Brian G. Murphy

Histological subtypes of oral non-tonsillar squamous cell carcinoma in dogs.

Several histological subtypes and grades of oral squamous cell carcinoma (SCC) are described in human literature and these subtypes have distinct morphological features and biological behaviour. This retrospective study (1990-2010) included 84 dogs diagnosed with SCC of the oral cavity and oropharynx, excluding the tonsils. Sixty-nine of the SCCs (82.1%) were further diagnosed as conventional SCC (CSCC) (33 [47.8%] well-differentiated, 31 [44.9%] moderately-differentiated and five [7.3%] poorly-differentiated), five (5.95%) each as papillary SCC and basaloid SCC, three (3.6%) as adenosquamous carcinoma and two (2.4%) as spindle cell carcinoma. Compared with the general hospital population, neutered female dogs, dogs aged 10 to <15 years, English springer spaniels and Shetland sheepdogs were overrepresented. The majority (78.1%) of SCCs were proliferative with or without associated ulceration, although no significant association was observed between the gross appearance and different SCC subtypes. 71.4% of SCCs were located in dentate jaws; however, well-differentiated CSCC more often affected the tongue and other non-dentate mucosal surfaces (P=0.0022). No significant association was found between any of the SCC subtypes and tumour-associated inflammation (TAI), perineural and lymphovascular invasion (PNI, LVI), or between gross appearance of the tumour and tumour location, PNI, LVI or TAI or PNI, LVI, TAI and tumour location.

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.

Tissue tropism and promoter sequence variation in caprine arthritis encephalitis virus infected goats.

Caprine arthritis encephalitis virus is a lentivirus that infects goats and is closely related to maedi-visna virus of sheep. Infection with CAEV results in multiple discrete disease manifestations in goats which can include chronic arthritis, mastitis, pneumonia or encephalomyelitis. Presently, no satisfactory mechanistic rationale for viral tropism has been put forward. We propose that specific sequences in the lentiviral promoter (U3 region of the viral long terminal repeat) are associated with viral tissue tropism and subsequent disease expression. A total of 41 distinct CAE viral promoter regions were amplified, sequenced and phylogenetically compared from the tissues of 24 CAEV-infected goats demonstrating a variety of disease manifestations. Phylogenetically, we identified no tendency for clustering of these promoter sequences into tissue-specific groups. These results therefore do not provide evidence for the study hypothesis. However, multiple motifs within the U3 promoter region were highly conserved both within the entire collection of sequences and within tissue-specific groups.

Levels of feline infectious peritonitis virus in blood, effusions, and various tissues and the role of lymphopenia in disease outcome following experimental infection.

Abstract Twenty specific pathogen free cats were experimentally infected with a virulent cat-passaged type I field strain of FIPV. Eighteen cats succumbed within 2–4 weeks to effusive abdominal FIP, one survived for 6 weeks, and one seroconverted without outward signs of disease. A profound drop in the absolute count of blood lymphocytes occurred around 2 weeks post-infection (p.i.) in cats with rapid disease, while the decrease was delayed in the one cat that survived for 6 weeks. The absolute lymphocyte count of the surviving cat remained within normal range. Serum antibodies as measured by indirect immunofluorescence appeared after 2 weeks p.i. and correlated with the onset of disease signs. Viral genomic RNA was either not detectable by reverse transcription quantitative real-time PCR (RT-qPCR) or detectable only at very low levels in terminal tissues not involved directly in the infection, including hepatic and renal parenchyma, cardiac muscle, lung or popliteal lymph node. High tissue virus loads were measured in severely affected tissues such as the omentum, mesenteric lymph nodes and spleen. High levels of viral genomic RNA were also detected in whole ascitic fluid, with the cellular fraction containing 10–1000 times more viral RNA than the supernatant. Replicating virus was strongly associated with macrophages by immunohistochemistry. Virus was usually detected at relatively low levels in feces and there was no evidence of enterocyte infection. Viral genomic RNA was not detected at the level of test sensitivity in whole blood, plasma, or the white cell fraction in terminal samples from the 19 cats that succumbed or in the single survivor. These studies reconfirmed the effect of lymphopenia on disease outcome. FIPV genomic RNA was also found to be highly macrophage associated within diseased tissues and effusions as determined by RT-qPCR and immunohistochemistry but was not present in blood.

Systemic Coxiella-like Infection With Myocarditis and Hepatitis in an Eclectus Parrot (Eclectus roratus)

A multiorgan infection with a Coxiella-like organism was determined to be the cause of death of a female eclectus parrot(Eclectus roratus). The diagnosis was based on gross lesions, histopathology, Gimenez and Gram special stains, immunohistochemistry, electron microscopy, and polymerase chain reaction amplification and sequencing of a bacterial 16s rRNA gene fragment isolated from hepatic and cardiac tissue. Gross postmortem examination revealed multifocal to coalescing foci of hepatic necrosis. The most significant histologic lesions included multifocal lymphohistiocytic necrotizing hepatitis, locally extensive lymphoplasmacytic myocarditis, and myocardial degeneration and necrosis. Intralesional cytoplasmic organisms were identified in cardiomyocytes, biliary epithelium, and pancreatic exocrine cells. This is the first description of a Coxiella-like organism with wide-ranging cellular tropisms in a psittacine bird. In addition, lymphoplasmacytic neuritis, myositis, splenitis, airsacculitis, and enteritis were detected. It is also the first report of a Coxiella-like infection in an eclectus parrot.

Risk factors associated with survival in dogs with nontonsillar oral squamous cell carcinoma: 31 cases (1990-2010)

OBJECTIVE To identify risk factors associated with survival in dogs with nontonsillar oral squamous cell carcinoma (OSCC) that were and were not treated with curative-intent surgery. DESIGN Retrospective case series. ANIMALS 31 dogs with OSCC. PROCEDURES Medical records for dogs with OSCC that were not treated, or were treated with curative-intent surgery only between January 1990 and December 2010 were reviewed. For each dog, data regarding signalment, clinical stage, treatment, tumor recurrence, and survival time were obtained from the medical record, and archived biopsy specimens were evaluated to identify the histologic subtype of the tumor and extent of tumor-associated inflammation (TAI), perineural invasion (PNI), and lymphovascular invasion (LVI). RESULTS Risk of death for the 21 dogs with OSCC that were surgically treated was decreased 91.4% (hazard ratio, 0.086; 95% confidence interval, 0.002 to 0.150), compared with that for the 10 dogs with OSCC that were not treated. The 1-year survival rate was 93.5% and 0% for dogs that were and were not surgically treated, respectively. Risk of death increased significantly with increasing TAI and increasing risk score (combination of TAI, PNI, and LVI). Tumor location, clinical stage, and histologic subtype were not associated with survival time. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the prognosis for dogs with OSCC was excellent following surgical excision of the tumor. Risk of death increased with increasing TAI, and combining TAI, PNI, and LVI into a single risk score may be a useful prognostic indicator for dogs with OSCC.

Transcriptional Regulation of Latent Feline Immunodeficiency Virus in Peripheral CD4+ T-lymphocytes

Feline immunodeficiency virus (FIV), the lentivirus of domestic cats responsible for feline AIDS, establishes a latent infection in peripheral blood CD4+ T-cells approximately eight months after experimental inoculation. In this study, cats experimentally infected with the FIV-C strain in the asymptomatic phase demonstrated an estimated viral load of 1 infected cell per approximately 103 CD4+ T-cells, with about 1 copy of viral DNA per cell. Approximately 1 in 10 proviral copies was capable of transcription in the asymptomatic phase. The latent FIV proviral promoter was associated with deacetylated, methylated histones, which is consistent with a condensed chromatin structure. In contrast, the transcriptionally active FIV promoter was associated with histone acetylation and demethylation. In addition, RNA polymerase II appeared to be paused on the latent viral promoter, and short promoter-proximal transcripts were detected. Our findings for the FIV promoter in infected cats are similar to results obtained in studies of human immunodeficiency virus (HIV)-1 latent proviruses in cell culture in vitro studies. Thus, the FIV/cat model may offer insights into in vivo mechanisms of HIV latency and provides a unique opportunity to test novel therapeutic interventions aimed at eradicating latent virus.

Feline Foamy Virus Adversely Affects Feline Mesenchymal Stem Cell Culture and Expansion: Implications for Animal Model Development

Mesenchymal stem cells (MSCs) are a promising therapeutic option for various immune-mediated and inflammatory disorders due to their potent immunomodulatory and trophic properties. Naturally occurring diseases in large animal species may serve as surrogate animal models of human disease, as they may better reflect the complex genetic, environmental, and physiologic variation present in outbred populations. We work with naturally occurring diseases in large animal species to better understand how MSCs work and to facilitate optimal translation of MSC-based therapies. We are investigating the use of MSC therapy for a chronic oral inflammatory disease in cats. During our efforts to expand fat-derived feline MSCs (fMSCs), we observed that∼50% of the cell lines developed giant foamy multinucleated cells in later passages. These morphologic alterations were associated with proliferation arrest. We hypothesized that the cytopathic effects were caused by infection with a retrovirus, feline foamy virus (FFV). Using transmission electron microscopy, polymerase chain reaction, and in vitro assays, we determined that syncytial cell formation and proliferation arrest in fMSCs were caused by FFV strains that were highly homologous to previously reported FFV strains. We determined that the antiretroviral drug, tenofovir, may be used to support ex vivo expansion and salvage of FFV-infected fMSC lines. MSC lines derived from specific pathogen-free cats do not appear to be infected with FFV and may be a source of allogeneic fMSCs for clinical application. FFV infection of fMSC lines may hinder large-scale expansion of autologous MSC for therapeutic use in feline patients.

Feline immunodeficiency virus latency

Despite highly effective anti-retroviral therapy, HIV is thought to persist in patients within long-lived cellular reservoirs in the form of a transcriptionally inactive (latent) integrated provirus. Lentiviral latency has therefore come to the forefront of the discussion on the possibility of a cure for HIV infection in humans. Animal models of lentiviral latency provide an essential tool to study mechanisms of latency and therapeutic manipulation. Of the three animal models that have been described, the feline immunodeficiency virus (FIV)-infected cat is the most recent and least characterized. However, several aspects of this model make it attractive for latency research, and it may be complementary to other model systems. This article reviews what is known about FIV latency and chronic FIV infection and how it compares with that of other lentiviruses. It thereby offers a framework for the usefulness of this model in future research aimed at lentiviral eradication.

Pharmacologic reactivation of latent feline immunodeficiency virus ex vivo in peripheral CD4+ T-lymphocytes.

FIV establishes a latent infection in peripheral CD4+ T-cells, and the latent FIV promoter is associated with deacetylated, methylated histones, consistent with a restrictive chromatin structure. Here we explored the use of 5 histone-modifying agents - 4 histone deacetylase inhibitors (HDACi) and 1 histone methyltransferase inhibitor (HMTi) - to reactivate latent FIV ex vivo. All compounds tested were able to alter histone lysine residue modifications in feline cells, both globally and at the FIV promoter locally. When latently FIV-infected peripheral CD4+ T-cells were cultured ex vivo in the presence of these inhibitors, viral transcription was significantly activated relative to no treatment controls. Transcriptional reactivation of virus mediated by the HDACi suberoylanilide hydroxamic acid (SAHA) was dose-dependent, detected after as little as 1h of exposure, and resulted in virion formation as evidenced by supernatant reverse transcriptase activity. A synergistic effect was not found when SAHA was combined with HMTi under the conditions tested. At low therapeutically relevant concentrations in primary feline PBMC, SAHA was found to be minimally cytotoxic and non-immune activating. HDACi and HMTi can reactivate latent FIV ex vivo, and SAHA, also known as the anticancer drug Vorinostat, in particular is a promising candidate for in vivo use because of its efficacy, potency, and relative safety. Use of the FIV/cat model of lentiviral latency to explore in vivo treatment with SAHA and other anti-latency therapeutics will allow investigations that are either ethically or logistically not addressable in patients persistently infected with human immunodeficiency virus (HIV-1).