Placental Umbilical Cord Sampling for Porcine Circovirus Type 2 and the effects of pooling on sensitivity of polymerase chain reaction results

Detection of Porcine Circovirus Type 2 (PCV2) in individual Placental Umbilical Cord Serum (PUCS) samples has been shown to be comparable to other sample types (e.g., colostrum, pre-suckle serum, fetal tissue) to monitor PCV2 sow herd stability. However, testing of individual samples is costly, and a frequently used method to reduce testing costs is sample pooling. The purpose of this study was to estimate the impact of PUCS sample pooling on PCV2 polymerase chain reaction (PCR) detection sensitivity.

Ninety-three PCR results per pool size were needed to detect a 20% difference in sensitivity (i.e., 50% vs 70%) as significant at an alpha probability < 0.05 and power > 0.8. All samples were collected from midwest United States breeding herds. Expelled placental tissues were gathered, placed in a refrigerator, at least three umbilical cords per placenta were collected and expressed into a serum seperator tube to make a single placental sample. A PCV2 rtPCR (HMC, Ames, IA) was run on each aggregate placental sample. Positive placental samples with Cq values ranging from 29.77- 39.96 were categorized into high virus concentration (Cq < 34.41), middle virus concentration (Cq between 34.42 and 36.22) and low virus concentration (Cq > 36.25). Using known placental sample results, pools consisting of one positive sample plus one negative sample (2:1), one positive sample plus two negative samples (3:1) and one positive sample plus four negative samples (5:1) were tested on PCV2 rtPCR. The experimental unit was the placental sample rtPCR result, and pools that contained a PCR positive placental sample was considered as the positive gold standard for calculating relative sensitivity.

Pooling 2:1, 3:1 and 5:1 resulted in a relative sensitivity of 81.7%, 76.3%, and 63.4%, respectively. All pools containing high virus concentration (low Cq) samples were positive. Pools containing middle virus concentration (middle Cq) samples were 100%, 80.6% and 54.8% positive for 2:1, 3:1 and 5:1 pools, respectively. Pools containing low virus concentration (high Cq) samples were 45.2%, 48.4%, and 35.5% positive for 2:1, 3:1 and 5:1 pools, respectively.

Pooling of PUCS-based placental samples reduced diagnostic detection sensitivity of PCV2 by rtPCR. As pooling increased, detection rates decreased. Further, the higher the initial Cq, the lower the detection rate for the middle and high Cq groups, i.e., pooling one middle or high Cq positive placental sample with one or more negative samples decreased detection rate in all pool sizes. These results can be used as a basis for further development of an optimal sampling protocol.

Disclosure of Interest: None Declared.