Laboratory Diagnostics: Porcine Reproductive and Respiratory Syndrome (PRRS)

Assays available

Polymerase chain reaction (PCR)

• Detects presence of specific sequence of viral nucleic acid (RNA).
• Sample types: tissues, whole blood, serum, oral fluids, etc.
• Pros:
  • Separate primers are used to detect both PRRS Type 1 (European) and Type 2 (North American) in the same sample at the same time.
  • Very high sensitivity (can detect small amounts of virus).
  • Early detection - acute cases should be positive.
  • Many different sample types can be used (tissue, blood, serum, oral fluids, etc).
  • Moderate cost:
    • Can often do pooling of 5 serum or tissue samples to lower cost while minimizing loss of sensitivity.
    • Often do not pool oral fluids due expected higher Ct values (lower virus concentrations) which can result in significant loss of sensitivity.
• Cons:
  • Laboratory must update primers periodically to avoid false negatives.
    • Both PRRS Type 1 and Type 2 primers must be updated.
  • Sequencing needed to differentiate vaccine virus vs. wildtype infection.

Enzyme-linked immunosorbent assay (ELISA)

• Detects presence of antibodies.
• Sample types: serum or oral fluids (some kits).
• Pros:
  • Most detect antibodies for both PRRS Type 1 and Type 2.
  • Animals remain positive for several months (3-12 months).
  • Can be used in chronic cases.
• Cons:
  • Specific antibodies detected and timing of detection may vary slightly between the different commercial kits available.
  • Takes 7 to 10 days for animals to become seropositive.
  • Unable to differentiate maternal antibodies vs exposure.
  • Unable to differentiate vaccine vs. wildtype infection.

Immunohistochemistry (IHC)

• Detects presence of viral antigen.
• Sample types: tissues.
• Pros:
  • Detects virus at site of lesion (good proof of causation).
  • Can identify low vs moderate vs high amounts of virus present.
• Cons:
  • Correct tissue sample must be submitted.
  • Requires significantly more virus to be present than PCR.
  • Only evaluating a small tissues sample.

Genetic Sequencing

• Sequences virus’s genetic nucleic acids (RNA).
• Sample types: tissues, whole blood, serum, oral fluids, etc.
• Pros:
  • Can differentiate wild type virus from vaccines.
  • Can help differentiate new virus introduction from existing or past viruses.
• Cons:
  • Expensive.
  • Often only sequence ORF5 which is 600 out of ~15,000 base pairs.
  • Samples with high CT values > 34 tend to be more difficult to sequence.

Table 1: Iowa State University Veterinary Diagnostic Laboratory sequence success based on PRRS PCR cycle threshold (Ct) values from oral fluid samples. Table from Chris Rademacher et al. 2016.

Specimen	PCR Ct range	Total samples tested	Number samples sequenced	% Samples positive sequenced
All samples	<30	2016	2013	99.85
	30.00-31.99	389	361	92.80
	32.00-33.99	324	265	81.79
	34.00-35.99	185	109	58.92
	36.00-37.00	65	26	40.00

Indirect fluorescent antibody (IFA)

• Detects presence of antibodies.
• Sample types: serum.
• Pros:
  • Along with a PCR test, can serve as a confirmatory test for unexpected ELISA positive samples.
    
• Cons:
  • Not feasible for large number of samples.
  • Results impacted by virus isolate used for assay.
  • Reliability is highly dependent on technician skills.

Result interpretation

PCR

• Positive – Virus is present/circulating highly suggestive of causation especially with lower Ct values and clinical signs are present. Recent vaccination with a modified live virus can result in positive PCR results.
• Negative – Negative or Virus could have been missed if testing occurs late after infection.

ELISA

• Positive – Maternal antibodies or past exposure (usually > 7-10 days post exposure) to vaccine or wildtype virus.
• Negative – Negative or Infection too early to detect (usually must be at least 7-10 days post exposure).

IHC

• Positive – Virus is present at site of lesion.
• Negative – Negative or virus could have been missed if testing occurs late after infection.

Genetic Sequencing

• Vaccine virus – Expect > 99% homology.
• Wildtype virus – Estimate about a 1-2% loss of homology per year.

IFA

• Positive – Maternal antibodies or past exposure (> 7-10 days post exposure) to vaccine or wildtype virus.
• Negative – Negative to vaccine or wildtype virus or infection too early to detect (must be at least 7-10 days post exposure).

Scenarios

Sow/Gilt abortions

• Aborted fetuses: Collect 6-8 fetuses and pool samples for PCR testing. Only about 50% of fetuses aborted will be PCR positive (so need to sample many fetuses), but those that are positive will have a large concentration of virus and therefore can pool in groups of 10 for PCR testing.
• Aborted sows/gilts: Collect serum from recently aborted (< 10 days) sows/gilts for PCR testing. Can pool in groups of 5. ELISA testing is not useful as it usually will take 7-9 days for naïve sows/gilts to test positive.

Sow/Gilts reproductive problems

• Collect 15 to 20 samples from affected and 15 to 20 samples from non-affected (30 – 40 samples total) gilts/sows and test via PCR (pools of 5 or 6) and ELISA (individually).

Weak piglets in farrowing

• Can collect family oral fluids from several litters with weak pigs and test via PCR.
• Can collect testicles (if castrated), tails, tongue tissue (dead pigs) from pigs from different litters in the farrrowing room. Large number of samples can be pooled together for testing.
• Collect serum samples from 10 affected litters by collecting 2 to 3 piglets per litter and test via PCR on pools of 5 or 6 piglets per pool. Ensure piglets have not been vaccinated for PRRS.

Growing pig with acute clinical signs of PRRS

• Collect oral fluids from 4 to 6 different pens and test individually via PCR. Do not pool samples for testing.
• Collect 15 to 30 serum samples from pigs with clinical signs or random sampling and test via PCR. Can pool in groups of 5 or 6 for PCR testing.

Growing pig with chronic clinical signs of PRRS

• Collect oral fluids from 4 to 6 different pens and test individually via PCR. Do not pool samples for testing. Can also test oral fluids samples via ELISA.
• Collect 30 serum samples from pigs with clinical signs or random sampling and test via PCR. Can pool in groups of 5 or 6 for PCR testing. Also test individual samples via ELISA to confirm exposure.