Detection of horses infected naturally with equine infectious anemia virusby nested polymerase chain reaction

A nested polymerase chain reaction (PCR) amplifying a region of the gag gen e of equine infectious anemia virus (EIAV) was developed for the rapid and direct detection of proviral DNA from the peripheral blood of naturally inf ected horses and was compared with the Coggins test. DNA prepared from whit e blood cells of 122 field horses from 15 stables with reported cases of EI AV and one seronegative: stable were analysed. Amplifications of expected s ize fragments were obtained by nested PCR for 88 horses using two different sets of primers targeting the gag region. The specificity of the amplified products was confirmed by hybridization using a digoxigenin-labeled probe. Gag-nested PCR-restriction fragment length polymorphism analysis distingui shed two different subtypes of gag gene, A and B. Subtype A was found to be the most prevalent among the infected horses that were tested. The PCR-gag amplified sequence of subtype A shared 84.6% nucleotide and 93%, deduced a mino acid sequence identities with the prototype Wyoming strain whereas sub type B sequence was almost 100%, identical to the prototype. Sequence analy sis of gag subtype A suggests the presence of a novel EIAV variant among in fected horses in Canada. The nested PCR assay developed in the present stud y detected more EIAV positive animals and was found as specific as the agar gel immunodiffusion (Coggins) assay and offers great potential a diagnosti c test for the detection of EIAV infections in field horses. (C) 2001 Elsev ier Science B.V. All rights reserved.