Research note: ELISA kit replacement leads to false negatives in ALV eradication-A case study in Chinese yellow-feathered broilers.

The ELISA kit for detecting the p27 antigen of avian leukosis virus (ALV) is a cornerstone reagent in avian leukosis (AL) eradication programs. Its analytical sensitivity is a key determinant of successful eradication. During an AL eradication campaign in a Chinese yellow-feathered broiler breeder flock, an unexpected shift in detection positivity rates occurred following a change in ELISA kit. The new positivity rate was inconsistent with the flock's clinical presentation, raising concerns that the lower sensitivity of the new kit may have resulted in false-negative results. In this study, we selected 200 breeding hens from the affected flock. Aseptically collected anticoagulated blood samples were subjected to virus isolation, and the cell culture supernatants were evaluated using two commercial ELISA kits (designated Kit A and Kit B) and ALV magnetic-particle chemiluminescent antigen detection kit. Kit B, the kit currently in use at the facility, yielded negative results for all 200 samples. In contrast, both Kit A and the magnetic-particle chemiluminescent kit consistently detected 12 positive samples, all of which corresponded to the same individuals. Further sequencing analysis of the env gene from positive plasma samples confirmed avian leukosis virus subgroup K (ALV-K), a strain with low replication capacity. This finding indicates that the insufficient analytical sensitivity of Kit B rendered it ineffective for detecting this particular subgroup. Collectively, this case study underscores the critical importance of pre-evaluating diagnostic kits for ALV eradication-especially when targeting low-replication strains such as ALV-K. For such strains, more sensitive detection platforms should be prioritized to ensure accurate identification and effective control.