A comparison of diagnostic tests for the diagnosis of equine influenza virus infections

The rapid diagnosis of Equine Influenza (EI) is critical to the implementation of control measures. In this study, two rapid antigen detection (RAD) kits and three ELISA kits were evaluated as diagnostic aids in the detection of EI. The Directigen Flu A, Espline Influenza A&B-N and the ID Screen Influenza A Antigen Capture ELISA were compared to the traditional ‘gold standard’ diagnostic tests, virus isolation (VI) and reverse transcription polymerase chain reaction (RT-PCR). A total of 75 nasopharyngeal swabs were collected from horses on premises where natural outbreaks had occurred, and a total of 104 swabs were collected from experimentally challenged foals and tested by all five methods. RT-PCR was the most sensitive diagnostic technique. Directigen Flu A was the most sensitive kit in both sample groups detecting 46.95% positive in comparison to 16.18% (Espline Influenza A&B-N) and 16.96% (ID Screen Influenza A Antigen Capture ELISA). The superiority of Directigen Flu A was confirmed by comparing the limit of sensitivity of the three assays using known concentrations of virus. Post challenge, Directigen Flu A detected positives at the time of peak virus shedding but was less effective when horses were excreting low levels of virus. Thus Directigen Flu A test is a useful screening technique but negative samples from suspect cases should be submitted to a laboratory for testing by RT-PCR. The ID Screen Influenza A Antibody Competition ELISA which detects antibodies against the viral nucleoprotein (NP) was compared to the single radial haemolysis (SRH) test and the haemagglutination inhibition (HI) test. Paired samples from 203 horses on 14 infected premises were tested. Fewer seroconversions were detected by ID Screen Influenza A Antibody Competition ELISA (25%) than by SRH (43%) or HI (41%). The acute samples from the majority of affected horses that were not detected by ID Screen Influenza A Antibody Competition ELISA were seropositive. Post challenge in seronegative foals, the ID Screen Influenza A Antibody Competition ELISA detected a rise in antibodies earlier than the SRH test suggesting that in naїve populations this ELISA could provide a faster diagnosis than traditional serology methods. Sera collected from 60 weanlings following primary vaccination with five different vaccines (two whole inactivated vaccines, two ISCOM based subunit vaccines and a canary pox recombinant vaccine) were tested by ID Screen Influenza A Antibody Competition ELISA and SRH. The ELISA did not detect the antibody response to vaccination with the canary pox recombinant vaccine (Proteq Flu Te), confirming the usefulness of the combination of this kit and vaccine to differentiate between naturally infected and vaccinated horses i.e. DIVA. The pattern of antibody response post vaccination detected with the ID Screen Influenza A Antibody Competition ELISA was similar to that detected by the SRH test for the other four vaccines. The antibody response to the other two subunit vaccines (Equip FT and Equilis Prequenza Te) was detected by ID Screen Influenza A Antibody Competition ELISA i.e. no DIVA capacity was evident. An ID Screen Influenza H7 Antibody Competition ELISA was also evaluated and demonstrated potential for measuring vaccinal response to H7N7 virus. In summary the results of this study suggest that sensitive RADs and ELISAs may be useful supplementary tests in the diagnosis and management of EI and in the monitoring of vaccine performance.