Neuronal and astrocytic involvement in striped dolphins (Stenella coeruleoalba) with morbilliviral encephalitis.

Dolphin morbillivirus (DMV), a highly pathogenic agent, may cause peculiar, "brain-only" forms of infection (BOFDI), in which viral antigen and/or genome is found exclusively in the brain from striped dolphins (Stenella coeruleoalba). These BOFDIs show morphopathological similarities with subacute sclerosing panencephalitis and old dog encephalitis (ODE) in measles virus-infected patients and in canine distemper virus-infected dogs, respectively. The brain tissue from 3 BOFDI-affected striped dolphins was investigated by means of double labelling-indirect immunofluorescence (DL-IIF) and ultrastructurally, in order to characterize the DMV-targeted neuronal and non-neuronal cell populations, along with the associated submicroscopic findings. Viral colonization of calbindin-immunoreactive (IR) and nitric oxide synthase-IR neurons was detected in the cerebral parenchyma from the 3 DMV-infected dolphins under study, associated with nuclear (chromatin) and cytoplasmic (mitochondrial) ultrastructural changes. Furthermore, a limited viral targeting of brain astrocytes was found in these animals, all of which exhibited a prominent astrogliosis/astrocytosis. To the best of our knowledge, those herein reported should be the first submicroscopic pathology and neuropathogenetic data about BOFDI in striped dolphins. In this respect, the marked astrogliosis/astrocytosis and the low viral colonization of brain astrocytes in the 3 DMV-infected dolphins under investigation are of interest from the comparative pathology and viral neuropathogenesis standpoints, when compared with ODE-affected dogs, in whose brain a non-cytolytic, astrocyte-to-astrocyte infectious spread has been recently documented. Further studies aimed at characterizing the complex DMV-host interactions in BOFDI-affected striped dolphins are needed.

Heterogeneous shedding of Brucella abortus in milk and its effect on the control of animal brucellosis.

AIMS: To ascertain whether in Brucella abortus-infected water buffalo herds, the number of newly infected animals could be reduced by culling superspreaders (the animals secreting > or =10(4) CFU per ml of milk). METHODS AND RESULTS: The number of B. abortus present in the milk (CFU per ml) from 500 water buffaloes was measured by the culture. Each animal was tested three times, at one month intervals. The presence or the absence of B. abortus in each milk sample was confirmed by PCR. A majority of infected animals shed the pathogen at a low level (< or =10(3) CFU ml(-1)). However, a few infected individuals (superspreaders) shed large numbers of B. abortus (> or =10(4) CFU ml(-1)). Quantitative PCR of B. abortus positive milk samples gave comparable results to culture. Culling of the superspreaders was sufficient to arrest the spread of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach described here can reduce significantly the cost of controlling brucellosis. Culture and quantitative PCR tests identify superspreaders and, compared with the serological tests in use to detect brucellosis, provide also a more accurate estimate of the disease incidence.

Activated platelet-derived growth factor beta receptor expression, PI3K-AKT pathway molecular analysis, and transforming signals in equine sarcoids.

The equine sarcoid is the most common dermatologic neoplasm reported in horses. Bovine papillomavirus (BPV) types 1 and 2 are associated with sarcoids, in which the expression of the major transforming oncoprotein (E5) is often recorded. The transformation activity of the virus is due to the binding of the E5 to the platelet-derived growth factor beta receptor (PDGFbeta-r). In the present study, we show by Western blot in 4 sarcoid samples and 3 normal equine skin samples that the PDGFbeta-r is more phosphorylated in sarcoid tissue than in normal skin (P < .001). Furthermore, the physical interaction between the activated receptor and the 85-kDa regulatory subunit (p85) of phosphatidylinositol-3-kinase (PI3K) is shown by coimmunoprecipitation. The PI3K-AKT-cyclin D3 molecular pathway downstream to the activation of the PDGFbeta-r is shown to be expressed, and the amount of the investigated molecules is higher than normal (P < .001), suggesting an activation of these effectors in sarcoids. Further, we demonstrate that phospho-JNK and phospho-JUN are more expressed in sarcoids than in normal skin. Our results provide new insights into the pathogenesis of equine sarcoids and support the validity of this in-vivo model to further characterize the molecular pathways underlying BPV E5-induced carcinogenesis.

Short communication: Detection of human Torque teno virus in the milk of water buffaloes (Bubalus bubalis).

Forty-four raw milk and 15 serum samples from 44 healthy water buffaloes reared in Caserta, southern Italy, the most important region in Europe for buffalo breeding, were examined to evaluate the presence of Torque teno viruses (TTV) using molecular tools. Furthermore, 8 pooled pasteurized milk samples (from dairy factories having excellent sanitary conditions) and 6 Mozzarella cheese samples were also tested. Four of the cheese samples were commercial Mozzarella cheese; the remaining 2 were prepared with TTV-containing milk. Human TTV were detected and confirmed by sequencing in 7 samples of milk (approximately 16%). No TTV were found in serum, pooled pasteurized milk, or Mozzarella cheese samples. The samples of Mozzarella cheese prepared with TTV-containing milk did not show any presence of TTV, which provides evidence that standard methodological procedures to prepare Mozzarella cheese seem to affect viral structure, making this food fit for human consumption. The 7 TTV species from water buffaloes were identified as genotypes corresponding to the tth31 (3 cases), sle 1981, sle 2031, and NLC030 (2 cases each) human isolates. Although cross-species infection may occur, detection of TTV DNA in milk but not in serum led us to believe that its presence could be due to human contamination rather than a true infection. Finally, the mode of transmission of TTV has not been determined. Contaminated of the food chain with TTV may be a potential risk for human health, representing one of the multiple routes of infection.

Expression of platelet-derived growth factor-beta receptor and bovine papillomavirus E5 and E7 oncoproteins in equine sarcoid.

Equine sarcoids are benign fibroblastic skin tumours that are recognized throughout the world. Infection with bovine papillomavirus (BPV) types 1 and 2 has been implicated as a major factor in disease development; however, the cellular mechanisms underlying fibroblast transformation remain poorly defined. The present study further characterizes aspects of the association with BPV in 15 equine sarcoids. BPV DNA was demonstrated in 12/15 tumours collected from different areas of Italy. Nine of these 12 tumours expressed the BPV oncoproteins E5 and E7, but these oncoproteins were not expressed by normal equine cells. The BPV E5 protein is known to bind to the platelet-derived growth factor-beta receptor (PDGF-betaR) and this molecule was expressed by 11 of the 12 sarcoids in which E5 was demonstrated. These findings add further weight to the theory that BPV and the PDGF-betaR may have a role in the pathogenesis of this disease.

Lymphoepithelioma-like carcinoma of the urinary bladder in a cow associated with bovine papillomavirus type-2.

Lymphoepithelioma-like carcinoma (LELCA) of the urinary bladder is reported in a 7-year-old cow that had grazed pasture rich in bracken fern and had suffered from severe intermittent haematuria from 3 to 4 years of age. On necropsy examination there were multiple haemorrhagic foci scattered over the mucosal surface of the urinary bladder. Microscopically there were nests, cords and sheets of neoplastic cells infiltrating the lamina propria and muscularis propria. These had a syncytial appearance with ill-defined cytoplasmic borders, large nuclei and prominent nucleoli. There was a prominent associated inflammatory infiltrate comprising lymphocytes and plasma cells with sparse histiocytes and granulocytes. Immunohistochemically, LELCA cells expressed cytokeratin but not vimentin. The LELCA was focally admixed with a concomitant papillary high-grade carcinoma that also infiltrated the lamina propria. A diffuse carcinoma in situ was also present. Bovine papillomavirus type-2 (BPV-2) DNA was amplified from frozen neoplastic tissue and from selected areas of formalin-fixed, paraffin wax-embedded tissue obtained by laser capture microdissection. Microbiological culture of a urine sample resulted in isolation of Weeksella virosa, Rhizobium radiobacter and Staphylococcus warneri. Flow cytometric analysis performed on blood mononuclear cells revealed down-regulation of a panel of markers including CD3, CD4, CD8alpha, CD45, MHC class I and MHC class II (HLA-DRalpha, HLA-DQ, HLA-DP). This report extends the spectrum of neoplastic urothelial lesions described in cattle and provides further evidence that some features of these tumours are similar to human counterparts.

Association of bovine papillomavirus type-2 and urinary bladder tumours in cattle from Romania.

In cattle, bracken fern toxicity is characterized by the presence of haematuria and tumours of the urinary bladder of both epithelial and mesenchymal origin. This syndrome is known as chronic enzootic hematuria (CEH) and is also present in Romania. From January 2006 to April 2007, 90 urinary bladders from slaughtered cows originating from hill-mountain area of Neamt county (Romania), where CEH is endemic, were collected. All samples were histologically examined and Bovine papillomavirus type 2 (BPV-2) DNA was detected by polymerase chain reaction (PCR) analysis in 68% of the analyzed tumours samples. BPV-2 positive urinary bladder tumours were also immunohistochemically analysed for the expression of the major viral oncoprotein E5. We found the expression of E5 intracytoplasmically with a typical juxtanuclear pattern. E5 expression was not observed in normal mucosa, suggesting a causal role for this protein in the neoplastic process. This is the first report of BPV-2 infection in Eastern European country, confirming the role of BPV-2 in naturally occurring bovine urothelial carcinogenesis.

Detection of bovine Deltapapillomavirus DNA in peripheral blood of healthy sheep (Ovis aries).

Blood samples from 65 sheep were tested for the presence of bovine Deltapapillomavirus (δPVs) DNA. The sheep were divided into three groups. Sheep in groups 1 and 2 were from Sardinia and Campania, respectively, and were in contact with cattle and grazed on lands contaminated with bracken fern. Sheep in Group 3 lived in closed pens and had no contact with cattle. These sheep were fed hay that did not contain bracken fern. Bovine δPV E5 DNA was detected in blood from 24 of 27 (89%) sheep in Group 1. A single bovine δPV type was detected in the blood from nine (33%) sheep, including the detection of bovine δPV-1 DNA in four sheep, bovine δPV-2 in four and δPV-13 in one sheep. Two δPV types were detected in 33% of the sheep, and three bovine δPV types were detected in 22% of the sheep. Bovine δPVs were detected in 17 of 20 (85%) sheep from Group 2. The detection rate by a single δPV type was 40% with just δPV-1 DNA amplified from two, just δPV-2 DNA from four, and just δPV-13 DNA from two sheep. Two and three δPVs were detected in 30% and 15%, respectively. All sequenced amplicons showed a 100% identity with papillomaviral E5 DNA deposited in GenBank. Bovine δPV-14 DNA sequences were not detected from any sheep. No bovine δPV DNA was revealed in blood samples from sheep in Group 3. The detection of bovine δPV DNA in the blood of sheep means that sheep may be able to be infected by these PVs. This suggests that bovine δPVs could potentially be a previously unrecognized cause of disease in sheep. Furthermore, it is possible that sheep could act as a reservoir for these viruses.

Bovine papillomavirus E5 oncoprotein binds to the activated form of the platelet-derived growth factor beta receptor in naturally occurring bovine urinary bladder tumours.

Studies regarding the functions of the bovine papillomavirus (BPV) E5 oncoprotein in vivo are lacking and no E5-mediated mechanism underlying epithelial carcinogenesis is known. We have shown that BPV-2 DNA is present in the majority of naturally occurring urinary bladder tumours of cattle and that E5 is expressed in the cancer cells. Here we show that the interaction between the platelet-derived growth factor (PDGF) beta receptor and BPV E5, described in vitro in cultured cells, takes place in vivo in bovine urinary bladder cancers. In these cancers, E5 and PDGF beta receptor colocalize, as shown by confocal microscopy, and physically interact, as shown by coimmunoprecipitation. Furthermore, the PDGF beta receptor associated with E5 is highly phosphorylated, suggesting the functional activation of the receptor upon E5 interaction. Our results demonstrate, for the first time, that E5-PDGF beta receptor interaction occurs during the natural history of bovine urinary bladder tumours, suggesting an important role for E5 in carcinogenesis. Finally, the system provides a suitable animal model of papillomavirus-associated cancer to test therapeutic vaccination against E5. Successful bladder tumour regression would provide a valuable model for therapeutic vaccination against papillomavirus-associated tumours.

Sialic acid and GM3 ganglioside expression in papillomavirus-associated urinary bladder tumours of cattle with chronic enzootic haematuria.

This study was based on 30 papillomavirus-associated urinary bladder tumours from cattle with chronic haematuria, the animals having been kept since birth on pasture rich in bracken fern. The ganglioside content was assessed and compared with that of normal bovine urinary bladders, which was shown to be 28.6+/-3.3 (mean+/-SD) microg of lipid-bound sialic acid per gram of fresh tissue. In neoplastic bladder samples this value was higher but variable (120.9+/-80.6 in benign tumours, and 94.7+/-45.7 in malignant tumours). The main ganglioside, GM3, represented ca 75% of the total ganglioside mixture in normal tissues and 50-80% in tumour samples. GM1, GM2, GD1a, GD3 and FucGM1 were found as minor components. The study suggested that GM3 ganglioside may have a crucial role in "downregulation" of the metastatic potential of bovine urothelial cancers.

Sigma 2 receptor expression levels in blood and bladder from healthy and bladder cancer cattle.

The expression of sigma-2 receptor (S2R) was assayed in blood and bladder samples from healthy cattle and in blood and bladder of cattle with deltapapillomavirus-associated urothelial tumors. Samples of bladder from cattle with neoplasia had significantly higher S2R than samples of bladder from healthy cattle (95% CI 0.31-0.82, P < 0.05). In addition, significantly higher S2R was detected in the blood of cattle with bladder cancer than blood from healthy cattle (95% CI 0.22-0.41, P < 0.05). The results provide evidence that increased expression of SR2 in blood could be useful as circulating biomarker for bladder cancer in cattle. PGRMC1 protein levels were also found to be increased in blood and bladder from cattle with cancer and increased expression of PGRMC1 transcripts was detected by quantitative real time PCR in samples from cattle neoplasia. Furthermore, electron microscopy revealed phagophores and numerous autophagosomes, ultrastructural hallmark of autophagy.

Selection of an Escherichia coli O157:H7 bacteriophage for persistence in the circulatory system of mice infected experimentally.

A bacteriophage lytic for Escherichia coli O157:H7 was isolated from bovine manure. Following in-vivo selection, the phage acquired the capacity to persist in the circulatory system of mice for at least 38 days. When mice were infected experimentally with E. coli O157:H7 (10(7) CFU/mouse), simultaneous injection of the mice with phage (10(8) PFU/mouse) cleared E. coli O157:H7 from the mice within 48 h.

Bovine Papillomavirus Type 13 Expression in the Urothelial Bladder Tumours of Cattle.

Bovine papillomavirus type 13 (BPV-13), a novel Deltapapillomavirus, has been found associated with urothelial tumours of the urinary bladder of cattle grazing on lands infested with bracken fern. BPV-13 was detected in 28 of 39 urothelial tumours. Diagnosis was based on sequencing of L1 and E5 amplicons from tumour samples. The nucleotide sequences generated from these amplicons showed a 100% homology with the sequences of BPV-13 L1 and E5 DNA found in Brazil from a fibropapilloma of the ear in a cow and from equine sarcoids in two horses. GenBank accession number of our representative BPV-13 sequences is JQ798171.1. Furthermore, mRNA encoding BPV-13 E5 oncoprotein was also documented, and its expression was also shown by immunohistochemistry and immunofluorescence in the basal and suprabasal urothelial tumour cells. In twenty-three tumours, BPV-13 was simultaneously found with BPV-2, a Deltapapillomavirus genus, species 4. The latter virus was detected by amplifying and sequencing a 154-bp-sized DNA fragment of BPV-2 E5. In addition, BPV-13 by itself was seen to be expressed in five BPV-2-negative urothelial tumours. This study shows that BPV-13 is present in urothelial tumour cells thus sharing biological properties with BPV-1 and BPV-2. Although further studies are needed, BPV-13 appears to be another worldwide infectious agent responsible for a distressing disease causing severe economic losses in cattle industry.

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells.

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.

Bovine papillomavirus type 4 in oesophageal papillomas of cattle from the south of Italy.

Oesophageal papillomas are known to occur in cattle infected with bovine papillomavirus type 4 (BPV-4), and BPV-4 papillomas may undergo malignant progression in cattle that feed on bracken fern. In the south of Italy, where bracken fern is common, examination of 1133 slaughterhouse cattle aged 4-12 years revealed oesophageal lesions (single or multiple peduncuolated proliferations, or mucosal thickening) in 147 (13%). These two types of lesion were consistent with exophytic and inverted papilloma, respectively. BPV-4 was detected by polymerase chain reaction (PCR) analysis in >60% of the samples in which oesophageal papilloma was diagnosed histopathologically. Nucleotide sequencing of the PCR amplicons confirmed the presence of BPV-4 in the papillomas. This is the first report of such infections in a European country other than Britain.

Chromosome aberrations in cattle with chronic enzootic haematuria.

Chromosomal aberrations were investigated in 56 cattle with chronic enzootic haematuria (CEH) raised on pastures giving access to bracken fern. Of these animals, 27 were slaughtered and showed neoplastic lesions of the urinary bladder. Tumour tissue from 11 of the 27 cattle contained bovine papillomavirus type 2 (BPV-2) DNA. Increased numbers of chromosomal aberrations were seen in all animals with CEH, as compared with 30 control cattle that had had no access to bracken fern. The highest clastogenic effect was observed in cattle with urinary bladder cancer and evidence of BPV-2 DNA, suggesting that BPV-2 and bracken fern act synergistically in the production of chromosomal instability. In 19 of 20 animals with CEH, two bracken fern toxic compounds (quercitin and ptaquiloside) were demonstrated in urine, serum and milk.

Efficacy of an inactivated canine coronavirus vaccine in pups.

The efficacy of an inactivated CCoV vaccine (Duramune PC) was evaluated in four pups. Two dogs were maintained non-vaccinated. Ten days after the booster shot all the pups were challenged with a field CCoV strain administered by oro-nasal route. The vaccinated pups did not display clinical signs and shed the challenge-virus for 11.25 days, evaluated by virus isolation, and 13.5 days, evaluated by PCR assay. The two non vaccinated pups displayed mild diarrhoea at day post-challenge 4 and shed the challenge-virus for 14 and 15 days respectively, by virus isolation, and for 22 and 24 days respectively, by PCR assay.

Clotting profile in cattle showing chronic enzootic haematuria (CEH) and bladder neoplasms.

Primary haemostasis (bleeding and blood clotting time), activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin III (ATIII), protein C, protein S, fibrinogen and D-dimer were determined in 13 cattle affected by chronic enzootic haematuria (CEH) and bladder neoplasms and 10 healthy cattle (control group). Increases in antithrombin III and protein S activities (P<0.01) and protein C and fibrinogen plasma levels (P<0.05) were observed in sick animals, while activated partial thromboplastin time, prothrombin time, and D-dimer did not show significant differences when compared to healthy animals. The clotting profile observed does not seem responsible for the chronic bleeding typical of CEH. The observed modification of some coagulation markers may derive from multiple interactions among cancer, inflammation and viral infection status typical of this syndrome.

Phosphatidylinositol-3-kinase-AKT pathway, phospho-JUN and phospho-JNK expression in spontaneously arising bovine urinary bladder tumours.

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). E5, the major BPV-1/2 oncoprotein, binds to the activated platelet-derived growth factor beta receptor (pPDGF-betaR), inducing cell transformation in vitro and spontaneously arising urinary bladder tumours. The aim of this study was to assess whether the 85 kDa regulatory subunit (p85) of the phosphatidylinositol-3-kinase (PI3K)-AKT pathway and other transforming signals phospho-JUN (pJUN) and phospho-JUN N-terminal kinases (pJNK) may be important in the development of BPV-associated urothelial carcinomas. A physical interaction between the pPDGF-betaR and PI3K was shown in four tumours and two samples of normal bladder tissue by co-immunoprecipitation and western blotting. There was greater expression of the PI3K-AKT-cyclin D3 molecular pathway downstream to the activation of pPDGF-betaR in neoplastic compared with normal tissue. pJNK and pJUN were overexpressed in samples of tumour compared with normal mucosal tissue. These findings provide new insights into the aetiopathogenic mechanisms underlying naturally occurring bovine urothelial carcinogenesis and contribute to understanding of the role of E5 oncoprotein in naturally occurring tumorigenesis.

Sigma-2 receptor expression in bovine papillomavirus-associated urinary bladder tumours.

The expression of sigma-2 receptors was investigated in nine urothelial tumours of the urinary bladder of cattle. Each tumour was associated with the presence of DNA of bovine papillomavirus type-2 (BPV-2) and expression of the E5 viral oncoprotein. Five tumours were classified as low-grade carcinoma on the basis of morphological criteria and calculation of mean nuclear area (MNA) and mean nuclear perimeter (MNP). Four tumours were classified as high-grade carcinoma. Sigma-2 receptors were overexpressed in both types of carcinoma. In control normal bovine bladder tissue the density of receptors (expressed as the B(max)) was 0.37 pmol/mg of protein. Low-grade carcinomas had a mean B(max) of 1.37+/-0.32 pmol/mg of protein (range 1.03-1.86) and in high-grade carcinomas the mean B(max) was 10.9+/-2.8 pmol/mg of protein (range 8.2-14). The difference in B(max) between low- and high-grade carcinomas was statistically significant (P=0.0001).