Unusual Necrotizing Encephalitis in Raccoons and Skunks Concurrently Infected With Canine Distemper Virus and Sarcocystis sp.

Canine distemper virus commonly infects free-ranging, terrestrial mesopredators throughout the United States. Due to the immunosuppressive effects of the virus, concurrent opportunistic infections are also common. Among these, secondary systemic protozoal infections have been described in a number of species. We report an unusual presentation of necrotizing encephalitis associated withSarcocystissp in four raccoons and one skunk concurrently infected with canine distemper virus. Lesions were characterized by variably sized necrotizing cavitations composed of abundant mineral admixed with inflammatory cells and protozoa.Sarcocystissp was confirmed via immunohistochemistry using a monoclonal antibody toSarcocystis neurona The pathologic changes are similar to lesions in human AIDS patients infected withToxoplasma gondii.

Localization of Bovine Papillomavirus Nucleic Acid in Equine Sarcoids.

Bovine papillomaviruses (BPV1/BPV2) have long been associated with equine sarcoids; deciphering their contribution has been difficult due to their ubiquitous presence on skin and in the environment, as well as the lack of decent techniques to interrogate their role in pathogenesis. We have developed and characterized an in situ hybridization (ISH) assay that uses a pool of probes complementary to portions of the E5, E6, and E7 genes. This assay is highly sensitive for direct visualization of viral transcript and nucleic acid in routinely processed histopathologic samples. We demonstrate here the visualization of BPV nucleic acid in 18 of 18 equine sarcoids, whereas no detectable viral DNA was present in 15 of 15 nonsarcoid controls by this technique. In nearly 90% (16/18) of the sarcoids, 50% or more of the fibroblastic cell nuclei distributed throughout the neoplasm had detectable hybridization. In the remaining 2 cases, fewer than half of the fibroblastic cells contained detectable hybridization, but viral nucleic acid was also detected in epithelial cells of the sebaceous glands, hair follicles and epidermis. A sensitive ISH assay is an indispensable addition to the molecular methods used to detect viral nucleic acid in tissue. We have used this technique to determine the specific cellular localization and distribution of BPV in a subset of equine sarcoids.

Equine Genital Squamous Cell Carcinoma: In Situ Hybridization Identifies a Distinct Subset Containing Equus caballus Papillomavirus 2.

Equus caballus papillomavirus 2 (EcPV2) has been proposed as an etiologic agent for genital squamous cell carcinoma (SCC), the most common malignant tumor of the horse penis. EcPV2 is commonly detected by polymerase chain reaction (PCR) on normal horse genitalia; therefore, unraveling the virus' role in oncogenic transformation requires other methods of detection. In this study, a highly sensitive multiple-probe chromogenic in situ hybridization (ISH) technique was designed to recognize the E6/E7 oncogenes of EcPV2. ISH demonstrated abundant virus within 6 of 13 penile and preputial SCCs, whereas evidence of solar damage was found in 6 cases that were negative for EcPV2 by ISH. The ISH technique is valuable for studies of pathogenesis, since it demonstrates for the first time that the vast majority of neoplastic cells contain virus. Moreover, hybridization was present in all metastases examined, implying stability of E6/E7 expression in these clonal populations of neoplastic cells. This study contributes to the accumulating evidence for a causal role of EcPV2 in a subset of genital SCCs in horses.

Identification of a novel papillomavirus in a Northern Fulmar (Fulmarus glacialis) with viral production in cartilage.

We report the identification of a novel papillomavirus, Fulmarus glacialis papillomavirus 1 (FgPV1), present within an interdigital foot mass of a Northern Fulmar (Fulmarus glacialis). The mass of interest was composed of normal stratified and keratinized epithelium and dense mesenchymal cells with central cartilaginous islands. Within the nuclei of many chondrocytes were loose aggregates or paracrystalline arrays of virions approximately 50 nm in size. Degenerate polymerase chain reaction was used to identify the virus as a putative papillomavirus, and the entire viral genome of 8132 base pairs was subsequently amplified and sequenced. Analysis revealed canonical papillomavirus architecture, including the early open reading frames E6, E7, E1, and E2 and the 2 late proteins L1 and L2. FgPV1 is most closely related to a cluster of avian and reptilian papillomaviruses as visualized by phylogenetic trees. This observation suggests that papillomavirus virion production can occur in mesenchymal cells.

Common and emerging infectious diseases in the animal shelter.

The beneficial role that animal shelters play is unquestionable. An estimated 3 to 4 million animals are cared for or placed in homes each year, and most shelters promote public health and support responsible pet ownership. It is, nonetheless, inevitable that shelters are prime examples of anthropogenic biological instability: even well-run shelters often house transient, displaced, and mixed populations of animals. Many of these animals have received minimal to no prior health care, and some have a history of scavenging or predation to survive. Overcrowding and poor shelter conditions further magnify these inherent risks to create individual, intraspecies, and interspecies stress and provide an environment conducive to exposure to numerous potentially collaborative pathogens. All of these factors can contribute to the evolution and emergence of new pathogens or to alterations in virulence of endemic pathogens. While it is not possible to effectively anticipate the timing or the pathogen type in emergence events, their sites of origin are less enigmatic, and pathologists and diagnosticians who work with sheltered animal populations have recognized several such events in the past decade. This article first considers the contribution of the shelter environment to canine and feline disease. This is followed by summaries of recent research on the pathogenesis of common shelter pathogens, as well as research that has led to the discovery of novel or emerging diseases and the methods that are used for their diagnosis and discovery. For the infectious agents that commonly affect sheltered dogs and cats, including canine distemper virus, canine influenza virus, Streptococcus spp, parvoviruses, feline herpesvirus, feline caliciviruses, and feline infectious peritonitis virus, we present familiar as well as newly recognized lesions associated with infection. Preliminary studies on recently discovered viruses like canine circovirus, canine bocavirus, and feline norovirus indicate that these pathogens can cause or contribute to canine and feline disease.

Temporal and geographic clustering of polyomavirus-associated olfactory tumors in 10 free-ranging raccoons (Procyon lotor).

Reports of primary nervous system tumors in wild raccoons are extremely rare. Olfactory tumors were diagnosed postmortem in 9 free-ranging raccoons from 4 contiguous counties in California and 1 raccoon from Oregon within a 26-month period between 2010 and 2012. We describe the geographic and temporal features of these 10 cases, including the laboratory diagnostic investigations and the neuropathologic, immunohistochemical, and ultrastructural characteristics of these tumors in the affected animals. All 9 raccoons from California were found within a localized geographic region of the San Francisco Bay Area (within a 44.13-km radius). The tight temporal and geographic clustering and consistent anatomic location in the olfactory system of tumor types not previously described in raccoons (malignant peripheral nerve sheath tumors and undifferentiated sarcomas) strongly suggest either a common cause or a precipitating factor leading to induction or potentiation of neuro-oncogenesis and so prompted an extensive diagnostic investigation to explore possible oncogenic infectious and/or toxic causes. By a consensus polymerase chain reaction strategy, a novel, recently reported polyomavirus called raccoon polyomavirus was identified in all 10 tumors but not in the normal brain tissue from the affected animals, suggesting that the virus might play a role in neuro-oncogenesis. In addition, expression of the viral protein T antigen was detected in all tumors containing the viral sequences. We discuss the potential role of raccoon polyomavirus as an oncogenic virus.

Ibex-associated malignant catarrhal fever-like disease in a group of bongo antelope (Tragelaphus eurycerus).

A private zoological facility experienced an outbreak of malignant catarrhal fever (MCF) in 3 bongo antelope (Tragelaphus eurycerus). All cases were periparturient bongos that presented acutely anorectic beginning ~6 weeks after being housed with a Nubian ibex. Disease quickly progressed to respiratory distress and death within 24-72 hours of onset of clinical signs. Consistent gross findings in affected bongos were pulmonary edema and small pale tan foci in the livers. Histological lesions included a nonsuppurative vasculitis in multiple tissues, which is well recognized in MCF, but additionally included necrotizing cholangiohepatitis and neutrophilic, necrotizing myocarditis. Ibex-associated viral DNA was detected by polymerase chain reaction and was identical in sequence whether derived from bongos or ibex. The sequence closely matched an MCF viral DNA fragment that had been amplified from an ibex and bongo in a previous case report.

Distribution of the feline calicivirus receptor junctional adhesion molecule a in feline tissues.

Junctional adhesion molecule A (JAM-A) is an immunoglobulin superfamily protein that plays an important role in the assembly and maintenance of tight junctions and the establishment of epithelial cell polarity. The feline JAM-A (fJAM-A) is a functional receptor for feline calicivirus (FCV). Among natural diseases associated with FCV infection, isolates that cause oral vesicular disease are detected in epithelial cells; however, isolates that cause systemic disease are detected in multiple cell types. The distribution of an FCV receptor or receptors in feline tissues is relevant to viral pathogenesis in that it should reflect the wide latitude of clinical sequelae associated with FCV infection. The authors examined the expression of feline JAM-A in the cat by using confocal immunofluorescence localization on normal tissues, with special regard to tissue targets of naturally occurring FCV. As described in the human and the mouse, fJAM-A was widely distributed in feline tissues, where it localized at cell-cell junctions of epithelial and endothelial cells. fJAM-A was highly expressed on feline platelets, with lower levels of expression on feline peripheral blood leukocytes. Additionally, FCV infection of a feline epithelial cell monolayer causes redistribution of fJAM-A to the cytosol of infected cells. It is reasonable to propose that the spectrum of lesions caused by FCV reflects disruption of intercellular junctions that rely on fJAM-A function and tight junctional integrity.

Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport.

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.

Gastrointestinal pythiosis in 10 dogs from California.

BACKGROUND: Pythium insidiosum is an aquatic oomycete that causes severe segmental thickening of the canine gastrointestinal (GI) tract, resulting in weight loss, vomiting, diarrhea, and death. Infection in dogs previously has been observed primarily in the southeastern United States. OBJECTIVE: To describe the clinicopathologic and epidemiologic findings associated with GI pythiosis in 10 dogs from California. METHODS: Dogs were initially identified on the basis of supportive clinical findings and routine histology. Pythiosis was confirmed in each dog with at least one of the following: immunoblot serology, enzyme-linked immunosorbent assay serology, immunohistochemistry, and culture followed by species-specific polymerase chain reaction, rRNA gene sequencing, or both. RESULTS: Between September 2003 and December 2006, GI pythiosis was confirmed in 1 dog from central California and 9 dogs that lived within a 30-mile radius of Davis, CA. Seven of 8 dogs for which environmental data were available had frequent access to flooded rice fields or other water sources. Esophageal lesions were present in 2 of 10 dogs. Common laboratory findings included eosinophilia (7/9), hypoalbuminemia (9/9), and hyperglobulinemia (8/9). Median survival time was 26.5 days (range, 0-122 days), and the disease was ultimately fatal in all 10 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: The geographic distribution of pythiosis has widened in recent years to include the western United States. Factors that may have contributed to this change include altered rice-farming practices and landscape irrigation. Veterinarians in California should be familiar with the clinicopathologic features associated with GI pythiosis to aid in early diagnosis and effective treatment.

Tumor necrosis factor alpha gene regulation in activated T cells involves ATF-2/Jun and NFATp.

The human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest genes expressed upon the activation of a T or B cell through its antigen receptor. Previous experiments have demonstrated that in stimulated T cells, a TNF-alpha promoter element, kappa 3, which binds NFATp, is required for the cyclosporin A-sensitive transcriptional activation of the gene. Here, we demonstrate that a cyclic AMP response element (CRE), which lies immediately upstream of the kappa 3 site, is also required for induction of TNF-alpha gene transcription in T cells stimulated by calcium ionophore or T-cell receptor ligands. The CRE binds ATF-2 and Jun proteins in association with NFATp bound to kappa 3. These proteins bind noncooperatively in vitro; however, the transcriptional activity of the CRE/kappa 3 composite site is dramatically higher than the activity of the kappa 3 site alone, indicating that the two sites cooperate in vivo. This study is the first demonstration of a role for ATF-2 in TNF-alpha gene transcription and of a functional interaction between ATF-2/Jun and NFATp. This novel pairing of NFATp with ATF-2/Jun may account for the specific and immediate pattern of TNF-alpha gene transcription in stimulated T cells.

A novel pulmonary polyomavirus in alpacas (Vicugna pacos).

Viral metagenomic analysis detected a novel polyomavirus in a 6-month old female alpaca (Vicugna pacos) euthanized after a diagnosis of disseminated lymphosarcoma. The viral genome was fully sequenced, found to be similar to other polyomaviruses in gene architecture and provisionally named Alpaca polyomavirus or AlPyV. Viral nucleic acid was detected by PCR in venous blood, spleen, thymus, and lung. AlPyV phylogenetically clustered in the "Wuki" group of PyVs, which includes WU and KI polyomaviruses, commonly found in human respiratory samples. In an ISH analysis of 17 alpaca necropsies, 7 had detectable virus within the lung. In animals without pneumonia, probe hybridization was restricted to the nuclei of scattered individual bronchiolar epithelial cells. Three of the ISH positive alpacas had interstitial pneumonia of unknown origin, and in these animals there was viral nucleic acid detected in bronchiolar epithelium, type II pneumocytes, and alveolar macrophages. The pattern of AlPyV distribution is consistent with a persistent respiratory virus that has a possible role in respiratory disease.

Intestinal Leiomyositis: A Cause of Chronic Intestinal Pseudo-Obstruction in 6 Dogs.

BACKGROUND: Intestinal leiomyositis is a suspected autoimmune disorder affecting the muscularis propria layer of the gastrointestinal tract and is a cause of chronic intestinal pseudo-obstruction in humans and animals. OBJECTIVE: To characterize the clinical presentation, histopathologic features, and outcome of dogs with intestinal leiomyositis in an effort to optimize treatment and prognosis. ANIMALS: Six client-owned dogs. METHODS: Retrospective case series. Medical records were reviewed to describe signalment, clinicopathologic and imaging findings, histopathologic diagnoses, treatment, and outcome. All biopsy specimens were reviewed by a board-certified pathologist. RESULTS: Median age of dogs was 5.4 years (range, 15 months-9 years). Consistent clinical signs included vomiting (6/6), regurgitation (2/6), and small bowel diarrhea (3/6). Median duration of clinical signs before presentation was 13 days (range, 5-150 days). Diagnostic imaging showed marked gastric distension with dilated small intestines in 4/6 dogs. Full-thickness intestinal biopsies were obtained in all dogs by laparotomy. Histopathology of the stomach and intestines disclosed mononuclear inflammation, myofiber degeneration and necrosis, and fibrosis centered within the region of myofiber loss in the intestinal muscularis propria. All dogs received various combinations of immunomodulatory and prokinetic treatment, antimicrobial agents, antiemetics, and IV fluids, but none of the dogs showed a clinically relevant improvement with treatment. Median survival was 19 days after diagnosis (range, 3-270 days). CONCLUSIONS AND CLINICAL IMPORTANCE: Intestinal leiomyositis is a cause of intestinal pseudo-obstruction and must be diagnosed by full-thickness intestinal biopsy. This disease should be considered in dogs with acute and chronic vomiting, regurgitation, and small bowel diarrhea.

Exposure to raccoon polyomavirus (RacPyV) in free-ranging North American raccoons (Procyon lotor).

There is evidence that raccoon polyomavirus is causative for neuroglial brain tumors in the western United States. It is unknown if infection is limited to geographic locales where tumors have been reported or is widespread, like human polyomaviruses. We demonstrate raccoons in western, eastern and midwestern states have been exposed to RacPyV by detection of antibodies to capsid protein, VP1. While raccoons in eastern and midwestern states are seropositive, exposure is lower than in the western states. Additionally, across geographic areas seropositivity is higher in older as compared to younger raccoons, similar to polyomavirus exposure in humans. Serum titers are significantly higher in raccoons with tumors compared to raccoons without. Unlike polyomavirus-associated diseases in humans, we did not detect significant sequence variation between tumor and non-tumor tissue in raccoons with tumors compared to those without tumors. This warrants further investigation into co-morbid diseases or genetic susceptibility studies of the host.

Post-vaccinal distemper encephalitis in two Border Collie cross littermates.

CASE HISTORY: One 4.5-month-old male Border Collie cross presented with aggression and seizures in October 2006. A 16-month-old, female, spayed Border Collie cross presented with hypersalivation and a dropped jaw and rapidly became stuporous in September 2007. The dogs were littermates and developed acute neurological signs 5 and 27 days, respectively, after vaccination with different modified live vaccines containing canine distemper virus. HISTOPATHOLOGICAL FINDINGS: Sections of brain in both dogs showed evidence of encephalitis mainly centred on the grey matter of brainstem nuclei, where there was extensive and intense parenchymal and perivascular infiltration of histiocytes and lymphocytes. Intra-nuclear and intra-cytoplasmic inclusions typical of distemper were plentiful and there was abundant labelling for canine distemper virus using immunohistochemistry. DIAGNOSIS: Post-vaccinal canine distemper. CLINCIAL RELEVANCE: Post-vaccinal canine distemper has mainly been attributed to virulent vaccine virus, but it may also occur in dogs whose immunologic nature makes them susceptible to disease induced by a modified-live vaccine virus that is safe and protective for most dogs.

Idiopathic granulomatous pneumonia in seven horses.

The history, clinical signs and pathological findings in seven adult horses with histologically confirmed idiopathic granulomatous disease, primarily of the lungs, are reviewed. They ranged in age from eight to 21 years, five were geldings and two were females, they belonged to five breeds and there were no seasonal or geographical associations. The primary clinical signs were chronic weight loss, exercise intolerance and respiratory distress which did not respond to conventional treatment. The most consistent physical findings were depression, anorexia, tachycardia, tachypnoea and adventitious lung sounds. Thoracic radiographs revealed a diffuse, structured, nodular, interstitial pulmonary pattern in each horse. Haematological measurements suggested a chronic inflammatory process and the cytology of transtracheal washes was consistent with a mild suppurative inflammation. Idiopathic granulomatous pneumonia was confirmed histologically in each of the horses, either postmortem or by a lung biopsy. The horses responded poorly to medical treatment and only one of three treated horses is still alive.

Listeria monocytogenes infection of the alimentary tract (enteric listeriosis) of sheep in New Zealand.

A retrospective study of the microscopical lesions of nine cases of enteric listeriosis of sheep was conducted. Lesions were present variably in the abomasum and the small and large intestines. The inflammation was multifocal to extensive, mainly neutrophilic and involved the lamina propria, muscularis mucosa and superficial submucosa, with intense focus on the muscularis mucosa. The mesenteric lymph nodes were also affected and, in some sheep, the liver. Large numbers of gram-positive rods were demonstrated within areas of inflammation in the gastrointestinal tract and mesenteric lymph nodes and Listeria spp. were identified immunohistochemically in these lesions. Ultrastructurally, bacteria were found free within the cytoplasm of myofibres of the muscularis mucosa.

A clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by Streptococcus equi subsp. zooepidemicus.

An outbreak of acute, fatal, hemorrhagic pneumonia was observed in more than 1,000 mixed breed dogs in a single animal shelter. The Department of Anatomic Pathology at the University of California at Davis School of Veterinary Medicine performed necropsies on dogs that were found moribund in acute respiratory distress or found dead with evidence of nasal bleeding. All dogs had hemothorax and an acute, fibrinosuppurative pneumonia. Large numbers of gram-positive cocci were observed within the lungs of all dogs and within septic thromboemboli of remote organs in about 50% of cases. Bacterial cultures from the dogs and their environment revealed widespread beta-hemolytic Streptococus equi subspecies zooepidemicus (Lancefield Group C). Extensive diagnostic testing failed to reveal the consistent presence of copathogens in individual cases. The clinical, epidemiologic, molecular biologic, and pathologic data indicate that a single clone of S. zooepidemicus was the cause of an acutely fatal respiratory infection in these dogs.

Cutaneous pythiosis in a nestling white-faced ibis.

A nestling white-faced ibis (Plegadis chihi) with multifocal skin ulcerations on the wings, neck, head, and limbs was found in a wetland agricultural region of the central valley in California. Pathologic, microbiologic, and molecular findings were consistent with restricted, cutaneous infection by the oomycete Pythium insidiosum. The microscopic features of the disease, including intense, necrotizing eosinophilic and granulomatous inflammation, are similar to those previously described in mammals. Pythiosis, which is most typical in tropical and subtropical climates, has recently emerged in California as a cause of cutaneous and enteric disease in horses and dogs, respectively. Environmental stability and persistence of a "water-mold" in the arid central valley of California could be associated with agricultural and community watering practices. To the best of the authors' knowledge, this is the first published report of pythiosis in birds.