Development of a panel of multiplex real-time polymerase chain reaction assays for simultaneous detection of major agents causing calf diarrhea in feces

• Published in: Journal of veterinary diagnostic investigation Vol. 22; no. 4; pp. 509 - 517
• Main Authors: Cho, Yong-Il, Kim, Won-Il, Liu, Siyuan, Kinyon, Joann M, Yoon, Kyoungjin J
• Format: Journal Article
• Language: English
• Published: United States 01.07.2010
• Subjects: Animals; bacterial infections; Bovine coronavirus; calves; Cattle; cattle diseases; Cattle Diseases - diagnosis; Cattle Diseases - microbiology; Coronavirus Infections - diagnosis; Coronavirus Infections - veterinary; Coronavirus Infections - virology; Coronavirus, Bovine - isolation & purification; cryptosporidiosis; Cryptosporidiosis - diagnosis; Cryptosporidiosis - parasitology; Cryptosporidiosis - veterinary; Cryptosporidium parvum; Cryptosporidium parvum - isolation & purification; diarrhea; Diarrhea - diagnosis; Diarrhea - microbiology; Diarrhea - veterinary; disease detection; Escherichia coli; Escherichia coli - isolation & purification; Escherichia coli Infections - diagnosis; Escherichia coli Infections - veterinary; Escherichia coli Infections - virology; Escherichia coli K99; feces; Feces - microbiology; group A Bovine rotavirus; microbial detection; Phylogeny; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - veterinary; Rotavirus; Rotavirus - isolation & purification; Rotavirus Infections - diagnosis; Rotavirus Infections - veterinary; Rotavirus Infections - virology; Salmonella; Salmonella - isolation & purification; Salmonella Infections, Animal - diagnosis; Salmonella Infections, Animal - microbiology; viral diseases of animals and humans
• ISSN: 1040-6387; 1943-4936
• DOI: 10.1177/104063871002200403

Calf diarrhea is a major economic burden to the bovine industry. Since multiple infectious agents can be involved in calf diarrhea, and the detection of each of the causative agents by traditional methods is laborious and expensive, a panel of 2 multiplex real-time polymerase chain reaction (PCR) assays was developed for rapid and simultaneous detection of the 5 major bovine enteric pathogens (i.e., Bovine coronavirus [BCoV; formally known as Betacoronavirus 1], group A Bovine rotavirus [BRV], Salmonella spp., Escherichia coli K99(+), and Cryptosporidium parvum). The estimated detection limit (i.e., analytic sensitivity) of the panel was 0.1 TCID(50) (50% tissue culture infective dose) for BCoV and group A BRV; 5 and 0.5 colony-forming units for E. coli K99(+) and Salmonella, respectively; and 50 oocysts for Cryptosporidium per reaction. In testing 243 fecal samples obtained from submissions to the Iowa State University Veterinary Diagnostic Laboratory or from experimental animals with known infection status, the newly developed multiplex real-time PCR panel simultaneously detected all 5 pathogens directly from fecal samples and was more rapid and sensitive than the traditional diagnostic tests. The PCR panel showed 89%-97% agreement with those conventional diagnostic tests, demonstrating diagnostic sensitivity equal to or better than that of the conventional tests. In conclusion, the multiplex real-time PCR panel can be a tool for a timely and accurate diagnosis of calf diarrhea associated with BCoV, group A BRV, E. coli K99(+), Salmonella, and/or Cryptosporidium.