Design of a multiplex quantitative reverse transcription‐PCR system to simultaneously detect 16 pathogens associated with bovine respiratory and enteric diseases

Abstract

Aim Bovine respiratory disease (BRD) and bovine enteric disease (BED) are two major diseases in cattle, resulting in severe economic losses in the dairy and beef industries. The two major diseases are associated with several factors such as viruses, bacteria, the health condition of the host and environmental factors. We aimed to design a new efficient diagnostic method, which rapidly detect causative pathogens, minimizing economic loss due to BRD and BED. Methods and Results We designed a multiplex quantitative reverse transcription‐PCR (qRT‐PCR) system for the simultaneous diagnosis of 16 pathogens, including 12 viruses and 4 bacteria related to BRD and BED, based on single qRT‐PCR assays in previous studies. The designed multiplex qRT‐PCR was highly sensitive and has minimal detection levels which will be no different from those of single qRT‐PCR. Moreover, the multiplex qRT‐PCR could more efficiently detect the causative pathogens than conventional RT‐PCR in test using a part of BRD and BED clinical samples. Furthermore, our data revealed that the multiplex qRT‐PCR had high performance in its specificity and reproducibility tests. Conclusions Our system can effectively detect multiple BRD or BED related pathogens from each animal while testing several clinical samples via the multiplex qRT‐PCR. It is more time‐, cost‐ and labour‐efficient than other diagnostic methods. Significance and Impact of the Study Rapid detection of infected animals from the herd using our system will greatly contribute to infection control and prompt treatment in field.

In vivo Microbiome Profiling of the Luminal and Mucosal Surface of the Duodenum Using a Cannulated Yearling Bovine Model

“…A 7.5-cm rumen cannula 2 was also surgically fitted in the rumen as previously described in the literature at the same time as duodenal cannulation for a concomitant rumen microbiota study (18). Post-operative treatment consisted of ceftiofur hydrochloride 3 , as an antibiotic, administered subcutaneously (2.2. mg/kg) once daily for 5 days and meloxicam 4 , as an antiflammatory, administered orally (1.0 mg/kg) once daily for 5 days. A 3month recovery period was observed following surgery to allow complete healing of the surgical sites, ensure appropriate drug withdrawal periods were met, and provide research animals a consistent diet prior to study initiation and sample collection.…”

“…Enteric diseases are known to be one of the major contributors, along with bovine respiratory disease, to decrease in feed consumption, weight gain, reduction in milk production, in dairy cattle, and deaths of youngstock, resulting in severe economic losses in the dairy and beef industries (3). The impact of such diseases extends to human health via the increased use of antimicrobial medications, risk of development of antimicrobial resistance, and the potential microbial contamination of human food products.…”

Genetic and Antigenic Characterization and Retrospective Surveillance of Bovine Influenza D Viruses Identified in Hokkaido, Japan from 2018 to 2020

“…These samples were subjected to conventional reverse transcription-PCR (RT-PCR) analysis specific for bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), and bovine coronavirus (BCoV) based on the recent prevalence of viruses associating with BRD, in Japan, reported in previous studies using the PrimesScript One Step RT-PCR Kit Ver. 2 (Takara, Shiga, Japan), according to previously reported methodologies [28][29][30][31][32]. In addition, all samples were subjected to real-time (RT)-PCR (qRT-PCR) specific for BIDVs, according to a previous report [31].…”

“…In addition, all samples were subjected to real-time (RT)-PCR (qRT-PCR) specific for BIDVs, according to a previous report [31]. Obtained samples were also tested for the presence of three bacterial species (Mycoplasma bovis, Pasteurella multocida, and Mannheimia haemolytica) based on the recent prevalence of bacteria associating with BRD in Japan in previous studies, according to a routine methodology [31][32][33][34].…”

Design of a High-Throughput Real-Time PCR System for Detection of Bovine Respiratory and Enteric Pathogens

“…A variety of laboratory tests, including culture and molecular methods, have been described and these methods all have their benefits and limitations in regard to sensitivity, specificity, predictive values, speed, and costs (23). During recent years, a range of multiplex real-time PCR (rtPCR) tests targeting pathogens involved in BRD and/or BED have been developed (24)(25)(26)(27)(28). The multiplex rtPCR test allows for simultaneous analysis of three-five pathogens in a single sample.…”