals, ensure good immunity and , mainly, does not interfere in diagnostic tests is featured on the scene of worldwide research on brucellosis. Since the creation of the first vaccine against brucellosis, several others have been created. However, few have been accepted and widely used. The perspectives for the future of vaccines against brucellosis can be seen as varied as they were in the past. In this context, the aim of this study is to reflect on brucellosis vaccines considering the main vaccines developed in the past, the ones in use today and the perspectives for the future of vaccinology againt brucellosis.

A brucelose bovina é uma das zoonoses mais difundidas no mundo e provoca prejuízos econômicos decorrentes de problemas reprodutivos. Diversos investimentos e esforços têm sido feitos para preveni-la em várias partes do mundo. Em destaque no cenário da pesquisa mundial sobre brucelose, está a busca por uma vacina que seja inócua aos humanos e animais, garanta uma boa imunidade e, principalmente não interfira nos testes de diagnóstico. Desde a criação da primeira vacina contra brucelose, diversas outras foram desenvolvidas, no entanto, poucas foram aceitas e usadas em larga escala. As perspectivas para o futuro das vacinas contra brucelose, demostram ser tão variadas quanto o seu passado. Nesse contexto, o objetivo desse trabalho é fazer uma reflexão sobre as vacinas contra brucelose considerando as principais vacinas desenvolvidas no passado, as utilizadas na atualidade e as perspectivas para a vacinologia contra brucelose. / Bovine brucellosis is one of the most widespread zoonoses in the world and it causes economical losses due to reproductive problems. Several investiments and efforts have been done to prevent bovine brucellosis in many parts of the world. The search for a vaccine which is harmless to both human and animals, ensure good immunity and , mainly, does not interfere in diagnostic tests is featured on the scene of worldwide research on brucellosis. Since the creation of the first vaccine against brucellosis, several others have been created. However, few have been accepted and widely used. The perspectives for the future of vaccines against brucellosis can be seen as varied as they were in the past. In this context, the aim of this study is to reflect on brucellosis vaccines considering the main vaccines developed in the past, the ones in use today and the perspectives for the future of vaccinology againt brucellosis.

Brucellosis, caused by Gram-negative bacteria of the genus <i>Brucella</i>, is a zoonotic disease with global impacts on human, livestock, and wildlife health. Around 500,000 cases of human brucellosis are reported by the World Health Organization annually. Even though brucellosis has been eradicated from domestic livestock in the United States of America, the causative bacterial pathogen is still present in elk, bison, and feral swine. With the growth of free-range farming, domestic livestock and wildlife populations come into close contact, spreading the disease. Feral swine interactions are of particular concern. They carry a number of zoonotic diseases including brucellosis. As there is no commercial vaccination protocol to prevent brucellosis in wildlife and swine, interactions with these populations are especially dangerous for public health. Feral swine population is increasing nationwide even with the current population control practices. There is an urgent need for efficient control of feral swine and preventing the spread of brucellosis. To aid in the prevention of the spread of feral swine across the USA, immunocontraceptives have been employed. Over the years several candidates have been tested, but the search for the perfect vaccine is still ongoing. The monumental task includes reversibly preventing one of life’s most basic necessities, reproduction, through an oral route with no effect on non-target species. One way that science is tackling both of these threats at once is through dual-purpose vaccines. Dual-purpose vaccines produce an immune response that targets two different pathogens, or in this case a pathogen and reproductive hormones. In the effort to produce this vaccine, more knowledge was needed in regards to <i>B. neotomae</i>. This dissertation showed that it has the ability to survive in a variety of cells from different species, in a similar manner to known virulent <i>Brucella</i> species. This is of concern when using <i>B. neotomae</i> as a vaccine, but it has also been shown that attenuated <i>B. neotomae</i> can provide protection against virulent <i>B. suis, B. abortus</i>, and <i>B. melitensis</i> challenge. This is a major finding in the effort towards a universal brucellosis vaccine. After genetic manipulation, cell culture assays, and mouse trials, several leucine auxotrophic <i>B. neotomae</i> and <i>B. abortus</i> strains show promise in the effort towards a dual-purpose vaccine. Strains of <i>B. neotomae</i> <i>ΔwboA ΔleuB</i> pNS4-trcD-fliC-Gonadotropins were discontinued in this effort towards a brucellosis immunocontraceptive dual-purpose vaccine due to lethality issues in mice. These stability and lethality issues are still under investigation. Instead, a proven stable strain of <i>B. abortus</i> RB51 (a USDA approved cattle vaccine) was used to investigate its effects on fertility in mice when expressing fliC-Gonadotropins. Strains <i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-porcineFSHβ (RB51LFSHβ) and <i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-GnRH (RB51LGnRH) confer reduced fertility characteristics in both male and female purpose bred mice. Strain RB51 ΔleuB has also been shown to protect against virulent <i>B. abortus</i> challenge in the literature. These findings warrant further investigation to determine the efficacy of these vaccine strains in swine as an oral vaccine. Ultimately, their ability to prevent brucellosis, while causing immunocontraception needs to be determined in feral swine. / Ph.D. / While brucellosis has been eradicated from domestic livestock in the United States, the causative agent is still present in wildlife like elk, bison, and feral swine. The interactions between these infected wildlife populations with domestic livestock and human populations pose a great health risk. Many tools are employed to mitigate these interactions including vaccination programs and population management. In particular, the feral swine population has proven difficult to control. It has quadrupled in the past ten years and continues to expand nationwide, making their population control an important national objective. Furthermore, feral swine are known carriers of zoonotic diseases, including hemorraghic colitis, leptospirosis, trichinosis, swine influenza, and brucellosis. Many cases of these diseases in humans have been traced back to interactions with feral swine. The current population control practices have failed to minimize the $1.5 billion of damage they cause to the agricultural industry per year. Thus, there is a need to effectively control the feral swine population and prevent the spread of zoonotic diseases like brucellosis. Rough leucine auxotrophic strains of <i>Brucella</i> expressing gonadotropin releasing hormone (GnRH) or porcine follicle stimulating hormone beta subunit (FSH) conjugated to <i>Salmonella</i> fliC show promise. They have been shown to provide protection from virulent Brucella strain challenge and reduce fertility characteristics in mice. Their effectiveness as an immunocontraceptive for feral swine management, while reducing the spread of brucellosis needs to be tested in swine. These vaccine strains [<i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-porcineFSHβ (RB51LFSHβ), <i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-GnRH (RB51LGnRH) and <i>B. neotomae ΔwboA ΔleuB</i> pNS4-trcD-fliC-GnRH (BNWLGnRH)] could pave the way for effective novel immunocontraceptive tools to be used in wildlife management.

Brucella abortus (BA) are gram-negative, facultative intracellular bacteria that cause abortions in cattle and debilitating illness in humans. The US is now virtually free of bovine brucellosis, but the disease is endemic in wildlife. The official brucellosis vaccine in the US is strain RB51 (RB51). It elicits protective cell-mediated immunity (CMI) against BA infections. Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis in ruminants. It is a slow growing intracellular parasite that requires CMI for its control, belongs to the genus Mycobacterium, and is closely related to M. avium avium (MA). Using RB51 as a vector that induces strong protective CMI may be useful to protect against MAP if it expresses MAP protective antigens. Therefore, MAP 85A and 35kDa proteins were expressed at low levels in RB51, and the immune responses elicited by these vaccines in BALB/c mice were evaluated. Strong anti-Brucella immunity was generated, but the anti-mycobacterial response was low. To evaluate protective efficacy, a BALB/c model using MA was developed. When mice were challenged with MA, protection was obtained in some experiments but was inconsistent. This may be due to the low expression of MAP antigens in RB51. Another objective was to evaluate the effect of an ongoing Brucella-infection on the efficacy of RB51 vaccination, and whether vaccination of already infected animals could have a curative effect. Mice acutely or chronically infected with virulent BA, rapidly cleared the RB51 vaccine organisms, but there was no significant decrease in the number of virulent BA. Brucella spp. have been developed as biological weapons, but there are no vaccines to protect humans. The development of a very attenuated protective vaccine is necessary to prevent human infections, as well as to protect wildlife. To generate such a vaccine, RB51 based vaccines were irradiated to render them non-replicative, but metabolically active. We demonstrated that in general, irradiated and non-irradiated RB51 vaccines remain protective at levels similar to those elicited by the live vaccines. Therefore, irradiation of strain RB51 is an effective means of attenuating the strain without affecting its protective characteristics, and could eventually be used as a vaccine for wildlife and humans. / Ph. D.

Brucella abortus is a zoonotic agent that primarily infects cattle and causes brucellosis. B. abortus strain RB51 is a live, attenuated vaccine licensed for cattle. However, there is no available vaccine to prevent human brucellosis. Outer membrane vesicles have been tested as potential vaccines to prevent diseases caused by bacterial species. OMV are constantly released from Gram-negative bacteria. They are comprised principally of the outer membrane components and periplasmic proteins from the bacterial cell envelope. The research in this thesis examined the adjuvant property of non-replicative, metabolically active irradiated strain RB51 and the protective ability of OMV derived from strain RB51. Irradiated B. abortus strain RB51 was assessed for its ability to act as an adjuvant to induce protection against malaria. It was found that irradiated B. abortus strain RB51 administered along with fasciclin related adhesive protein (FRAP) to mice induced a protective immune response and a significant decrease in parasitemia after challenge with Plasmodium berghei. Strain RB51 and strain RB51 over-producing Cu/Zn superoxide dismutase (Cu/Zn SOD) were used to produce OMV. Western blotting and SDS-PAGE gel staining confirmed the presence of OMV and the over-production of Cu/Zn SOD. OMV were delivered to mice using an intraperitoneal route and, in some cases, with aluminum hydroxide adjuvant. The immune response was assessed by antibody isotyping with respect to OMV and measuring splenic clearance (i.e. protection) from a B. abortus strain 2308 challenge. The results demonstrate that OMV from B. abortus strain RB51 or strain RB51 over producing Cu/Zn SOD produced a Th1 polarized immune response as measured by specific OMV antibodies and cytokines but no statistically significant protection was observed. / Master of Science

Brucella are Gram-negative intracellular bacteria that cause abortion and infertility in livestock and chronic disease in humans. The Centers for Disease Control and Prevention (CDC) categorizes them as class B pathogens due to their zoonotic potential. Currently, there are no efficacious Brucella vaccines for humans available. Very few studies have focused on identifying protective vaccines against respiratory exposure. Protection by B. abortus rough vaccine strains RB51 and RB51SOD is through strong CD4⁺ Th₁ and CD8⁺ Tc₁ adaptive immunity. However, limited information is available on how they stimulate innate immunity. This knowledge is critical for improving these vaccines for their potential use in humans. Dendritic cells (DCs) play a crucial role bridging innate and adaptive immunity. Therefore, enhancing the ability of rough vaccine strains to induce DC maturation and function could be critical for upregulating protective T-cell responses. Herein, we demonstrated that live vaccine strain RB51 induced significantly better (p≤0.05) DC maturation and function in vitro and upon intranasal inoculation in vivo compared to strain RB51SOD or strain 2308. Due to safety concerns of live vaccines, irradiated and heat killed vaccines were also tested; only live strain RB51 infected DCs induced significant (p≤0.05) DC function based on TNF-α and IL-12 secretion. DC activation occurs through Toll-like receptors (TLRs) 2, 4 and 9. Our study reported that strain RB51 induced significant (p≤0.05) DC activation compared to strain 2308, which was not dependent on a specific TLR. However, strain RB51 induced TNF – α production was TLR2 and TLR9 dependent and IL-12 production was TLR2 and TLR4 dependent. TLR4 KO mice had significantly (p≤0.05) higher number of strain RB51 colonies present at day 14 post infection. By unraveling the innate immune responses to Brucella, the ultimate goal of these studies is to develop a protective vaccine for animals and people against respiratory challenge. As such, we tested several vaccination strategies. Despite enhanced DC activation and function achieved by vaccine strains, they failed to protect mice against intranasal challenge with strain 2308. Future experiments will address host-pathogen interaction at the lung microenvironment and elucidate immune mechanisms that will enhance protection against aerosol exposure. / Ph. D.

Livestock across the world contract several infectious diseases of both bacterial and viral origin. Swine brucellosis caused by Brucella suis and swine influenza caused by Influenza A virus affect both domestic and feral swine populations. Both the diseases have zoonotic potential to cause disease in humans with serious complications apart from inflicting huge economic losses. Infected feral swine can also act as a source of spread and outbreak where the disease is not endemic. At present, there is no vaccine available for swine brucellosis. The currently used swine influenza vaccine may not be effective against influenza strains like the recent H1N1 strain that caused a pandemic. To develop an effective bivalent vaccine for swine against these two diseases, a leucine auxotroph of the USDA approved vaccine B. abortus strain RB51 was constructed along with leuB gene complementing plasmid pNS4 to over-express antigens from Brucella and influenza. This antibiotic resistance free system over-expressed Brucella derived antigens SOD, L7/L12 and WboA in three different constructs. Against a virulent challenge of B. suis, the candidate vaccine strain over-expressing both SOD and WboA protected mice more significantly than the control group and was also found to be better protective than other candidate vaccine strains over-expressing either SOD and L7/L12 together or SOD alone. Immunoassays (ELISA) suggested that the protection afforded is Th1 type mediated immune response, as cytokine IFN-γ and IgG2a antibody sub-isotype was observed in the splenocyte culture supernatant and serum samples respectively. The strain RB51leuB platform was not expressing influenza derived antigens Hemagglutinin (HA) and Nucleoprotein (NP) when screened for expression by immunoblot. Influenza antigens, HA, NP and ectodomain of matrix protein M2e, were not found to be expressing even after optimizing their codon usage to suit Brucella tRNA preference. However, RT-PCR showed that the influenza genes mRNA were produced. In conclusion, this dissertation describes the construction of an environmentally safe antigen over-expression platform and successful employment of the system as a candidate vaccine in protecting mice against B. suis challenge. This new platform is a potential candidate for developing vaccines against other infectious diseases of livestock. This document also discusses alternate strategies for expressing influenza antigens in a Brucella platform. / Ph. D.