[Show abstract][Hide abstract] ABSTRACT: Abstract Avian influenza has emerged as one of the most ubiquitous viruses within our biosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses; however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understanding which and how many species can be infected. Moreover, novel influenza virus sequences were recently isolated from New World bats. Crocodilians have a slow rate of molecular evolution and are the sister group to birds; thus they are a logical reptilian group to explore susceptibility to influenza virus infection and they provide a link between birds and mammals. A primary American alligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenic avian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggs supported virus replication, as evidenced by the influenza virus M gene and infectious virus detected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cells were also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining; however, the requirement for trypsin to support replication in cell culture limited replication. To assess influenza virus replication in culture, primary alligator cells were inoculated with H1N1 human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both viruses replicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. This research demonstrates the ability of wild-type influenza viruses to infect and replicate within two crocodilian substrates and suggests the need for further research to assess crocodilians as a species potentially susceptible to influenza virus infection.
Journal of wildlife diseases 01/2015; 51(1):187-198. · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Four healthy adult dogs (Golden Retrievers aged 6 years and 9 years, Dalmatian aged 13 years, and Mastiff aged 5 years) developed clinical signs of acute respiratory disease and died within 2 to 7 days of onset of clinical signs. The lungs of the 3 dogs submitted for necropsy were diffusely and severely reddened due to hyperemia and hemorrhage. Microscopic lesions in all dogs were suggestive of acute viral or toxic respiratory damage and varied from acute severe fibrinonecrotic or hemorrhagic bronchopneumonia to fibrinous or necrotizing bronchointerstitial pneumonia. Necropsied dogs also had hemorrhagic rhinitis and tracheitis with necrosis. Virus isolation, transmission electron microscopy, and polymerase chain reaction were used to confirm the presence of canid herpesvirus 1 (CaHV-1) in the lung samples of these dogs. Lung tissues were negative for influenza A virus, canine distemper virus, canine parainfluenza virus, canine respiratory coronavirus, and canine adenovirus 2. Canid herpesvirus 1 has been isolated from cases of acute infectious respiratory disease in dogs but has only rarely been associated with fatal primary viral pneumonia in adult dogs. The cases in the current report document lesions observed in association with CaHV-1 in 4 cases of fatal canine herpesvirus pneumonia in adult dogs.
[Show abstract][Hide abstract] ABSTRACT: A 1.5-year-old mixed-breed dog was examined because of a 1-month history of anorexia, vomiting, diarrhea, and weight loss.
The dog was very thin on physical examination (body condition score, 3/9). Results of all diagnostic tests were within reference limits except intestinal thickening and lymphadenopathy were identified on abdominal ultrasound examination. During exploratory laparotomy, thickening at the ileocecal-colic junction and within the transverse colon and mesenteric lymphadenopathy were identified, and the ileocecal-colic junction was resected. Histopathologic evaluation of the ileocecal-colic junction and full-thickness biopsy specimens from other sites as well as results of a serum ELISA were diagnostic for gastrointestinal Pythium insidiosum infection.
Pythiosis was initially treated medically with administration of itraconazole and terbinafine by mouth, but the colonic lesion was progressive with this regimen. Two months after diagnosis, a subtotal colectomy was performed; marginal excision (0.6 cm) was obtained at the aboral margin. The dog was treated with 3 doses of a pythiosis vaccine beginning approximately 2 weeks after surgery and was continued on itraconazole and terbinafine for 5 months. Parenteral and enteral nutrition as well as considerable general supportive care were required postoperatively. Six months after treatment, the dog had a normal serum ELISA titer. Two years after treatment, the dog had returned to preoperative weight and was clinically normal.
This patient had an unusually positive therapeutic response to chronic, extensive, marginally excised gastrointestinal pythiosis.
Journal of the American Veterinary Medical Association 08/2012; 241(3):358-63. · 1.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Borrelia burgdorferi is the causative agent of Lyme disease, which is mainly characterized by lameness in dogs. More than 95% of naturally infected dogs are asymptomatic or subclinical; however, in experimental studies, histologic synovial lesions are consistently observed in asymptomatic dogs inoculated with B. burdgorferi. This study investigates the ability of a synovial histopathologic scoring system, clinicopathologic data, and polymerase chain reaction (PCR) testing to differentiate between B. burgdorferi-infected and uninfected dogs. Eighteen 18-week-old beagles were subject to challenge with B. burgdorferi-infected wild-caught ticks (Ixodes scapularis), and 4 uninfected dogs served as controls. Infection was confirmed by serology (ELISA) and PCR amplification of B. burgdorferi-specific DNA of skin biopsies taken at the tick attachment site. A synovial scoring system from human medicine was adapted and implemented on postmortem synovial samples to discriminate infected and noninfected animals. Application of this system to elbows and stifles with a cumulative joint score cutoff > 4 showed a sensitivity of 88.2% and a specificity of 100%, with a positive likelihood ratio of infinity and a negative likelihood ratio of 0.12. Complete blood count, serum biochemistry, urinalysis, urine protein:creatinine, urine PCR, synovial and lymph node cytology, and synovial PCR were evaluated but were not reliable indicators of clinical disease.
[Show abstract][Hide abstract] ABSTRACT: Canine lymphoma is a common spontaneous tumor with many similarities to human lymphoma, and thus has potential to be an important animal model of lymphomagenesis. This study determined that microRNA (miRNA) expression in canine tumors can be assessed using a commercially available human cancer miRNA qPCR array. miRNA expression in six different canine lymphoid cell lines and in naturally occurring canine B- and T-cell lymphomas was compared using RNA harvested from normal canine peripheral blood mononuclear cells (PBMC) and normal lymph nodes (LN) as controls. We found that false discovery rate (FDR) correction for multiple testing after quantile normalization controlled for variation across arrays and that they were the best methods for normalization and statistical analysis. Increases in miRNAs known to upregulate oncogenes (miR19a+b, miR17-5p) and decreased expression of miRNAs with tumor suppressor functions (miR-203, miR-218, and miR-181a) also seen in human lymphoid malignancies were observed. However, there were few similarities between canine groups. The results of this study indicate that the use of both PBMC and LN cells as controls provides different, but potentially equally important targets for further analysis. Our findings of miRNA dysregulation in canine lymphoid cell lines and clinical cases of lymphoma emphasize the potential of canine lymphoma as an important spontaneous, large animal model of human B- and T-cell lymphomas.
Genes Chromosomes and Cancer 11/2011; 50(11):950-67. · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recent development of porcine induced pluripotent stem cells (piPSCs) capable of generating chimeric animals, a feat not previously accomplished with embryonic stem cells or iPSCs in a species outside of rodents, has opened the doors for in-depth study of iPSC tumorigenicity, autologous transplantation, and other key aspects to safely move iPSC therapies to the clinic. The study of iPSC tumorigenicity is critical as previous research in the mouse showed that iPSC-derived chimeras possessed large numbers of tumors, rising significant concerns about the safety of iPSC therapies. Additionally, piPSCs capable of generating germline chimeras could revolutionize the transgenic animal field by enabling complex genetic manipulations (e.g., knockout or knockin of genes) to produce biomedically important large animal models or improve livestock production. In this study, we demonstrate for the first time in a nonrodent species germline transmission of iPSCs with the live birth of a transgenic piglet that possessed genome integration of the human POU5F1 and NANOG genes. In addition, gross and histological examination of necropsied porcine chimeras at 2, 7, and 9 months showed that these animals lacked tumor formation and demonstrated normal development. Tissue samples positive for human POU5F1 DNA showed no C-MYC gene expression, further implicating C-MYC as a cause of tumorigenicity. The development of germline-competent porcine iPSCs that do not produce tumors in young chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals.
[Show abstract][Hide abstract] ABSTRACT: Collectively, these presentations introduced the audience to the roles of ES cells in generating phenotypes of transgenic animals,and they provided examples where the GEMs were used to define molecular mechanisms of disease or where ES cells were used as a therapeutic modality. Points of discussion among audience members reinforced the importance of strain-associated background lesions in animal models, technological advances in imaging functional biology, opportunities for stem cell therapies, and ubiquitination in regulation of cell proliferation. The 2012 American College of Veterinary Pathologists symposium ‘‘Evolutionary Aspects of Animal Models’’ will focus on the proper selection of a relevant animal model in biomedical research as critical to investigative success. Recent work characterizing rapid evolutionary changes and differences in physiology between species questions the validity of some comparative models. Dr. Robert Hamlin will be speaking on cardiovascular disease in ‘‘Animals as Models of Human Cardiovascular Disease: Or the Search to Overcome Outdated Evolutionary Homeostatic Mechanisms.’’ Dr. Stefan Niewiesk will discuss evolutionary factors that affect modeling the human immune system in ‘‘Of Mice and Men: Evolutionarily, What Are the Best Rodent Models of the Human Immune System for Infectious Disease Research?’’ Dr. Steven Austad will consider evolution in ‘‘Evolutionary Aspects of Animal Models of Aging.’’Finally, Dr. Elizabeth Uhl will conclude the session with ‘‘Modeling Disease Phenotypes: How an Evolutionary Perspective Enhances the Questions.’’
[Show abstract][Hide abstract] ABSTRACT: Although Lew/Crl rats are central to a classic model of renal transplantation and may provide a valid system for evaluating the effect of obesity on transplantation outcomes, their response to high-fat diet has not been evaluated sufficiently. The objective of this study was to evaluate biometric and basic metabolic data of Lew/Crl rats fed a 60% kcal, lard-based, very high-fat diet (HFD) compared with those fed a 10% kcal fat control diet (CD). Rats were maintained for 17 wk; body parameters and caloric intake were monitored weekly. Biometric data were collected and calculated before and after euthanasia. Serum was evaluated for liver enzyme activity and total bilirubin, glucose, triglyceride, cholesterol, insulin, leptin, and creatinine concentrations, and urine was evaluated for protein, glucose, specific gravity, and ketones. Tissues were harvested, weighed, and evaluated histologically. Compared with CD rats, HFD rats consumed more calories and weighed more after 3 wk. After 17 wk, HFD rats had significantly increased body weight, girth, volume, epididymal fat pad weight, omental weight, and body fat. In addition, HFD rats had mild elevations in some liver enzymes and a lower serum triglyceride concentration than did CD rats. Histologic assessment and other metabolic markers of disease were not different between the 2 groups. Lew/Crl rats fed a 60% kcal HFD become obese, but they lack significant metabolic abnormalities frequently associated with obesity in other rat strains.
Comparative medicine 04/2011; 61(2):131-7. · 0.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 15-year-old female red-tailed hawk (Buteo jamaicensis) was evaluated because of dyspnea, anorexia, and coelomic distension. Diagnostic imaging results confirmed severe coelomic effusion and revealed a markedly dilated right ventricle. The diagnosis was right-sided congestive heart failure. Results of measurements of vitamin E, selenium, lead, zinc, and cardiac troponin levels were normal or nondiagnostic. The hawk was treated with furosemide, antifungal and antimicrobial agents, and supplemental fluids and oxygen, but euthanasia was elected because of the poor prognosis and the practical difficulties associated with intensive case management. To our knowledge, this is the first described case of cardiomyopathy and congestive heart failure in a captive red-tailed hawk.
Journal of Avian Medicine and Surgery 03/2011; 25(1):32-9. · 0.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 7-year-old spayed female domestic shorthair feline presented with tachycardia and was later euthanized due to a declining condition. On gross examination, the thoracic cavity contained an expansile, multiloculated mass that displaced the lungs dorsocaudally. The mass, within the pericardial sac, compressed adjacent myocardium. Cut surface revealed variably sized, fluid-filled spaces with multiple foci of hemorrhage and necrosis. Histologically, the mass was composed of solid foci of polygonal cells admixed with colloid-containing follicles. Immunohistochemical staining for thyroglobulin was positive, and staining for calcitonin was negative. Grossly, thyroid glands were normal, and serum thyroxine was within reference intervals.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 11/2010; 22(6):1010-3. · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Weanling Brown Norway (BN) rats are susceptible to persistent steroid-responsive pulmonary abnormalities following resolution of an acute respiratory virus infection. In contrast, Fischer 344 (F344) rats recover without complications. Previous studies determined that NF-κB activation and subunit composition were markedly different between these 2 rat strains. This study examined whether viral infection also resulted in altered pulmonary expression of IκBα and IκBβ, 2 inhibitory regulators of NF-κB. Western blot analyses of total pulmonary protein extracted from BN and F344 rats at 7, 10, and 14 days after inoculation (n = 5 per group) did not reveal virus-induced differences in IκBβ expression. In contrast, a lower molecular weight form of IκBα appeared in the BN rats at 14 days postinfection, and it was still present at 21 days after infection (n = 5 per group). The change in IκBα expression observed in the susceptible BN but not the resistant F344 animals occurs when the epithelium is proliferating during the repair phase, and it correlates with the development of the persistent virus-induced airway inflammation and pulmonary functional abnormalities. These results further implicate differential regulation of NF-κB in the pathogenesis of virus-induced asthma.
[Show abstract][Hide abstract] ABSTRACT: Confirmed reports of large domesticated cats becoming infected with highly pathogenic avian influenza (HPAI) H5N1 virus have raised questions about both the risk of infection for these animals, and their potential as vector or reservoir hosts in an influenza pandemic. With this in mind, we examined the immunogenicity of the hemagglutinin (HA) of H5N1 strain A/Vietnam/1203/04 using several different vaccination strategies. Data from ELISA assays showed that vaccination with a single dose of recombinant H5 HA protein induces a robust antibody response against both whole inactivated virus and recombinant HA antigen. Moreover, a single dose of the recombinant H5 HA protein induced hemagglutination inhibition titers >or=40, which is indicative of protective immunization. Cats receiving the IND H5N1 vaccine required two doses before similar H5 HA-specific antibody titers were observed, and despite boosting, these animals had HIA titers that were lower than or equivalent to those in the group receiving one injection of recombinant protein. In contrast, cats vaccinated with plasmid DNA encoding HA failed to develop HA-specific antibody responses above those seen in cohorts receiving an unrelated control plasmid. The results of this study indicate that recombinant H5 HA protein-based vaccines can rapidly induce high serum antibody titers, and may be more effective than either inactivated influenza virus or DNA vaccines in cats.
[Show abstract][Hide abstract] ABSTRACT: Osteogenic melanoma is a rare variant of metaplastic malignant melanoma in human medicine and appears to be a similarly rare variant in dogs. Two dogs with oral malignant melanoma with neoplastic bone formation are reported in this study. Both tumors were characterized by malignant melanocytes that transitioned into neoplastic bone at the deep margins of the neoplasm. Immunohistochemical analysis revealed S100- and Melan-A-positive neoplastic cells adjacent to, and occasionally embedded within, an osteoid and chondroblastic matrix. Scattered clusters of neoplastic cells were also positive for osteocalcin. The findings indicate that in dogs, as in humans, neoplastic melanocytes have metaplastic potential and can be osteogenic.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 01/2010; 22(1):147-51. · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 17-month-old 7-kg (15.4-lb) Shih Tzu was evaluated because of progressive thoracic limb weakness of 3 months' duration.
Neuroanatomic diagnosis was consistent with a lesion affecting the cervicothoracic (C6 through T2) spinal cord segments. Electrophysiologic testing revealed abnormal spontaneous activity in the thoracic limbs. Via magnetic resonance (MR) imaging, a lesion in the spinal cord that extended from the C5 through C7 vertebrae was detected, as were symmetric lesions in the cranial portion of the cervical spinal cord, caudal colliculi, and vestibular and cerebellar nuclei. Tests to detect metabolites indicative of inborn errors in metabolism revealed no abnormalities.
Prior to undergoing MR imaging, the dog received clindamycin (14 mg/kg [6.4 mg/lb], PO, q 12 h), trimethoprim-sulfadiazine (17 mg/kg [7.7 mg/lb], PO, q 12 h), and prednisone (1 mg/kg [0.45 mg/lb], PO, q 24 h). Because of its deteriorating condition, the dog was euthanized. During necropsy, gross lesions were identified in the cervical spinal cord, caudal colliculi, and vestibular and cerebellar nuclei (corresponding to lesions detected via MR imaging). Microscopic evaluation of the brain and spinal cord revealed polioencephalomyelopathy; there was severe spongiosis of the neuropil with reactive astrocytes (many with high numbers of swollen mitochondria) and preservation of large neurons.
The form of polioencephalomyelopathy in the Shih Tzu of this report was similar to that described for Australian Cattle dogs; the similarity of findings in dogs with those in humans with Leigh disease is suggestive of a mitochondrial defect.
Journal of the American Veterinary Medical Association 10/2009; 235(5):551-7. · 1.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infections with respiratory pathogens such as respiratory syncytial virus and rhinovirus have been associated with the development of long-term chronic airway disease. To better understand the events responsible for this clinical outcome, a rodent model of virus-induced chronic airway disease has been characterized. Upon infection with Sendai virus (parainfluenza virus type-1), Brown Norway (BN) rats develop an asthma-like clinical syndrome, while Fischer 344 (F344) rats fully recover. Our previous studies demonstrated that after infection, tumor necrosis factor-alpha (TNF-alpha) expression is substantially higher in BN rats compared to F344 rats, and this may at least partially mediate the virus-induced airway abnormalities. To investigate the underlying mechanism(s) for the increased TNF-alpha expression, the role of nuclear factor-kappaB (NF-kappaB), an important regulator of TNF-alpha gene transcription, was examined. Supershift electrophoretic mobility shift assays (EMSAs) indicate that normal F344 rats predominantly express the p65 subunit of NF-kappaB in the lungs, and virus infection temporarily increases expression of the p50 subunit. In contrast, normal BN rats have higher expression of the p50 subunit in the pulmonary tract. Upon infection, p50-subunit expression in BN rats increases to levels higher than those observed in virus-infected F344 rats. Interestingly, treatment of infected BN rats with dexamethasone at doses known to prevent virus-induced airway abnormalities increases pulmonary expression of the p65 subunit, and decreases TNF-alpha mRNA levels in the lungs. Furthermore, direct inhibition of TNF-alpha also increases pulmonary expression of p65 in virus-infected BN, but not F344, rats. Taken together, these results suggest that differential expression of NF-kappaB subunits may play an important role in the development of post-viral chronic airway abnormalities.
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidiosis is an emerging problem in reptile medicine and has been associated with a wasting syndrome in leopard geckos (Eublepharis macularius). This study determined the prevalence of infection in a breeding colony of leopard geckos to be 9.8%. Two groups of 20 geckos, one that was fecal positive for oocysts of Cryptosporidium sp., and one, whose individuals were fecal negative at the inception of the study, were followed for 2 mo. Fecal samples were tested for oocysts every 2 wk, body weights were measured, and a body condition score was assigned for each gecko. Selected geckos from these two groups were euthanized and necropsied. There were statistically significant differences (P < 0.05) between the two groups for mean body weight, mean body condition score, and prevalence of infection. Cryptosporidium sp. infection is endemic in this breeding colony, and there were a large number of geckos with a subclinical or carrier state of infection. These animals continued to be infected with Cryptosporidium sp. but gained weight and remained in good body condition. Only one gecko in the entire group of 40 was confirmed to be negative for oocysts or developmental stages by repeated fecal exams and histopathology. An additional 37 severely emaciated geckos from the breeding colony were euthanized, and all were positive for Cryptosporidium sp. on histopathologic examination of the gastrointestinal tract. The results of this study indicate that although some animals can recover from a clinical infection, if a gecko is severely wasted, it should be euthanized because of the poor prognosis and possible source of infection to other geckos.
Journal of Zoo and Wildlife Medicine 12/2008; 39(4):600-7. · 0.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances in vaccine technology are occurring in the molecular techniques used to develop vaccines and in the assessment of vaccine efficacy, allowing more complete characterization of vaccine-induced immunity correlating to protection. FIV vaccine development has closely mirrored and occasionally surpassed the development of HIV-1 vaccine, leading to first licensed technology. This review will discuss technological advances in vaccine designs, challenge infection assessment, and characterization of vaccine immunity in the context of the protection detected with prototype and commercial dual-subtype FIV vaccines and in relation to HIV-1.
Veterinary Immunology and Immunopathology 06/2008; 123(1-2):65-80. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.
[Show abstract][Hide abstract] ABSTRACT: Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self-renewing, undifferentiated, human ESCs (hESCs). These cells are a valuable model of human development in vitro and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. The extracellular markers that have been used to characterize hESCs are primarily carbohydrate epitopes on proteoglycans or sphingolipids, such as stage-specific embryonic antigen (SSEA)-3 and -4. The expression of SSEA-3 and -4 is tightly regulated during preimplantation development and on hESCs. Although this might imply a molecular function in undifferentiated cells, it has not yet been tested experimentally. We used inhibitors of sphingolipid and glycosphingolipid (GSL) biosynthesis to block the generation of SSEA-3 and -4 in hESCs. Depletion of these antigens and their precursors was confirmed using immunostaining, flow cytometry, and tandem mass spectroscopy. Transcriptional analysis, immunostaining, and differentiation in vitro and in teratomas indicated that other properties of pluripotency were not noticeably affected by GSL depletion. These experiments demonstrated that the GSLs recognized as SSEA-3 and -4 do not play critical functional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellular differentiation.