This is our Friday rubric: every week a new Science Page from the Bob Morrison’s Swine Health Monitoring Project. The previous editions of the science page are available on our website.
In this week’s Science Page researchers Igor A.D. Paploski, Mariana Kikuti, Xiaomei Yue, Marcello Melini, Albert Canturri, Stephanie Rossow, and Cesar A. Corzo take a look at PRRS detection using different testing protocols.
- Tongue tips from dead 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 samples and conditions to optimize viral detection via molecular tests is crucial for a wider and more rational adoption of this specimen in the swine industry.
Objective:
We aimed to describe the impact of different sample 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 piglet mortality (n=20) were collected at each farm and tested by RT-PCR at the University of Minnesota Veterinary Diagnostic Laboratory. Samples from 5 farms were tested using different pooling strategies (pools of 20, pools of 5, or individual testing) and laboratory processing techniques (tongue tip fluid – TTF vs. tongue tissue homogenate – TTH). Samples from other 2 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). The sensitivity of different pooling protocols was calculated compared to individual sample testing, both for TTF and TTH. The cycle threshold (Ct) 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). Linear regression model was used to assess the expected increase in Ct for each day elapsed since sample collection.
Results:
A total of 140 tongue tips were collected from seven sow farms. The within-farm PRRSV prevalence on piglet mortality ranged from 0 to 100% when testing TTF individually and from 0 to 45% when testing TTH, also individually. Assuming that TTF testing is the gold standard, testing of TTH exhibited 36% sensitivity (Se), 100% specificity (Sp) and positive predictive value (PPV), and 76% negative predictive value (NPV) in samples tested individually. When tested in pools of 5, TTH showed 75% Se, 100% Sp and PPV and 86% NPV. Both TTF and TTH performed similarly when tested in pools of 20. From a Ct standpoint, values obtained from TTF were on average 2 units lower than values obtained from TTH. Assuming that individual testing of the tongue tips is the gold standard, testing TTF in pools of 5 led to an agreement with individual testing of 85%; while testing TTH in pools of 5 led to an agreement with individual testing of 95. On the other hand, testing TTF in pools of 20 led to an agreement of 75; while testing TTH in pools of 20 led to an agreement of 100%. Overall, Ct values in frozen samples were lower than in refrigerated specimens kept for the same length of time (Figure 1). It was also observed that for each day elapsed since collection, Ct values of the samples increased by 0.2 units on average.
Conclusions and implications:
- Testing TTF rather than TTH allowed for more sensitive detection of PRRSV, and that Ct values detected on TTF were lower.
- Testing samples in pools did reduce the ability to detect PRRSV when compared to testing individually.
- These results reinforce the importance of storing samples frozen during shipping. Additionally, delays in handling/ shipment should be avoided, as those impact measurably the Ct value of the samples for each elapsed day.
This study showed that processing choices and conditions surrounding RT-PCR testing of tongue tips for PRRSV significantly affect test results. It is recommended that veterinarians discuss specific testing objectives with pathologists, as pooling samples may still be viable depending on the exact question being addressed with the submission. Minimizing the time between sample collection and testing is critical for further sample usage, such as sequencing or viral isolation. Tongue tips are a sample that can be easily collected, which targets animals potentially more likely to be infected (piglet mortality), eliminating welfare concerns during sample collection. This study provides valuable insights into how testing choices and submission conditions may impact RT-PCR PRRSV testing results of tongue tips.
This project was funded by the Swine Health Information Center (SHIC), project number #23-068.