With the goal of improving the diagnostic value of tongue tips for PRRS surveillance, a study led by Dr. Onyekachukwu Henry Osemeke from Iowa State University aimed to optimize protocols to increase the likelihood of recovering live PRRSV isolates from tongue tip fluids. Successfully isolating PRRS viruses from post-mortem tongue tips enhances their diagnostic value as a sample type for herd surveillance. Funded by the Swine Health Information Center, the study evaluated four different sample collection protocols across 597 tongue tips from stillborn and dead piglets. Samples with the lowest Ct values on PRRSV PCR testing were selected for virus isolation on two cell lines and primary alveolar macrophages. Overall, the highest rates of successful virus isolation were found in fluids extracted from fresh tongue tissues using PBS (22.6%) and on ZMAC cells (21%).
Find the industry summary of this study here.
Porcine reproductive and respiratory syndrome virus surveillance is particularly challenging in low-prevalence scenarios due to the cost of testing representative units and the complexities associated with PRRSV ecology (Muñoz-Zanzi et al., 2006). Recently, the post-mortem tongue tip or tongue tip fluids sample was demonstrated to be a cost-effective and population-sensitive approach for PRRSV surveillance in swine herds. Although the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of PRRSV RNA is the most requested test for PRRSV surveillance in the US (Trevisan et al., 2019), isolating the live virus remains crucial for confirming the presence of infectious PRRSV in a herd. There is currently no documented evidence of live PRRSV being isolated specifically from tongue tip samples.
Thus, the goal of the study described herein was to identify what protocols for collection, processing, and cell culture enhance the recovery of live PRRSV isolates from tongue tip fluids collected from perinatal mortalities. By optimizing collection techniques and testing procedures, this study aimed to improve the diagnostic value of tongue tips as a sample type for PRRSV surveillance. As a field application, this could offer swine practitioners a more herd-representative tool for formulating live vaccines.
To complete this study, samples were collected from 597 perinatal mortalities from a 5,000-head PRRSV-positive breeding herd over a four-day period. Tongue tissues were grouped into 20 batches (approximately 30 mortalities or tongues per batch). Each tongue was divided into four quarters, with each quarter randomly assigned to one of four collection protocols: 1) tongue fluids extraction from fresh tissues using phosphate buffered saline, 2) tongue fluids extraction from fresh tissues using virus transportation medium, 3) tongue fluids extraction in phosphate buffered saline after one freeze-thaw cycle, and 4) tongue tissue homogenate.
The result was 80 total samples (20 batches x 4 protocols) that were sent to a NAHLN-approved veterinary diagnostic laboratory for RT-qPCR testing. The RT-qPCR cycle threshold values were averaged across the four protocols in each of the 20 batches, and the 10 batches with the lowest mean Ct values were selected for virus isolation (VI). Two cell lines (ZMAC and MARC-145) and one batch of primary alveolar macrophages were tested for their ability to successfully isolate PRRSV.
All samples tested positive for PRRSV by RT-qPCR, with the average Ct values for the phosphate buffered saline, virus transportation medium, freeze-thaw, and homogenate groups being 21.9, 21.8, 22.6, and 24.8, respectively. PRRSV was isolated successfully from tongue tissues in all groups with variable success rates. The virus isolation success rate was 22.6% in the phosphate buffered saline group, 12.1% in the virus transportation medium group, and 2.8% in both the freeze-thaw and homogenate groups. The probability of successful viral isolation was 3.1% in MARC-145 cells, 21.0% in ZMAC cells, and 4.8% in primary alveolar macrophages cells. Mortality batches with only stillborn piglets had a 35.5% probability of successful PRRSV isolation, while batches with stillborn and dead piglets had a 1.0% probability.
Investigators concluded that live PRRSV can be isolated from postmortem tongue tip fluids. Extracting tongue tip fluids from fresh stillborn piglets using phosphate buffered saline or virus transportation medium increases the chances of successful virus isolation. The ZMAC cell line outperformed the MARC-145 cell line and primary alveolar macrophages cells in this study. Ensuring a cold chain from sample collection until arrival at the laboratory maintains the diagnostic quality of the samples. Isolating PRRSV from aggregate samples such as tongue tip fluids provides several surveillance and vaccine development benefits. As virus isolation using aggregate samples allows for the efficient co-detection of multiple PRRSV strains within a herd, there are great advantages for surveillance and developing autogenous vaccines.
Because there is no predicting when or where the next emerging disease will appear, the critical evaluation of convenient sampling protocols for applicability and effectiveness for disease surveillance will help the industry monitor and detect diseases as they emerge. This study provides objective information about diagnostic testing applications and best practices for obtaining tongue tip fluids from perinatal mortalities.
The Swine Health Information Center, launched in 2015 with Pork Checkoff funding, protects and enhances the health of the US swine herd by minimizing the impact of emerging disease threats through preparedness, coordinated communications, global disease monitoring, analysis of swine health data, and targeted research investments. As a conduit of information and research, SHIC encourages sharing of its publications and research. Forward, reprint, and quote SHIC material freely. For more information, visit http://www.swinehealth.org or contact Dr. Megan Niederwerder at mniederwerder@swinehealth.org or Dr. Lisa Becton at lbecton@swinehealth.org.