Establishment and preliminary application of an ELISA method for detecting antibodies to field strains of bovine infectious rhinotracheitis virus.

Infectious bovine rhinotracheitis (IBR), caused by IBR virus (IBRV), is an acute, febrile, and highly contagious disease affecting cattle. Infected animals become lifelong carriers and shed the virus intermittently, complicating disease control and eradication, thereby compromising herd health and inflicting substantial economic losses on the cattle industry. Although vaccination can mitigate clinical signs, it does not eliminate the virus from infected individuals. A more effective control strategy integrates immunization with the detection and culling of infected animals. Given that only inactivated vaccines are approved for use in China, this study aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) capable of specifically detecting IBRV infection by targeting antigenic epitopes derived from the non-structural immediate-early transactivator protein BICP0, which is expressed exclusively upon host infection. Bioinformatics analysis identified epitope 3 of BICP0 as highly specific. The coding sequence contained several rare codons for Escherichia coli; following codon optimization, the gene was cloned into an expression vector, transformed into E. coli, and expressed at a large scale. The purified recombinant protein was subsequently used to establish an optimized BICP0-ELISA. The optimal assay conditions were determined as follows: antigen coating concentration, 1 μg/mL; blocking buffer, 5% skim milk; serum dilution, 1:10; horseradish peroxidase (HRP)-conjugated goat anti-bovine IgG dilution, 1:2500; and chromogenic substrate incubation, 15 min. Using sera from non-vaccinated cattle and a commercial ELISA kit as the reference standard, receiver operating characteristic (ROC) curve analysis established a cut-off value of 0.225. At this threshold, the BICP0-ELISA achieved 100.0% sensitivity, 98.6% specificity, and 98.6% agreement with the commercial kit. Field evaluation of serum samples collected from cattle farms in selected areas of Liaoning Province revealed IBRV infection rates of 28.5% in non-vaccinated herds and 50.9% in vaccinated herds, with an overall seropositivity rate of 40.1%. These findings indicate a high prevalence of IBR among cattle farms in Liaoning. In summary, the BICP0-ELISA developed in this study exhibits excellent specificity and sensitivity. Based on the non-structural nature of BICP0, this method demonstrates potential for differentiating infected from vaccinated animals (DIVA) in herds immunized with inactivated vaccines. The preliminary data support its application as a candidate serological tool for IBR surveillance and eradication programs.