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 (PI) animals with the intent of eliminating them from the herd or preventing their introduction into the herd. In some fetuses infection can be established within the uterus during the first trimester and the early part of the second trimester. If this fetus survives to term, it will have an immunotolerance to BVDv and remain persistently infected 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). This is a whole blood sample in an EDTA tube (lavender top) 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 tissue samples. 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. Sensitive techniques using fluorescence based Beacon technologies can now be used for routine diagnostic tests. 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 in serum, the virus is neutralized and will not grow.

As a 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 PI-BVD 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 PI-iBVD microplate assay. In both the PI-BVD 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 some herds can be in the range of 2 to 5%. The PADLS-PSU lab tests for both Type I and Type II when doing PCR on pools. BVD Antigen (Ag) Capture ELISA assay can be used on serum to test for the virus even if the virus in the serum is not viable.

In animals less than 5 months of age maternal antibodies can interfere with the use of the PI-BVD microplate virus isolation assay. As a result the Immunohistochemistry (IHC) ear notch test is one 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) (any skin tag can be used: such as skin from the tail if tail docking is being used as a management tool).

Skin tags can also be used in adult individuals, as a substitute for the PI-BVD microplate assay. In addition to the IHC ear notch test the BVD Antigen (Ag) Capture ELISA can be used on ear notch (or skin tag) 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. In some cases where large groups are being screened, skin tag samples can be pooled at the laboratory into groups of approximately 30. These pooled samples can be screened by Ag Capture or PCR. Pooling can make screening tests more cost effective. Pool sizes are designed by the lab personnel and with consultation of the herd veterinarian such that sensitivity and specificity are not compromised.

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 the first milking is put in an empty tank. Max of 400 animals in milking herd or group. A single negative result is inconclusive as to herd status. 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 a faculty member of PADLS or Dr. Wolfgang if they have questions regarding the detection of BVDv in herds, immunization, or prevention strategies.

Source: David Wolfgang, VMD, Diplomate ABVP - Dairy PADLS and Field Investigations, Dept. of Veterinary and Biomedical Sciences, Penn State University