Adrian Philbey

Serological evidence for the presence of a calicivirus in Australian wild rabbits, Oryctolagus cuniculus, before the introduction of rabbit haemorrhagic disease virus (RHDV): its potential influence on the specificity of a competitive ELISA for RHDV

The objective of the study was to determine for Australian wild rabbits the specificity of a competitive enzyme-linked immunosorbent assay (cELISA) developed in Italy for detecting antibodies to RHDV. Analysis of 657 sera collected before the arrival of RHDV (pre-RHD) indicated that between 17 and 38% appeared to give false positive results depending on the cut-off criteria used. The finding of pre-RHD sera reacting positively in the cELISA prompted the testing of sera in a cELISA using as antigen smooth forms of RHDV (cELISA-sf) and a solid-phase ELISA (spELISA), both of which detect reactivity to an epitope shared by the lagomorph caliciviruses. Testing of a subset of the pre-RHD sera in the cELISA-sf and the spELISA revealed that 86 and 91%, respectively, were positive. Similar results were obtained for a set of sera collected pre-RHD in the Australian Capital Territory (ACT). Sera collected from wild-stock rabbits born and raised in isolation in an animal house in the ACT were all negative in the cELISA, 6% were positive in the cELISA-sf and 13% in the spELISA. It was concluded that a calicivirus related to RHDV and European brown hare syndrome virus (EBHSV) was present in the rabbit population before the arrival of RHDV, and may still be present. The potential consequences of these findings for epidemiological studies on RHD in Australia are discussed.

Analysis of Integration Sites of Jaagsiekte Sheep Retrovirus in Ovine Pulmonary Adenocarcinoma

ABSTRACT Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.

Lymphocyte deficiency limits Epstein-Barr virus latent membrane protein 1 induced chronic inflammation and carcinogenic pathology in vivo

BackgroundThe importance of the malignant cell environment to its growth and survival is becoming increasingly apparent, with dynamic cross talk between the neoplastic cell, the leukocyte infiltrate and the stroma. Most cancers are accompanied by leukocyte infiltration which, contrary to an anticipated immuno-protective role, could be contributing to tumour development and cancer progression. Epstein-Barr virus (EBV) associated cancers, including nasopharyngeal carcinoma and Hodgkins Disease, show a considerable leukocyte infiltration which surrounds the neoplastic cells, raising the questions as to what role these cells play in either restricting or supporting the tumour and what draws the cells into the tumour. In order to begin to address this we have studied a transgenic model of multistage carcinogenesis with epithelial expression of the EBV primary oncoprotein, latent membrane protein 1 (LMP1). LMP1 is expressed particularly in the skin, which develops a hyperplastic pathology soon after birth.ResultsThe pathology advances with time leading to erosive dermatitis which is inflamed with a mixed infiltrate involving activated CD8+ T-cells, CD4+ T-cells including CD4+/CD25+/FoxP3+ Treg cells, mast cells and neutrophils. Also significant dermal deposition of immunoglobulin-G (IgG) is observed as the pathology advances. Along with NF-kappaB activation, STAT3, a central factor in inflammation regulation, is activated in the transgenic tissue. Several inflammatory factors are subsequently upregulated, notably CD30 and its ligand CD153, also leukocyte trafficking factors including CXCL10, CXCL13, L-selectin and TGFβ1, and inflammatory cytokines including IL-1β, IL-3 and the murine IL-8 analogues CXCL1, CXCL2 and CXCL5-6, amongst others. The crucial role of mature T- and/or B-lymphocytes in the advancing pathology is demonstrated by their elimination, which precludes mast cell infiltration and limits the pathology to an early, benign stage.ConclusionsLMP1 can lead to the activation of several key factors mediating proliferation, angiogenesis and inflammation in vivo. With the initiation of an inflammatory programme, leukocyte recruitment follows which then itself contributes to the progressing pathology in these transgenic mice, with a pivotal role for B-and/or T-cells in the process. The model suggests a basis for the leukocyte infiltrate observed in EBV-associated cancer and its supporting role, as well as potential points for therapeutic intervention.

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERα and p53.

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor alpha (ERalpha), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERalpha expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.

Lymphoid hyperplasia and lymphoma in transgenic mice expressing the small non-coding RNA, EBER1 of Epstein-Barr virus

Background Non-coding RNAs have critical functions in diverse biological processes, particularly in gene regulation. Viruses, like their host cells, employ such functional RNAs and the human cancer associated Epstein-Barr virus (EBV) is no exception. Nearly all EBV associated tumours express the EBV small, non-coding RNAs (EBERs) 1 and 2, however their role in viral pathogenesis remains largely obscure. Methodology/Principal Findings To investigate the action of EBER1 in vivo, we produced ten transgenic mouse lines expressing EBER1 in the lymphoid compartment using the mouse immunoglobulin heavy chain intronic enhancer Eμ. Mice of several of these EμEBER1 lines developed lymphoid hyperplasia which in some cases proceeded to B cell malignancy. The hallmark of the transgenic phenotype is enlargement of the spleen and mesenteric lymph nodes and in some cases enlargement of the thymus, liver and peripheral lymph nodes. The tumours were found to be of B cell origin and showed clonal IgH rearrangements. In order to explore if EBER1 would cooperate with c-Myc (deregulated in Burkitts lymphoma) to accelerate lymphomagenesis, a cross-breeding study was undertaken with EμEBER1 and EμMyc mice. While no significant reduction in latency to lymphoma onset was observed in bi-transgenic mice, c-Myc induction was detected in some EμEBER1 single transgenic tumours, indicative of a functional cooperation. Conclusions/Significance This study is the first to describe the in vivo expression of a polymerase III, non-coding viral gene and demonstrate its oncogenic potential. The data suggest that EBER1 plays an oncogenic role in EBV associated malignant disease.

Histopathologic, immunohistochemical, and cytologic analysis of feline myeloma-related disorders: further evidence for primary extramedullary development in the cat.

Feline myeloma-related disorders (MRD) are rare neoplasms of plasma cells. The multistep transformation model of myeloma in humans is based on the premise that plasma cells undergo neoplastic transformation primarily within the intramedullary compartment and that over time they become poorly differentiated and metastasize to extramedullary locations. Historically, diagnostic criteria used for human multiple myeloma have been applied to the cat, with the assumption that feline MRD commonly arises in the intramedullary compartment. Our objectives were to describe the features of feline MRD confirmed by cytology, histopathology, histochemistry, and immunohistochemistry and to categorize these tumors. A priori hypotheses were 1) tumor category predicts survival and 2) cats with well-differentiated tumors commonly have extramedullary involvement in contrast to human myeloma patients. This multicenter, retrospective study identified 26 MRD cases. There was good agreement between histopathologic and cytologic tumor categorization. Histochemistry and immunohistochemistry were shown to be valuable adjunct tests in the diagnosis of MRD. Cats with well-differentiated tumors had increased median survival relative to those with poorly differentiated tumors (254 versus 14 days). We have reported that marked extramedullary involvement at initial clinical presentation is significantly more common in the cat than in human MRD patients. In this study, we demonstrate that cats with well-differentiated tumors more commonly have extramedullary involvement than human myeloma patients with well-differentiated tumors (90% versus 20%, P < 0.0002). These results contrast strongly with the human myeloma model of primary intramedullary neoplastic transformation and suggest that primary extramedullary neoplastic transformation may be more common in feline MRD.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

OBJECTIVE To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. DESIGN Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). PROCEDURE Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. RESULTS Neutralising antibodies (VNT titres > or = 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. CONCLUSION Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Clinicopathological presentation of cardiac disease in cattle and its impact on decision making

The records of 116 cattle suffering from cardiac disease were examined retrospectively. On the basis of the results of postmortem examinations there were 52 cases of endocarditis, 39 of pericarditis and 25 congenital cardiac defects. The most useful clinical tool for differentiating between these conditions was auscultation of the heart. The cases of pericarditis were characterised by muffled heart sounds, and the cases of endocarditis and congenital cardiac defects were characterised by a cardiac murmur. Endocarditis could be differentiated from congenital cardiac defects by the presence of a jugular pulse, venous distension, oedema, a reduced appetite, pain and polyarthritis, whereas congenital defects were associated with conformational abnormalities. These two conditions could also be differentiated by differences in the plasma sodium concentration, the albumin:globulin ratio, red blood cell count, lymphocyte count and haematocrit. The ability to differentiate between these three groups of cardiac diseases can help the veterinary practitioner in deciding whether treatment, economic salvage (slaughter for human consumption) or disposal (slaughter not for human consumption) is likely to be the best option.

Infectious canine hepatitis in red foxes (Vulpes vulpes) in the United Kingdom

The pathological findings are described in three cases of infectious canine hepatitis in free-ranging red foxes (Vulpes vulpes) in England. The foxes died after short periods of clinical illness. Mild jaundice and hepatic congestion were evident grossly. On histopathological examination, intranuclear inclusion bodies were visible in hepatocytes, in association with hepatocyte dissociation and necrosis, as well as in renal glomeruli, renal tubular epithelial cells and vascular endothelial cells. Canine adenovirus type 1 (CAV-1) was isolated from all three foxes. In a serological study, antibodies to CAV-1 were detected in tissue fluid extracts taken from 11 of 58 (19 per cent) frozen red fox carcases from England and Scotland.

Isolation of avian strains of Salmonella enterica serovar Typhimurium from cats with enteric disease in the United Kingdom

AVIAN-ADAPTED strains of Salmonella enterica serovar Typhimurium have been detected in wild birds, particularly finches (family Fringillidae), in Europe, Scandinavia and North America ([Daoust and others 2000][1], [Tauni and Osterlund 2000][2], [Pennycott and others 2006][3]). In the uk, these