Um PCR/RT-PCR multiplex para detecção de cinco agentes da diarreia neonatal bovina

Abstract

Neonatal calf diarrhea (NCD) is a complex disease that affects calves, especially in the first month of life, and is responsible for high economic losses, being a major health challenge in beef and dairy cattle herds. NCD has a multifactorial etiology and is often frequently associated with single or mixed viral, bacterial and protozoal infections. Consequently, laboratory diagnostic of NCD usually requires specific tests for each agent, a time-consuming, laborious, complex and expensive process. Herein, we describe an end-point multiplex PCR/RT-PCR (one-step RT-PCR-based) for individual or simultaneous detection of five major NCD agents: bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (E. coli K99), Salmonella enterica (S. enterica) and Cryptosporidium parvum (C. parvum). Initially, we selected and/or designed high-coverage primers. Subsequently, we standardized the multiplex PCR/RT-PCR, optimizing the mix (primer concentration and enzyme volume) and assay conditions (initial denaturation, annealing and extension temperature, and number of cycles). After careful and rigorous optimization, we evaluated the analytical sensitivity of the multiplex for the five targets and then assessed the assay's diagnostic performance by testing 95 clinical samples of diarrheaic calf feces. The analytical specificity was evaluated against other bovine pathogens: bovine viral diarrhea virus (BVDV), E. coli heat-stable enterotoxin (STa) and Eimeria spp. The detection limit of our multiplex was 10 infectious units of BRV, 10-2 dilution of a BCoV positive sample pool, 5 x 10-4 CFU for S. enterica, 5 x 10-6 CFU for E. coli K99 and 50 oocysts for C. parvum. No nonspecific amplification of other potential bovine diarrhea agents was detected in the assay. Out of 95 samples analyzed, 50 (52.6%) were positive for at least one target, being 35 and 15 single and mixed infections, respectively. BRV was the most frequent agent detected in single infections (16/35), followed by Cryptosporidium spp. (11/35), which was also the most frequent agent in mixed infections (11/15). Importantly, positive and negative multiplex results were confirmed in individual reactions for each agent. In conclusion, we described an end-point multiplex PCR/RT-PCR for faster, easier and more efficient NCD diagnosis, which may be useful in future clinical and surveillance studies.