Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Derek A. Mosier / The bovine respiratory disease complex (BRDC) is a major concern for cattle producers in the United States and worldwide. One of the most costly and deadly components of BRDC is bovine pneumonic pasteurellosis (BPP) caused by Mannheimia haemolytica. The initial pulmonary inflammation associated with BPP is a characteristic serofibrinous exudation into the lung, which is believed to be induced by M. haemolytica virulence factors such as lipopolysaccharide (LPS) and leukotoxin (LKT) and host cytokines and chemokines such as tumor necrosis factor – α, interleukin – 1β, and interleukin – 8. However, these pulmonary changes often occur before virulence factors or cytokines are substantial components of the pulmonary microenvironment. Other proinflammatory molecules such as substance P (SP) may be involved in the pathogenesis of the peracute serofibrinous exudation of BPP. SP is an 11 amino acid long neuropeptide that is a neurotransmitter of pain that can be released from sensory nerves into tissues to cause neurogenic inflammation. Neurogenic inflammation is characterized by serofibrinous exudation and leukocyte activation. SP-like immunoreactivity was present in the airways, alveolar septa, macrophages, endothelium, and peribronchial nerves in both pneumonic and normal bovine lung; however, SP-like immunoreactivity was increased in pneumonic compared to normal bovine lung due to increased immunoreactivity in macrophages. SP and the combination of SP with histamine and LPS increased the permeability of a calf pulmonary arterial endothelial cell line to Evans blue dye labeled albumin by 12.34%, 13.57%, and 22.03%, respectively compared to a cell control. Similarly, SP and the combination of SP and histamine increased the monolayer permeability of a bovine adrenal gland capillary endothelium by 8.27% and 16.69% compared to a cell control. The increase in permeability was due to endothelial cell shape change and the formation of intercellular gaps rather than cell death. However, SP does not increase the surface expression of the β2 integrin CD18 (the M. haemolytica LKT receptor) on bovine neutrophils nor does it increase LKT-induced leukocytotoxicity of bovine peripheral blood leukocytes. These findings indicate that SP may be a contributor to BPP in association with other cytokines.

Master of Science / Department of Animal Sciences and Industry / Terry A. Houser / Nursery age pigs (n=198) were used to evaluate the difference in the occurrence of injection site abscesses between needle-free jet injection and conventional needle-and-syringe injection systems. Pigs were fed for 21 d prior to treatment administration to acclimate the pigs to the environment of the Kansas State University Segregated Early Weaning (SEW) unit. On d 21 each pig was injected with aluminum hydroxide adjuvant in the neck and ham with needle-free jet injection (Pulse Needle-Free Systems, Lenexa, KS) and conventional needle-and-syringe injection. Needle-free and conventional needle-and-syringe injections were randomly assigned to pig side yielding a total of 396 injections per treatment with a total of 792 injections sites. Immediately prior to injection, the external surface of the injection sites were contaminated with an inoculum of Arcanobacterium pyogenes, a bacterium commonly associated with livestock abscesses. The pigs were then fed for a period of 27 or 28 d. On d 27 or d 28 the pigs were humanely euthanized and sent to the Kansas State Veterinary Diagnostics Laboratory where necropsies were performed and the injection sites harvested for histopathological evaluation. The needle-free jet injection system was associated with more injection site abscesses than the conventional needle-and-syringe injection method for both neck (P=0.0625) and ham (P=0.0313) injection sites. Twelve abscesses were found at injection sites administered via needle-free jet injection method while only 1 abscess was found with the conventional needle-and-syringe injection method. 5 abscesses were found at the neck injection sites and 8 abscesses were found at ham injection sites. There were no significant differences seen in tissue granulation resulting from reaction to the adjuvant. In summary, the implementation of needle-free jet injection systems in market hog production will be beneficial to eliminate needles and needle fragments in meat products but, when in the presence of Arcanobacterium pyogenes, it may increase the occurrence of injection site abscesses in pork carcasses that will need to be trimmed in pork processing plants. Although more abscesses were associated with needle-free jet injection, their occurrence was observed at a very low rate given that all injection sites were intentionally contaminated prior to injection.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / David G. Renter / Bradley J. White / The objective of my research was to generate novel information concerning the epidemiology, diagnosis and prevention of bovine respiratory disease complex (BRDC), a common pre-weaning and post-weaning beef calf disease. To reach my objective, I conducted three prospective field trials within post-weaned calf populations, and one retrospective study of pre-weaned calves utilizing survey data. I evaluated differences in behavior, health and performance in calves receiving multiple component health programs. Calves in a minimally invasive program, which included primarily non-injectable products, displayed less aversion to initial product administration but experienced higher BRDC morbidity (P = 0.02) and poorer performance (P = 0.04) compared to calves in a more invasive (all injectable products) program. Secondly, in a study of Mannheimia haemolytica inoculated calves, I found that no parameter included in physical examinations, or common blood component evaluations could discern health from disease. However, disease recognition was aided by the measurement of the number of steps taken by a calf in a 24 hour period. None of the parameters that were evaluated predicted the severity of lung pathology. Thirdly, I conducted a study in post-weaned feeder calves that determined prevalence estimates for Mollicutes in general, and Mycoplasma bovis specifically, and their respective associations with health and performance. Nasal Mollicutes prevalence was high on arrival, and differences in calf performance were associated with (P < 0.01) nasal prevalence. More than half of the calves seroconverted to M. bovis; calves not seroconverting gained more weight (0.49 kg/head/day) during the study than those calves that did seroconvert (0.35 kg/head/day). Finally, I conducted a retrospective analysis of national U. S. cow-calf survey data to identify herd level management practices associated with pre-weaned calf BRDC. I found feeding antibiotics to pre-weaned calves, importing cattle, the number of outside visitors, economic purpose of the cow-calf operation, and breeding management of the herd were associated with herd-level pre-weaning BRDC rates. My research projects generated unique information concerning the epidemiology of important pathogens, differences among preventive health programs, objective BRDC diagnostic parameters, and pre-weaning BRDC risk factors. These research studies reinforce the complexity of BRDC and demonstrate the pathogen, animal and management factors affecting BRDC risk in pre- and post-weaned beef calves.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Larry C. Hollis / Mark F. Spire / Two studies were undertaken to evaluate the use of Near Infrared Spectroscopy (NIRS) to determine arterial oxygen saturation (StO[subscript]2) in cattle with naturally-occurring Undifferentiated Bovine Respiratory Disease (UBRD) and experimentally-induced UBRD utilizing Mannheimia haemolytica. The first study was a natural infection model utilizing 679 beef heifers weighing approximately 227 kg (500 pounds) originating from a southeastern U.S. salebarn. Heifers were evaluated for UBRD upon feedlot arrival, at revaccination, at day 35 on feed, at re-implant time, and two weeks prior to shipment for slaughter. Animals deemed to have UBRD were treated for UBRD and data was collected for 5 days following treatment, while a comparable healthy cohort was also evaluated at the time of treatment. There was a trend for NIRS to be able to predict the incidence of subsequent UBRD when cattle were evaluated on arrival (p=0.0552). However, the ability to detect UBRD in clinically ill cattle was not significantly different (p>0.1690) when compared to healthy cohorts in this model. When carcass characteristics were evaluated at each time point, NIRS StO[subscript]2 values were able to differentiate between yield grades of animals with UBRD and healthy cohorts when evaluated at revaccination, day 35, re-implant, and pre-shipping (p<0.0199). NIRS tended to be able to differentiate yield grades at initial processing (p=0.0513). StO[subscript]2 was not a predictor of quality grade at any time point (p>0.1023), nor was there any correlation between lung lesions at slaughter and StO[subscript]2 (p>0.2292). The second study involved 12 head of 181 kg (400 pound) heifers which were subjected to an experimental challenge model of Mannheimia haemolytica. Animals were evaluated daily and StO[subscript]2 readings recorded 12 hours pre-inoculation, at inoculation, 6, 12 and 24 hours post inoculation and daily for the next 12 days. While NIRS could not definitively differentiate healthy cohort cattle from challenge cattle (p>0.0713), there were trends toward challenge cattle having lower StO[subscript]2 values than healthy controls. The authors conclude that while these studies did not provide conclusive evidence of the ability of NIRS to detect UBRD, further studies with a machine that is specifically calibrated and designed for use with cattle should be performed.

Master of Science / Department of Clinical Sciences / James D. Lillich / Objective: To assess the in vitro ability of a novel lipopolysaccharide binding compound (EVK063) to inhibit cytokine production in lipopolysaccharide-stimulated equine peripheral blood mononuclear cells Animals: Eight healthy horses were sources for mononuclear cells. Procedures: Replicate aliquots (concentrated at 4-5 million cells/mL) were stimulated with S. typhimurium lipopolysaccharide (LPS) (100ng/mL), treated with graded concentrations of EVK063, (0.01µM, 0.1µM, 1µM, 10µM), Polymyxin B (PMB) (10µM) and incubated at 37°C for 6 hours. Media and cell samples were collected and stored at -80°C for evaluation of Tumor necrosis factor (TNF) using an equine specific ELISA and Interleukin-6 (IL6) via qRT-PCR. NanoDrop confirmed RNA quantity and primer sets designed for equine IL6 and the housekeeping gene 18s were used. EVK063 toxicity was evaluated with propidium iodide staining as determined by flow cytometry. Data was normalized, expressed as percent inhibition of cytokine up-regulation by LPS, and statistically evaluated by analysis of variance. Statistical significance was set at P ≤ 0.05. Results: Samples incubated in media with 0% serum demonstrated the following results: 0.01µM and 0.1µM EVK063 maintained >90% cellular viability yet failed to significantly inhibit TNF production or IL6 expression. The 1µM and 10µM EVK063 concentrations exhibited 25% and 70% cell death respectfully and therefore an interpretation as to their efficacy to inhibit TNF production or IL6 expression could not be made. Samples incubated in media with 10% serum demonstrated the following results: 0.01µM, 0.1µM and 1µM concentrations of EVK063 maintained >90% cellular viability yet failed to inhibit TNF production or IL6 expression. The 10µM EVK063 concentration exhibited 35% cell death and therefore an interpretation as to the efficacy to inhibit TNF production or IL6 expression could not be made. In a whole blood preparation, all samples evaluated maintained >90% cellular viability. The 10µM EVK063 significantly reduced TNF production and IL6 expression. Conclusion: This in vitro study confirms the ability of EVK063 to inhibit TNF production and IL6 expression in LPS stimulated equine mononuclear cells with comparable results to PMB.

Master of Science / Department of Clinical Sciences / James D. Lillich / Objective - To compare the biomechanical characteristics of the currently recommended (CR) technique and a less invasive (LI) surgical approach for arthrodesis of the proximal interphalangeal joint (PIPJ). Additionally, to describe a technique for cartilage removal and disruption of the subchondral bone. Study design - Randomized paired limb design for biomechanical comparison. Cartilage removal and subchondral bone disruption was accomplished using an orthopedic drill bit. Sample Population – 76 cadaver limbs. Methods - Cadaver PIPJs were drilled using a 3.5mm, 4.5mm or 5.5mm drill bit. Articular surfaces were digitally photographed and analyzed. Other paired PIPJs were arthrodesed using either the CR or the LI surgical technique. Implants consisted of a 3-hole DCP and two 5.5mm transarticular screws. Constructs were tested to failure in dorso-palmar/plantar and latero-medial in single cycle 3-point bending. The maximum load and yield load was measured and composite stiffness was calculated and statistically compared. Results - The LI technique had significantly greater mean yield load (11.3 ± 2.8 kN vs. 7.68 ± 1.1 kN, P=0.008) and mean maximum load (13.5 ± 3.1 kN vs. 10.1 ± 1.94 kN, P= 0.02) under latero-medial bending. Under dorso-palmar/plantar bending there was no statistical difference between the surgical approaches (P=0.5). The 4.5mm drill bit removed 42% ± 7.3 of the cartilage and disrupted subchondral bone. The LI technique had a decreased surgical time (19 ± 3 min.) when compared with the CR (31 ± 3 min.) technique. Conclusion – The LI technique results in a stronger composite as measured in 3-point bending, loaded to failure. Clinical Relevance – The LI surgical technique may be considered for clinical cases requiring arthrodesis of the PIPJ as there is no reduction in composite strength.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen that causes infection in people and several vertebrate animals. One of the striking features of E. chaffeensis infection is the prolonged persistence in its vertebrate and tick hosts. The mechanism of persistent infection and the reasons for the host immune system failure to clear the infection are not well understood. One hypothesis is that differential gene expression serves as an important adaptive mechanism used by E. chaffeensis in support of its continued survival in both tick and vertebrate hosts. One way to test this hypothesis is by performing mutational analysis. However, the methods for introducing mutations in this pathogen have not yet been documented and are challenging, possibly due to its obligate, intraphagosomal growth requirement. Recently, a novel gene mutation method called ‘TargeTron Gene Knockout System’ that is based on the modified group II intron insertion strategy has been developed. This method appears to be effective in creating mutations in a wide range of gram positive and gram negative bacterial organisms. The group II intron can be programmed for insertion into virtually any desired DNA target with possibly high frequency and specificity. In this study, I focus on creating mutations in E. chaffeensis using the TargeTron gene knockout system. I prepared modified group II intron constructs retargeting for insertion into three E. chaffeensis genes: Ech_0126 (a transcriptionally silent gene), macrophage-specific expressed gene (p28-Omp 19, Ech_1143) and tick cell-specific expressed gene (p28-Omp 14, Ech_1136). In support of driving the expression of the modified group II introns in E. chaffeensis, the pathogen- specific high-expressing gene promoter (tuf) was inserted upstream to the transcription start site. In addition, a chloramphenicol acetyltransferase gene with E. chaffeensis rpsl promoter was introduced for use as a selection marker. The constructs were then evaluated by transforming into E. chaffeensis. Transformants with mutations, introduced in two of the three genes (Ech_0126 and Ech_1143), were identified by PCR and Southern blot methods. Although the mutants are detectable for up to 48 hours, establishment of stable transformants remains to be challenging. The outcomes of this project will have important implications in defining the pathogenesis of E. chaffeensis, particularly to assess the differences in the organism in tick and vertebrate hosts.

In the research reported here, the pathology, virulence, and strain differences of Taura syndrome virus (TSV) was studied. Initial studies on TSV pathogenesis compared the survival of juveniles of a highly Taura syndrome (TS) susceptible line of Penaeus vannamei, a line of TS resistant P. vannamei, and an innately TS resistant P. stylirostris line following TSV challenge by feeding (per os) or injection methods, in the absence of horizontal transmission via cannibalism and/or absorption from the water. Per os_TSV challenge resulted in I00% survival in P. stylirostris, but challenge by per os exposure produced significant mortality commencing on about the same post-exposure day in both SPF and SPR P. vannamei (P < 0.001), suggesting that P. stylirostris is significantly (P < 0.001) more resistant toper os TSV infection and presentation of TS disease than either SPF or SPR P. vannamei. The potential roles of the cuticular lining of the stomach and hindgut and unlined portions of the gut in TSV resistance in penaeid shrimp are discussed as factors where an innate resistance mechanism was postulated to explain the observed differences between the different species and populations of shrimp in TSV susceptibility. To investigate apparent TSV strain differences, three geographic and year isolates of TSV from naturally occurring TS epizootics of cultured penaeid shrimp were obtained from Mexico (SIN98TSV and MX99TSV from P. vannamei and SON2KTSV from P. stylirostris) and one TSV isolate from Belize, Central America (BLZ02TSV from P. vannamei) were analyzed and compared to the reference TSV isolate (HI94TSV) by selected TSV diagnostic and genetic analysis methods. The results show that screening of penaeid shrimp broodstock and postlarvae by MAb I Al testing will not detect all TSV isolates, possibly leading to false negative results, further spread of TSV and re-emergence of TS in regions where it has been eradicated. The putative VP1 antigenic epitope recognized by TSV MAb 1A1 is identified, with SIN98TSV and BLZ02TSV having 70.0% and 80.0% AA homology, respectively, within the 10 AA region. There are three distinct electropherotypes and 'serotypes' of TSV, with electropherotype A (TSV Etype-A) and serotype A (TSV-A) representing those TSV isolates conforming to VP1 properties of the Hawaiian 1994 TSV isolate, electropherotype B (TSV Etype-B) and serotype B (TSV-B) representing those TSV isolates conforming to the VP1 properties of the Sinaloan 1998 TSV isolate, and electropherotype C (TSV Etype-C) and TSV serotype C (TSV-C), representing those TSV isolates conforming to the VP1 properties of the Belize 2002 TSV isolate. In a parallel activity, the University of Arizona (UAZ) Aquaculture Pathology Diagnostic Laboratory (APL) Case Database (DB) and the UAZ Aquaculture Pathology Diagnostic Laboratory Client Address Book Database (AB), relational databases, were created using FileMaker Pro software, are used to keep an up to date and accurate record of all UAZAPL diagnostic and research case and client information and may be searched and sorted to find case data and/or client information of interest.

Clostridium difficile-associated disease (CDAD) in neonatal pigs has emerged as a serious economic concern for swine producers throughout North America. The disease has been diagnosed clinically and reproduced in experimental inoculation trials in pigs, but little is known of the epidemiology or pathogenesis of the disease in pigs. Strain characteristics and distribution of C. difficile isolates from pigs, calves, dogs, horses, and humans were assessed by PCR-ribotyping. Porcine and bovine isolates were dominated by a single ribotype. This ribotype was uncommon among isolates from other host species; it was particularly uncommon from humans, suggesting there is little transfer of isolates between humans and calves or pigs. The reason for a single common ribotype circulating among distinct bovine and porcine populations is unknown. The intragastric inoculation of newborn pigs demonstrated their sensitivity to C. diffcile toxins. Toxin B (TcdB) surprisingly resulted in more severe lesions than Toxin A (TcdA). The two toxins together acted synergistically. Colon explants were sensitive to TcdA in a dose-dependent manner. However, TcdB did not cause significant lesions in the explants, nor was there any synergism with TcdA. Electron microscopy of colon explants treated with TcdA revealed severe, ultrastructural lesions that accrued in a dose-dependent manner by two h post infection. Direct immunohistochemistry assays demonstrated specific binding of biotinylated TcdA throughout the gastrointestinal tract of neonatal pigs. The density of bound toxin in different segments correlated with the severity of lesions in those segments from pigs gavaged with TcdA. TcdB did not bind any tissues, though it was fully active in cell-culture assays. A monoclonal antibody to Galalpha1-3beta1-4GlcNAc-R (alpha-Gal epitope), a putative receptor for TcdA in pigs, specifically bound the brush border of enterocytes, but the distribution of binding did not correlate with the distribution of TcdA binding. Specific TcdA binding to the plasmallema of microvilli was also confirmed by immunoelectron microscopy. By five min post inoculation some toxin was already visible in endosomes or free in the cytoplasm. TcdA localized to the mitochondria of epithelial cells and, less frequently, to the nuclei. Endothelial cells and leucocytes in the superficial lamina propria were similarly labeled by toxin.

The genome of a Korean isolate of Hepatopancreatic parvovirus (HPV) was partially cloned, sequenced and characterized. Random PCR amplification of viral DNA was combined with conventional cloning methods to generate three clones named HPV8 (2,136 bp insert), HPV3 (1,560 bp insert), and CP1139 (413 bp insert). DNA sequencing demonstrated overlapping regions between HPV8 and HPV3 and between HPV3 and CPII39. The combined sequence of these three clones encompass approximately 3,350 bp of the total 5,000 bp estimated for the HPV genome. A large open reading frame (1,692 bp) was found within clones HPV3/CPII39 encoding a polypeptide of 549 residues (∼60 kDa) whose amino terminus shows 100% homology with the first 12 residues sequenced from an apparently single 54 kDa (by SDS-PAGE) structural protein found in a Korean isolate of HPV. Two new gene probes EC.592 (592 bp) and EC.350 (350 bp) were developed by PCR amplification of previously constructed HPV (Korean) clones and labeled with DIG11-dUTP. These probes recognize different regions of the HPV genome. The specificity of both probes was confirmed by in situ hybridization using HPV-infected Penaeus chinensis (Korean) as a positive control and specific-pathogen free P. vannamei and IHHNV-infected P. stylirostris, as negative controls. Both probes were used in in situ hybridization to compare their reaction to HPV-type lesions detected by conventional H&E histology in 7 species of HPV-infected shrimp, and one of freshwater prawn, from 13 countries. The results of this comparison strongly suggest the existence of genomic differences among these geographic isolates. A post-embedding in situ hybridization assay at the electron microscope level was developed to detect HPV nucleic acids on HPV-infected hepatopancreata from P. chinensis and P. monodon . Hybridized probe (EC.592) was detected with an anti-DIG sheep antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal was observed within HPV-infected hepatopancreatic cells, which was specifically located within intranuclear viral inclusions, cytoplasm, microvillous border, and associated to necrotic debris within the lumen of hepatopancreatic tubules. A non-destructive method, based on the PCR, was developed to detect HPV by examination of crude fecal samples from HPV-infected shrimp.