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Short Communications
Difficulties in the interpretation of bluetongue RT-PCR results in France
  1. S. Zientara1,
  2. J. P. Amat1,
  3. C. Sailleau1,
  4. C. Viarouge1,
  5. A. Desprat1,
  6. D. Vitour1 and
  7. E. Bréard1
  1. 1ANSES Alfort, UMR 1161 ANSES/INRA/ENVA, 94703 Maisons-Alfort Cédex, France
  2. 1ANSES, Risk Assessment Department, 27-31 Avenue du Général Leclerc, Maisons-Alfort, France
  1. E-mail for correspondence Stephan.Zientara{at}anses.fr Provenance: not commissioned; externally peer reviewed

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BLUETONGUE virus (BTV) is the cause of bluetongue (BT), an insect-transmitted disease of domestic and wild ruminants. BTV is transmitted by Culicoides biting midges that act as biological vectors of the virus.

From 1998 to 2006, five different BTV serotypes (1, 2, 4, 9 and 16) have spread throughout extensive portions of Mediterranean Europe (Mellor and others 2008). In 2006, BTV serotype 8 emerged unexpectedly in the North of Europe involving Belgium, France, Germany, Luxembourg and the Netherlands (Toussaint and others 2006). In 2008, two other BTV serotypes were detected in Northern Europe: BTV-6 in the Netherlands and BTV-11 in Belgium (De Clercq and others 2009, Maan and others 2010).

To limit direct losses and in an effort to minimise the circulation of BTV and allow safe movements of animals, authorities from affected European countries undertook vaccination of livestock with inactivated BTV-8 and BTV-1 vaccines. The vaccination of livestock has had a major role in reducing virus circulation and even eradicating the virus from some areas of Europe (Zientara and others 2010).

In 2011, the high vaccination coverage achieved across many European countries has resulted in the successful control of BT in Europe. No virus has been isolated in 2011 in the North of Europe (Belgium, Luxembourg, The Netherlands, Germany) and in France. More than 80 per cent of cattle, sheep and goats were vaccinated in France throughout 2009 and 2010. This high vaccination coverage resulted in a dramatic reduction of BTV cases and the likely eradication of BTV from France.

The detection of BTV was routinely undertaken during diagnostic and surveillance operations using a real-time quantitative RT-PCR (rt-RT-PCR) assay (Toussaint and others 2007).

In 2007, in France, more than 14,000 cases were reported, more than 38,000 were reported in 2008, less than 90 in 2009 and only one positive case was reported in 2010. These cases corresponded to animals with low Ct (below 25) and showing clinical signs or for which epidemiological links were in favour of BTV infection.

FIG 1:

Numbers of positive rt-RT-PCR results obtained between June 2009 and December 2010 in France

FIG 2:

Geographical distribution of the samples which have been typed between June and December 2010 in France

This short communication reports a number of incidents of positive results obtained by rt-RT-PCR. All the positive RT-PCR results obtained between June 2009 and December 2010 were weak (high Ct) and it was not possible to isolate virus from these samples.

In winter 2010, during the vector-free season, from January to February, although the temperature was very low and that no culicoides vectors were identified by vector traps, about a hundred EDTA-blood samples of cattle with Ct-cycle threshold values mostly >33 were received in the NRL laboratory of Anses. The animals (cattle or sheep) had no clinical signs. No virus was isolated from these positive samples by the classical method; that is, inoculation of embryonated chicken eggs or KC cells (Culicoïdes cells).

When the samples were first tested, weak positive results were obtained (Ct values ranging from 33.5 to 39.8). When rt-RT-PCR was repeated using RNAs isolated from a second extraction, similar Ct values were obtained. Some of these samples amplified by a serotype-specific rt-RT-PCR also gave similar results. Subsequent resampling (15 days after the first sampling) of each of the cattle yielded negative results, suggesting that the BTV genome was cleared more quickly in these animals than in BTV8-infected animals in 2007, before the compulsory vaccination campaigns (data not shown).

From June 2009 to December 2010, 1.792 positive rt-RT-PCR results were obtained from samples collected from animals (obtained by active surveillance based on random sampling, sampling at slaughterhouses and sampling of sentinel animals in herds) (Anses 2011).

Although some of the positive rt-RT-PCR results obtained from samples collected in the winter and spring of 2009 to 2010 could be attributed to viral circulation in 2009, it is also possible that some of these positive results were a consequence of vaccination. Steinrigl and others 2010 have previously reported that the genome of the vaccine virus could be detected in sheep for as long as 61 days after vaccination. However, Oura and others 2009 were unable to detect the BTV genome by rt-RT-PCR in blood samples collected after vaccination with an inactivated vaccine. In 20 goats vaccinated twice with BTV-8 vaccines from Merial or Intervet, no vaccine RNA was detected in animals 21 days after vaccination (Bréard and others 2011) using the same extraction and rt-RT-PCR kits as Steinrigl and others 2010.

Between May and December 2010, the persistence of viral RNA from the previous year is unexpected. These positive rt-RT-PCR results could represent detection of a viral RNA component of the vaccine antigen, or detection of low levels of virus circulation. Low levels of virus circulation in unvaccinated animals would be predicted to give rise to clinical signs of disease in some animals and to substantial variation in viral RNA load: this was not observed. Low levels of viral circulation in vaccinated animals would be the result of vaccine failure. This is unlikely, as most reports indicate that the vaccines used ­prevent viral circulation.

For the 1792 positive (group-specific) rt-RT-PCR results, molecular typing was performed. It was not possible to type 95 per cent of the samples probably because of the low viral load. Among the samples which have been typed, 83 per cent were BTV-8, 16.4 per cent were BTV-1 and 0.6 per cent were co-infections of BTV1 and BTV-8. In 2011, the same observation was reported: 73 positive rt-RT-PCR results were seen in particular in winter in the first few months of the year (absence of midges, no vaccination performed and no evidence of laboratory contamination).

It is difficult to determine whether the positive rt-RT-PCR results arose from a low level of viral circulation, by persistence of viral RNA or from some other cause such as the detection of the BTV genome in the inactivated vaccine.

Although only weak positive results were obtained by rt-RT-PCR in samples collected throughout 2009 and 2010, these results were the cause of significant alarm in a country that had just experienced its first outbreak of BTV and where control and eradication efforts were in place and had been successful. This highlights the problems arising when vaccination and surveillance are carried out contemporaneously.

Acknowledgements

The authors would like to thank DGAl (Direction Générale de l'Alimentation, Dr Jérome Languille) for providing the data obtained by the active surveillance.

References

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