Experimental transmission of enzootic nasal adenocarcinoma in sheep.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the secretory epithelial cells of the nasal mucosa of sheep and goats. It is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), but a causative relationship has yet to be demonstrated. In this study, 14-day-old lambs were experimentally infected via nebulization with cell-free tumor filtrates derived from naturally occurring cases of ENA. At 12 weeks post-infection (wpi), one of the five infected lambs developed clinical signs, including continuous nasal discharge and open mouth breathing, and was euthanized. Necropsy revealed the presence of a large bilateral tumor occupying the nasal cavity. At 45 wpi, when the study was terminated, none of the remaining infected sheep showed evidence of tumors either by computed tomography or post-mortem examination. ENTV-1 proviral DNA was detected in the nose, lung, spleen, liver and kidney of the animal with experimentally induced ENA, however there was no evidence of viral protein expression in tissues other than the nose. Density gradient analysis of virus particles purified from the experimentally induced nasal tumor revealed a peak reverse transcriptase (RT) activity at a buoyant density of 1.22 g/mL which was higher than the 1.18 g/mL density of peak RT activity of virus purified from naturally induced ENA. While the 1.22 g/mL fraction contained primarily immature unprocessed virus particles, mature virus particles with a similar morphology to naturally occurring ENA could be identified by electron microscopy. Full-length sequence analysis of the ENTV-1 genome from the experimentally induced tumor revealed very few nucleotide changes relative to the original inoculum with only one conservative amino acid change. Taken together, these results demonstrate that ENTV-1 is associated with transmissible ENA in sheep and that under experimental conditions, lethal tumors are capable of developing in as little as 12 wpi demonstrating the acutely oncogenic nature of this ovine betaretrovirus.

Development of a PCR assay to detect papillomavirus infection in the snow leopard.

BACKGROUND: Papillomaviruses (PVs) are a group of small, non-encapsulated, species-specific DNA viruses that have been detected in a variety of mammalian and avian species including humans, canines and felines. PVs cause lesions in the skin and mucous membranes of the host and after persistent infection, a subset of PVs can cause tumors such as cervical malignancies and head and neck squamous cell carcinoma in humans. PVs from several species have been isolated and their genomes have been sequenced, thereby increasing our understanding of the mechanism of viral oncogenesis and allowing for the development of molecular assays for the detection of PV infection. In humans, molecular testing for PV DNA is used to identify patients with persistent infections at risk for developing cervical cancer. In felids, PVs have been isolated and sequenced from oral papillomatous lesions of several wild species including bobcats, Asian lions and snow leopards. Since a number of wild felids are endangered, PV associated disease is a concern and there is a need for molecular tools that can be used to further study papillomavirus in these species. RESULTS: We used the sequence of the snow leopard papillomavirus UuPV1 to develop a PCR strategy to amplify viral DNA from samples obtained from captive animals. We designed primer pairs that flank the E6 and E7 viral oncogenes and amplify two DNA fragments encompassing these genes. We detected viral DNA for E6 and E7 in genomic DNA isolated from saliva, but not in paired blood samples from snow leopards. We verified the identity of these PCR products by restriction digest and DNA sequencing. The sequences of the PCR products were 100% identical to the published UuPV1 genome sequence. CONCLUSIONS: We developed a PCR assay to detect papillomavirus in snow leopards and amplified viral DNA encompassing the E6 and E7 oncogenes specifically in the saliva of animals. This assay could be utilized for the molecular investigation of papillomavirus in snow leopards using saliva, thereby allowing the detection of the virus in the anatomical site where oral papillomatous lesions develop during later stages of infection and disease development.

Development of a SYBR Green-based real-time quantitative polymerase chain reaction assay to detect enzootic nasal tumor virus in goats.

Enzootic nasal adenocarcinoma is a contagious respiratory disease in goats that is caused by the enzootic nasal tumor virus 2 (ENTV-2). In order to increase the number of available detection methods for ENTV-2, we developed a SYBR Green real-time polymerase chain reaction (SGrPCR) assay that targets the gag gene of ENTV-2. The low limit of detection of the assay was 3.68 × 101 copies/µL, a hundredfold more sensitive than conventional PCR. The melt curve showed a single sharp melt peak at 83°C, which indicated that there was no non-specific amplification or primer dimer formation. The intra-assay and inter-assay coefficients of variation were 1.58% and 1.82%, respectively. There was no cross-reactivity with closely related goat viruses (i.e., orf virus, peste des petits ruminants virus, goatpox virus, foot-and-mouth disease virus) and endogenous retroviruses. In conclusion, the SGrPCR assay is specific for the gag gene of ENTV-2 and provides a rapid and sensitive approach for detecting ENTV-2 in clinical samples.

L'adénocarcinome nasal enzootique est une maladie respiratoire contagieuse chez les chèvres qui est causé par le virus de la tumeur nasale enzootique 2 (ENTV-2). Afin d'augmenter le nombre de méthodes de détection disponibles pour ENTV-2, nous avons développé un test de réaction en chaîne par polymérase en temps réel SYBR Green (SGrPCR) qui cible le gène gag de ENTV-2. La limite basse de détection du test était de 3,68 × 101 copies/µL, cent fois plus sensible que la PCR conventionnelle. La courbe de fusion montrait un seul pic de fusion net à 83 °C, ce qui indiquait qu'il n'y avait pas d'amplification non spécifique ou de formation de dimère d'amorce. Les coefficients de variation intra-essai et inter-essai étaient respectivement de 1,58 % et 1,82 %. Il n'y avait pas de réactivité croisée avec les virus caprins étroitement apparentés (c'est-à-dire le virus orf, le virus de la peste des petits ruminants, le virus de la variole caprine, le virus de la fièvre aphteuse) et les rétrovirus endogènes. En conclusion, le test SGrPCR est spécifique du gène gag de l'ENTV-2 et fournit une approche rapide et sensible pour la détection d'ENTV-2 dans des échantillons cliniques.(Traduit par Docteur Serge Messier).

Pan-European Study on the Prevalence of the Feline Leukaemia Virus Infection - Reported by the European Advisory Board on Cat Diseases (ABCD Europe).

Feline leukaemia virus (FeLV) is a retrovirus associated with fatal disease in progressively infected cats. While testing/removal and vaccination led to a decreased prevalence of FeLV, recently, this decrease has reportedly stagnated in some countries. This study aimed to prospectively determine the prevalence of FeLV viraemia in cats taken to veterinary facilities in 32 European countries. FeLV viral RNA was semiquantitatively detected in saliva, using RT-qPCR as a measure of viraemia. Risk and protective factors were assessed using an online questionnaire to report geographic, demographic, husbandry, FeLV vaccination, and clinical data. The overall prevalence of FeLV viraemia in cats visiting a veterinary facility, of which 10.4% were shelter and rescue cats, was 2.3% (141/6005; 95% CI: 2.0%-2.8%) with the highest prevalences in Portugal, Hungary, and Italy/Malta (5.7%-8.8%). Using multivariate analysis, seven risk factors (Southern Europe, male intact, 1-6 years of age, indoor and outdoor or outdoor-only living, living in a group of ≥5 cats, illness), and three protective factors (Northern Europe, Western Europe, pedigree cats) were identified. Using classification and regression tree (CART) analysis, the origin of cats in Europe, pedigree, and access to outdoors were important predictors of FeLV status. FeLV-infected sick cats shed more viral RNA than FeLV-infected healthy cats, and they suffered more frequently from anaemia, anorexia, and gingivitis/stomatitis than uninfected sick cats. Most cats had never been FeLV-vaccinated; vaccination rates were indirectly associated with the gross domestic product (GDP) per capita. In conclusion, we identified countries where FeLV was undetectable, demonstrating that the infection can be eradicated and highlighting those regions where awareness and prevention should be increased.

Comparison of three feline leukaemia virus (FeLV) point-of-care antigen test kits using blood and saliva.

Feline leukaemia virus (FeLV) can be a challenging infection to diagnose due to a complex feline host-pathogen relationship and occasionally unreliable test results. This study compared the accuracy of three point-of-care (PoC) FeLV p27 antigen test kits commonly used in Australia and available commercially worldwide (SNAP FIV/FeLV Combo, Witness FeLV/FIV and Anigen Rapid FIV/FeLV), using detection of FeLV provirus by an in-house real-time polymerase chain reaction (qPCR) assay as the diagnostic gold standard. Blood (n=563) and saliva (n=419) specimens were collected from a population of cats determined to include 491 FeLV-uninfected and 72 FeLV-infected individuals (45 progressive infections [p27 and qPCR positive], 27 regressive infections [p27 negative, qPCR positive]). Sensitivity and specificity using whole blood was 63% and 94% for SNAP Combo, 57% and 98% for Witness, and 57% and 98% for Anigen Rapid, respectively. SNAP Combo had a significantly lower specificity using blood compared to the other two kits (P=0.004 compared to Witness, P=0.007 compared to Anigen Rapid). False-positive test results occurred with all three kits using blood, and although using any two kits in parallel increased specificity, no combination of kits completely eliminated the occurrence of false-positive results. We therefore recommend FeLV proviral PCR testing for any cat that tests positive with a PoC FeLV antigen kit, as well as for any cat that has been potentially exposed to FeLV but tests negative with a FeLV antigen kit, before final assignment of FeLV status can be made with confidence. For saliva testing, sensitivity and specificity was 54% and 100%, respectively, for all three test kits. The reduced sensitivity of saliva testing compared to blood testing, although not statistically significant, suggests saliva testing with the current generation of PoC FeLV antigen kits is unsuitable for screening large populations of cats, such as in shelters.

A review of virus infections of cataceans and the potential impact of morbilliviruses, poxviruses and papillomaviruses on host population dynamics.

Viruses belonging to 9 families have been detected in cetaceans. We critically review the clinical features, pathology and epidemiology of the diseases they cause. Cetacean morbillivirus (family Paramyxoviridae) induces a serious disease with a high mortality rate and persists in several populations. It may have long-term effects on the dynamics of cetacean populations either as enzootic infection or recurrent epizootics. The latter presumably have the more profound impact due to removal of sexually mature individuals. Members of the family Poxviridae infect several species of odontocetes, resulting in ring and tattoo skin lesions. Although poxviruses apparently do not induce a high mortality, circumstancial evidence suggests they may be lethal in young animals lacking protective immunity, and thus may negatively affect net recruitment. Papillomaviruses (family Papovaviridae) cause genital warts in at least 3 species of cetaceans. In 10% of male Burmeister's porpoises Phocoena spinipinnis from Peru, lesions were sufficiently severe to at least hamper, if not impede, copulation. Members of the families Herpesviridae, Orthomyxoviridae and Rhabdoviridae were demonstrated in cetaceans suffering serious illnesses, but with the exception of a 'porpoise herpesvirus' their causative role is still tentative. Herpes-like viruses and caliciviruses (Caliciviridae) give rise to cutaneous diseases in Monodontidae and Delphinidae. Antibodies to several serotypes of caliciviruses were found in odontocetes and mysticetes. An unrecognized Hepadnaviridae was detected by serology in a captive Pacific white-sided dolphin Lagenorhynchus obliquidens with chronic persistent hepatitis. Adenoviruses (Adenoviridae) were isolated from the intestinal tracts of mysticeti and a beluga Delphinapterus leucas but were not associated with any pathologies. We discuss the potential impact of Paramyxoviridae, Poxviridae and Papovaviridae on the dynamics of several odontocete populations.

Induction of virus-producing tumours in athymic nude mice by bovine papillomavirus type 4.

Bovine papillomavirus type 4 (BPV-4), the causative agent of alimentary papillomatosis, has been used to infect, in vitro, fragments of palatine mucosa from late term bovine fetuses. These small explants were placed beneath the renal capsule of athymic nude mice where they grew to produce, at first, squamous epithelial cysts containing BPV-4 genomic DNA and, later, papillomas which were morphologically identical to those of cattle and which contained large amounts of replicating virus. The possible utility of this technique in assessing neutralising antibodies in vaccine development is discussed.

Prevalences of feline leukaemia virus and feline immunodeficiency virus infections in cats in Sydney.

OBJECTIVE: To determine prevalences of feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) infections in 'healthy' cats that, through acute misadventure or other circumstance, were presented to veterinary practitioners. Prevalences of FeLV and FIV in this population were compared to those in a population of predominantly sick cats. DESIGN AND PROCEDURES: Serum specimens were obtained over a 2-year period from 200 cats older than 1 year of age presented to veterinary clinics for routine procedures, including cat fight injuries or abscesses, vehicular trauma, neutering, dental scaling, vaccination, grooming or boarding. An additional 894 sera were obtained over approximately the same period from specimens submitted by veterinarians to a private clinical pathology laboratory, mainly from sick cars suspected of having immune dysfunction, but including some sera from healthy cats being screened prior to FeLV vaccination. FIV antibody and FeLV antigen were detected in samples using commercial enzyme immunoassays. RESULTS: Amongst 200 'healthy' cats, the prevalence of FeLV infection was 0 to 2%, and the prevalence of FIV was 6.5 to 7.5%, depending on the stringency of the criteria used to define positivity. FIV infection was significantly more prevalent in cats which resided in an inner city environment (P = 0.013). Of the 894 serum specimens submitted to the laboratory by practitioners, 11/761 (1.4%) were FeLV positive, while 148/711 (20.8%) were FIV positive. The prevalence of FIV was significantly higher in these predominantly 'sick' cats than in cats seen for routine veterinary procedures (P < 0.00001), while there was no difference in the prevalence of FeLV (P = 0.75) CONCLUSIONS: The prevalence of FeLV and FIV in healthy cats may have been substantially overestimated in some previous Australian surveys. FeLV infection would appear to be a rare cause of disease in Australian cats. The higher prevalence of FIV positivity in sick as opposed to healthy cats infers that FIV infection contributes to the development of disease.

Association between oral health status and retrovirus test results in cats.

OBJECTIVE: To determine associations between oral health status and seropositivity for FIV or FeLV in cats. DESIGN: Cross-sectional survey. ANIMALS: 5,179 cats. PROCEDURES: Veterinarians at veterinary clinics and animal shelters completed online training on oral conditions in cats and then scored oral health status of cats with no known history of vaccination against FIV. Age, sex, and results of an ELISA for retroviruses were recorded. Results were analyzed by means of standard logistic regression with binary outcome. RESULTS: Of 5,179 cats, 237 (4.6%) and 186 (3.6%) were seropositive for FIV and FeLV, respectively, and of these, 12 (0.2%) were seropositive for FIV and FeLV. Of all 5,179 cats, 1,073 (20.7%) had gingivitis, 576 (11.1%) had periodontitis, 203 (3.9%) had stomatitis, and 252 (4.9%) had other oral conditions (overall oral disease prevalence, 2,104/5,179 [40.6%]). Across all age categories, inflammatory oral disease was associated with a significantly higher risk of a positive test result for FIV, compared with the seropositivity risk associated with other oral diseases or no oral disease. Stomatitis was most highly associated with risk of FIV seropositivity. Cats with any oral inflammatory disease were more likely than orally healthy cats to have a positive test result for FeLV. Increasing age was associated with a higher prevalence of oral disease in retrovirus-seronegative cats. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory oral disease was associated with an increased risk of seropositivity for retroviruses in naturally infected cats. Therefore, retroviral status of cats with oral inflammatory disease should be determined and appropriate management initiated.

Survey of the feline leukemia virus infection status of cats in Southern Germany.

Most studies that investigate the prevalence of infections with feline leukemia virus (FeLV) are based on the detection of p27 antigen in blood, but they do not detect proviral DNA to identify the prevalence of regressive FeLV infections. The aim of the present study was to assess the prevalence and status of FeLV infection in cats in Southern Germany. P27 antigen enzyme-linked immunosorbent assay (ELISA), anti-p45 antibody ELISA, DNA polymerase chain reaction (PCR) of blood and RNA PCR of saliva were performed. Nine out of 495 cats were progressively (persistently) infected (1.8%) and six were regressively (latently) infected (1.2%). Cats with regressive infections are defined as cats that have been able to overcome antigenemia but provirus can be detected by PCR. Twenty-two unvaccinated cats likely had abortive infections (regressor cats), testing FeLV antigen- and provirus-negative but anti-p45 antibody-positive. Most of the FeLV-vaccinated cats did not have anti-FeLV antibodies. Both progressive, as well as regressive infections seem to be rare in Germany today.

Longitudinal patterns of viremia and oral shedding of rhesus rhadinovirus and retroperitoneal fibromatosis herpesviruses in age-structured captive breeding populations of rhesus Macaques (Macaca mulatta).

Rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), 2 closely related γ2 herpesviruses, are endemic in breeding populations of rhesus macaques at our institution. We previously reported significantly different prevalence levels, suggesting the transmission dynamics of RRV and RFHV differ with regard to viral shedding and infectivity. We designed a longitudinal study to further examine the previously observed differences between RRV and RFHV prevalence and the potential influence of age, season, and housing location on the same 90 rhesus macaques previously studied. Virus- and host-genome-specific real-time PCR assays were used to determine viral loads for both RRV and RFHV in blood and saliva samples collected at 6 time points over an 18-mo period. Proportions of positive animals and viral load in blood and saliva were compared between and within viruses by age group, location, and season by using 2-part longitudinal modeling with Bayesian inferences. Our results demonstrate that age and season are significant determinants, with age as the most significant factor analyzed, of viremia and oral shedding for both RRV and RFHV, and these pathogens exhibit distinctly different patterns of viremia and oral shedding over time within a single population.

Therapeutic effects of fig tree latex on bovine papillomatosis.

The effects of fig tree latex in treating teat papillomatosis in cow in comparison with salicylic acid were evaluated. For this purpose, 12 cows of 1-3 years of age (average 2.25) affected by teat papillomatosis were divided into three groups. In group A, four cows were treated by fig tree (Ficus carica) latex; in group B, four cows were treated with 10% salicylic acid solution and in group C, four cows were kept as control animals receiving no treatment. Animals in each treatment group received their treatment once every 5 days. In groups A and B, de-epithelialization and shrinking of the warts began from the fifth day of treatment and all the warts disappeared within 30 days. However, in the control group no changes in the number of warts were observed until day 15 but thereafter a number of warts disappeared spontaneously in some of the animals. Both salicylic acid and fig tree latex were evaluated as having similar therapeutic effects in treating teat papillomatosis in cow.

Persistent Epstein-Barr virus infection in the common marmoset (Callithrix jacchus).

Epstein-Barr virus (EBV) infection of the common marmoset causes long-term infection, with production of antibodies to virus-induced antigens, without clinical illness. Attempts to show the presence of EBV DNA in saliva of infected animals by PCR were initially unsuccessful, although slot-blot hybridization analysis demonstrated that viral DNA was present. Further investigations showed that most samples of pilocarpine-induced saliva, and 33% of the samples of whole mouth fluids (WMF) tested, were inhibitory to PCR. Similar results were found using human WMF. A method of assessing samples of marmoset WMF for the presence of EBV, by PCR using an EBV BamHI W probe, and removing inhibition with Chelex 100, is described. A total of 202 samples from 21 EBV infected, and seven non-infected animals was tested. Five seropositive animals shed virus on every occasion, and 15 intermittently. Two marmosets, infected as neonates, showed progressively increasing humoral responses to viral antigens, and shed virus on every occasion tested over 3 years. When mated with uninfected animals, the latter seroconverted 4 and 6 weeks later, respectively, and later shed virus into their WMF. The naturally infected animals were paired with naive marmosets, and were able to pass on infection. These results establish that long-term, permissive EBV infection occurs in the common marmoset, and demonstrate again the similarities in the response to EBV between marmoset and man.

Evaluation of a saliva test kit for feline leukemia virus antigen.

An enzyme-linked immunosorbent assay (ELISA) test kit for the detection of feline leukemia virus (FeLV) antigen in saliva was evaluated in 150 cats. Saliva and blood samples from all cats were tested for FeLV using the saliva ELISA kit and a plasma ELISA kit, respectively. These results were compared with indirect immunofluorescent antibody (IFA) testing of blood smears also obtained from each cat. The proportion of cats that tested positive were 10%, 7%, and 8% for each test, respectively. Using the IFA test as the gold standard, the saliva FeLV test had a sensitivity of 91.7% and specificity of 97.1%, while the plasma ELISA test had a sensitivity of 91.7% and specificity of 100%.

The epithelial neoplasm observed in the cultured coho salmon, Oncorhynchus kisutch.

The epithelial neoplasms were observed on the mouth of the cultured coho salmon, Oncorhynchus kisutch. Histopathologically, the tumors were formed to be proliferative epithelial cells, but no change was observed in other organs. The virus from this tumor was isolated in RTG-2 and CHSE-214 cells and developed the cytopathic effect which characterized to be the formation of syncytia and the migration of chromatin. This virus was neutralized with anti-Oncorhynchus masou virus (OMV) rabbit serum.

Adoptive immunotherapy of feline leukemia virus infection using autologous lymph node lymphocytes.

Adoptive immunotherapy using autologous cells expanded ex vivo from lymph nodes was examined in cats infected with the retrovirus feline leukemia virus (FeLV). Cells were obtained from popliteal lymph nodes from 18 FeLV-antigen-positive cats without complications; a mean of 6.2 x 10(7) cells were obtained. Lymph node cells were cultured with 600 IU/ml interleukin-2 (IL-2) for 7 days. Cells expanded 0.8- to 11-fold (mean, 2.7; median, 2.4); were 80% +/- 8.0% CD3+, 29% +/- 8.1% CD4+, and 41% +/- 7.0% CD8+, and exhibited cytolytic activity against FeLV-transformed FL74 cells. Sixteen cats received a single intravenous infusion of 0.13 to 3.9 x 10(8) cells. Cell infusion was well tolerated; fever developed approximately 1 hour postinfusion. Clinical activity, antiviral activity, or both was observed in 10 cats. Nine cats had clinical responses with improvement in weight, activity, appearance, or a combination of these that began 2 to 4 weeks after cell infusion and that lasted for up to 13 or more months. FeLV antigen became undetectable in 4 cats. These results indicate that adoptive immunotherapy using autologous lymph node cells, activated and expanded ex vivo in short-term cultures with low concentrations of IL-2, can modulate the course of a retroviral infection.