High genetic diversity of equine infectious anaemia virus strains from Slovenia revealed upon phylogenetic analysis of the p15 gag gene region.

REASONS FOR PERFORMING STUDY: The equine infectious anaemia virus (EIAV), which belongs to the Retroviridae family, infects equids almost worldwide. Every year, sporadic EIAV cases are detected in Slovenia. OBJECTIVES: To characterise the Slovenian EIAV strains in the p15 gag gene region phylogenetically in order to compare the Slovenian EIAV strains with EIAV strains from abroad, especially with the recently published European strains. STUDY DESIGN: Cross-sectional study using material derived from post mortem examination. METHODS: In total, 29 EIAV serologically positive horses from 18 different farms were examined in this study. Primers were designed to amplify the p15 gag gene region. Amplicons of 28 PCRs were subjected to direct DNA sequencing and phylogenetic analysis. RESULTS: Altogether, 28 EIAV sequences were obtained from 17 different farms and were distributed between 4 separate monophyletic groups and 9 branches upon phylogenetic analysis. Among EIAV strains from abroad, the closest relatives to Slovenian EIAV strains were European EIAV strains from Italy. Phylogenetic analysis also showed that some animals from distantly located farms were most probably infected with the same EIAV strains, as well as animals from the same farm and animals from farms located in the same geographical region. CONCLUSIONS: This is the first report of such high genetic diversity of EIAV strains from one country. This led to speculation that there is a potential virus reservoir among the populations of riding horses, horses kept for pleasure and horses for meat production, with some farmers or horse-owners not following legislation, thus enabling the spread of infection with EIAV. The low sensitivity of the agar gel immunodiffusion test may also contribute to the spread of infection with EIAV, because some infected horses might have escaped detection. The results of the phylogenetic analysis also provide additional knowledge about the highly heterogeneous nature of the EIAV genome.

Detection and molecular characterisation of equine infectious anaemia virus from field outbreaks in Slovenia.

REASONS FOR PERFORMING THIS STUDY: In 2009, a surprisingly high number of animals seropositive for equine infectious anaemia virus (EIAV; 26 horses from 13 farms) were detected in Slovenia. OBJECTIVES: To develop a polymerase chain reaction (PCR) assay for the detection of the proviral nucleic acid, to phylogenetically characterise the Slovenian EIAV strains and to investigate whether transmission in utero occurred. STUDY DESIGN: Cross-sectional clinical study. METHODS: In total, 26 horses (including 2 foals and 4 pregnant mares) and 4 fetuses were examined in this study. A PCR assay using the EIAV F1 and EIAV R1 primers was designed and tested using genomic DNA extracted from 28 spleen samples, 18 whole blood samples and 17 peripheral blood leucocyte samples. Amplicons of 22 PCRs obtained from the spleen samples were subjected to direct DNA sequencing and phylogenetic analysis. RESULTS: All spleen samples from 22 adult animals were positive for EIAV by PCR, whereas whole blood and the peripheral blood leucocyte samples were positive from only 4 animals. Spleen samples from foals and fetuses were negative by PCR. The Slovenian EIAV sequences could be mapped to 9 different branches of the phylogenetic tree. CONCLUSIONS: The PCR was able to detect different EIAV strains from spleen samples of seropositive animals detected in Slovenia. Phylogenetic analysis revealed high genetic diversity of the EIAV strains detected in Slovenia, with their closest relatives being European strains. In utero transmission in pregnant mares did not occur.