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

Abstract

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.

Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection ofBluetongue virusandEpizootic hemorrhagic disease virus

“…Primer and probe sequences for detection of BTV, EHDV, and XIPC were adopted from previous publications. 14,16,31 The synthetic construct clone NISTag38 external RNA control sequence (GenBank accession no. DQ883679) is a unique artificial antigenomic sequence with no significant homology to annotated species sequences.…”

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

“…Primer and TaqMan probe sequences for detection of ASFV, CSFV, FMDV, and XIPC were taken from previous publications, 1,4,20,21 and optimal oligonucleotide concentrations were determined through empirical testing. Fluorescence and/or quencher molecules for oligonucleotide probes for ASFV and FMDV were modified from their original USDA designs in order to make the mRTqPCR compatible for simultaneous discrimination of 4 different targets.…”

Coronavirus Pnömoenteritis Sendromlu Buzağılarda Ultrastrüktürel ve İmmunohistokimyasal İncelemeler

“…As early as in 1972, Stair et al [2] reported pneumoenteritis in calves induced by coronaviruses. The first isolation of BоCV from bronchial secretion and nasopharyngeal washings of calves with signs of pneumonia occurred in 1982 and later, it was determined as bovine respiratory coronavirus (BRCoV) [1,2] . Strains isolated from diarrhoeic newborn calves and cattle have been classified as enteric or enteropathogenic coronaviruses (BECoV) [3] .…”

“…Bоvine Corona Virus -BoCV is outlined as one of primary etiological agents causing gastrointestinal diseases in newborn calves (calf diarrhoea -CD), winter dysentery (WD) in adult cattle and respiratory infections in calves and cattle in fattening farms -pneumoenteritis syndrome [1] . As early as in 1972, Stair et al [2] reported pneumoenteritis in calves induced by coronaviruses.…”