Francisella asiatica is a Gram negative, facultative intracellular pathogen that causes fish francisellosis. In this project, homologues to the F. tularensis iglABCD genes were found present in F. asiatica. As few as 23 F. asiatica bacteria injected in the peritoneum were found capable of causing mortalities in tilapia nilotica (Oreochromis niloticus), and even fewer were enough to cause pathological changes. We examined the in vivo and in vitro interaction of F. asiatica wild type (WT) and a ÄiglC strain with tilapia and tilapia head kidney derived macrophages (HKDM). The ÄiglC was found to be attenuated following intraperitoneal and immersion challenges in tilapia. The WT was found to be able to invade HKDM and replicate vigorously within them, causing apoptosis and cytotoxicity in the macrophages. The ÄiglC, however is defective for survival, replication and the ability to cause cytotoxicity in HKDM. We further characterize the efficacy of the ÄiglC as a live attenuated vaccine against subsequent immersion challenge with the WT. Tilapia vaccinated by immersion with a suspension of the ÄiglC and subsequently challenged with WT were protected (90% survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the ÄiglC were subsequently found to protect tilapia against WT challenge in passive immunization experiments. The lack of effective treatments led us to investigate the efficacy of florfenicol for treatment of F. asiatica in vitro and in vivo. Addition of florfenicol to the medium at 10 µg/ml resulted in uptake of the drug by HKDM and significantly reduced bacterial loads in vitro. Fish fed medicated feed 1 and 3 days post infection showed significantly higher survival rates, and significantly lower numbers of bacteria than controls. Finally, a real time polymerase chain reaction assay was developed to rapidly and accurately detect and quantify F. asiatica from fish tissue. Probe specificity was confirmed by the lack of signal and cross-reactivity with twelve common fish pathogens, two subspecies of F. tularensis, F. noatunensis, and tilapia tissue. The limit of detection was 50 fg of DNA (~25 genome equivalents).

Ticks are the only disease vectors for spotted fever group (SFG) Rickettsia which are obligate intracellular bacteria belonging to the genus Rickettsia. In nature, ticks maintain the infection of SFG Rickettsia via vertical and horizontal transmission. However, the prevalence of rickettsial transmission is limited to certain species of ticks, and this limitation is known as a specific tick/Rickettsia relationship. Due to the continuous increase of tick-borne rickettsial disease cases in the United States, which contrasts with very low prevalence of Rickettsia in tick vectors, the study of vector competence of tick to Rickettsia is needed in order to understand the ecology and epidemiology of tick-borne rickettsioses. Here we characterized the role of Dermacentor variabilis á-catenin during rickettsial infection in tick ovaries suggesting a role in rickettsial infection in tick ovaries. We demonstrated that the typical nonpathogenic (R. montanensis) and typical pathogenic (R. rickettsii) Rickettsia persistently infect Dermacentor variabilis compared to atypical Rickettsia (R. amblyommii), and only R. montanensis is able to disseminate to tick ovaries. D. variabilis glutathione S-transferase1 (DvGST1) has been identified as a tick immune-like molecule that specifically responds to atypical rickettsial challenge in tick midguts suggesting a role in controlling atypical rickettsial infection in tick midguts. DvGST1 is highly upregulated in tick midguts during bloodmeal acquisition. The function of GST is known to be involved with detoxification and oxidative stress reduction, and acaricide resistance in ticks. Silencing of DvGST1 gene demonstrates significant reduction of mRNA and enzyme activity of DvGST1 in tick midguts; however, further characterization of DvGST1 is needed due to the off-target effect of negative control dsRNA. Continued study on the tick/Rickettsia interaction influencing tick vector competence for Rickettsia will lead to a better understanding of ecology and epidemiology of tick-borne rickettsioses.

Extrinsic and environmental factors are known to affect the transmission of arthropod-borne viruses (arboviruses), including variations in the arthropod vector populations. Differences among these factors have been associated with differential transmission and are sometimes used to control the spread of an arbovirus through a vertebrate population in an effort to prevent or disrupt an outbreak. However, diversity in intrinsic viral populations, such as genetic and phenotypic variability, is not often accounted for when considering alterations in transmission. Presented in this dissertation are four experimental studies that explore the contribution of viral intrinsic factors, especially phenotypic variability, to the transmission potential of arboviruses as judged by modeling parameters such as vectorial capacity (VC) and the basic reproductive number (R0). The overall hypothesis of this research is that phenotypic differences of arboviruses alter the transmission potential of these arboviruses by conferring fitness advantages in either the vector or the vertebrate. Further, these phenotypic differences need not be large in magnitude to affect the relative transmission potential. To investigate this hypothesis, this research determined 1) whether intrinsic viral characteristics can lead to differential transmission in a given locale, 2) whether variability of viral fitness in the mosquito vector can lead to significant differential transmission potential, 3) how our newly formulated methods from our preceding aim could aid in the explanation of a currently puzzling phenomenon in the field of arbovirology, 4) whether phenotypic differences in the vertebrate host alters the potential for transmission, and 5) how identified phenotypic differences in both the vector and vertebrate hosts could act synergistically or antagonistically to alter transmission potential of arboviruses. The research in this dissertation offers a more accurate tool for assessing transmission potential in the vector, provides a new model assessing transmission potential in the vertebrate, and provides several of the necessary steps towards a more appropriate calculation of R0. Our use of R0 based on dynamic phenotypic differences provides a framework for a more dynamic formulation of transmission models, and provides an accessible framework for output validation and reporting to public health stakeholders.

There are many contradictory reports in the literature involving the culture of Spiroplasma mirum, its resistance to disinfectants and antibiotics, and its potential role in Transmissible Spongiform Encephalopathies (TSEs). These contradictions led to an interest in a possible link between spiroplasma and TSEs and the development of a research plan to elucidate this connection. It was hypothesized that Spiroplasma was associated with a neurodegenerative disease such as TSE. In this work we further characterized S. mirum laboratory strains and continued to evaluate the possible correlations they have with TSE infections and the prion. To optimize recovery of S. mirum from experimental infections, we established reproducible culture conditions in M1D media, embryonated eggs, and SP4 plates. Overall, results indicated that SP4 plates are the most accurate quantitative method for S. mirum. However, plate counts should be accompanied by qualitative assessments of cultures in liquid M1D as well. A susceptibility profile for physical and environmental disinfectants and antibiotics for spiroplasma was also determined. The three species of Spiroplasma tested were susceptible to minimal dilutions of common laboratory disinfectants. They were also susceptible to many of the antibiotics in use for other mollicutes. Although Spiroplasma and prions, the presumed infectious agent of TSEs, may both persist in neurologic tissues for extended periods of time, the two do not share the same properties of resistance. Experimental neonatal goat infections with a S. mirum laboratory strain SMCA using three different inoculation routes did not result in pathology, clinical signs, or an immune response over a two year time period. In addition, the organism was not detected via PCR or culture. The same strain did cause minimal clinical signs in one animal when inoculated into five month old white-tailed deer and was recovered from the cortex of the clinically-affected deer after multiple passages in culture. Although the goat experiments did not result in TSE-like disease, an improved methodology for a spiroplasmosis animal model was developed, and further research should be conducted using similar methods in neonatal white-tailed deer.

Accurately defining disease distributions and calculating disease risk is an important step in the control and prevention of diseases. This study used geographical information systems and remote sensing technologies within the MaxEnt ecological niche modeling program to create predictive risk maps for leprosy and Schistosomiasis in Brazil and Chagas disease in both Brazil and Bolivia. New disease cases were compiled for leprosy, Schistosomiasis, and Chagas disease from the Brazilian ministry of Health for 2001 to 2009 and the data was stratified to a 10,000 population for each municipality. Bolivian Chagas prevalence rates were calculated from 2007 to 2009 survey data. Environmental data was compiled from MODIS satellite imagery, and WorldClim data for both countries. Socioeconomic data was compiled from the Brazilian IBGE and the Bolivian INE. Leprosy results showed that areas of lower moisture and specific temperature ranges were related to areas of high leprosy case detection especially in the central western, north eastern and northern regions of the country. The states of Bahia and Minas Gerais continue to show the highest levels of new Schistosomiasis cases and also were predicted to have some of the highest risks for the disease in our study. This study confirmed the importance of sanitation and educational level in relation to Schistosomiasis, which has been previously established in other studies. Chagas disease models identified altitude as being important, as well as lower levels of precipitation, and higher ranges of temperature which correspond to the biological requirements of the insect vectors. Information for housing materials was only found for Bolivia, but demonstrated the importance of improved housing materials. Adobe wall materials were found to be highly related to the disease while areas with hardwood floors demonstrated a direct negative correlation. These studies demonstrated that MaxEnt can be successfully adapted to disease prevalence and incidence studies and provides governmental agencies with an easily understandable method to define disease risk area for use in resource planning, targeting, and implementation. This study emphasizes the need for more refined socioeconomic data to create better socioeconomic and smaller regional study areas to better elucidate region specific disease characteristics.

Edwardsiella ictaluri encodes a urease operon and an arginine decarboxylase (AdiA) that are required for virulence in head kidney derived macrophages (HKDM). The urease produces ammonia in amounts sufficient to alter environmental pH from acid to neutral. A hypothetical model was proposed, involving arginine metabolism in E. ictaluri infected HKDM, focusing on bacterial urease, AdiA, a second arginine decarboxylase (SpeA), and agmatinase (SpeB). Using fluorescence based ratiometric pH determination of E. ictaluri in live HKDM, it was shown that E. ictaluri modulates HKDM phagosome pH to above six. Urease and AdiA mutants failed to up-regulate vacuole pH, while vacuole pH for the SpeA and SpeB mutants was similar to the wild-type. These mutants could also replicate in HKDM similar to wild type E. ictaluri. These data show that urease and AdiA are required for phagosome pH neutralization. To determine the source of urea for E. ictaluris urease, an arginase inhibitor, L-norvaline, was used to partially block HKDM urea production. In arginase inhibited HKDM, E. ictaluri could not neutralize phagosome pH, nor could it replicate. Nitric oxide production in HKDM was not significantly different between controls and experimental groups. This indicates that HKDM have limited capacity to produce NO. Levels of urea produced in infected and control HKDM were at the lowest limit of assay detection and were not significantly different from one another. Together, these data show that E. ictaluri uses its urease and AdiA to neutralize phagosome pH, and that it uses urea derived from HKDM arginase to do so.

Rickettsia parkeri, a member of the spotted fever group of Rickettsia, is the agent of an emerging rickettsiosis in the southeastern United States and South America. Despite increased recognition of human cases, limited information is available regarding infection of invertebrate and vertebrate hosts for this emerging tick-borne disease. Towards development of a viable transmission model and to further characterize the pathology associated with R. parkeri infection, inbred mouse strains (A/J, Balb/C, C3H/HeJ, and C3H/HeN) were intravenously and intradermally inoculated with R. parkeri. The C3H/HeJ strain of mice were identified as the most susceptible to R. parkeri infection and were found to develop eschar-like lesion at the site of intradermal inoculation in the tail. These mice were further utilized to test the effect of tick feeding on the proliferation of R. parkeri at the intradermal inoculation site. Ticks were allowed to feed over the site of intradermal inoculation of R. parkeri at the nape of the neck. Rickettsial proliferation was significantly increased by tick feeding, suggesting a role for ticks as more than just fomites. Finally, the natural ecology of R. parkeri was investigated by screening domestic dogs in temporary housing situations for the presence of Rickettsia using PCR for the genus specific 17 kDa antigen gene. The more specific primers for rompA were utilized for PCR on the 12 positive samples identified by the screening PCR. After sequencing, the rompA amplicons were identified as R. parkeri, indicating a role for dogs in the ecology of R. parkeri and as a potential risk factor for development of human disease. Continued study into the pathogenesis of R. parkeri rickettsiosis in the murine model, the influence of tick saliva on rickettsial proliferation, and the role of dogs in the natural ecology of R. parkeri will lead to a better understanding of this emerging tick-borne rickettsiosis.

Acute gram-negative bacterial infections are a leading cause of mortality among the nosocomial infections. Increasing numbers of immunosuppressed individuals and growing numbers of antibiotic resistant strains make antibiotic treatment difficult. Neutrophils are the first cells recruited to the site of infection and are critical players in the host defense against gram-negative bacterial pneumonia. Therefore, identification of targets that boost neutrophil-associated host defense in the lung is essential in designing better therapies to control pulmonary infections. Production of chemokines is an important step for neutrophil recruitment. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is important for monocyte and T-lymphocyte influx. It is important for the host defense during Listeria monocytogenes and Streptococcus pneumoniae infection. However, the role of MCP-1 during pulmonary gram-negative infections is not known. We hypothesized that MCP-1 is essential for the host defense during a gram-negative infection. To test the hypothesis, we infected MCP-1 gene-deficient (MCP-1-/-) mice and controls intratracheally (i.t.) with E. coli (106 CFUs/mouse) and Klebsiella pneumoniae (103 CFUs/mouse). We found that MCP-1 is critical for host defense against gram-negative infections, mainly by recruiting neutrophils to the site of infection. MCP-1 utilizes its receptor, CCR2, to recruit neutrophils directly and indirectly by regulating the expression of cytokines (IL-6, TNF-a) and chemokines (KC, MIP-2) through activation of NF-kB and MAPKs. We also observed that MCP-1 can regulate expression of G-CSF and thereby neutrophil numbers in circulation during Kp infection. In addition, exogenous administration of rG-CSF can restore the defects in host defense in MCP-1-/- mice following gram-negative Kp infection. This study demonstrates an unrecognized role of MCP-1 in host defense during gram negative bacterial pneumonia. These findings bolster pleiotropic effects of MCP-1 in the host defense and demonstrate a potential role as a therapeutic agent to augment host defense during acute bacterial pneumonia.

Biomonitoring has become an important component in bioassessment programs. It is used to maintain high water quality standards, and determine contaminant levels and biological affects in areas that have been heavily disturbed. The objectives of this research were 1) to improve and apply certain modern biomonitoring techniques and 2) to locate possible contaminant sources affecting the flora and fauna of the Green River and of Mammoth Cave. Actinonaias ligamentina (Lamarck, 1819), a freshwater mussel, was used for interpretation of these impacts as well as refinement of biomonitoring techniques. The mussels were collected in the Lawler Bend region of the Green River, an area upstream from the Mammoth Cave System, and from Haynes Shell Midden (dating 4000 - 6000 years before present) 45 miles downstream. Analyzing the shell nacre of these mussels, and the soft tissue of recently collected specimens, produced an abundance of information including high tissue concentrations of organochloride pesticides, significant concentrations of several metals including Cadmium, Copper, Mercury, Nickel, Silver and Zinc (with Mercury and Silver being found at the impact site), and numerous shell nacre stains. These results indicated possible impact from agriculture in the region and past and present contamination from local industries, and demonstrated the importance of the nacreous shell to biomonitoring programs.

Equine Herpesvirus 1 (EHV-1) is an important ubiquitous enzootic equine pathogen and one of the most common pathogens of the horse, causing, respiratory disease, epidemic abortion, occasionally neurological disease in horses, which leads to significant economic losses to the horse industry. EHV-1 induces several clinical signs of disease ranging in severity, from mild respiratory disease to abortion in pregnant mares, neonatal foal death and neuropathogenic disorders. Natural EHV-1 infection stimulated short lived protective immunity and had both humoral and cellular immune responses. Currently vaccination remains the best option to prevent EHV-1 infection and different applications of vaccination have been investigated and developed over the past decades. The objective of this research was the design of a safe and effective virus-vectored vaccine to prevent EHV-1 infections. In this research, EHV-1 glycoprotein D (gD) gene was cloned into the Herpes Simplex Virus Type-1 (HSV-1) VC2 vector, which contains the gK∆31-68 deletion and UL20∆2-22 deletion. The VC2 strain cannot infect axonal neurons of mice and rats and has been shown to produce protective immune responses against both HSV-1 and HSV-2 viruses in mice and guinea pigs. Vaccination of mice with the HSV-VC2-EHV-gD increased virus neutralizing activities against EHV-1 (33.6%) in mice after three vaccinations, which was similar to commercial whole virus vaccine group (32.6%) and significantly higher than VC2 and Unvaccinated control groups (p<0.01 or p<0.001). Mice vaccinated with the VC2-EHV-gD group exhibited significantly higher humoral and cellular immune responses as detected by polychromatic flow cytometry when compared to the other groups (p<0.01 or p<0.001). Induction of IgG1 and IgG2a antibodies were significantly higher in the VC2-EHV-gD group than other groups after three vaccinations (p<0.001). Its interesting that induction of IgM antibody in the Vetera group was significantly higher than other groups before and after challenge (p<0.01 or P<0.05). Vaccination with the VC2-EHV-gD also stimulated strong cellular immune response (IFN-γ and TNF). Additional studies are needed to assess the VC2-EHV-gD vaccine efficacy in generating protective humoral and cellular immune responses in horses.