Diagnostic performance of the canine influenza A virus subtype H3N8 hemagglutination inhibition assay

Canine Influenza A virus subtype H3N8 (H3N8 CIV) was recognized in 2004 as a novel respiratory pathogen for dogs. To date, infections have been diagnosed in thousands of dogs in 38 U.S. states. Diagnostic techniques such as reverse transcription polymerase chain reaction (RT-PCR) and virus isolation may yield false-negative results if samples are collected after virus shedding has ceased. Therefore, serology is often necessary to confirm diagnosis. The hemagglutination inhibition (HI) assay is the test of choice for serological diagnosis of influenza infections in animals. However, discrepancies exist between diagnostic laboratories and research groups in some of the test parameters for the H3N8 CIV HI assay and the cutoff antibody titer for seropositivity. The objectives of the current study were 1) to assess the diagnostic performance of a H3N8 CIV HI assay using field sera from canine infectious respiratory disease outbreaks and 2) to evaluate the effect of test parameter variations on test performance, including the use of different red blood cell (RBC) species, serum treatment methods, and virus isolates. Based on a receiver operating characteristic analysis using serum microneutralization assay titers as the gold standard, the H3N8 CIV HI assay described in the present study is highly sensitive (99.6%) and specific (94.6%) when the cutoff antibody titer for seropositivity is 32. Evaluation of parameter variations determined that the sensitivity and specificity of the H3N8 CIV HI assay depend on serum pretreatment with a receptor-destroying enzyme or periodate, use of 0.5% turkey or chicken RBCs, and use of antigenically well-matched H3N8 virus strains.