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Facilitating the diagnosis of Q fever using FTA cards to store and ship bulk tank milk samples
Treilles, M.; Charollais, P...
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Objectives: Coxiellosis, also named Q fever, is an infectious disease caused by an intra-cellular bacterium: Coxiella burnetii (Cb). In cattle, Q fever, when symptomatic, is mainly responsible for abortions, stillbirth, birth of weak calves, retained placenta and metritis/endometritis. But it is also a zoonotic disease, ruminants being the main reservoir of Cb.
Bulk tank milk (BTM) is an easy, inexpensive, and representative sample to detect Cb infections in dairy herds using RT-PCR. But one major limitation under field conditions is the need to deliver the BTM samples in adequate conditions (quickly, refrigerated and safely) to a qualified laboratory. In addition, sending non-inactivated biological material via regular post may be forbidden. A new innovative, easy, and accurate diagnostic tool (QTest) for Q fever was developed to overcome these constraints. Farmers or veterinarians simply place some drops of BTM on a WHATMAN FTA Elute Micro Card (FTA card) and let it dry before posting the card to the laboratory.
The objective of this study was to validate the reliability of this innovative technique.
Material and methods: This study had two complementary objectives, carried out in two steps. The first step aimed at assessing the preservation of Cb DNA detection from BTM spotted on a FTA card over time under two different temperatures (20-22°C and 37°C) to mimic field conditions. A milk sample was artificially contaminated with Cb to reach a load of ~5x106 Genome Equivalent (GE)/ mL which became the master sample. The master sample was then successively diluted at different dilutions (10-1, 10-2, 10-3, 10-4, and 10-5). Each diluted milk was then sampled several times on FTA cards and stored for up to 29 days either at 20- 22°C or at 37°C. RT-PCR was performed for each dilution and each storage temperature on days 1, 4, 6, 8, 11, 15, 20 and 29.
The second step aimed at comparing, after ageing, the detection of Cb DNA by RT-PCR either when directly applied on BTM or on BTM preserved on FTA cards. 70 BTM field samples previously tested positive for Cb by RT-PCR were stored as raw milk for 17 days before extraction or inoculated onto FTA card on day 15 and extracted on day 21. On day 21, a RT-PCR was performed for all the samples (raw milk and FTA card) and the results were compared between the two techniques for each sample.
Results: The first step showed that regardless of the duration of FTA card storage, all samples with a dilution below 10-3 (approximately 103 GE/mL) were detected to contain Cb DNA. Also, no significant loss of detectability was noted from d1 to d29, regardless of dilution or storage temperature. This means that the FTA card system ensures a stable preservation of Cb DNA in BTM samples stored at 20-22°C and 37 °C for at least 29 days.
For the second step, of the original 70 positive samples, we had 58 samples that were tested positive using one of both of the storage option. Of these 58 samples, 45 raw BTM samples tested positive and, of these, five tested negative when using FTA cards. In other words, there were 13 false negatives with older raw BTM samples while there were only 5 false negatives for older BTM on FTA cards. These five remaining were all with Ct value >35 indicating low quantities of Cb DNA. We can assume that the non-detection was likely due to the lack of reproducibility of the PCR technique for weak positive samples.
Importantly, for 13 samples, FTA cards produced positive PCR results while the equivalent raw BTM samples tested gave negative PCR results. This indicates that the detection rate was higher using FTA cards with aged BTM (91.4%) than with raw aged BTM (77.6%) samples.
Conclusion: Our study showed that the use of QTest makes BTM sampling, shipment, and storage very easy and cheap, while results do not seem to be impaired by the preservation/transportation method. Indeed, the stability of Cb DNA on an FTA card is maintained for at least 29 days at either 20-22°C or 37 °C. Therefore, this technique would facilitate an easier and more practical approach to diagnosis of Q fever at herd level and would be supportive of Q fever control strategies.
Keywords: Q fever, Coxiella burnetii, bulk tank milk, diagnosis, PCR.
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Affiliation of the authors at the time of publication
Laboratoire Qualyse, Champdeniers, France;
INRAE, Oniris, BIOEPAR, Nantes, France;
Ceva Santé Animale, Libourne, France.
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