BVD Testing at the PA Animal Diagnostic Laboratory System

Posted: April 4, 2006

Bovine Virus Diarrhea virus (BVDv) is a bovine enteric virus with a worldwide distribution.

In preliminary studies in 2002 on the development of BVD control programs for Europe, losses due to BVD were estimated at between $ 5 and $ 20 per calving. The Scandinavian countries along with a few other regions in Europe have had success with control of BVD without the use of vaccines and are moving towards total eradication. As many producers within Pennsylvania are maintaining control or eradication programs for BVD within their herds, it is important to understand the various test methods and systems that are available within PADLS. The nine different test methods currently available through PADLS can be used in three different applications. The first is for the diagnosis of acute infections in infected herds, the second is to determine if BVDv is causing abortion problems within a herd, and the third is for surveillance systems directed at the detection of persistently infected animals with the intent of eliminating them from the herd or preventing their introduction into the herd. When the fetus is infected in the first trimester and the early part of the second trimester, the potential exists for the fetus to develop an immunotolerance to BVDv and remain persistently infected (pi) for the rest of its life.

The detection of acute clinical infections in the live animal is most effectively done through the use of virus isolation methods to identify the presence of the virus in the ‘buffy coat’ (white blood cells) obtained from an EDTA (lavender top tube) blood sample which was kept cool and forwarded to the laboratory by overnight courier. These ‘buffy coat’ cells are co-cultured in sterile flasks with tissue cultures capable of replicating the virus. In instances of acute death, tissues from the Peyer’s patches in the small intestine along with samples from lung, spleen and mesenteric lymph nodes can be collected. These can be evaluated for the presence of BVDv by virus isolation and conventional PCR techniques in conjunction with the assessment of histological lesions seen in the individual. In addition to these conventional virus isolation and PCR methods, new approaches such as Real Time PCR techniques have been developed in PADLS - PSU for use on various tissue and blood samples. Plans are in progress to introduce these fluorescence based Beacon technologies as routine diagnostic tests within the next six months. These methods are capable of differentiating BVDv Type I and Type II infections.

The serum neutralization serological test can also be employed in the detection of exposure to BVDv both in vaccinated and non-vaccinated individuals. This test measures the antibody titer to BVD. Serum should be collected in a red top tube or serum separator tube (grey marble top), kept refrigerated and received by the laboratory within 48 hours of collection. Serial two fold dilutions of serum from individual cows are incubated with a known quantity of virus. The serum and virus mixture is then incubated with indicator cells for 72 hours to assess virus growth. If antibodies are present general guideline for interpretation, antibody titers of 1:1028 or higher are indicative of exposure to live virus, either modified live vaccine or field strains. Cattle inoculated multiple times with killed vaccine can sometimes reach this level. However, usual titers for cattle vaccinated with killed vaccines more typically range from 1:128 to 1:512. Lower titers indicate declining vaccination titers, poorly vaccinated animals, or exposure to BVD virus strains that are very different from the type I BVD virus strain (Singer strain) used for the SN test. These titers are only offered as guidelines, remembering that great variation occurs from cow to cow. Acute and convalescent (paired) serum samples are of value for investigation of abortion and for diagnosis of acute infections. The testing of paired samples should be done a minimum of 14 days apart. Changes in titer must be four fold or greater to be considered significant due to inherent minor variation in the test. Single serum samples from multiple herd cohorts may be useful for assessing vaccination or exposure of cattle to field strains of BVD. This test should not be used to screen for persistently infected cows since persistently infected animals can have antibody titers to antigenically different strains of BVD virus.

The identification of BVDv as the cause of an abortion involves the testing of tissue samples obtained from the fetus and/or the placenta. Both conventional virus isolation and PCR test methods are currently used for the detection of the
virus. Due to the state of deterioration often seen with these fetal and placental samples virus, isolation is not always possible as the virus may be dead due to the impact of the enzymes and pH changes associated with the autolysis. As a result the use of PCR techniques can often be more rewarding when testing these samples.

The detection of persistently infected animals in the herd is an essential part of any BVD control program even when vaccination programs are in use. The persistently infected individuals do not routinely produce a significant antibody response either in the face of vaccination or exposure to field virus, however they are not always serologically negative. As a result alternate test methods need to be employed. The serum piBVD microplate virus isolation assay is the principal assay used to identify individual animals. The test utilizes an enzyme conjugated antibody and substrate to identify the presence of live cell free BVD virus in serum of animals 5 months of age or older. Serum should be collected in red top tubes or grey marble topped serum separator tubes, kept refrigerated and received by the laboratory within 48 hours of collection. This test detects requires a repeat test of positives three weeks later to distinguish transient and persistently infected animals. In herds of 100 head or more costs can be reduced by the initial testing of pools of 20 to 30 animals each using Real Time PCR. The individual animals within the positive pools are then tested by means of the more labor intensive piBVD microplate assay. In both the piBVD microplate and Real Time PCR assays the blood samples should be individually numbered and forwarded to the laboratory without separation. Sterility is required in the handling of the samples so it is best that they not be opened prior to shipment. Studies have shown the incidence of persistently infected animals in most herds to be in the range of 2 to 5 %. The fee for individual piBVD microplate assays is $ 5.00 per sample. In most instances the use of Real Time PCR for preliminary screening reduces the cost to between $2.50 and $ 4.50 depending herd size and the number of PCR positive pools identified. The PADLS-PSU lab tests for both Type I and Type II when doing PCR on pools. In instances where the serum was delayed in shipment or not adequately cooled the alternate more costly BVD Antigen (Ag) Capture ELISA assay is used as the virus in the serum may not be viable.

interfere with the use of the piBVD microplate virus isolation assay. As a result the Immunohistochemistry (IHC) ear notch test is the preferred means of detection. Samples for the IHC ear notch test are submitted in individual tubes submerged in a small volume of formalin (available on request).This test can also be used in adult individuals, as a substitute for the piBVD microplate assay, however the labor intensive nature of the test makes it difficult for the laboratory to maintain a large case load. In addition to the IHC ear notch test the BVD Antigen (Ag) Capture ELISA can be used on larger ear notch samples of at least 1.0 cm (0.5 inches) square. Ear notch samples for the test can be submitted in individual tubes with or without sterile phosphate buffered saline (PBS). In breeding herds caution needs to be exercised in the collection of the ear notch samples as the potential exists to transmit Bovine Leukosis between individual animals by means of the ear notch or punch tools. The fee for both the IHC and Ag RELISA ear notch assays is ~ $5.50 per sample.

Bulk milk samples for BVDv are sent to Cornell (practitioners are encouraged send them directly to Cornell). Sampling method is from the outlet valve (200 mls preferred from bottom of tank with no agitation after first milking put in an empty tank. Max of 400 animals in milking herd or group can not use the same sample as used in bulk tank mastitis testing). A single negative result is inconclusive as to herd status. Better correlation with 3 separate negative tests. It is important to remember that this procedure only tests adult lactating animals and most piBVD animals in a herd are youngstock.

Practitioners and dairy producers are encouraged to contact the authors if they have questions regarding the detection of BVDv in herds, immunization, or prevention strategies.

Doug Key DVM, MSc, DVSc, Dave Wolfgang VMD, Diplomate ABVP - Dairy
Animal Diagnostic Laboratory, Pennsylvania State University, University Park, PA