Bluetongue virus serotype 8: abortion and transplacental transmission in cattle in the Burgundy region, France, 2008-2009.

During the incursion of bluetongue virus (BTV) serotype 8 in France in 2007, an increase in the number of abortions in cattle was observed, but the cause was not clearly established. A survey of all the reported cases of abortion in cattle from November 2008 to April 2009 was conducted in the Nièvre district (Burgundy region) to determine the percentage of abortions as a result of BTV-8 and to study factors that could have played a role in BTV-8 transplacental transmission. BTV-8 was present in 16% of the fetuses or newborn calves that died within 48 h, from 780 dams. Dams inseminated before the BTV epizootic peak recorded from July to September 2008 were more likely to have BTV-positive abortions (OR=5.7, P<0.001) and those vaccinated in May or June 2008 were less likely to have BTV-positive abortions (OR=0.3, P=0.01 and OR=0.4, P=0.001, respectively). The gestational month was not a predictor of BTV abortion. In blood or spleen, fetuses/calves from RT-PCR-positive dams had significantly higher RNA concentrations than fetuses/calves from RT-PCR-negative dams. Of the 128 dams that had BTV-positive fetuses or calves, 60% were RT-PCR-negative. BTV-8-positive fetuses/calves were significantly more frequent (n=42 vs n=21, P=0.082) amongst those showing clinical signs or lesions suggestive of cerebral damage.

Prevalence of Mycoplasma bovis udder infection in dairy cattle: preliminary field investigation in southeast France.

AIMS: To update epidemiological data on Mycoplasma bovis infection in dairy herds from six departments in the southeast of France, in order to obtain a first estimate of the prevalence of M. bovis infection through bacteriological investigations on bulk tank milk, and estimate the prevalence of M. bovis in clinical mastitis in this population of cattle. METHODS: To estimate a prevalence of M. bovis of 2%, with 95% confidence, a sample of >270 herds was required. Bulk tank milk samples were collected from herds between January and February 2005 and milk samples from cows with clinical mastitis were collected between January 2007 and March 2008. Bulk tank milk and composite mastitic samples were analysed for M. bovis using culture and/or PCR. RESULTS: Mycoplasma bovis was not detected by either technique in any of the 345 bulk tank milk samples. The prevalence of M. bovis infection in this population of dairy herds was <1%, with 95% confidence. Mycoplasma bovis was not isolated from any of the 166 composite samples obtained from 828 samples of mastitic milk. The prevalence of M. bovis in clinical mastitis was <0.44%, with 95% confidence. CONCLUSIONS: These preliminary results suggest that the prevalence of udder infections with M. bovis is very low in dairy herds in the southeast of France. These two studies provide preliminary data, that can be used to derive working hypotheses for future statistically representative investigations at the national level.

Detection by real-time RT-PCR of a bovine respiratory syncytial virus vaccine in calves vaccinated intranasally.

Seventeen four- to five-week-old calves that were not shedding bovine respiratory syncytial virus (BRSV) were vaccinated intranasally against the disease and sampled by nasal swabbing on 16 different days for up to 20 days after vaccination. BRSV vaccine virus was detected in 15 of the 17 calves. Five of the calves were PCR positive on only one swab, eight were PCR positive on two to five swabs and two were PCR positive on more than five swabs. Twelve of the calves were positive only before day 14 and three were positive after day 14. The nasal shedding of BRSV vaccine virus was very variable.

Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

Assessing the within-herd prevalence of cows antibody-positive to bovine viral diarrhoea virus with a blocking ELISA on bulk tank milk.

Milk samples from 135 herds in Brittany were tested by a blocking ELISA for antibodies to bovine viral diarrhoea virus (BVDV) and used to assess the relationship between the bulk milk result and the within-herd prevalence of antibody-positive lactating cows. This relationship was first quantified by using a general linear model and controlling for the number of cows contributing milk to the bulk tank, for the percentage of primiparous cows in the herds and for the number of milkings contributing to the bulk tank. Receiver operating characteristic (ROC) analysis was then used to define classes of percentage inhibition in the bulk milk associated with minimum intraclass and maximum between-class variances of the within-herd prevalence. Only the percentage inhibition of bulk milk had a significant positive effect on within-herd prevalence (R2 = 0.85). The ROC analysis provided three classes of bulk milk results corresponding to different expected levels of within-herd prevalence. Herds with bulk milk percentage inhibitions of 0 to 35 per cent, 35 to 60 per cent and 60 to 100 per cent had mean (sd) observed prevalences of 4.8 (5.7) per cent, 21.6 (14.6) per cent and 66.0 (29.3) per cent, respectively. Herds with a bulk milk inhibition of 0 to 35 per cent were expected to be BVDV-free. A herd with two consecutive bulk milk results four months apart of 60 per cent or more was likely to have a very high prevalence (median of 93 per cent) and could be suspected of harbouring an active infection.

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk.

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappa value=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.