Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow.
Calf diarrhea (scours) is a primary cause of illness and death in young calves. Significant economic losses associated with this disease include morbidity, mortality, and direct cost of treatment. Multiple pathogens are responsible for infectious diarrhea, including, but not limited to, Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp. Identification and isolation of carrier calves are essential for disease management. Texas Veterinary Medical Diagnostic Laboratory current methods for calf diarrhea pathogen identification include electron microscopy (EM) for BCV and BRV and a direct fluorescent antibody test (DFAT) for organism detection of Cryptosporidium spp. A workflow was developed consisting of an optimized fecal nucleic acid purification and multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) for single tube concurrent detection of BCV, BRV, and Cryptosporidium spp., and an internal control to monitor nucleic acid purification efficacy and PCR reagent functionality. In "spike-in" experiments using serial dilutions of each pathogen, the analytical sensitivity was determined to be <10 TCID(50)/ml for BCV and BRV, and <20 oocysts for Cryptosporidium spp. Analytical specificity was confirmed using Canine and Feline coronavirus, Giardia spp., and noninfected bovine purified nucleic acid. Diagnostic sensitivity was ≥98% for all pathogens when compared with respective traditional methods. The results demonstrate that the newly developed assay can purify and subsequently detect BCV, BRV, and Cryptosporidium spp. concurrently in a single PCR, enabling simplified and streamlined calf diarrhea pathogen identification.
Texas Veterinary Medical Diagnostic Laboratory, 1 Sippel Road, College Station, TX 77843, USA.
Megan E
ME
Schroeder
Mangkey A
MA
Bounpheng
Sandy
S
Rodgers
Rocky J
RJ
Baker
Wendy
W
Black
Hemant
H
Naikare
Binu
B
Velayudhan
Loyd
L
Sneed
Barbara
B
Szonyi
Alfonso
A
Clavijo
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D017938:Coronavirus, Bovine / Q000302:isolation & purification*; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000473:pathology / Q000662:veterinary; D003458:Cryptosporidium / Q000302:isolation & purification*; D003967:Diarrhea / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary* / Q000821:virology; D055032:Electron Microscope Tomography; D005243:Feces / Q000469:parasitology / Q000821:virology; D009696:Nucleic Acids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus / Q000145:classification / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D009696:Nucleic Acids