By Gaurav Rawal, Paulo Arruda, Christopher Rademacher and Daniel C. L. Linhares, Iowa State University Veterinary Diagnostic and Production Animal Medicine; and Ran Bi, Iowa State University Department of Statistics
Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia, and a major causative agent of the porcine respiratory disease complex. This study summarizes and describes the general diagnostic trends on Mhp DNA detection by quantitative polymerase chain reaction in cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2004 to 2016.
Selected variables including sample type and age group were included in the analysis. The most common sample types were lung, followed by oral fluids, nasal swab and broncho-alveolar lavage. The lung homogenate, bronchial swab was more sensitive than other sample types while nasal swab and oral fluids were least sensitive by PCR. There was no significant interaction found between age group and sample type.
Materials and methods
To summarize and describe the diagnostic trends of Mhp, a retrospective study was performed with data from the ISU-VDL from 2004 to 2016. All porcine cases of which Mhp qPCR was performed were gathered. The data was screened, and cases with non-conventional sample types for Mhp diagnostics including semen, conjunctival swab, inoculum, liver, cell cultures, environmental, extract and vaccine were excluded from the analysis. Cases with inconclusive or suspect results were also excluded from the data analysis.
Results were further divided by age group (cases from pigs with less than 21 days of age were defined as suckling, 21 days to 6 weeks of age as nursery, 6 weeks to 11 weeks of age as growing, 11 weeks to 200 days as finishing, and greater than 200 days as adult), and by sample type which included lung, oral fluid, nasal swab, broncho-alveolar lavage, bronchial swab, non-specific swab, tracheal swab, laryngeal swab, lung homogenate and other. Samples labeled as saliva were included under the oral fluid category and assorted, lung swab, serum, pleural swab, pharyngeal swab, oropharyngeal swab and tonsil swab were included under other. Infrequent sample types (those comprising less than 1% of submissions) such as fluid, fibrin, lymph node, tonsil scrapings, tonsil, fresh tissue, fibrin swab were excluded from the analysis.
SAS 9.4 and Tableau were used to aggregate data, and to produce figures and tables. For statistical analysis, the frequency of positive results was compared across groups (age, season, sample type) with Pearson’s Chi-square test. The significance level was considered at alpha 0.05. The K-means clustering method was implemented to categorize the Mhp detection rate as “low,” “medium” or “high” for variables of interest, such as sample type.
The Mhp qPCR detection rate was 66% in lung homogenate, 64% in bronchial swab, 49% in broncho-alveolar lavage, 34% in tracheal swab, 33% in lung, 27% in other, 18% in laryngeal swab, 18% in non-specific swab, 10% in nasal swab, and 8% in oral fluids (Figure1). Lung homogenate and bronchial swab had significantly higher Mhp qPCR detection rate than other sample types (P < 0.001), followed by broncho-alveolar lavage (P < 0.001), while oral fluids had the lowest Mhp detection rate (P < 0.001).
Overall, there was a positive correlation between pig age and Mhp detection rate (Figure 2). In other words, Mhp detection rate was lower in suckling pigs, and increased in growing, finishing and adult pigs. Regardless of age group, bronchoalveolar lavage had the highest detection rate, and nasal swabs along with oral fluids had the lowest.
In suckling pigs, there was no significant difference of Mhp qPCR detection rate among broncho-alveolar lavage, non-specific swab, lungs, laryngeal swab, bronchial swab, tracheal swab and others (p = 0.077), while nasal swab had lowest rate (P < 0.001).
In nursery age pigs, broncho-alveolar lavage had the highest Mhp detection (p = 0.0436).
In growing age pigs, broncho-alveolar lavage and bronchial swab had significantly higher Mhp detection (P < 0.001), while non-specific swab and oral fluids had the lowest (P < 0.001).
In finishing age group lung homogenate had the highest Mhp detection rate (n=8) followed by broncho-alveolar lavage and bronchial swab (P < 0.001), and nasal swab had significantly lower detection rate than all other sample types (P < 0.001).
In adult age pigs, broncho-alveolar lavage had significant higher prevalence (P < 0.001).
Mhp detection rate by PCR was highest in lung homogenate, bronchial swab and broncho-alveolar lavage. Tracheal swab and lung had medium detection rate. Non-specific swab, laryngeal swab, nasal swab, and oral fluid had lowest detection rate.
The most frequent sample types submitted to ISU-VDL from 2004 to 2016 for Mhp testing by PCR were lung and oral fluids, followed by nasal swab and broncho-alveolar lavage.