Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples

Authors: POETA SILVA, ANA PAULA - IOWA STATE UNIVERSITY; STORINO, GABRIEL - SAO PAULO STATE UNIVERSITY (UNESP); MATIAS FERREYRA, FRANCO - IOWA STATE UNIVERSITY; ZHANG, MIN - IOWA STATE UNIVERSITY; MILLER, JESSICA - IOWA STATE UNIVERSITY; HARMON, KAREN - IOWA STATE UNIVERSITY; GAUGER, PHILLIP - IOWA STATE UNIVERSITY; WITBECK, WENDY - IOWA STATE UNIVERSITY; DOOLITTLE, KENT - IDEXX LABORATORIES; ZIMMERMAN, SILVIA - IDEXX LABORATORIES; WANG, CHONG - IOWA STATE UNIVERSITY; DERSCHEID, RACHEL - IOWA STATE UNIVERSITY; CLAVIJO, MARIA - IOWA STATE UNIVERSITY; Arruda, Bailey; ZIMMERMAN, JEFFREY - IOWA STATE UNIVERSITY

Submitted to: Preventive Veterinary Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2022
Publication Date: 5/13/2022
Citation: Poeta Silva, A.S., Storino, G.Y., Matias Ferreyra, F.S., Zhang, M., Miller, J.M., Harmon, K.M., Gauger, P.C., Witbeck, W., Doolittle, K., Zimmerman, S., Wang, C., Derscheid, R.J., Clavijo, M.J., Arruda, B.L., Zimmerman, J.J. 2022. Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples. Preventive Veterinary Medicine. 204.Article 105670. https://doi.org/10.1016/j.prevetmed.2022.105670.
DOI: https://doi.org/10.1016/j.prevetmed.2022.105670

Interpretive Summary: Mycoplasma hyopneumoniae (MHP) is an important cause of respiratory disease in swine. Commonly, tracheal swabs, which are labor-intensive and require a trained individual, are used to detect MHP in the respiratory tract of swine. Therefore, the objective of this study was to compare the probability of detecting MHP in oral fluid as a function of within pen MHP prevalence and test protocol (DNA extraction and PCR) using oral fluid samples of known MHP infection status. PCR protocols performed differently suggesting that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.

Technical Abstract: Four Protocols (1 – 4) were compared in terms of the probability of detecting Mycoplasma hyopneumoniae (MHP) DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Under experimental conditions, 39 7-week old pigs were randomly assigned to a negative control pen (3 pigs) and 4 pens containing 9 pigs each but differing in the proportion of MHP-inoculated pigs (1, 3, 6, or 9). Deep tracheal swabs were collected twice weekly to establish individual pig MHP infection status and derive within-pen prevalence. Oral fluids (n = 322) were collected daily between -4 to 59 days post inoculation (DPI). On DPI 3, tracheal swabs from 17 of 19 inoculated pigs were MHP DNA positive. In the aggregate, MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 109 with Protocols 4, 2, and 1, respectively. Using mixed-effect logistic regression coupled with a piecewise exponential model to account for within-pen transmission dynamics, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.05) and differed among test protocols. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (90.8%), followed by Protocols 4 (77.5%), 2 (73.8%), and 1 (63.6%). The fact that PCR protocols performed differently suggests that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.