Samples sizes required to accurately quantify viral load and histologic lesion severity at the maternal-fetal interface of PRRSV-inoculated pregnant gilts

Porcine reproductive and respiratory syndrome virus (PRRSV) is transmitted vertically, causing fetal death in late gestation. Spatiotemporal distribution of virus at the maternal-fetal interface (MFI) is variable, and accurate assessment of viral concentration and lesions is thus subject to sampling error. Our objectives were: 1) to assess whether viral load and lesion severity in a single sample of endometrium (END) and placenta (PLC), collected near the base of the umbilical cord (the current standard), are representative of the entire organ; and 2) to compare sampling strategies and evaluate if spatial variation in viral load can be overcome by pooling of like-tissues. Spatially distinct pieces of END and PLC of 24 fetuses from PRRSV-2-infected dams were collected. PRRSV RNA quantified by RT-qPCR was compared in 5 individual pieces per fetus and in respective pools of tissue and extracted RNA. Three distinct pieces of MFI were assessed for histologic severity. Concordance correlation and kappa inter-rater agreement were used to characterize agreement among individual samples and pools. The viral load of individual samples and pools of END had greater concordance to a referent standard than did samples of PLC. Larger pool sizes had greater concordance than smaller pool sizes. Average viral load and lesion severity did not differ by location sampled, and no technical advantages of pooling tissues versus RNA extracts were found. We conclude that multiple pieces of MFI tissues must be evaluated to accurately assess lesion severity and viral load. Three pieces per fetus provided a reasonable balance of cost and logistic feasibility.