Assessing the use of oral fluid samples for identification of Mycoplasma hyopneumoniae using a quantitative Real-Time PCR assay

Introduction: Mycoplasma hyopneumoniae (Mhyo) is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs. Mhyo is also considered as one of the main agents contributing to the porcine respiratory disease complex. Diagnosis of Mhyo is key for the control of this pathogen. Evaluation of clinical signs, slaughterhouse lung checks, serological analysis or direct identification of Mhyo by PCR can be part of the diagnostic approach. It has been showed recently that tracheo-bronchial swabbing (TBS) is one of the best sampling techniques for direct identification of Mhyo. To our knowledge, studies investigating the use of oral fluids (OF) for direct identification of Mhyo are scarce. The aim of this study was to compare TBS and OF sampling for direct identification of Mhyo by quantitative Real-Time PCR assay. Materials and Methods: Two farrow-to-finish farms positive for Mhyo were selected for this study, both located in Brittany, France. In farm 1, sucklers were sampled just before weaning (21 days old) and weaners around 70 days of age. In farm 2, only weaners were sampled at 33 days of age. For each farrowing or post-weaning pen, OF samples were collected using a cotton rope (Ø0.8 cm, 30 min/pen) and for each piglet, TBS were collected individually. Direct identification of Mhyo was then performed using a quantitative Real-Time PCR assay (Marois et al., 2009). Results: In farm 1, at 21 days of age, all the piglets sampled (n=114) were negative for Mhyo by TBS. The OF samples collected in the corresponding farrowing pens were also negative. Using TBS at 70 days of age, 19 pigs out of 100 were positive for Mhyo, results ranging from 5.5x102 to 5.1x107 CFU/ml. At the pen level, 4 pens were negative and 2 pens were positive using TBS, with 10 pigs out of 16 and 9 pigs out of 27 being positive respectively. All OF samples were negative for Mhyo. In farm 2, at 33 days of age, 21 piglets out of 33 and 2 piglets out of 18 were Mhyo positive using TBS in two different pens, results ranging from 5.6x102 to 4.5x103 CFU/ml. The corresponding OF samples of these two pens were negative. Conclusion: Contrary to TBS, pooled OF sampling did not allow direct identification of Mhyo using a quantitative Real-Time PCR assay. This difference in sensitivity could be explained by the individual sampling performed for TBS compared to OF samples taken at pen level and by the sampling material/site for Mhyo (saliva/mouth versus mucus/lower respiratory tract). DNA damage by enzymes in the saliva, low yield of DNA extraction from saliva and/or the presence of PCR inhibitors in the saliva can play a role in the lower sensitivity of OF samples compare to TBS.