[Diagnosis of bovine viral respiratory diseases]. / Diagnostik viral bedingter Atemwegserkrankungen beim Rind.

Enzootic bronchopneumonia (EBP) is an infectious, multifactorial respiratory disease of cattle. Different viruses may be involved in its pathogenesis. In this study an adapted method of endoscopic bronchoalveolar lavage (BAL) of caudal parts of the right cranial lung lobe was established and evaluated. The obtained bronchoalveolar lavage fluid (BALF) served as template for the detection of BRSV, BPIV-3 and BCoV specific nucleic acids by RT-PCR. BALF samples of 44 cattle affected with respiratory disease were compared to nasal swabs in their reliability to detect the causative agent(s). In 6/7 animals tested positive for BRSV, RNA of this virus was detected in the BALF, in 4 animals it could be found in the nasal swabs. In two of the three BPIV-3 positive animals, the BALF was the only material that tested positive. The most reliable samples for detection of 15 BCoV positive animals were the nasal swabs. BAL was easy to perform, it led to severe coughing in one case and moderate worsening of dyspnoe in three cases. In conclusion this study shows that BAL of the right cranial lung lobe is in many cases the only tool to detect BRSV and BPIV-3, major viral triggers of EBP.

Phylogenetic analysis of Austrian canine distemper virus strains from clinical samples from dogs and wild carnivores.

Austrian field cases of canine distemper (14 dogs, one badger [Meles meles] and one stone marten [Martes foina]) from 2002 to 2007 were investigated and the case histories were summarised briefly. Phylogenetic analysis of fusion (F) and haemagglutinin (H) gene sequences revealed different canine distemper virus (CDV) lineages circulating in Austria. The majority of CDV strains detected from 2002 to 2004 were well embedded in the European lineage. One Austrian canine sample detected in 2003, with a high similarity to Hungarian sequences from 2005 to 2006, could be assigned to the Arctic group (phocine distemper virus type 2-like). The two canine sequences from 2007 formed a clearly distinct group flanked by sequences detected previously in China and the USA on an intermediate position between the European wildlife and the Asia-1 cluster. The Austrian wildlife strains (2006 and 2007) could be assigned to the European wildlife group and were most closely related to, yet clearly different from, the 2007 canine samples. To elucidate the epidemiological role of Austrian wildlife in the transmission of the disease to dogs and vice versa, H protein residues related to receptor and host specificity (residues 530 and 549) were analysed. All samples showed the amino acids expected for their host of origin, with the exception of a canine sequence from 2007, which had an intermediate position between wildlife and canine viral strains. In the period investigated, canine strains circulating in Austria could be assigned to four different lineages reflecting both a high diversity and probably different origins of virus introduction to Austria in different years.

Pestivirus infection in sheep and goats in West Austria.

Blood samples from 3112 sheep (185 flocks) and 1196 goats (163 flocks) from the Western region of Austria were tested for pestivirus-specific RNA. In this area, communal Alpine pasturing of sheep, cattle and goats is an important part of farming. The prevalence of sheep persistently-infected (PI) with pestivirus was 0.32% (10 animals) and the PI animals originated from five flocks (2.7% of those investigated). In goats, only one PI animal (0.08%) was detected. Sequence analysis of the 5'-end untranslated region (UTR) revealed that the strains of Border disease virus (BDV) detected were closely related to genotype 3 but the PI animals did not show any clinical signs of Border disease. The goat was PI with bovine viral diarrhoea virus-1 (BVDV-1). On one farm a high abortion rate among sheep had been observed 1year before the study was carried out but the other farms did not show any evidence of reproductive failures. Pestiviruses are endemic in small ruminants in some Alpine regions of Austria and PI healthy animals as described here have a key epidemiological role. A successful BVDV eradication programme in Austria will create highly pestivirus-susceptible cattle populations. Sheep and goats present a high risk for the reintroduction of pestiviruses to cattle herds because they are less likely to be considered to be PI. The results underline the need for the immediate consideration of small ruminants in eradication programmes.

Investigation of the role of Austrian ruminant wildlife in the epidemiology of malignant catarrhal fever viruses.

Malignant catarrhal fever (MCF) is an ubiquitous disease of cattle and other ruminants caused by Ovine herpesvirus 2 (OvHV-2), which is endemic in sheep and transmitted from healthy carriers. Further viruses of the MCF group are also able to induce MCF in ruminants. As alpine pasturing is very common in Austria, possible contact with ruminant wildlife carrying and excreting MCF viruses might be suspected as an infection source. To investigate the epidemiologic role of Austrian deer and chamois, spleen samples were collected from 55 red deer (Cervus elaphus), 72 roe deer (Capreolus capreolus), four fallow deer (Dama dama), and five chamois (Rupicapra rupicapra) during the hunting seasons 2001-2003. Samples were tested by both herpesvirus consensus and OvHV-2-specific polymerase chain reaction. As all spleen samples tested negative, there is no indication that in the region and period investigated, MCF viruses circulated in wild ruminants.

Simultaneous canine distemper virus, canine adenovirus type 2, and Mycoplasma cynos infection in a dog with pneumonia.

The present case is the first description of a triple infection with canine distemper virus (CDV), canine adenovirus (CAV) type 2, and Mycoplasma cynos in a dog. The 5-month-old female Miniature Pinscher was euthanized because of dyspnea, croaking lung sounds, weight loss, and lymphopenia. Pathologic examination revealed a fibrinous necrotizing pneumonia with large amphophilic intranuclear and acidophilic intracytoplasmatic inclusion bodies in different lung cells. Immunohistochemically, CDV antigen was present in lung and many other organs. In situ hybridization for detection of CAV nucleic acid showed positive signals in the lung only. Polymerase chain reaction of lung tissue and consecutive sequencing of the amplification product identified CAV type 2. Bacteriologic examination of lung tissue yielded large amounts of M cynos. This infection was confirmed by immunohistochemistry detecting abundant positive signals in the lung tissue.

Influence of communal alpine pasturing on the spread of pestiviruses among sheep and goats in Austria: first identification of border disease virus in Austria.

The purpose of this investigation was to determine the influence of communal Alpine pasturing on the spread of pestivirus infections among sheep and goats. The study included 481 sheep from 23 farms and 131 goats from 26 farms pastured on separated Alpine meadows in the western part of Austria. At the starting of pasturing on the sheep meadow, 325 (67.6%) animals were seropositive, on the goat meadows in 16 (12.2%) samples antibodies to pestiviruses were detected. At the end of pasturing, 74 seronegative sheep and two seronegative goats had seroconverted. Between the beginning and the end of pasturing the seroprevalence in sheep increased significantly from 67.6% to 83% (P<0.05). Moreover, in the peripheral blood mononuclear cells of four sheep, pestivirus-specific RNA was detected before as well as after pasturing; these animals remained serologically negative throughout the investigation. They were, therefore, identified as persistently infected. Sequence analysis in the N(pro) region revealed that the detected pestiviruses were the same at genetic level and they were grouped into the Border disease virus (BDV)-3 genotype. No pestivirus RNA was found in goat samples. The results of this survey indicate that communal Alpine pasturing does play a key role in the spread of BDV. Moreover, BDV has been identified and characterized for the first time in sheep in Austria, which until then had been regarded as being free from BD.

M gene analysis of atypical strains of feline and canine coronavirus circulating in an Austrian animal shelter.

Coronavirus-positive samples of faeces collected in an Austrian animal shelter from 12 cats and 10 dogs were analysed by reverse transcriptase-pcr with primers amplifying a segment of the M protein gene, and by sequence analysis. In addition, the samples were subjected to S gene typing, using primers that differentiated between feline coronavirus (fcov) types I and II. A phylogenetic analysis of the M gene sequences revealed not only clearly segregating canine coronavirus (ccov) in the dogs, typical ccov sequences and the recently described fcov-like ccov, but also at least two genetic clusters of fcov in the cats, one species-specific, the other more closely related to fcov-like ccov. The M gene sequences of these new feline strains had at most 88 per cent identity with the fcov-like ccov strain 259/01 and only up to 85 per cent with any fcov sequence available in GenBank. In the phylogenetic tree they occupy an intermediate position between feline and canine coronaviruses.

Detection of bovine torovirus in neonatal calf diarrhoea in Lower Austria and Styria (Austria).

Faeces of 230 calves with and without diarrhoea collected during the winter period 2004/2005 in 100 Austrian farms (Styria and Lower Austria) were examined for viral, bacterial and parasitic enteropathogens. Torovirus-specific nucleic acid confirmed by reverse transcriptase-polymerase chain reaction was found in 12 of 230 calves (5.2%). Ten of these calves were clinically ill, several of them showing signs of dehydration and abnormal faecal consistency at the time of sampling. Computer assisted analysis of two nucleotide sequences obtained from Austrian bovine samples revealed 93% similarity to Breda strain, but only 71% or 52% similarity to Equine Berne or Porcine Markelo torovirus strains respectively. Phylogenetic analysis grouped Austrian torovirus samples into the Bovine torovirus cluster indicating the first detection of Bovine torovirus in Austria. In addition, the following agents were detected in bovine faecal samples: Bovine coronavirus, 25.7%; Escherichia coli, 17%; Cryptosporidium spp., 11.7%; Eimeria spp., 10.4%; Rotavirus, 9.1%; Clostridium perfringens, 9.1% and Giardia spp., 6.1%. Salmonella spp. was not detected.

Oesophagoscopy and detection of viral nucleic acids in oesophageal biopsies--A contribution to BVDV diagnosis.

The endoscopic appearance of the oesophagus of animals infected with bovine virus diarrhoea virus (BVDV) but without signs of acute mucosal disease (MD) was investigated for any common or 'early warning' lesions. Thirty-seven BVDV-infected animals [36 persistently infected (PI) and one transiently infected] were examined clinically and endoscopically for typical erosions of the oronasal and oesophageal mucosa, respectively. During oesophagoscopy, mucosal biopsies were taken and tested for pestivirus-specific nucleic acids by reverse transcription polymerase chain reaction (RT-PCR). The results were compared with those of RT-PCR with various sample materials (blood, nasal and conjunctival swabs and faecal samples) usually used for routine diagnosis of BVDV infection. The average age of the 36 PI animals was 13.5 months, eight animals were 24 months or older. None of the PI animals had clinically visible erosions of the oronasal mucous membranes. During oesophagoscopy, all animals showed a reddening but unexpectedly no erosions or ulcerations of the oesophageal mucosa. Although in 21 of 36 PI animals all samples tested positive, the oesophageal biopsies were with no exception constantly RT-PCR positive in contrast to all other materials investigated. Remarkably, in the transiently infected animal all samples tested negative except for the oesophageal biopsy. Hence, this infection would have been missed by conventional diagnostic sampling.

Canine adenovirus type 2 infection in four puppies with neurological signs.

Four nine- to 11-week-old puppies developed respiratory and neurological signs due to an infection with canine adenovirus type 2 (cav-2); three of these were euthanased. They had moderate, diffuse pneumonia but there were no histological abnormalities in the central nervous system. Adenovirus-specific nucleic acid was detected by pcr in samples of lung and brain and the amplified product was 99.8 per cent homologous with the cav-2 reference strain Toronto a26/61. The positive pcr result was confirmed by in situ hybridisation in samples of lung, liver and spleen, but not in brain, and cav was isolated in cell culture from lung material; pcrs for canine distemper virus and canine herpesvirus-specific nucleic acids were negative, but large amounts of Bordetella bronchiseptica were isolated from lung material.