Seroprevalence of turkey coronavirus in North American turkeys determined by a newly developed enzyme-linked immunosorbent assay based on recombinant antigen

Abstract

Turkey coronavirus (TCoV) causes diarrhea in young turkey poults, but little is known about its prevalence in the field. To address this, the complete nucleocapsid gene was cloned and expressed in Escherichia coli. Expressed nucleocapsid gene produced two distinct proteins (52 and 43 kDa); their specificity was confirmed by Western blotting using two different monoclonal antibodies. Recombinant N protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay (ELISA) for the serological detection of TCoV that was then validated using experimentally derived turkey serum. The N-based ELISA showed (97%) sensitivity and (93%) specificity for TCoV, which was significantly higher than an infectious bronchitis coronavirus-based commercial test for TCoV. To assess the utility of this recombinant ELISA, 360 serum samples from turkey farms in Ontario, Canada, and 81 serum samples from farms in Arkansas were tested for TCoV-specific antibodies. A high seroprevalence of TCoV was found in turkeys from the Ontario farms with 73.9% of breeders and 60.0% of meat turkeys testing seropositive using the N-based ELISA. Similarly, a high field prevalence was found in the turkeys from Arkansas, for which 64.2% of the serum samples tested seropositive.

FIG. 1.

Reverse transcriptase PCR amplification of the TCoV N gene (1,227 bp) from TCoV used for the cloning and expression of recombinant protein.

FIG. 2.

Production and confirmation of antigenicity of TCoV N recombinant fusion protein produced by E. coli BL-21 transfected with pGEX-4T3-TCoV-N using 12% discontinuous SDS-PAGE (lanes 1 to 3) and subsequent Western blotting with anti-GST and anti-TCoV N-protein MAbs 1.01 and 4.23 (lanes 4 to 7). Lane M, molecular mass marker in kilodaltons; lane 1, nonpurified bacterial lysate after induction with IPTG; lane 2, TCoV N fusion protein purified with GST beads from bacterial lysate after induction with IPTG; lane 3, the same as lane 2 but from noninduced bacterial culture; lanes 4 and 5, Western blot using anti-GST antibodies against the TCoV N fusion protein expressed and purified as in lanes 2 and 3, respectively; lanes 6 and 7, Western blot using MAbs 1.01 and 4.23, respectively, against the TCoV N fusion protein expressed and purified as described for lane 2.

FIG. 3.

ROC analysis of TCoV N-based ELISA. The cutoff value derived from the curves was 0.180 (indicated by ⊗) that resulted in 97% sensitivity and 93% specificity for the TCoV N-based ELISA.

FIG. 4.

Comparison of the detection of TCoV in turkey serum samples obtained from breeder turkey farms (84 samples) and meat turkey farms (84 samples) in Ontario, Canada, examined in duplicate using TCoV N-based (gray bars) and IBV whole-virus-based (black bars) ELISAs. The recombinant TCoV N-based ELISA detected an overall seroprevalence of TCoV of 70.24% that was about fourfold higher than the 16.67% overall seroprevalence reported by the commercial IBV whole-virus-based ELISA.

FIG. 5.

Scatter plot of the OD₄₀₅ values obtained from 168 paired turkey serum samples from Ontario turkey farms using TCoV N- and a whole-virus IBV-based commercial ELISA. Only a modest correlation (R = 0.2623) was observed between the recombinant antigen TCoV N-based ELISA and the commercial IBV whole-virus-based ELISA. The usual OD₄₀₅ cutoff value for the N-based ELISA is 0.180 (horizontal dotted line), and the usual OD₆₅₀ cutoff value for the whole-virus IBV-based ELISA is 0.100 (vertical dotted line).