By Igor A.D. Paploski, Mariana Kikuti, Xiaomei Yue, Marcello Melini, Albert Canturri, Stephanie Rossow and Cesar A. Corzo, University of Minnesota College of Veterinary Medicine
Porcine reproductive and respiratory syndrome virus causes significant economic losses in the U.S. — approximately $1.2 billion annually — due to reproductive failure, abortion and high pre-weaning mortality among piglets. Thirty percent of the U.S. breeding herd experiences annual PRRSV outbreaks. A farm’s positive stable status is confirmed by sampling serum from piglets or using processing fluids.
Tongue tips from deceased animals are being considered as an alternative specimen to support a farm’s stability status. Tongue tip samples may contain contaminants and PCR inhibitors. Better understanding how to process and optimize conditions to facilitate viral detection via molecular tests is crucial for a wider and more rational adoption of this specimen in the swine industry.
Our object was to describe the impact of different processing and testing protocols for PRRSV detection on tongue tips that optimizes the sensitivity and specificity of PRRSV detection in sow herds.
Materials and methods
- Seven conveniently selected sow farms were visited between 2 and 5 months after the onset of a PRRSV outbreak and tongue tips from 20 dead piglets were collected on each farm.
- Samples from five farms were tested using different pooling strategies (pools of n=20, pools of n=5, or individual testing) and laboratory processing techniques (tongue tip fluid vs. tongue tissue homogenate )
- Samples from the other two farms were exposed to different storage and shipping conditions (frozen vs. refrigerated) and were tested at different time points since sample collection (1, 4 and 7 days).
- Samples were tested by RT-PCR at the University of Minnesota Veterinary Diagnostic Laboratory.
- The sensitivity of different pooling protocols was calculated when compared to testing samples individually, both when testing TTF and TTH.
- The cycle threshold values of samples from TTF and TTH were compared, along with the effects of sample storage (frozen at -20°C vs. refrigerated at 4°C).
- A linear regression model was used to measure the expected increase in Ct for each day elapsed since sample collection.
Results
A total of 140 tongue tips were collected from seven visited farms. The within-farm PRRSV prevalence on dead piglets ranged from 0 to 100% when testing TTF individually and from 0 to 45% when testing TTH also individually. Five farms were used to assess the diagnostic accuracy as a function of the tissue type and of the pooling strategy:
- Under the assumption that TTF is the gold standard, the testing of TTH had 36% sensitivity, 100% specificity and positive predictive value, and 76% negative predictive value when samples were tested individually. When tested in pools of n=5, TTH had 75% Se, 100% Sp and PPV and 86% NPV. Both TTF and TTH performed similarly when tested in pools of n=20. From a Ct standpoint, those obtained from TTF were on average 2 units lower than those obtained from TTH. These results suggest that testing the TTF rather than the TTH allows for more sensitive detection of PRRSV, and that Cts detected on TTF are lower.
- Under the assumption that the individual testing of the tongue tips is the gold standard, testing TTF in pools of n=5 led to an agreement with individual testing of 85%, with three cases of false negative results; while testing TTH in pools of n=5 led to an agreement with individual testing of 95%, with one false positive result. On the other hand, testing TTF in pools of n=20 led to an agreement of 75%, with one false negative result; while testing TTH in pools of n=20 led to an agreement of 100%. These results suggest that testing samples in pools does reduce the ability to detect PRRSV when compared to testing them individually.
Tongues from pigs originating from two farms were used to simulate shipping conditions and assess the effects of two factors: storage conditions (frozen at -20°C vs. refrigerated at 4°C) and time elapsed since collection (1, 4 and 7 days) on the Cts. Overall, Cts of frozen samples were lower than refrigerated ones kept for the same length of time (Figure 1). We also observed that for each day that elapsed since collection, Cts of the samples increased by 0.2 units on average. These results reinforce the importance of keeping tongue tips frozen during shipping, when possible, but also show that refrigerated samples still yield positive results. Additionally, delays in handling/shipment should be minimized, as those impact the Ct of the samples in a measurable way for each elapsed day.
Conclusions and implications:
This study demonstrates that processing choices and circumstances surrounding RT-PCR testing of tongue tips for PRRSV significantly affect test results. While pooling samples reduces costs for producers, it also impacts diagnostic accuracy. We recommend that veterinarians discuss their specific testing objectives with pathologists, as pooling samples may still be viable depending on the exact question being addressed with the submission.
Our study also indicates that testing tongue tip fluids yields more sensitive results with lower Cts compared to tongue tissue homogenates. We advocate for using tongue tip fluids as the primary specimen for PRRSV diagnostics, and this information helps guiding and optimizing laboratory processing workflow of tongue tips.
Additionally, our results highlight the importance of storage conditions on test performance, which is crucial when samples are shipped from farms and may face shipping delays. Minimizing the time between sample collection and testing is critical for further potential sample usage, such as sequencing or viral isolation.
Tongue tips are an easy-to-collect sample type that targets animals potentially more likely to be infected (dead piglets), eliminating welfare concerns during sample collection. This study provides valuable insights into how testing choices and submission circumstances impact RT-PCR PRRSV testing results of tongue tips.
The project was funded by the Swine Health Information Center, project number #23-068.
To read the original article, visit the National Hog Farmer website.