Simultaneous detection of major pathogens causing bovine diarrhea by multiplex real-time PCR panel

Bovine diarrhea is a major economical burden to the bovine industry in Korea. Since multiple infectious agents can be involved in bovine diarrhea, differential diagnosis is essential for effective treatment. Therefore, a panel of two multiplex real-time PCR assays which can simultaneously detect six major bovine enteric pathogens [i.e., bovine viral diarrhea virus (BVDV), bovine coronavirus (BCoV), group A bovine rotavirus (BRV), Salmonella spp., Escherichia coli (E. coli) K99⁺, and Cryptosporidium parvum] was developed and applied to test 97 fecal samples collected from cattle farms in Korea. In addition, microscopic examination was also preformed on the samples to detect Coccidium oocyst. The estimated sensitivity of the multiplex PCR was 0.1 TCID₅₀ for BVDV, BCoV and group A BRV, 5 and 0.5 CFU for E. coli K99⁺ and Salmonella, respectively, and 50 oocysts for Cryptosporidium. The amplification efficiency of the multiplex PCR ranged between 0.97 and 0.99 for each pathogen. Among 97 samples, 36 samples were positive for at least one of the 6 major pathogens and 6 samples were simultaneously positive for 2 pathogens by the multiplex PCR assay. Coccidium oocysts were also detected in 48 samples, which were all collected from over 1 month old calves. In conclusion, the multiplex real-time PCR panel can be a useful tool for fast and accurate diagnosis of calf diarrhea associated with BVDV, BCoV, group A BRV, E. coli K99⁺, Salmonella, and/or Cryptosporidium and Coccidium may be an important target which needs to be included in the multiplex PCR panel in the future.