Researchers: Carles Vilalta, Juan Sanhueza, Montserrat Torremorell, Cesar Corzo and Robert Morrison, University of Minnesota; Julio Alvarez, VISAVET Health Surveillance Center and Universidad Complutense; and Deb Murray, New Fashion Pork
With porcine reproductive and respiratory syndrome costing the U.S. swine industry more than $600 million each year, making sure newborn pigs are not at risk for prolonged infections in the herd is vital.

Molecular diagnostic tests have become an important tool to declare herds as stable, and they have been used in due-to-wean pig serum samples to generate evidence of absence of viral replication and transmission in pigs prior to weaning. The recommended sampling protocol consists of bleeding at least 30 due-to-wean pigs 30 days apart for a minimum of 90 days and testing them by reverse transcription polymerase chain reaction in pools of five (Holtkamp et al., 2011).

Pooling allows a larger number of animals to be tested, thus increasing herd-level sensitivity while keeping diagnostic costs low (Rovira et al., 2007). However, cost-effective sampling approaches that can be integrated in the farm routine procedures are still needed.

Samples are often collected during tail docking and castration, but tools used in these procedures have tested positive in PRRS virus in prior studies (O’Connor et al., 2014; Thompson et al., 2012). Litters handled after processing PRRS virus-infected litters have a higher risk of testing RT-PCR positive (Thompson et al., 2012), suggesting that these procedures are associated with PRRS virus dissemination.

During tail docking and castration, it is a common practice to place tails and testicles obtained in a pail to avoid the spread of bloodborne pathogens within the farrowing room. Serosanguineous fluids originating from these tissues, which are known as processing fluids, accumulate at the bottom of the pail and can be used as a sensitive sample to determine the presence of PRRS virus in processed pigs (Lopez et al., 2018).

However, there are scarce data on the use of PF to determine herd-level sensitivity and PRRS virus infection dynamics in newborn pigs.

That’s why researchers with the University of Minnesota recently set out to better understand the sensitivity of the use of PF at litter level, more specifically at 3 days old.

Past history with PRRS virus
A commercial 6,000-sow breed-to-wean farm in Illinois that had become infected with PRRS virus in April 2015 was chosen for the study. It was not until June 2016 that the pigs, prior to wean, tested PRRS virus- and RT-PCR-negative, were considered stable (Holtkamp et al., 2011).

In February 2017, the herd experienced reproductive clinical signs compatible with PRRS virus infection, and samples were submitted to the diagnostic laboratory that confirmed another PRRS virus infection with the same strain.

A new PRRS virus immunity homogenization plan was initiated by ensuring exposure of PRRS virus to all breeding animals and incoming gilts eight weeks after the outbreak, following industry standard protocols (Torremorell et al., 2003). However, during the time of the study, it was decided to not perform herd closure due to remodeling of the facilities.

Sampling protocol
Sampling started 10 days after the outbreak had been confirmed with diagnostics and was repeated every three weeks for a total of eight sampling events, representing 24 weeks since infection was confirmed. The sampling protocol consisted of bleeding all the pigs within a litter, and collecting all the tails and testicles of the castrated piglets for each litter.

Every pig was bled using a new sterile needle. Processing tissues were collected for each litter and placed in 4- by 6-inch reclosable zip-close bags. The tissues remained in the bags for at least three hours before the fluids were removed with a sterile pipette and placed in sterile sera tubes. Both individual serum samples and PF were centrifuged at the farm and transported to the laboratory while refrigerated. Pig gender was recorded to assess, quantify and evaluate its association with litter PRRS virus status (positive or negative). 

Gloves were changed between litters when bleeding. However, no specific instructions to change gloves were given to farm employees during tail docking and castration, as this study aimed to represent field conditions. Serum and PF samples were individually tested at the University of Minnesota Veterinary Diagnostic Laboratory for PRRS virus by RT-PCR (Rovira et al., 2007).

A sample was considered positive if the cycle threshold value was ≤35 or ≤37 for a serum or PF, respectively, as a result of the receiver operating characteristic curve analysis conducted as part of this study.

Detecting PRRS virus
A total of 78 litters with 945 piglets (480 males and 465 females) were sampled. One litter, which had six pigs and was sampled on Week 11 post-outbreak, was removed from the analysis due to loss of the associated PF. Of the remaining 77 litters, 23 (29.8%) litters and 100 out of 939 (10.6%) pigs tested PRRS virus- and RT-PCR positive. There were also 23 out of 77 (29.8%) PF samples that yielded PRRS virus- and RT-PCR positive results.

The proportion of positive pigs and litters changed over time, with more samples testing positive earlier in the outbreak. However, a transitory increase was seen in Week 8 post-outbreak, coinciding with virus inoculation with the same outbreak strain as an intervention strategy in breeding females. The proportion of PF RT-PCR-positive samples followed a similar pattern as the one observed with the serum samples.

At least one positive litter was detected in each sampling event during the whole study regardless of sample type tested, except in Week 11 post-outbreak for PFs. The parity distribution of the sows included in the study followed the expected parity distribution in a commercial farm, with 23 (29.9%), 14 (18.2%), 14 (18.2%), 10 (13.0%) and 16 (20.8%) of litters born to first, second, third, fourth and fifth-to-ninth parity sows, respectively.

The percentage of RT-PCR-positive PF samples was higher in lower parities. It was not possible to detect any positive pigs in parities above five, although some of the PF were RT-PCR-positive. There were significant differences between the proportion of positive litters from Parity 1 and 2 sows, 78.3% (18/23), compared to litters from Parity 3 or higher 21.7% (5/23).

A significant higher proportion of positive serum samples was observed in males (61/476, or 12.8%) compared to females (39/463, or 8.4%). A similar trend was obtained when comparing positive cycle threshold values by gender with values from males. Additionally, a significant association between parity and probability of detecting a positive pig was observed.

The probability to detect a positive PF significantly increased with the number of pigs infected in a litter, although a high uncertainty in the estimate for the association between positive PF results and presence of >2 serum-positive piglets was found due to the lack of negative PF samples. False negative PFs were identified in litters having two or fewer PRRS virus-positive piglets.

PF is practical
These results show PF samples are a valuable, practical and time-efficient way to monitor PRRS virus in breeding herds at processing. PF had a sensitivity of 87% to detect a PRRS virus-positive litter, compared with sampling all the piglets. That loss in sensitivity can be compensated for by the larger sampling. Each time processing fluids are tested from one litter, 10 to 12 piglets are actually being tested. This means a larger number of animals are being tested for the same budget in diagnostics.

Other important findings from this study are the association between positive litters with gilts and the longitudinal description of the outbreak. Those findings led to another study lead by Juan Sanhueza, University of Minnesota, to try to understand if gilts could be a delaying factor for PRRS virus stability in breeding herds. 

For more information, contact Carles Vilalta.