Master of Veterinary Biomedical Science / Department of Diagnostic Medicine/Pathobiology / Michael W. Dryden / Patricia A. Payne / Giardiasis is a common intestinal protozal parasitic infection of the pet dog and cat population. Veterinarians often have difficulty correctly diagnosing this parasite. Studies were conducted to compare the zinc sulfate double centrifuge fecal flotation to the SNAP (registered trademark) Giardia fecal ELISA test manufactured by IDEXX laboratories Inc. in purpose bred beagles and shelter and commercial kennel dogs. In these evaluations the zinc sulfate double centrifuge fecal flotation and fecal ELISA test performed similarly. Both tests performed better in the shelter and commercial kennel dog population than the chronically infected purpose bred beagles. There was an increase in number of positive animals identified when 3 consecutive daily samples were evaluated as compared to any one individual day for either test method. Post treatment evaluation of the diagnostic tests was performed in 23 laboratory beagles. Each beagle was treated for 3 consecutive days with Drontal plus and then bathed on the last day of treatment and fecal samples were collected from the treated dogs every other day starting one day post treatment for 21 days. It was found that all beagles were negative on zinc sulfate double centrifugation fecal flotation, fecal ELISA and IFA within 24 hours of treatment and nineteen (82.6%) of the beagles did not re-shed cysts during the 21 day post-treatment evaluation period. Four beagles returned to shedding cysts (Flotation or IFA positive) between days 17 and 21. These findings suggest that a positive test within a week of treatment is likely the result of inappropriate treatment. After the prepatent period, positive results may occur due to a return to shedding, reinfection or inappropriate treatment. Chronically infected laboratory beagles may not be a good model for acute Giardia infections as these dogs are rarely clinically ill and detection is more difficult.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Shafiqul I. Chowdhury / Bovine herpesvirus type 1 (BHV-1) is an important pathogen of cattle that can cause severe respiratory tract infection known as infectious bovine rhinotracheitis (IBR), abortion in pregnant cows, and is an important component of the Bovine Respiratory Disease Complex (BRDC, “Shipping fever”). The ability of BHV-1 to transport anterogradely from neuron cell bodies in trigeminal ganglia to axon termini in the nasal and ocular epithelia of infected cattle complicates the control of the disease in both vaccinated and infected cattle populations. In calves and rabbits, Us9 deleted viruses have defective anterograde neuronal spread from cell bodies in the trigeminal ganglia to nerve termini in the nose and eye but retrograde spread remains unaffected. To characterize the neuronal spread of BHV-1, we developed primary neuronal cultures using the dorsal root ganglia (DRG) of rabbit embryos. We successfully used microfluidic chamber devices to isolate DRG in the somal compartment and allowed for efficient growth of axons into the axonal compartment. This enabled us to study axon mediated neuronal spread of infection as well as viral transport in axons. Thus, rabbit DRG neuronal culture was susceptible to BHV-1 mutant and wild-type infection, and the method allowed visualization of viral spread in chamber cultures using live cell imaging and fluorescent microscopy. Lastly, using the microfluidic chamber compartmentalized neuron culture system we showed that Us9 acidic domain-deleted and Us9 null mutant BHV-1 viruses had defective anterograde neuronal transport relative to BHV-1 wild type and/or Us9 rescued viruses.

The spontaneous rate of congenital cardiac defects in the commonly used Sprague-Dawley rat has not been extensively researched. The incidence of general congenital defects has been studied thoroughly and reported as very low incidence in this rat. Teratology studies using mammalian models are of increasing importance as the number of environmental contaminants increases. It is essential to know if the spontaneous congenital cardiac defect rate of this most common mammalian model in order to plan statistically appropriate research protocols. Using a thorough and detailed method of evaluating the structure of the Sprague-Dawley fetal rat heart, 624 fetal rat hearts were examined at term, just prior of delivery. The overall incidence of spontaneous congenital cardiac defects is 2.3%. This is similar to the incidence in man and thus enhances the suitability of the Sprague-Dawley rat as a small mammalian model for research into cardiac teratogenesis.

I studied Trichomonas gallinae, a flagellated protozoan that is the causative agent of the avian disease trichomoniasis. The purpose of my study was to assess (1) the incidence of trichomonads in wild birds, (2) the prevalence of trichomonads in water sources utilized by wild birds, and (3) possible methods to control the transmission of trichomonads in water sources utilized by wild birds. I trapped 403 birds during 1994 and 1995. Approximately 1/3 of these birds tested positive for T. gallinae, however, none exhibited any signs of lesions. I collected water samples from 10 bird baths, isolating flagellated protozoa from 2 of them. I could not identify the species of flagellated protozoa. I determined that high temperatures (50°C), near ultra-violet radiation, and natural sunlight are effective against trichomonads. In addition, the highest effective dilutions of Chlorox, Nolvasan, and distilled white vinegar active against trichomonads were determined.

Arcanobacterium pyogenes is a gram-positive, fastidious, facultative anaerobe that can persist in a number of animal species both as a commensal and as a pathogen. The economic impact from A. pyogenes -related bovine mastitis and liver abscess infections makes the characterization of possible targets for vaccine development important. One such target, pyolysin (PLO), is a member of the cholesterol-dependent cytolysin (CDC) family and a major virulence factor and host protective antigen in A. pyogenes pathogenesis. Here, sequence analysis of the chromosomal region surrounding plo, which encodes PLO, indicated that the plo gene and an upstream open reading frame, orf121, constitute a genomic islet conserved across geographically diverse isolates. Two genes downstream of plo, ftsY and ffh, encode homologues of the protein components of the signal recognition particle (SRP) pathway which may be involved in plo expression and secretion. However, the role of ftsY and ffh in plo expression could not be defined as they were found essential in A. pyogenes, and ffh was unable to complement a heterologous system. Analysis of plo mRNA and PLO levels across a growth curve indicated that plo is transcriptionally or post-transcriptionally regulated. Sequence analysis of the plo promoter region identified three 11-mer repeats, R1, R2 and R3, and two putative sigma70-like promoter sequences, P1 and P2. Primer extension experiments suggested active transcription from P1 and P2 in vitro. Analysis of transcriptional fusions of successive truncations of the plo promoter region and site-directed mutations in the promoter motifs to a chloramphenicol acetyl transferase (cat) gene, indicated a role for each motif in the regulation of plo expression in vitro, with R2 required for activation and P2 being the regulated promoter. While a null mutation in orf121 did not have a significant effect on plo expression in vitro, gel shift analysis confirmed the ability of some factor within A. pyogenes soluble cellular extracts to bind specifically to the plo promoter region. The identification of cis- and trans-acting factors involved in plo expression provides a basis for characterizing PLO expression in vivo, and may lead to a better understanding of the global regulation of pathogenesis in A. pyogenes.

<p> In recent years, large-scale 'omic' studies have helped to understand disease pathogenesis; however, these studies were done largely at the 'transcriptome' level. Understanding the biological processes at the protein level is also equally important. In general, mass spectrometry (MS)-based quantitative proteomic strategies are used to study protein alterations in different biological states. Among these strategies, stable isotope labeling by amino acids in cell culture (SILAC) is most widely used for comparative proteomics. In SILAC, proteins in cell populations are metabolically encoded with 'heavy' isotopes of lysine and arginine and are used as internal standards for relative quantification of differentially altered proteins. In this dissertation, the use of SILAC was extended to study <i>in vivo</i> proteomic modulations in mice. A stable isotope (¹³C-lysine)-labeled 'SILAC mouse' was generated in order to quantify and compare protein alterations between normal and pathological conditions. </p><p> The primary objective of this dissertation was to identify novel and disease specific pathogenic mechanisms implicated in duchenne muscular dystrophy (DMD: a genetic muscle disease) and myositis (an autoimmune muscle disease) <i> in vivo</i> considering the entire complexity of the tissue. More importantly, the goal is to study global protein alterations in an unbiased manner in the affected skeletal muscle tissue. However, it is impractical to study in-depth disease pathology at tissue level using human muscle biopsy samples due to heterogeneity, complexity, and limited availability of muscle tissues at different stages in the disease process. To overcome these issues, mouse models that closely mimic the human disease phenotype i.e. a dystrophin deficient 'mdx' for DMD and a conditional major histocompatibility complex (MHC) class-I transgenic mice for myositis, were used. It was hypothesized that identification of precise proteomic alterations in the affected muscle, using mass spectrometry based untargeted-stable isotope labeled-proteomic strategy, would help to discover disease specific pathogenic mechanisms. </p><p> Firstly, the untargeted-labeled-proteomics approach using <i>in vivo</i> SILAC mouse proved to be a robust technique to uncover previously unidentified pathological pathways in mouse models of human skeletal muscle diseases. With respect to DMD, SILAC mouse proteomic profiling identified 73 significantly altered proteins in the early stage of the disease in mdx muscle compared to healthy muscle. Bioinformatics analyses of the altered proteins identified that integrin-linked kinase (ILK), actin cytoskeleton signaling, and mitochondrial energy metabolic pathways are significantly altered very early in the disease process in dystrophin deficient muscle. Disease specific protein modulations were further validated using an independent set of samples, SILAC spike-in strategy and specific antibody based biochemical assays. Moreover, the potential candidates of ILK pathway such as vimentin, desmin and ILK were confirmed to be significantly up-regulated in dystrophin-deficient human DMD samples suggesting the importance of these findings in relation to human disease pathology. </p><p> A novel association between the reduced mitochondrial activity and impaired sarcolemmal healing was identified in dystrophin deficient muscle. Live imaging of isolated single muscle fibers determined that reduced mitochondrial translocation to the site of injury negates the membrane repair processes even in the presence of compensatory up-regulation of repair proteins (dysferlin and annexin). Thus, current studies provided the first comprehensive understanding of the dystrophic muscle pathologies at the proteomic level. </p><p> In auto-immune myositis, SILAC mouse proteomic profiling identified significant alteration in the levels of 179 proteins. These proteins belong to the endoplasmic reticulum (ER) stress response, ubiquitin proteasome pathway (UPP), ER associated degradation (ERAD), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. A significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) was observed in myositis, but not in normal or other dystrophic muscles. Furthermore, inhibition of UPP using a specific proteasome inhibitor, bortezomib, significantly improved muscle function and also significantly decreased TNF-&alpha; (pro-inflammatory cytokine) expression in the skeletal muscle of myositis mice. Treatment with bortezomib, decreased GRP-78 (ER stress sensor) levels and also enhanced muscle regeneration. Thus, it can be concluded that UPP and ERAD activation in myositis muscle contribute to muscle degeneration. UCHL-1 is a potential biomarker for myositis disease progression and inhibition of UPP offers a potential therapeutic strategy for myositis. </p><p> These studies not only provided information on the implicated pathways both in 'dystrophin-deficient' and 'myositis' muscle but also identified potential therapeutic targets. Nevertheless, future experiments will help to associate pathways identified using this proteomic strategy with gene expression profiling to comprehensively understand disease specific as well as general pathogenic pathways. These studies will pave the way to enhance development of improved therapies for these rare muscle diseases.</p>

<p> The purpose of this research was to better understand the effects of abdominopelvic irradiation in dogs. Three studies were performed to that end. The first was a clinical investigation, performed by retrospective data analysis, of safety and activity of intensity-modulated and image-guided radiation therapy (IM-IGRT) for treatment of genitourinary (GU) carcinomas in dogs. The second was a prospective study which developed dogs as a novel animal model for studying radiation-induced erectile dysfunction (RI-ED). The third study reviewed pathological changes associated with unexpected colorectal toxicities encountered in the development of the RI-ED model. </p><p> As mentioned, the objective of the first study was to assess local tumor control, overall survival and toxicosis following IM/IGRT for treatment of genitourinary carcinomas (CGUC) in dogs. Medical records of patients for which there was intent to treat with a course of definitive-intent IM/IGRT for CGUC were reviewed. Primary tumors were located in the prostate, urinary bladder or urethra of 21 dogs. The total radiation dose ranged from 54-58 Gy, delivered in 20 daily fractions. Grade 1 and 2 acute gastrointestinal toxicoses developed in 33% and 5% of dogs, respectively. Grade 1 and 2 acute genitourinary, and grade 1 acute integumentary toxicoses were documented in 5%, 5% and 20% of dogs, respectively. Four dogs experienced late grade 3 gastrointestinal or genitourinary toxicosis. The subjective response rate was 60%. The median event-free survival was 317 days; the overall median survival time was 654 days. Neither local tumor control nor overall survival were statistically dependent upon location of the primary tumor. In conclusion, IM/IGRT is generally well-tolerated and provides an effective option for locoregional control of CGUC. And, as compared with previous reports in the veterinary literature, inclusion of IM/IGRT in multimodal treatment protocols for CGUC can result in superior survival times. </p><p> The etiopathology of RI-ED is poorly understood, though this is a common complication of men treated for prostate cancer. Purported mechanisms include cavernosal, arteriogenic and neurogenic injuries. Radiation dose to the posterolateral prostatic neurovascular bundles (NVB) and penile bulb (PB) have been associated with RI-ED. Herein, a canine model is described that has been developed to study the pathogenesis of RI-ED. Stereotactic body radiotherapy (SBRT) was used to irradiate the prostate gland, NVB and/or PB of purpose-bred, intact male dogs. Manual evaluation was used to characterize erectile function and quality. Ultrasound of the internal pudendal arteries, prostate and penis, dynamic contrast-enhanced MRI of the NVB and prostate, and electrophysiology of sensory and motor nerves as well as muscle were performed before and after irradiation. Gross necropsy and histopathology was also performed. Erectile dysfunction was a repeatable finding in subjects for whom the prostate, neurovascular bundles and penile bulb were irradiated with 50 Gy, as documented via subjective and objective manual evaluations following SBRT. Irradiated dogs were found to have a decreased extravascular, extracellular volume in the glans penis, longer systolic rise times in the pudendal artery following papaverine injection, abnormal spontaneous EMG activity in the bulbocavernosus muscle, and slower pudendal nerve motor conduction velocities. Radiation dose-dependent changes in internal pudendal arterial function and dysfunction of the pudendal nerve due to axonal loss may contribute to RI-ED. Measurable endpoints have been developed for evaluation of RI-ED in dogs, that should be used in future studies to refine this novel animal model and perform additional studies aimed at further elucidating the etiopathologic processes underlying RI-ED. </p><p> The objective of the final study was to describe the dose-response relationship and time-dependency of late radiation-induced colorectal complications endured by dogs in the RI-ED study. The prostates of nineteen intact male mixed breed hounds were irradiated with one of four different dose/fractionation schemes. Subjects were monitored for signs of colorectal toxicosis for up to one year following irradiation. Gross necropsy and histopathology were performed upon euthanasia. All toxicoses were graded according to the RTOG criteria for gastrointestinal toxicity. The frequency and severity of colorectal ulceration were higher in dogs treated 5 fractions of 10 Gy delivered on consecutive days, as compared with those treated on an every other day schedule. The mechanism for this time-dependency is unclear, but likely related to completeness of epithelial regeneration. Vascular sclerosis and serosal thickening occurred in all treatment groups, in a dose-responsive fashion.</p>

No description available.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3518. Adviser: Milton M. McAllister. Includes bibliographical references (leaves 73-99) Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0869. Adviser: Uriel D. Kitron. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.