Validation of an indirect in-house ELISA using synthetic peptides to detect antibodies anti-gp90 and gp45 of the equine infectious anaemia virus.

BACKGROUND: Equine infectious anaemia (EIA) is controlled by the identification of seropositive animals. The official diagnostic method is the agar gel immunodiffusion (AGID) test, which detects antibodies against a viral core protein (p26). Although AGID is inexpensive and specific, the report of results takes considerable time and the test has low analytical sensitivity. OBJECTIVE: To validate our in-house indirect ELISAgp90/45 , following the World Organization of Animal Health (OIE) criteria. STUDY DESIGN: Test validation. METHODS: Synthetic peptides gp90 and gp45 were used as antigens in ELISAgp90/45 . Tests used for validation, calibration and linear working operating range, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility were assessed by comparing them with the AGID test and using 1844 equine sera grouped into five different panels. RESULTS: We were able to replace the National References Sera with our Internal Reference Sera. ELISAgp90/45 had acceptable repeatability and reproducibility. Analytical sensitivity of the ELISAgp90/45 was 800 times greater than that of AGID test for positive sera and 400 times greater for weak positive sera. ELISAgp90/45 also showed optimal analytical specificity, since no cross-reactivity was detected with antibodies against other equine viruses. One sample was positive by AGID test and negative by ELISAgp90/45. ELISAgp90/45 was performed using 243 EIA positive and 878 negative equid sera, and showed a diagnostic sensitivity of 99.59% [CI 97.73%-99.99%] and a diagnostic specificity of 90.32% [CI 88.17%-92.19%], compared to AGID test; thus, it was demonstrated to be a robust test. MAIN LIMITATIONS: Samples were derived from naturally infected equid populations showing heterogeneous clinical states: therefore, their status was uncertain and some horses were sampled more than once. The AGID test may not be the most useful gold standard. CONCLUSION: ELISAgp90/45 is a useful tool for the diagnosis of EIAV infection and meets validation requirements established by the OIE.

Identification and genetic characterization of equine infectious anemia virus in Western Balkans.

BACKGROUND: Equine infectious anemia (EIA) is a viral disease, caused by the Equine Infectious Anemia virus (EIAV) belonging to the Retroviridae family, genus Lentivirus. Horses (or equids) infected with EIAV are lifelong carriers and they remain contagious for other horses even in the absence of clinical signs. So far, EIAV infection has been reported among horses in North and South America, France, Germany, Italy, Hungary and Romania, with no publication regarding the presence of EIAV in horses in Serbia. To determine the circulation of EIAV among, approximately, the 5000 horses of the Vojvodina region, northern part of Serbia, 316 serum undergone serological testing for EIA. Then, identification and full genome sequencing using next generation sequencing was performed from one EIA positive horse. RESULTS: the 316 sera were tested with 3 different commercial agar gel immunodiffusion (AGID) tests and two different commercial enzyme-linked immunosorbent assay (ELISA). With the three AGID kits, 311 (98.4%) among the 316 tested sera were negative and only five (1.6%) sera were positive for EIA. Some discrepancies were seen for the two ELISA kits tested since one exhibited the same results as AGID test and the second gave 295 sera with negative results, five with a positive result and 16 with doubtful outcome. Phylogenetic analysis performed using the full genome sequence showed that EIAV characterized from a horse in Serbia is different from those identify so fare around the world and form a distinct and separate group together with another EIAV strain. CONCLUSIONS: This study demonstrate for the first time that EIAV is circulating at a low level in the horse population from the Northern part of Serbia. Interestingly, phylogenetic data indicates that this EIAV from the western Balkan region of Europe belongs to a new cluster.

Occurrence of seropositive sheep (Ovis aries) to bovine leukemia virus in Brazil / Ocorrência de ovinos (Ovis aries) soropositivos ao vírus da leucemia bovina no Brasil

Occurrence of seropositive sheep (Ovis aries) to Bovine Leukemia Virus (BLV) by agar-gel immunodiffusion test (AGID) using the antigen gp51 was surveyed for the period 2005-2007. Samples were collected from sheep in the Brazilian states of Rio Grande do Sul, Paraná, São Paulo, Pernambuco, Maranhão, Pará, Bahia, Mato Grosso, Rondônia, and Acre. Two of 35 (5.7%) flocks were seropositive to BLV, and the rate of seropositive animals was 0.077% (two of 2,592). The two seropositive sheep were female, one 13-month old Santa Inês breed and other of unknown age and breed, both from the state of São Paulo. Distribution of BLV in the ovine population studied proved to be a rare event in Brazil.

A ocorrência de ovinos sororreagentes ao vírus da Leucemia Bovina (VLB) pelo teste de imunodifusão em gel de ágar (IDGA) utilizando o antígeno gp51 foi avaliada no período de 2005-2007, em diferentes regiões do Brasil. Amostras foram colhidas de ovinos dos Estados do Rio Grande do Sul, Paraná, São Paulo, Pernambuco, Maranhão, Pará, Bahia, Mato Grosso, Rondônia e Acre. Duas em 35 cabanhas (5,7%) e dois em 2592 ovinos (0,077%) foram soropositivos. Os únicos animais com anticorpos contra o VLB eram fêmeas, uma com 13 meses de idade e da raça Santa Inês e a outra não se conhecia a idade e raça, ambas provenientes do Estado de São Paulo. A distribuição da soropositividade na população estudada demonstrou ser rara a infecção pelo VLB em ovinos no Brasil.