SHIC Calls for Proposals for Improved Oral Fluids PCR Sensitivity

( Swine Health Information Center )

Recent unpublished, observational research indicates oral fluids (OF), when collected by rope as an aggregate sample, could be a good sample for rapid detection of African swine fever virus (ASFv). However this experimental data also suggests that there is potential for false negative testing results.  Therefore, the Swine Health Information Center (SHIC) is calling for proposals to develop methods to improve the detection of low levels of nucleic acid in OF through enhancements to sample pre-extraction treatment(s) or through improved extraction methodologies compatible with the high throughput testing currently done in NAHLN laboratories.

With thousands of OF samples coming into diagnostic labs each month, the value of this type of testing for swine disease monitoring is irrefutable. The objective of this SHIC-funded research is to address concerns about OF testing sensitivity. Then, with this knowledge, the potential for an official option for ASF testing beyond whole blood, which is the antemortem sample presently proposed by USDA for surveillance, opens. Due to the urgency of preparing for ASF and other foreign animal disease testing protocols, SHIC will favor proposals reflecting this immediate need.

In a negative cohort study, no false positives for ASFv were reported. Experimental, unpublished, observational work indicated that ASFv may be detected in OF prior to onset of clinical signs even if only a small proportion of animals are infected. However, as the viral load, as measured by PCR CT comparison, was much lower in OF than individual tissue samples, the study indicated false negatives do occur. This limits the potential use of swine OF as an official monitoring and surveillance tool in the event of an ASF outbreak in the US without further study and refinement.

OF samples are widely used in swine surveillance for porcine reproductive and respiratory syndrome virus (PRRSV), Influenza A virus in Swine (IAV-S), and Mycoplasma hyopneumoniae (MHP) nucleic acids on US farms and have shown the potential for use in foreign animal disease (FAD) surveillance, as well. To date, more than 27 pig pathogens have been shown to produce detectable levels of nucleic acid in OF.  The proposed research could lead to increased sensitivity of PCR testing for these domestic and foreign animal diseases.

SHIC is currently sponsoring OF sensitivity research in Vietnam related to ASF as well. Building on those projects, it is hoped the research related to this new request for proposals will result in methods sensitive enough to support OF PCR diagnostic validation.

The following detailed points are included in SHIC’s request for proposal.

  • The objective of the research is to identify sample treatments (for example centrifugation, nucleic acid concentration, or other sample treatments) or extraction methods that improve the quantitative detection of PRRSV, IAV-S, and MHP nucleic acids in swine oral fluids.
  • The research should include comparisons of the proposed sample treatments or extraction methods with the high throughput protocols currently in use in NAHLN veterinary diagnostic laboratories.
  • The proposal should include a clear description of the experimental design and statistical methods to be used in data analyses and provide for enough replicates and statistical power to be able to clearly show if the probability of detection x concentration differs among treatments or extraction methodologies for a particular PCR test.
  • Since research has shown that it is easier to recover nucleic acids from intentionally spiked samples than from samples collected from infected pigs, the experimental design should include OF samples collected from infected pigs. Proposals that leverage partnerships with pork producers and their veterinarians to use those samples from naturally infected pigs, i.e., control costs, will be prioritized.
  • With the importance of improving swine OF PCR sensitivity so it can be used with confidence as an official monitoring and surveillance tool, proposals with a timeline reflecting that urgency will also be prioritized.

The deadline for submitting an RFP to SHIC for this project is August 25, 2020, at 5:00 pm CDT. For more information on the RFP process, please contact SHIC Executive Director Dr. Paul Sundberg at [email protected].