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.

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.

HSV-1 facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in gB, gK, UL20 and other viral genes, drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, which is regulated by interactions with viral glycoproteins gD, gH/gL, gK, membrane protein UL20 and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl terminal 28 amino acids of gB, or the dominant syncytial mutation in gK (Ala-to-Val at amino acid 40), was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletions substantially affected gB, gC, gD, gE and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM, while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells slower than their parental wild-type virus. Moreover, the mouse eye model study, revealed that although the UL11-null virus replicated less efficiently in mouse corneas, it infected ganglionic neurons with greater efficiency than either the gM or gE-null viruses. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread in vivo as well as in vitro, and also that gK plays the most important role in corneal and ganglionic infection in the mouse eye model followed by gM, gE, and UL11 in a descending order of importance relative to the wild-type virus.

Among the soil-transmitted helminths (STH), hookworms are a worldwide problem in both humans and animals. They cause non-specific gastrointestinal symptoms, and in young children and animals, they can cause stunting, malnutrition and anemia. Canine hookworms have significant zoonotic potential as a cause of cutaneous larvae migrans and eosinophilic enteritis in humans. To determine the ecological niche of human hookworm in Brazil, two risk models were developed based on the Growing Degree Day-Water Budget (GDD-WB) concept, one based on accumulation of monthly temperatures above a base temperature of 15oC and threshold WB value >0.4. The second was based on a gradient index of the product of monthly accumulated GDD and WB values. It was determined that both environmental temperature and moisture are important in the distribution of hookworm. This study supports the validity of the GDD-WB concept for mapping risk of hookworm at a national scale. A cross-sectional survey was conducted in human outpatients in Mutuípe municipality, Brazil, to determine prevalence of soil-transmitted helminths, including hookworm. Mutuípe falls within the permissive zone for the transmission of hookworm. A sucrose double centrifugation flotation technique was used for the concentration of helminth eggs in fecal samples. Hookworm infection was the most prevalent of the STH and the prevalence was highest in adults and males. PCR was then used to determine the species of hookworm present. Necator americanus was confirmed by PCR as the predominant hookworm species. A single case of Ancylostoma ceylanicum was identified. A study on the prevalence of hookworms and other gastrointestinal parasites in shelter dogs in south Louisiana and the anthelminthic protocols used in the shelters was conducted. Fecal samples examined by direct smear, flotation and sedimentation methods revealed that hookworm had the highest prevalence (53.6%) followed by Trichuris vulpis (28.7), Cystoisospora ohioensis (17.2%), Giardia duodenalis (12.0%), C. canis (7.7), Toxocara canis (6.2%), Dipylidium caninum, Alaria spp and Capillaria sp A PCR-RFLP developed to differentiate A. caninum and A. braziliense revealed A. caninum as the only species found. Evaluation of the anthelminthic protocols used in nine shelters showed current methods were inadequate for control of hookworms in shelter dogs.

Leishmaniasis is a public health problem in Brazil, and despite the control programs in place, Bahia remains one of the states with the highest incidence rates of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). This study proposed to develop and evaluate the applicability of ecological niche models (ENM) for leishmaniasis, to determine the influence of environmental and socioeconomic factors on the incidence of these diseases at three geographic scales: national, statewide and community. Water availability, garbage collection, precipitation and temperature were the most important variables in predicting areas suitable for VL and CL in the country. The extensive geography of Brazil and the coarse scale of the data used to evaluate both socioeconomic and environmental variables revealed the need of a more refined scale to define the role of these factors in risk area identification. At the statewide scale, the models were developed for Bahia state and data on vector occurrence was added to the analysis. Three environment structural indices were evaluated in addition to the environmental variables explored in the national model. Water content of vegetation was a very strong predictor of CL and VL incidence followed by NDVI. The sand fly species found in Bahia were sensitive to variations in temperature and rainfall related variables. The occurrence of Lutzomyia longipalpis, the vector of VL in the state, was most influenced by precipitation and vegetation. The district of Monte Gordo, in Bahia, was selected for development of a community level ENM using high resolution WorldView-2 imagery. CDC light traps were used to collect sand flies for a period of three months. Sand flies were tested by polymerase chain reaction (PCR) to determine host feeding preferences and natural infection by Leishmania spp. The sand flies preferably fed on chickens and humans. No natural Leishmania spp. infections were detected. NDVI was the most influencing factor in the ENM model (99.4% contribution). Implementation of a multi-scale geospatial surveillance and risk modeling capability to monitor disease incidence and their vectors, with the addition of molecular analysis, into the actions of the control program can help reduce the impact of endemic leishmaniasis in Bahia.