Prevalence of antibodies to bovine viral diarrhoea virus in bulk tank milk and associated risk factors in Scottish dairy herds.

Bulk tank milk samples were collected from 374 dairy farms in Scotland in 2007/2008 along with questionnaire data relating to the management of the farm. Milk samples were tested for antibodies to bovine viral diarrhoea virus (BVDV) using a commercially available (Svanova) kit and percentage positivity scores calculated according to the manufacturer's guidelines. There were 220 farms that did not routinely vaccinate for bovine viral diarrhoea (BVD), and these were distributed according to the Swedish BVD eradication classes as 12.7 per cent, 22.3 per cent, 44.5 per cent and 20.5 per cent for Classes 0, 1, 2 and 3, respectively. A more sophisticated statistical method (finite mixture modelling) which does not depend on arbitrary thresholds and categories suggested a 73 per cent prevalence of herds with high mean levels of antibodies. Risk factor analysis suggested that routine vaccination for BVD, suspicion of BVD, housing of pregnant cows with calves, total number of cows and the proportion of cows that were dry were all associated with increased BVDV antibodies in bulk milk. The inclusion of BVD within the farm's health plan was associated with decreased BVDV antibodies in the bulk milk.

Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs.

(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [corrected] (HPMPC, cidofovir, CDV, Vistide) is an acyclic nucleoside analogue with a potent and selective activity against a broad spectrum of DNA viruses including the poxviruses. In this study we present the results of different treatment regimens in lambs experimentally infected with orf virus with different cidofovir formulations prepared in Beeler basis and Unguentum M. Our results show that choice of excipient, concentration of codofovir [corrected] and treatment regimen were all important to the clinical outcome of the therapy. Whilst one particular regimen appeared to exacerbate the lesion, treatment with 1% (w/v) cidofovir cream, prepared in Beeler basis, for 4 consecutive days did result in milder lesions that resolved in milder lesions that resolved [corrected] more quickly than untreated lesions. Furthermore the scabs of the treated animals contained significantly lower amounts of viable virus meaning there should be less contamination of the environment with virus than would normally occur.

Pestiviruses in wild animals.

Pestiviruses are not strictly host-species specific and can infect not only domestic but also wild animals. The most important pestivirus, CSFV, infects domestic pigs and wild boars, which may cause a major problem for successful CSFV eradication programmes. Mainly BVDV specific antibodies have been reported in captive and free-living animals. Virus has been isolated from some of these animal species, but since BVDV can contaminate cell cultures and foetal calf serum, early reports of BVDV isolation have to be considered with caution. Genetic typing of early pestivirus isolates from wild species revealed that the majority were BVDV-1. Of the pestiviruses identified so far three species (CSFV, BVDV-1, giraffe pestivirus) and three genotypes (BDV-2, BDV-4, pronghorn) appear to circulate in wildlife animal populations. The potential for pestiviruses to spread between farm animals and free-living animals is discussed as are epidemiological and technical problems, and the future direction of research.

Detection of Louping ill virus in clinical specimens from mammals and birds using TaqMan RT-PCR.

The identification of Louping ill virus (LIV) in clinical specimens has been routinely achieved by virus isolation using susceptible pig kidney cells and subsequent serological analysis. While this method is sensitive and detects infectious virus, it is relatively labour intensive and time-consuming. In view of the veterinary and potential medical importance of LIV, a rapid and precise detection method for routine use that employs the TaqMan reverse transcription polymerase chain reaction (RT-PCR) has been developed to detect LIV RNA extracted from field samples. The TaqMan assay was evaluated against virus isolation using 22 cell culture grown LIV isolates, which had previously been partially characterised by sequencing, and material from 63 suspect field cases. Histopathological and/or serological reports were available for 39 of the suspect cases, providing additional diagnostic information to evaluate the results obtained from the TaqMan RT-PCR assay. The TaqMan assay was as sensitive as the cell culture infectious virus assay currently used and had the advantage that it was able to detect LIV in clinical specimens from which infectious virus could not be isolated possibly due to the presence of high levels of LIV antibody.

Development of an enzyme-linked immunosorbent assay for the detection of antibodies against malignant catarrhal fever viruses in cattle serum.

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cell-associated herpesviruses of the subfamily gammaherpesvirinae. In this study, an ELISA (WC11-ELISA) was developed to detect antibody to malignant catarrhal fever virus (MCFV) in cattle serum and compared to the commercially produced competitive-inhibition ELISA (CI-ELISA). Crude lysate antigen from alcelaphine herpesvirus-1 strain WC11 was bound to 96-well microplates and used to capture antibodies to MCFV. Dilutions of test sera were added to wells containing bound MCF antigen and control wells containing uninfected cell lysates. A horseradish peroxidase-labelled rabbit-anti-bovine IgG conjugate detected antibodies to MCF, and the results were expressed as absorbance readings at 450 nm. Samples were selected blind from cattle sera which had been sent to the laboratory for diagnostic testing for MCFV antibodies and were tested in both the WC11-ELISA and the CI-ELISA. Good agreement between the WC11-ELISA and CI-ELISA test (k=0.86, n=95) results was found.

Development of a real time RT-PCR to detect and type ovine pestiviruses.

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp.

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.

Recent isolates of bovine respiratory syncytial virus from Britain are more closely related to isolates from USA than to earlier British and current mainland European isolates.

Eight isolates of Bovine respiratory syncytial virus (BRSV) were made from calves with severe respiratory disease on seven farms in one region of Britain. Genetic analysis of the viruses showed that seven, which were isolated between 1997 and 1999 were almost identical and were distinguishable from an earlier 1991 isolate. When compared with the available sequences of BRSVs from other countries, the recent British isolates were more closely related to US isolates than to earlier British and current mainland European isolates.

Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure.

The first report of a vascular endothelial growth factor (VEGF)-like gene in Orf virus included the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41.1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates of Orf virus derived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30.8% with an average amino acid identity between pairs of NZ2-like sequences of 86.1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.

Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions.

In sheep infected with the parapoxvirus orf virus, primary infection orf skin lesions developed and resolved within 8 weeks. Reinfection lesions were smaller and resolved within 3 weeks. The host response in the skin was characterized by an accumulation of neutrophils, dendritic cells, CD4+ T cells, CD8+ T cells, B cells and T19+ gammadelta T cells. The magnitude of this accumulation paralleled orf virus replication in the skin. In situ hybridization was used to detect cells expressing interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) mRNAs in orf skin. Cells expressing IL-4 mRNA were not detected at any time after infection. Cells expressing IFN-gamma mRNA were detected after reinfection but not after primary infection. Cells expressing TNF-alpha mRNA included epidermal cells, vascular endothelium and uncharacterized cells that increased more rapidly in the skin after reinfection compared to primary infection. The results are consistent with a prominent role for IFN-gamma in the host immune response controlling the severity of the disease.

[Seroprevalence of maedi-visna and border disease in Switzerland]. / Seroprävalenz von Maedi-Visna und Border Disease in der Schweiz.

3866 sheep from 226 flocks of breeding associations and 1218 sheep from 15 independent sheep owners were tested for the presence of serum antibodies against Maedi-Visna and Border Disease viruses. The flocks were randomly selected based on the relative proportion and the geographical distribution of the 4 predominant Swiss sheep breeds (Braunköpfiges Fleischschaf, Schwarzbraunes Berg- und Juraschaf, Walliser Schwarznasenschaf, Weisses Alpenschaf). Additionally two smaller breeds were included in the study (Charollais Suisse, Milchschafe). Sera of all sheep older than 1 year were collected together with data characterizing host and management factors. The sera were tested using established ELISAs for detection of antibodies to Maedi-Visna and Bovine Virus Diarrhea/Border Disease viruses. ELISA results of Maedi-Visna serology were confirmed by immunoblotting. 9% of the sheep of breeding associations were antibody-positive for Maedi-Visna virus. The results of the different breeds varied between 0.4% and 36%. A multiple logistic regression procedure identified breed, age, airing in barns, herd size, pasturing on alps and way of keeping the animals during winter as associated factors with individual serostatus. The prevalence of antibodies to Border Disease was 20% in sheep of breeding associations and 65% in those of independent sheep owners.

Parapoxviruses are strongly inhibited in vitro by cidofovir.

Three parapoxviruses which cause orf or related diseases in humans and animals and the orthopoxvirus, vaccinia virus, were tested for their in vitro sensitivity to cidofovir. The 50% inhibitory concentration for the three parapoxviruses was between 0.21 and 0.27 microg/ml and for vaccinia was 1.32 microg/ml. The selectivity index varied from 198 to 264 for the parapoxviruses and was 42 for vaccinia virus. Virus yield assays confirmed the ability of cidofovir to reduce ortho- and parapoxvirus replication. The efficacy of cidofovir against parapoxviruses justifies its evaluation as a candidate drug for the treatment of parapoxvirus infections in humans and animals.

Expression of the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) elicits virus-type specific neutralising antibodies.

A region of genome from the NADL strain of BVDV corresponding to the coding sequence for the E2 glycoprotein has been molecularly cloned using RT-PCR. The viral cDNA sequence was used to construct vaccinia virus recombinants that expressed either the entire E2 coding sequence or fragments of it. These recombinants were used to immunise mice of three H-2 haplotypes to investigate their ability to elicit a neutralising antibody response against BVDV. Sera from mice immunised with the recombinant expressing full length E2 contained high levels of virus neutralising antibodies that in addition to giving neutralisation of the homologous NADL strain were also able to neutralise the Oregon C24V reference strain. These sera failed to give any neutralisation of the Osloss reference strain providing evidence for the division of BVDV isolates into at least two distinct E2 serotypes. These results were confirmed in gnotobiotic lambs. Expression of E2 fragments revealed the presence of at least two distinct neutralising epitopes, one of which was localised within the carboxy terminal 90 amino acids of the protein.

The control of bovine virus diarrhoea virus in Shetland.

A scheme to control and eradicate bovine virus diarrhoea (BVD) was initiated in 1994 in the Shetland Islands by local veterinary surgeons and funded by the Shetland Islands Council and Shetland Enterprise Company. Over a 3-year period every bovine animal on the islands was blood-sampled (heparinised) and laboratory tested using MAb-based ELISAs for BVD virus antibody and antigen detection for evidence of disease. A number of BVD virus positive animals (40) were found and culled. A total of 6150 animals were tested from 213 herds and 43% herds were found to be BVD naive. The remaining herds had experienced infection and contained many BVD antibody positive animals. Some repeat sampling of stock in infected herds determined further virus positive animals which were slaughtered and in 1997 the scheme ceased since it appeared that there were no persistent excretors present. The major risk to the Shetland Islands is from bought-in stock, especially animals which are imported in calf. It is vital that all bought-in animals are tested and proven to be free of BVD virus if these animals are in calf, the calves must be tested a birth to determine status. It is strongly advised that only bulls and bulling heifers or cows are bought into Shetland in future, thus, protecting the present stock. Continued surveillance will be required to claim eradication of BVD from Shetland.

Restriction endonuclease profiles of orf virus isolates from the British Isles.

A comparison of DNA profiles of representative isolates of orf virus, obtained using four different restriction endonucleases (RE), showed that the enzyme EcoRI could be used to discriminate between wild-type virus isolates and vaccine strains. The enzyme was used to compare the RE profiles of orf virus isolates from 43 outbreaks of orf that occurred in vaccinated flocks between 1988 and 1993; 21 outbreaks yielded wild-type virus, 10 yielded vaccine viruses, three produced both vaccine and wild-type viruses and no clear result was obtained from nine of the outbreaks. From the 21 outbreaks yielding wild-type viruses, 28 orf virus isolates had clear RE profiles and 15 distinct RE profiles were recorded. Usually only one virus type was associated with each outbreak but from two farms, two different wild-type viruses were recovered. No predominant genotype was identified, with four RE profile types being recovered for more than one outbreak. From the more severe form of orf involving the buccal cavities of lambs only wild-type viruses were recovered, with at least four different genotypes being represented.