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Bovine Viral Diarrhea Virus

Making Sure Bovine Viral Diarrhea Virus Testing Answers the Right Questions for You and Your Clients

From: Russ Daly, Chris Chase, Travis Clement, Matt Dammen - SDSU Veterinary and Biomedical Sciences Department

Bovine Viral Diarrhea Virus (BVDV) is among the most important pathogens affecting today’s beef and dairy cattle operations.  Associated with reproductive, digestive, and respiratory illnesses in cattle, the virus can also create a congenital, persistent infection in calves, greatly aiding the virus’ spread within and between herds.  Testing and removal of these persistently infected (BVDV-PI) calves is the hallmark of BVDV control and prevention.  However, there are several different test options and potential pitfalls to avoid. 

Q.  What are the triggers to initiate a whole-herd BVDV test?

A.  Evidence that BVDV has infected cattle within an operation is perhaps the best indication that a BVDV-PI animal may be present in the herd, and efforts should be made to identify and remove the individual(s).  The most apparent evidence is detection of the virus in diagnostic submissions such as sick calves or aborted fetuses.  Indirect evidence could come in the form of persistent unexplained illness rates (due to immunosuppression) or reproductive failure. 

Q.  What is the goal of a whole-herd BVDV test?

A.  In cow-calf and dairy operations, whole-herd testing ensures that BVDV-PI animals are not present in the herd during breeding, where they could infect pregnant cows and create more BVDV-PI animals.  Testing calves entering a feedlot will ensure BVDV-PI animals do not infect susceptible animals. 

To accomplish this in cow-calf operations, the BVDV-PI status of each animal must be determined, and BVDV-PI animals removed prior to the breeding season.  If a calf is confirmed to be BVDV-PI, its mother should be tested due to the small risk that she is BVDV-PI herself.  All bulls, replacement heifers, and held-over open cows should be tested with no exceptions. 

Q.  What is the best test to use for BVDV herd screening?

Real Time PCR on pooled ear notches or blood serum (“Pooled PCR”).  If submitting ear notches, they should be about 1 cm x 1 cm in size, placed in individual plain red-top tubes with no additive.  The lab will pool the notches in groups of up to 20 at their discretion, or according to your grouping instructions.  If submitting serum, the lab will pool samples in groups of up to 10. 

If pools test positive for BVDV, then each individual sample that comprised the pool needs to be tested individually to identify BVDV-PI animals.  Typically this is accomplished using an antigen-capture ELISA (ACE) procedure.  However, when using serum samples from calves less than 3 months of age, ACE should not be used due to the potential for maternal antibody interference and false negative tests.  In those cases, individual PCR testing, which detects viral nucleic acid, should be used to identify BVDV-PI animals. 

Q.  Why do I get a BVDV PCR positive on a pooled sample but BVDV ACE negative results on the individual tests?

A.   A couple possible reasons exist:

1.  Differences in test sensitivity between the PCR and ACE ELISA test.  The PCR test typically has a higher sensitivity, making it more possible to detect transient infections or vaccine virus.

2.  The presence of maternal antibodies in a serum or blood sample.  The BVDV ACE test utilizes antibodies in the detection of the virus.  When maternal antibodies are present in the sample, they could interfere with the test antibody binding to the BVDV antigen, causing false negative results.  Therefore, animals younger than 3 months old should not be tested with the BVDV ACE test.  PCR techniques do not use antibodies for detection – thus the possibility of a positive PCR, but negative ACE, test.  

Q.  What is another sampling pitfall to avoid that could cause a false negative BVDV PCR test? 

A.  Sample storage conditions.  Certain additives, such as buffered saline or formalin, make sample storage conditions critical.  Ear notches in these solutions must be frozen immediately to maintain the integrity of the sample.  BVDV RNA is highly susceptible to degradation via enzymes that destroy the RNA and cause a negative PCR test result.  These compounds do not affect the viral proteins detected by the ACE test.  In this scenario, you might have a BVDV ACE positive result and then a negative PCR results.  Bottom line: please do not use additives in your ear notch sample submissions.  

Q.  Is there a test that can confirm an animal’s BVDV-PI status on the basis of a one-time sample?

A.  No.  BVDV test procedures can detect transient or vaccine-related exposures as well as persistently-infected animals.  BVDV-positive animals should be isolated and sampled again 3 weeks later.  The second BVDV test should preferentially use a different methodology than the first (e.g., serum PCR if the first test was an ear-notch ACE). 

Q.  Will the PCR or ACE test detect vaccine in herds using a modified live BVDV vaccine?

A.  Yes, PCR and the ACE test detect BVDV virus, even if the antigen is in a vaccine.  Waiting to sample cattle until at least 3 weeks after BVDV MLV vaccine use will help avoid this interference.

Q.  Is there a test to differentiate BVDV vaccine from field strains?

A.  Currently the SDSU-ADRDL does not have a test to differentiate vaccine from field strains.  BVDV strains can be typed through sequencing techniques, but these procedures don’t conclusively identify a strain as vaccine-related.

Q.  Under what circumstances should I consider getting a BVDV isolate sequenced?  What is needed to do this? 

A.  Sequencing will tell you the subtype (1a, 1b, 2) present in the animal tested.  This can help you determine the vaccines that would be most efficacious for the exposed herd. 

Whole (EDTA) blood (preferred) and serum are good samples to obtain sequencing results.  Regardless of the sample, sufficient virus must be present in order to be sequenced.  This means that the sample’s real-time PCR result should be positive with a Ct value less than 35.  Ear notches are not an ideal sample for BVDV sequencing because the low amount of recoverable virus compared to serum or whole blood makes accurate sequencing difficult.

Q.  Where can I get more information before embarking on a BVDV-PI testing plan? 

A.  A phone call to the SDSU ADRDL at 605-688-5171 before taking samples can help identify the sampling and testing strategy that avoids unwanted consequences and helps ensure your best chance of success for a testing strategy for your clients.