One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

Mintu Nath,Madeleine Maley,Kim Willoughby,Ellie Laming,Dylan Turnbull,Janice Gilray,Leenadevi Thonur,Tara Crook

doi:10.1186/1746-6148-8-37

Abstract

BackgroundDetection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD.ResultsA mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT).ConclusionsThe mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

Highlights

Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests
Integrity of clinical material A b-actin signal was detected in all clinical samples tested in the mRT-qPCR indicating no evidence of extraction failure or PCR inhibition
On well characterised archived isolates for each of the viruses (Table 1), only the intended target virus was amplified by the mRTqPCR

Summary

Introduction

Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. Bovine respiratory disease (BRD) is a major disease problem for the cattle industry, causing huge economic losses; research on BRD has been a longstanding global priority. While usually considered a respiratory pathogen, infection with BoHV-1 can cause abortion in pregnant cattle [7]. Infection with these viruses can facilitate invasion of opportunistic secondary pathogens such as Mannheimia haemolytica, Pasteurella multocida, Haemophilus somni and a number of mycoplasma species such as M. bovis and M. dispar [1,2,8]. Permanent lung damage can result following an episode of BRD, making animals more susceptible to subsequent episodes of respiratory disease compromising growth rates and economic returns for the farmer [4,9,10]

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