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Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid
Ana Paula S Poeta Silva1, Gabriel Y Storino2, Franco S Matias Ferreyra1
1Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA, USA.
Abstract:
Combinations of 2 nucleic acid extractions and 3 Mycoplasma hyopneumoniae (MHP) PCRs (namely Protocol 1, 2, 3, and 4) were compared in terms of the probability of detecting DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Oral fluid samples were created by randomly assigning 39 7-week old pigs to one of 5 pens, i.e., negative control pen (3 pigs) and 4 pens of 9 pigs each that differed 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 estimation. On DPI 3, tracheal swabs from 15 of 19 inoculated pigs were MHP DNA positive. Oral fluids (n = 320) were collected daily from - 4 to 59 days post inoculation (DPI). Using a piecewise exponential model to account for within-pen transmission dynamics followed by a mixed-effect logistic regression, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.0001) and differed among test protocols. MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 101 with Protocols 4, 2, and 1, respectively. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (95.7%), followed by Protocols 4 (70.1%), 2 (60.1%), and 1 (34.0%). 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.
Insights
This study compared Mycoplasma hyopneumoniae (MHP) DNA detection in pig oral fluids using different PCR protocols. Protocol 3 demonstrated the highest detection probability, highlighting the importance of assay selection for effective MHP surveillance.
Area of Science:
- Veterinary Microbiology
- Diagnostic Assays
- Swine Disease Surveillance
Background:
- Mycoplasma hyopneumoniae (MHP) is a significant respiratory pathogen in swine.
- Accurate detection of MHP is crucial for effective herd management and disease control.
- Oral fluid sampling offers a less invasive alternative to traditional methods for pathogen detection.
Purpose of the Study:
- To evaluate the diagnostic performance of different nucleic acid extraction and PCR protocols for MHP DNA detection in swine oral fluids.
- To determine the influence of within-pen MHP prevalence on the probability of MHP DNA detection in oral fluid samples.
- To identify the optimal protocol for maximizing MHP detection in oral fluid-based surveillance programs.
Main Methods:
- A study involving 39 pigs across 5 pens with varying MHP inoculation proportions was conducted.
- Deep tracheal swabs were used to confirm individual pig MHP infection status and estimate within-pen prevalence.
- Oral fluid samples were collected daily, and analyzed using 2 nucleic acid extraction methods combined with 4 different MHP PCR protocols.
Main Results:
- The probability of detecting MHP DNA in oral fluids was significantly associated with within-pen MHP prevalence (P < 0.0001).
- Protocol 3 yielded the highest MHP DNA detection rate (173 positive samples), outperforming Protocols 4, 2, and 1.
- At 100% within-pen prevalence, Protocol 3 achieved a 95.7% probability of detection, compared to 70.1% (Protocol 4), 60.1% (Protocol 2), and 34.0% (Protocol 1).
Conclusions:
- The choice of PCR protocol significantly impacts the sensitivity of MHP DNA detection in swine oral fluids.
- Protocol 3 is recommended for maximizing MHP detection in oral fluid samples, particularly in high-prevalence scenarios.
- Future improvements in extraction and PCR methods could further enhance diagnostic capabilities for MHP surveillance.
