Diagnostic Tests and Procedures for Alpacas Persistently Infected with Bovine Viral Diarrhea Virus
Introduction:
An alpaca that was persistently infected (PI) with bovine viral diarrhea virus (BVDV) has been recognized recently in Canada. Some cases have been diagnosed in the USA and there is much concern among alpaca producers as to the proper methods and procedures used to identify this condition.
Since this is a relative new challenge for the camelid industry, diagnosticians, and veterinarians who serve this industry do not have a great deal of experience dealing with this problem. Accordingly, we must use the information gathered from cattle in establishing criteria for making a diagnosis.
Tests used for detecting BVDV:
Virus isolation (VI) is the gold standard for detecting BVDV. Serum, white blood cells (WBCs), and tissue from infected animals taken at necropsy can all be used for VI. While feces from an animal with diarrhea may appear to be a good sample, there are a number of reasons why this material is not satisfactory. After a sample is received and processed, it is inoculated into cell cultures and virus allowed to replicate. Other procedures are used to detect presence of virus. This test is very sensitive for identifying virus-infected animals if samples are taken properly.
Polymerase chain reaction (PCR) is also a commonly used test for diagnosing BVDV. The same types of samples as listed for VI can be submitted for this test. However, the sample is tested directly without first replicating the virus in cell cultures. The PCR test reacts with a specific segment of the viral genetic material. Since it is so sensitive, it is more prone to giving a false-positive reaction. However, diagnostic laboratories go to great lengths to control this problem; very rarely is a false diagnosis made. It is a very rapid test with results available within a day after the sample is processed.
The enzyme-linked immunosorbent assay (ELISA) test is used by some diagnostic laboratories; the test identifies antigens of the virus. Similar samples as listed for VI are used with the ELISA test. In addition, this test can be used to detect virus from skin biopsies. It is a moderately sensitive test and results can be obtained in a rapid fashion. As of February 2006, this test has proven unreliable in diagnosing BVDV infection in alpacas and cannot presently be recommended. Should that situation change, this information will be updated.
The immunohistochemical (IHC) staining test is used extensively in identifying viral antigens in infected cells. As virus replicates in the animal and gains access to the blood, (a characteristic of PI animals), it infects a number of cell types including cells in the skin and hair follicles. When a sample is subjected to IHC staining and viewed with a microscope, virus-infected cells can be identified. This test is used extensively in identifying PI cattle. However, a low percentage of animals undergoing an acute infection with BVDV (and are not PI) . may give positive test results for a variable period of time after the acute infection is over. One form of the IHC test is called the immunoperoxidase test or IPX.
Procedures for detecting PI animal using the above-listed tests:
- A blood sample is taken and placed in a tube with an anticoagulant. In the laboratory, white blood cells (WBCs) or serum are separated and examined for presence of virus. Tests used for this procedure include VI, PCR or ELISA. This initial procedure will detect virus from acutely infected as well as PI animals. A second sample must be submitted 3 to 4 weeks later. An acutely infected animal will test negative on the second sample while a PI animal will remain positive. This is the traditional method for identifying PI animals.
- A skin biopsy is taken, placed in formalin, and sent to the laboratory where it is subjected to IHC staining. If virus-infected cells are observed and the animal shows typical disease signs of being PI, many veterinarians will make a diagnosis on this basis. This is often the practice when dealing with bovines. However, to verify that the animal was not just acutely infected and viral antigens lingered in skin cells, a second confirmatory test should be submitted. A confirmatory test consists of submitting a blood sample (serum or WBCs) 3 to 4 weeks later and testing for virus by VI, PCR, or ELISA.
- There is a third procedure that has been used to identify PI bovines. It consists of a skin biopsy that is refrigerated, sent to the laboratory and subjected to ELISA testing. This procedure just demonstrates presence of virus in the sample so a second confirmatory test, as listed for IHC, should be taken 3 to 4 weeks later. Continued presence of virus confirms a PI diagnosis.
Comments and recommendations:
Diagnosis of a PI alpaca is an important issue. It has far-reaching implications in that the animal readily sheds virus to other members of the herd where it can induce a variety of disease conditions. Of special concern is the pregnant animal that becomes infected and passes the PI condition to its fetus. It should be noted that a fetus is vulnerable to becoming PI only during a certain phase of its development. Generally, this period starts when the embryo implants onto the uterus and continues to just a little over the end of the first trimester. An animal can not develop the PI condition at any other time. The Veterinary Diagnostic Laboratory at Oregon State University has recently tested a PI alpaca using the IHC skin testing protocol. The veterinarian submitted skin from both an ear notch (a common practice with bovines) and a region of the skin ventral and laterally from the rectum (perineal area). Both samples were strongly positive. With the understanding that alpaca owners will not want to take notches from the ear of their animals, this serves to verify that skin samples may be taken in locations other than the ear. A sample taken from the perineal area would not disfigure the animal. We must address the issue of PI animals openly and aggressively. This problem may or may not be a major issue with camelids. If it is a problem, the more aggressively we diagnose cases the less impact it will have. It is hoped that detecting a PI animal and controlling spread of the virus will not blemish the reputation of any specific producer. In fact, it should serve to identify those individuals who are progressive leaders in health issues related to their animals. It is not an unreasonable request to have your veterinarian submit samples to another veterinary diagnostic laboratory to reconfirm a PI diagnosis. It is extremely rare for an accredited diagnostic laboratory to err in their testing procedures. I recommend that, while the animal is being reconfirmed for being PI, that it be isolated from the rest of the herd. A PI animal will shed thousands of viruses from every secretion and excretion and is a health threat to the rest of the herd.
Donald E. Mattson DVM PhD
Emeritus Virologist
Veterinary Diagnostic Laboratory
College of Veterinary Medicine
Oregon State University
Corvallis, OR 97331