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

Nicole B Goecke,Bodil H Nielsen,Lars E Larsen,Mette B Petersen

doi:10.3389/fvets.2021.677993

Nicole B Goecke, Bodil H Nielsen + Show 2 more

Open Access

https://doi.org/10.3389/fvets.2021.677993

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Abstract

Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. A vast number of viruses and bacteria are associated with the diseases. Pathogen detection using real-time PCR (rtPCR) assays performed on traditional rtPCR platforms are costly and time consuming and by that limit the use of diagnostics in bovine medicine. To diminish these limitations, we have developed a high-throughput rtPCR system (BioMark HD; Fluidigm) for simultaneous detection of the 11 most important respiratory and enteric viral and bacterial pathogens. The sensitivity and specificity of the rtPCR assays on the high-throughput platform was comparable with that of the traditional rtPCR platform. Pools consisting of positive and negative individual field samples were tested in the high-throughput rtPCR system in order to investigate the effect of an individual sample in a pool. The pool tests showed that irrespective of the size of the pool, a high-range positive individual sample had a high influence on the cycle quantification value of the pool compared with the influence of a low-range positive individual sample. To validate the test on field samples, 2,393 nasal swab and 2,379 fecal samples were tested on the high-throughput rtPCR system as pools in order to determine the occurrence of the 11 pathogens in 100 Danish herds (83 dairy and 17 veal herds). In the dairy calves, Pasteurella multocida (38.4%), rotavirus A (27.4%), Mycoplasma spp. (26.2%), and Trueperella pyogenes (25.5%) were the most prevalent pathogens, while P. multocida (71.4%), Mycoplasma spp. (58.9%), Mannheimia haemolytica (53.6%), and Mycoplasma bovis (42.9%) were the most often detected pathogens in the veal calves. The established high-throughput system provides new possibilities for analysis of bovine samples, since the system enables testing of multiple samples for the presence of different pathogens in the same analysis test even with reduced costs and turnover time.

Highlights

Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity
A wide range of viruses and bacteria are involved in bovine respiratory disease (BRD), including bovine adenovirus, bovine coronavirus (BCoV), bovine herpesvirus 1 (BHV1), bovine parainfluenza virus type 3, bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis [4,5,6]
We describe the design, optimization, validation, and use of a similar high-throughput real-time PCR (rtPCR) system consisting of 11 rtPCR assays targeting 11 respiratory and enteric viral and bacterial bovine pathogens known to be involved in BRD and bovine enteric disease (BED)

Summary

Introduction

Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. The two major calf disease syndromes are bovine respiratory disease (BRD) and bovine enteric disease (BED) which are multifactorial diseases associated with presence of a range of pathogens, environmental factors, stress conditions, and health and immunological status of the animal. Bovine respiratory disease and BED can have substantive economic consequence due to reduced productivity, increased mortality, and/or morbidity, as well as decreased animal welfare and increased use of antibiotics [1,2,3]. A wide range of viruses and bacteria are involved in BRD, including bovine adenovirus, bovine coronavirus (BCoV), bovine herpesvirus 1 (BHV1), bovine parainfluenza virus type 3, bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis [4,5,6]. The progression of BRD is believed to be related to suppression of the immune system, allowing for inflammation and damage of the respiratory tissue, which in severe cases can lead to pneumonia or even death [13]

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