Bovine rotavirus (BRV) is major cause of neonatal diarrhea in calves and cause substantial economic loss in dairy and livestock production. The tissue culture infectious dose (TCID₅₀) is a cytopathic effect (CPE)-based microscopic examination assay designed to measure virus titers almost a century ago. It is difficult to determine the virus titers when replicating viruses do not induce CPEs in cell cultures and more accurate and reliable methods need to be developed.

Fecal samples were obtained from newborn calves and a RT-PCR assay was performed to detect BRV. A cell culture isolation method was performed to isolate BRV strains in fecal suspensions and confirmed by ELISA. All BRV strains were employed to develop and validate ALPSpot assay. Spots become visible after the addition of a chromogenic substrate that catalyzes alkaline phosphatase-conjugated antibodies.

A total of 17 fecal samples tested positive for BRV in one-step RT-PCR. Only 6 BRV isolates were successfully rescued through cell culture virus isolation studies and tested positive in ELISA (Cut-off: 0.247). Assay sensitivity was measured with a higher correlation rate (R²=0.9833) when compared to the TCID₅₀ titer method and there are no significant differences between TCID₅₀ and ALPSpot methods in terms of virus titer. In addition, ALPSpot also determines the lowest virus titer when compared to TCID₅₀. In this study, an alkaline phosphatase spot (ALPSpot) detection assay has been optimized to determine BRV titers.