Due to the complex pathogenesis and insidious nature of BVDV infections in the cattle population, the laboratory diagnosis is an essential component of developing measures for the control and prevention of BVDV infections. The positive isolation of BVDV from animal tissue submissions or from aborted fetuses, should provide a strong indication for further epidemiological investigations within the identified infected herds. In these situations, the ultimate goal should be the establishment of sound control and prevention methods by prevention of any potential exposure to BVDV (ie. removal of persistently infected animals and preventing the introduction of infected animals) and by protecting animals by using effective vaccination programs.
The diagnosis of acute infections must be done during the window of opportunity for virus isolation, as early as 3 days postinfection to 8 or 10 days postinfection. Some animals may be virus isolation positive for only 2 to 3 days during the course of BVDV infection. A whole blood sample is the best sample to collect and submit for BVDV isolation to identify animals acutely infected.
A viremia would be detectable in serum by virus isolation during a more narrow window of opportunity than from whole blood. In addition, swabs from mucosal or nasal surfaces can be collected and submitted for virus isolation. Since nucleic acid methods are not influenced by the development of neutralizing antibodies, these methods may remain positive for longer periods during the convalescent stage when neutralizing antibody would interfere with virus isolation. In acute outbreaks in dairy herds, the collection of samples from other herdmates in addition to the severely affected animals may aid in the ability to make positive diagnoses. Whole blood and clotted blood samples should be collected from any animals with mild diarrhea, slight increase in temperature, decrease in feed consumption, or decrease in milk production. Virus isolation should be done on whole blood samples and the serum stored for submission with a paired sample 30 days later for serology. To properly asses serology, paired acute and convalescent samples collected 30 days apart are required to identify four fold increases in serum antibody titers following convalescence.
Specimens collected at postmortem examinations from lymphoid organs as mentioned previously should be submitted for virus isolation from any animals that die or fetuses from any abortions that may occur. Many BVDV associated abortions are virus isolation negative. Some aborted fetuses may already have produced antibodies, the presence of which will confirm intrauterine infection. Maternal serology is only seldom helpful because seroconversion has often taken place before the abortion. If the dam on the other hand is antibody negative BVDV can be ruled out as the cause of abortion. Most BVDV associated congenital defects occurring following infection after the onset of immunological competence and therefore calves with these defects have BVDV antibody. The diagnosis of BVDV induced congenital defects in calves should included both virus isolation and serology to detect BVDV-specific antibody prior to uptake of colostrum.
The identification of persistently infected animals is most routinely done by virus isolation. The level of viremia in persistently infected animals is generally quite high (106 CCID50/ml of serum) but may vary from 102 to 107 CCID50/ml. In addition, the level of viremia may decline in individual animals over time. In most cases, for routine identification of persistently infected animals, serum is adequate for virus isolation. In young calves, maternal antibody will decrease the level of free virus in serum and virus isolation may be falsely negative. Due to colostral antibody, in young calves less than 3 months of age the best sample remains to be whole blood in which the mononuclear cells are separated for virus isolation. It has been suggested that serology can be used to identify seronegative animals as candidates for testing by virus isolation to identify persistent infections. Although persistently infected animals are immunotolerant, they may develop neutralizing antibody titers to BVDV. Therefore, serology should not be used as a screening method to identify animals to test for virus isolation. Since the number of persistently infected animals in any one herd will generally be low, any potentially infected animals should not be excluded from being tested. Persistent infections should only be determined by identification of BVDV by virus isolation in sequential samples collected 30 days apart. By testing the animal 30 days apart it is possible at the same time to test for a four-fold increase in antibody titer should the first virus isolation have been due to acute infection.
If a laboratory diagnosis of BVDV infection has been made from any submitted clinical samples, then there is an indication and an obligation for further investigation to be conducted at the herd level. Currently, herd screening involves individual animal testing by virus isolation which requires a certain amount of time and expense. Therefore, herd screening should not be done unless a commitment is made to establish and continue long term plans for the control and prevention of BVDV. If possible, due to the time and expense of herd screening, a positive laboratory diagnosis should be sought to indicate the infection status of a herd prior to herd screening.
The infection status of a herd may be established by additional methods such as testing bulk milk for anti-BVDV antibody or the testing of the bulk milk sample for BVDV by RT-PCR amplification. These types of assays are best utilized as an initial screening test to identify herds in which further diagnostic testing should be done to identify the source of virus spread (ie. persistently-infected animals). In some situations it is difficult to convince herd owners that the expense of herd testing is warranted. Therefore, positive identification of BVDV by preliminary PCR screening from a bulk milk sample would give good justification for further herd testing by virus isolation. Although the PCR bulk milk test could not be used to rule out BVDV infection, a positive result would provide useful information. Positive PCR results would be weighted more heavily than negative results. Therefore, the use of this assay may be most beneficial as a method of focusing on or identifying BVDV positive herds for development of control strategies and not as a definitive test to ensure that a herd is negative for BVDV. Other screening methods such as serology on groups of nonvaccinated calves at 6 months of age also are useful as discussed previously.
The most common approach to herd screening would be to obtain serum samples from all the animals in the herd over 3 months of age. In addition, whole blood samples should be collected from calves less than 3 months of age. Basically, all animals in the herd should be tested; therefore pregnant animal must be considered as 2 animals. Virus isolation using a microtiter immunoperoxidase detection methods is the most common method used for testing such large numbers of samples. Using this method, results from 2 serial passages are generally available within 5 to 9 days. In addition, any calves born for the next 9 months must be tested to ensure that no additional persistently infected animals are born that were in utero at the time of testing. Gestating animals may be convalescent from an acute infection and virus isolation negative while a persistently infected fetus remains in utero. Due to the interference of maternal antibody, it is recommended that calves be retested at 3 to 4 months of age, prior to vaccination, to ensure that no persistently infected animals would be advanced and placed in the replacement heifer breeding groups. During the 9 to 12 month period of testing, segregation of age groups and prevention of exposure of young replacements by preventing direct contact and controlling traffic of personnel and equipment with any breeding females is essential to prevent the cycle of fetal exposure and infection.
In some cases following herd testing, no persistently infected animals are identified. In these herds the persistently infected animals may have been culled or have died previously. Another possible explanation is that the herd infection with the particular strain of BVDV may have likely resulted in abortions than the production of persistently infected calves. In any case, it is essential to establish that no persistently infected animals are present in the herd and that the cycle of maternal-fetal transmission is broken. In these herds, it is important that calf testing be continued and that the importance of management decisions are stressed which reflect sound control and prevention measures to prevent exposure to BVDV. If persistently infected animals are identified these animals should be culled and removed from the herd. The potential exposure of pregnant animals to any of the identified persistently infected animals should be noted for future screening of new born animals. Vaccination
Due to the high prevelance of BVDV in the cattle population it is manditory that BVDV vaccination be done due decrease potential losses due to BVDV infection. The use of killed or modified-live vaccines can provide protection by decreasing the consequences of acute infections. However, it is questionable whether killed or modified-live vaccines provide complete fetal protection from the development of in utero fetal infections.
Kenny V. Brock, DVM, MS, PhD