Reagents for Detection of Viral Pathogens

Final Report

Generation of reagents to be used in the detection and control of viral pathogens in alpaca with particular emphasis on BVD virus

Principal investigator: Dr. Julia Ridpath

National Animal Disease Center, USDA, Ames, Iowa

Alpaca Research Foundation — Agreement #58-3625-8-507

There were two objectives associated with this agreement. They were as follows:

Objective 1—Development of alpaca derived cell lines to be used in virus isolation, propagation and characterization of viruses that infect alpacas.

Objective 2—Development of ELISA test(s) that detect antibodies to BVDV in alpaca sera. Such tests can be used to detennine BVDV herd exposure status.

In support of Objective 1, two alpaca fetuses were harvested and primary cell lines were derived from turbinate, testes, lung and kidney tissue. Eleven BVDV isolated from alpacas (here after referred to as alpaca BVDV) were characterized. In preliminary experiments some alpaca BVDV did not grow to high titers in bovine turbinate cells but did grow to high titers in ovine turbinate cells. Future experiments will be conducted that will compare growth rates of all eleven isolates in primary cell cultures derived from alpacas, cattle, sheep, and deer.

In support of Objective 2, six alpaca were inoculated, in succession, to a BVDV lb strain, a BVDVl a strain, and a BVDV2 strain. The primary purpose of these inoculations was to generate antisera with high levels of neutralizing antibodies against BVDV subgenotypcs currently circulating in the U.S. However, in order to maximize the information generated in these studies, we also monitored basal temperatures, blood counts, platelet counts, viral replication (as determined by virus isolation from buffy coat samples), and development of neutralizing serum antibodies. Significant drops in circulating total white blood cells (> 40% decline), lymphocytes (> 60% decline), and platelets (> 30% decline) were observed between 3 and 6 days following infection. These results suggest that acute infection with BVDV results in depletion circulating immune cells which could leave infected alpacas vulnerable to infection by secondary pathogens.

Sera with high titers of BVDV neutralizing antibodies were generated. These titers were detectable with commercial kits designed to detect antibodies against BVDV in bovine sera suggesting that commercial products now on the market could be used in surveillance for BVDV exposure in alpaca populations. A preliminary ELISA test to detect BVDV antigens based on binding by alpaca antibodies was assembled. This test is a prototype for tests to be used in screening of alpaca populations for the presence of animals persistently infected with BVDV.