Estimation of the performance of two real-time PCR assays for detection of Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dysgalactiae in pooled milk samples in a field study

The early detection of major mastitis pathogens is crucial for the udder health management of dairy herds. Testing of pooled milk samples, either individual test-day cow samples (TDCSs) or aseptically collected pre-milk quarter samples (PMQSs) may provide an easy to use and cost-effective group level screening tool. Therefore, the aim of this study was (1) to evaluate the sensitivity (Se) and specificity (Sp) of 2 commercial multiplex real-time PCR test kits applied to pooled milk samples using a Bayesian latent class analysis and (2) to estimate the probability of detection in relation to the pool size and the number of cows positively tested by BC within a pool.

Pools of 10, 20 and 50 cows were assembled from 1,912 test-day samples and 7,336 PMQSs collected from a total of 2,045 cows from 2 commercial dairy farms. Two commercial quantitative real-time PCR kits were applied to detect Staphylococcus (Staph.) aureus, Streptococcus (Strep.) agalactiae and Strep. dysgalactiae in the pooled samples, and a bacteriological culture (BC) was applied to PMQSs yielding a cumulative pool result. A pool was considered BC-positive if it contained at least one BC-positive PMQS.

Pathogens were more frequently detected in the PMQS pools than in the TDCS pools. Pools of 10 cows showed the highest probability of detection irrespective of sample type or type of PCR kit compared with larger pool sizes. Estimation with a Bayesian latent class analysis resulted in a median Se in PMQS pools of 10 cows for S. aureus of 63.3% for PCR kit I, 78.1% for PCR kit II and 95.5% for BC; the Sp values were 97.0, 97.6% and 89.1%, respectively. The estimated median Se for Strep. species for PCR kits ranged between 77.5 and 85.6% and for BC between 73.7 and 79.2%; the median Sp values ranged between 93.6 and 99.2% for PCR kits, and between 96.9 and 97.4% for BC. In addition, the probability of detection increased with an increasing number of BC-positive cows per pool. To achieve a probability of detection of 90%, the estimated number of positive cows in PMQS pools of 10 cows for Kit I was 4.1 for Staph. aureus, 1.5 for Strep. agalactiae and 1.3 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 6.9, 1.9 and 2.0 positive cows were required, respectively. For Kit II and PMQS pools, the number of positive cows required was 2.8 for Staph. aureus, 1.4 for Strep. agalactiae and 1.2 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 5.3, 1.8 and 2.0 positive cows were required, respectively.

In conclusion, the type of samples used for pooling, the pool size and the number of infected cows per pool determine the probability of detecting an infection with major mastitis pathogens within a pool by PCR testing.