Detection of equine infectious anemia nucleic acid in asymptomatic carrier horses by nested PCR

Abstract

Abstract Background: Equine infectious anemia virus (EIAV) is a lentivirus with an almost worldwide distribution, infecting equids. It causes a persistent infection that is characterized by recurring episodes of fever, anemia, and thrombocytopenia. Most of the horses may control EIAV replication within a year, remaining persistently infected without clinical signs of disease. Objective: Detect EIA nucleic acid from peripheral blood of asymptomatic horses using nested PCR. Materials and method: We used nested PCR, amplifying P26 gag gene of EIAV, for direct detection of viral RNA in plasma and proviral DNA from PBMC in asymptomatic carrier horses in comparison with the Coggins test. EIA nucleic acid was prepared from 20 seropositive and five EIAV seronegative horses. Amplification of 246 bp expected size fragments was obtained using two different sets of primers targeting the P26 gag gene. Results: Among 20 seropositive horses, nine samples were positive for RNA and DNA. The five samples were positive for DNA but not for RNA, which indicates that the virus integrated into the host cell genome with a low level of viral replication. However, six samples were negative for both DNA and RNA. False negative could result due to primer failure caused by gag sequences variation among strains circulating in Thailand when compared with various strains from other parts of the world. EIAV antigens may also be prepared from cell cultures contaminated with other retroviruses thus causing false positives with the Coggins test. Conclusion: Nested PCR can be a useful tool for detecting the presence of EIAV in asymptomatic carrier horses. This may be especially true during the acute stage of the disease where the viremia levels are usually at the highest levels before detectable antibodies appear.