Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow

Area of Science:

• Veterinary Diagnostics
• Molecular Biology
• Animal Health

Background:

• Calf diarrhea (scours) causes significant morbidity, mortality, and economic losses in young cattle.
• Infectious agents like Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp. are primary causes.
• Current diagnostic methods (EM, DFAT) are often separate and less efficient for concurrent detection.

Purpose of the Study:

• To develop and validate a streamlined workflow for simultaneous identification of key calf diarrhea pathogens.
• To improve the efficiency and accuracy of diagnosing infectious diarrhea in calves.
• To establish a reliable method for identifying carrier calves.

Main Methods:

• Development of an optimized fecal nucleic acid purification protocol.
• Creation of a multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay.
• Inclusion of an internal control for quality assurance of purification and PCR.

Main Results:

• The assay achieved high analytical sensitivity (<10 TCID50/ml for BCV/BRV, <20 oocysts for Cryptosporidium).
• High diagnostic sensitivity (≥98%) was confirmed for all targeted pathogens compared to traditional methods.
• Analytical specificity was validated against related and unrelated organisms.

Conclusions:

• The developed multiplex RT-qPCR assay enables concurrent detection of BCV, BRV, and Cryptosporidium spp. in a single reaction.
• This streamlined approach simplifies and enhances the identification of calf diarrhea pathogens.
• The assay offers a valuable tool for disease management and reducing economic losses in calf production.