Validation of an immunoblot assay employing an objective reading system and used as a confirmatory test in equine infectious anaemia surveillance programs.

Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As for the in-house competitive ELISA (c-ELISA) and the AGIDT, the Italian National Reference Laboratory for EIA (NRL) validated the IB according to the OIE guidelines, employing eight panels containing positive sera, including those from EIA virus (EIAV) proven infected horses, and negative horse, mule and donkey sera collected from different geographical areas. In addition, two international reference image panels were employed for the optimization and the validation of the digital image reading system adopted that allows an impartial measurement of the serum reactivity in the IB assay. The immunological reactivity to EIAV antigens, p26, gp45 and gp90 adsorbed on the IB membrane, determines the serological status of the animal and for EIA, a p26 positive band together with at least one of the other antigen defines a subject as serologically positive for EIAV. For validation, the parameters assessed were threshold values, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility. These parameters were evaluated for each antigen as well as in combination, according to the diagnostic algorithm established above. The validation data defined the IB as having a satisfactory sensitivity, specificity, repeatability and reproducibility for all antigens and species tested. An instrumental recording of the results improves the confidence in using IB as a confirmatory test for EIAV, differently from the AGIDT that is read by an operator. The advantages of using the IB are its higher sensitivity, to that of the AGIDT, which allows an earlier detection of infection that reduces the risk of transmission and therefore the incidence of the EIA, and its higher specificity to that of the ELISA which is based on the discrimination of subjects reacting only against the p26, the antigen used by all ELISAs available, which are not considered as infected by EIAV. In particular, when this assay is used in outbreaks it can detect new cases earlier than the AGIDT, and therefore reduce the restriction period with an economic benefit for the animal owners and the public veterinary sanitary system.