Channel catfish farming is the largest aquaculture industry in the U.S., and virulent Aeromonas hydrophila is an important re-emerging bacterial pathogen of farmed catfish. A. hydrophila is ubiquitous in the aquatic environment and causes motile Aeromonas septicemia (MAS) in farmed catfish. A. hydrophila infection is challenging to treat due to rapidly progressive mass mortalities. Although florfenicol has been used in managing outbreaks, acute onset of anorexia, drug cost, and emergence of antimicrobial-resistant strains are significant concerns. Prevention of MAS by vaccination could be a promising approach, but a commercial vaccine is not available. Thus, it is critical to develop effective vaccines against A. hydrophila to prevent losses in the catfish industry. The overall objective of this study is to develop effective live attenuated A. hydrophila vaccines and understand macrophage-A. hydrophila interactions. To achieve this, potential virulence-related genes from T6SS, secretion pathway, tat pathway, and flagellar system were deleted by in-frame, and mutants’ virulence and protection were evaluated in channel catfish. Also, macrophage-A. hydrophila interactions were explored by studying global gene expression to understand macrophage responses to A. hydrophila and A. hydrophila virulence mechanisms

Malignant catarrhal fever (MCF) is a lymphoproliferative and inflammatory syndrome affecting primarily ruminant species. The disease, which is often fatal, is most often described as affecting bovids and cervids. No vaccines are available for prevention of MCFV infection. The primary method to control spread of disease is to prevent contact between carriers and clinically susceptible species. There is no known method to control infection of malignant catarrhal fever virus-white-tailed deer variant (MCFV-WTD), as the carrier animal of this virus is unknown. To determine the prevalence of malignant catarrhal fever viruses in Tennessee ruminant populations, blood and/or lymph node samples were collected from farms, animal processing and disposal facilities, and hunter check-in stations from 2006-2008 from several species of animals including cervids, cattle, and goats. Strain-specific real time PCR was developed to detect ovine herpesvirus-2 (OvHV-2), caprine herpesvirus-2 (CpHV-2), and MCFV-WTD DNA. MCFV DNA was detected in all species of ruminants sampled. Although disease related to infection with MCFV-WTD and CpHV-2 has not been reported in Tennessee cattle or cervid populations, MCFV-WTD DNA was detected in 3 percent of cervid samples, and MCFV-WTD and CpHV-2 DNA was detected in 27 and 3 percent respectively of cattle samples from animal disposal facilities that process dead or debilitated animals. One hunter harvested deer (n=781) and 25 cattle (n=165) tested from animal disposal facilities were positive for OvHV-2 DNA. This study demonstrated that healthy cattle and cervids can be infected with OvHV-2 and MCFV-WTD without apparent disease, and dead or debilitated cattle were infected with OvHV-2, MCFV-WTD and CpHV-2 at a higher percentage than healthy herd animals. Prevalence of CpHV-2 in Tennessee goat populations (7%) was significantly lower than reported in other goat populations (73%). Low prevalence of CpHV-2 in Tennessee goat populations likely explains why no evidence of infection was found in cervids tested, and the low prevalence of CpHV-2 infection in dead or debilitated cattle compared to prevalence of infection with OvHV-2 and MCFV-WTD. The discovery of infection in cattle with CpHV-2 and MCFV-WTD opens a new avenue of investigation into the pathology and virulence of MCFV’s in domestic cattle.

Bovine viral diarrhea (BVD) is an economically important disease of cattle. Calves persistently infected (PI) with the bovine viral diarrhea virus (BVDV) are a powerful agent for spread of the virus. A total of 24,423 southeast origin beef cattle comingled at three Warren County, Kentucky locations were tested from November 2007 to June 2010 for PI BVDV. A total of 97 head tested positive for PI BVDV, giving an average overall prevalence of 0.397%. Calves tested were subdivided into categories for additional calculations of dependence. A total of 8,910 were categorized by weight range upon testing (300-399 lbs, 400-499 lbs, 500-599 lbs, and 600-699 lbs). Prevalence does show a dependence on weight, with a higher prevalence found in lower weight classes, especially 300-399 lb calves (P<0.001). A total of 24,423 were categorized by season at time of testing (Fall, Winter, Spring, Summer). Prevalence does not show a dependence on season (P>0.05). Although eradication programs are not likely to be organized in the United States, several control programs have been developed. These findings can be used as additional support for PI testing of calves, especially those in lighter weight classes, as part of a BVD control program.

Lawsonia intracellularis and the disease it causes in horses, equine proliferative enteropathy (EPE), is an emerging pathogen of increasing importance to the horse industry from both an economic and welfare standpoint. Long recognized as an economically important disease of swine, the hallmark of EPE is a protein-losing enteropathy, where affected horses suffer weight loss and some ultimately succumb to the disease despite aggressive treatment. There are currently no known EPE preventative measures and the epidemiology of the disease remains poorly defined. While EPE is a sporadic disease affecting less than 25% of exposed horses, some farms experience clinical cases year after year. Further, weanlings are uniquely susceptible to this disease, although no conclusive reason for this predisposition has been identified. The overall hypothesis is that the host immune response plays a significant role in the susceptibility of weanlings to L. intracellularis infection and the occurrence of clinical equine proliferative enteropathy. To test this hypothesis, four individual hypotheses were proposed: (H1) previous farm history of EPE does not have an effect on weanling seroprevalence, (H2) passively-acquired antibodies do not have an effect on susceptibility to L. intracellularis and the occurrence of EPE, (H3) the serological status of mares can be used to determine the role they play in the epidemiology of EPE on endemic farms, and (H4) L. intracellularis-specific IFN-g expression is not associated with increased resistance to EPE.

Despite advances in mosquito management, mosquito-borne disease in the United States is still of relevant public health concern and vector control is a top priority in preventing transmission of pathogens. Insecticide barrier sprays have become a common tool for suppression of mosquitoes in single-homeowner backyards. The application of the synthetic pyrethroid, lambda-cyhalothrin to perimeter vegetation with a backpack sprayer has been shown to significantly suppress mosquito levels for around 6 weeks. In an attempt to lengthen the effective duration of treatment, the IGR, pyriproxyfen, was added to a backpack mist blower with lambda-cyhalothrin, as adult mosquitoes exposed to pyriproxyfen have been shown to disseminate it to oviposition sites and to experience lowered fecundity. This treatment was compared to lambda-cyhalothrin alone and to a water control. Mosquito populations were sampled using CO2-baited CDC light traps, CDC gravid traps, human landing rates, and ovitraps. Leaf bioassays were performed. The following summer, the same treatments were applied with a truck-mounted mist blower to tree lines in Central Kentucky, to test the efficacy of an application method that could be used on large properties. Finally, bioassays were performed with water sampled from pyriproxyfen-treated containers, exposed to field conditions to test for residual efficacy.

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.

Staphylococcus pseudintermedius affecting dogs is analogous to S. aureus on humans, acting as both normal flora and opportunistic pathogen. Methicillin resistance in S. pseudintermedius is recent, with the first documented occurrence of an isolate bearing the methicillin resistance gene, mecA, in 1999. This gene encodes penicillin binding protein 2a, which renders all beta-lactam drugs ineffective and functions as a “gateway” antibiotic resistance determinant. In the presence of ineffective antibiotics, opportunities for mutational events and acquisition of mobile genetic elements increase as microbial densities increase, often leading to multi-drug resistance. Methicillin-resistant S. pseudintermedius (MRSP) infections have become increasingly common. For example, approximately 30% of the S. pseudintermedius isolates tested by the University of Tennessee College of Veterinary Medicine Clinical Bacteriology Laboratory are resistant to methicillin. An increasing number of MRSP isolates are also resistant to most clinically useful antibiotics available to veterinarians except for chloramphenicol, and resistance to this antibiotic is common among European MRSP isolates. Chloramphenicol resistance has begun to appear in the US and if this trend continues there may soon be few viable antibiotic treatment options. Compared with the arrival of methicillin-resistant S. aureus in the 1960s, the opportunity currently exists to apply advanced molecular methods early in this recognized emergence of MRSP. To that end I have pursued projects utilizing multilocus sequence typing, pulsed-field electrophoresis, and SCCmec characterization of both susceptible and resistant S. pseudintermedius. The initial result was the detection of a clonal population of MRSP in the southeastern United States. Further characterization of this and other clonal lineages using genomic sequencing and real-time RT-PCR expression analysis of antibiotic resistance and quorum sensing genes revealed a marked difference in the regulation of antibiotic resistance between regional clones. These discoveries have interesting epidemiological implications and provide a foundation for the development of novel therapeutics to circumvent the expanding antibiotic resistance repertoire of MRSP. Potential targets identified by this work include membrane-bound beta-lactamase receptors responsible for the regulation of mecA, non-cognate auto-inducing peptides, and synthetic antisense oligonucleotides.

Experimental infection (EI) studies, involving the intentional inoculation of animal or human subjects with an infectious agent under controlled conditions, have a long history in infectious disease research. Longitudinal infection response data often arise in EI studies designed to demonstrate vaccine efficacy, explore disease etiology, pathogenesis and transmission, or understand the host immune response to infection. Viral loads, antibody titers, symptom scores and body temperature are a few of the outcome variables commonly studied. Longitudinal EI data are inherently nonlinear, often with single-peaked response trajectories with a common pre- and post-infection baseline. Such data are frequently analyzed with statistical methods that are inefficient and arguably inappropriate, such as repeated measures analysis of variance (RM-ANOVA). Newer statistical approaches may offer substantial gains in accuracy and precision of parameter estimation and power. We propose an alternative approach to modeling single-peaked, longitudinal EI data that incorporates recent developments in nonlinear hierarchical models and Bayesian statistics. We begin by introducing a nonlinear mixed model (NLMM) for a symmetric infection response variable. We employ a standard NLMM assuming normally distributed errors and a Gaussian mean response function. The parameters of the model correspond directly to biologically meaningful properties of the infection response, including baseline, peak intensity, time to peak and spread. Through Monte Carlo simulation studies we demonstrate that the model outperforms RM-ANOVA on most measures of parameter estimation and power. Next we generalize the symmetric NLMM to allow modeling of variables with asymmetric time course. We implement the asymmetric model as a Bayesian nonlinear hierarchical model (NLHM) and discuss advantages of the Bayesian approach. Two illustrative applications are provided. Finally we consider modeling of viral load. For several reasons, a normal-errors model is not appropriate for viral load. We propose and illustrate a Bayesian NLHM with the individual responses at each time point modeled as a Poisson random variable with the means across time points related through a Tricube mean response function. We conclude with discussion of limitations and open questions, and a brief survey of broader applications of these models.

The objectives of this thesis were: (1) to evaluate the downregulation of the stallion hypothalamic-pituitary-gonadal (HPG) axis by a GnRH antagonist (acyline) based upon endocrine, seminal, testicular and behavioral effects, and (2) to assess recovery after treatment. Stallions were treated for 50 days (n=4; 330µg/kg acyline q 5d) and controls (n=4) received vehicle alone. Stallions were assessed pre-treatment and for 72 days after last treatment. Treatment induced declines (p<0.05) in FSH, LH, testosterone (to castrate levels) and estrone sulfate. Gonadotropins and testosterone returned to control values within nine days and estrone sulfate by 14 days after treatment discontinuation. Acyline-treated stallions failed to respond with FSH, LH and testosterone increase after exogenous GnRH stimulation (25µg gonadorelin, IV) compared to pre-treatment and control stimulation. Total sperm numbers and motility were reduced in acyline-treated stallions, as well as total seminal plasma protein and testicular volume (p<0.05). Time to ejaculation was increased in acyline group (p<0.5). Testicular, sexual behavior and most seminal parameters regained normal levels within 72 days after treatment ceased. Sperm output of acyline-treated stallions was regained within seven months after ending treatment. Acyline reversibly suppressed the stallion HPG axis, thus has potential for treating the androgen-dependent Equine-Arteritis-Virus carrier state and as behavior modulator.

Influenza virus is one of the major respiratory pathogens of humans as well as animals, including equines. There is an increasing evidence that bacterial infections are the most common cause of the death during influenza. In horses also, secondary bacterial pneumonia can lead to death, and surviving horses may take up to six months for the complete recovery resulting in heavy economic loss to the equine industry. Interleukin (IL)-23 mediated innate immune response has been shown to protect the host from various respiratory bacterial infections. However, studies to investigate the role of host and viral factors in the regulation of IL-23 are limited. Endoplasmic reticulum (ER) stress-induced transcription factor CHOP-10 and IFN-β has been shown to participate in the regulation of IL-23. Primary hypothesis for the current study was that influenza A virus (IAV) NS1 protein downregulates the IL-23 expression via inhibition of CHOP-10. In order to test our hypothesis, we infected the RAW264.7 cells - a murine macrophage cell line, and primary murine alveolar macrophage cells either with the wild type Influenza A virus (PR/8/34, PR8) or isogenic mutant virus lacking NS1 (delNS1). Quantitative analysis of mRNA expression revealed a significantly higher mRNA expression of IL23p19, IFN-β and CHOP-10 in delNS1 virus infected cells as compared the PR8 virus infected cells. Additionally, overexpression of CHOP-10 partially restored the expression of IL-23p19 in PR8 virus infected cells and knockdown of CHOP-10 resulted in downregulated expression of IL-23p19 in delNS1 infected cells. Taken together, these results suggest that IAV NS1 protein mediated inhibition of CHOP-10 expression leads to downregulation of IL-23 expression in macrophage cells in-vitro. Similar results were also observed in-vivo using IAV and Streptococcus zoooepidemicus (S. ze) co-infection model. In a co-infection mouse model delNS1 virus co-infection resulted in significantly higher expression of the IL-23 and IL-17. Considering the role of IL-23 in protection against respiratory bacterial pathogens, effect of exogenous supplementation of IL-23 was also investigated in the influenza and S. ze co-infection mouse model. We found that a single intranasal dose of recombinant murine IL-23 significantly improved the survival of mice co-infected with PR8 and S .ze. Overall, our study suggests that IAV infection subverts the IL-23 mediated respiratory innate immune response and restoration of IL-23 could protect from influenza-associated respiratory bacterial infections.