| Avian respiratory pathogens,including avian influenza virus(AIV),infectious bronchitis virus(IBV),infectious laryngotracheitis virus(ILTV),Newcastle disease virus,Escherichia coli,mycoplasma,are the primary causes of morbidity and mortality in the poultry industry worldwide.Avian respiratory viruses have been identified as the most economically important agents of acute and highly contagious diseases.Co-infections of multiple agents in poultry are common and have resulted in more severe clinical signs,for example,tracheal obstruction and dyspnea etc,when compared to single agent infections.Likewise,co-infection of LPAIV H9N2 circulating in China with other respiratory pathogens,particularly with IBV,ILTV,can exacerbate LPAIV H9N2 infections and result in severe clinical disease such as tracheal obstruction with different rates of mortality.IBV has many serotypes and poor protection without cross-immunity between serotypes,which often leads to immunization failure.Infectious laryngotracheitis occasionally outbreak in poultry flocks.These pathogens are of major significance and have a large economic impact because they are able to induce disease independently or in association with other avian pathogens.Many fluorescent quantitative PCR methods have been developed for the detection of avian respiratory viruses,which provide references for people engaged in the diagnosis of avian respiratory virus infections.However,most of these detection methods can only detect one virus,and the reaction conditions of different methods are not the same.Therefore,there is an urgent need for a reliable multiplex fluorescence quantitative PCR method covering a variety of important pathogens to detect unknown samples.The aim of this study is to develope a triplex real-time TaqMan PCR method for detection of H9N2 AIV,IBV and ILTV,and to determine the co-infection rate of the viruses.A total of 660 samples collected from respiratory diseased chicken in 17 provinces of China in 2019 were tested using the multiplex fluorescence quantitative PCR method established.1.Development of a triplex real-time quantitative PCR for detecting H9N2 AIV,IBV and ILTVH9N2 AIV,IBV and ILTV cause respiratory diseases in poultry.Clinical diagnosis of these viruses is challenging given the different disease presentations and the frequent occurrence of co-infections with other pathogens.Here,we standardized and validated triplex real-time fluorescence quantification PCR(RT-qPCR)assays for the straightforward detection of the viruses.The primers and TaqMan probes of this assay are based on the target genes:HA gene of H9N2,5' non-coding region of IBV and ICP4 gene of ILTV.Analytical sensitivity tests on 10-fold serial dilutions containing 5100-5109 copies of recombinant plasmids contain target gene indicated that the simplex assays have good determination coefficients and efficiency and detect a wide range of virus doses(100 to 109 molecules copies/reactions).The limitation of detection for H9AIV,IBV and ILTV are 50copies/reactions,500copies/reactions and 500copies/reactions.The relatively small values of intra-and inter-assay variability ensure the repeatability.The assays have an excellent specificity and absence of cross-reactivity with negative samples,or with other common avian viruses.The simplex H9N2 AIV,IBV and ILTV assays used probes labelled with different dyes(FAM,HEX and Cy5)and could be multiplexed for the simultaneous detection of three viruses.The determination coefficients,PCR efficiencies,and relatively small intra-and inter-assay variability were comparable to the simplex assays.This is the first time that triple RT-qPCR has been used to detect H9N2,IBV and ILTV simultaneously in trachea and lung samples or cotton swab samples,and the limitation of detection of triplex RT qPCR is the same as that of single RT-qPCR.Therefore,this method is time saving,provides quantitative results for three targets without any cross-reaction.The qPCR assays here developed can be used in simplex and triplex formats for detection and quantification of large number of samples with reliable sensitivity and specificity.These tools are expected to improve surveillance and control of these ubiquitous viruses.2.Application of TaqMan triplex real-time PCR in clinical detectionIn this study,660 clinical samples were collected from 17 provinces in China,including Jiangsu,Shandong and Hebei.Nucleic acids were extracted from homogenized or cotton-squeezed swabs,and finally detected by TaqMan triplex real-time PCR.The results showed that the total positive rate of IBV was the highest as 30.91%,followed by 19.39%of ILTV and 14.7%of H9N2 AIV.The rate of co-infection with H9 and IBV was 3.79%,the rate of co-infection with H9 and ILTV was 3.79%,and the rate of mixed infection of IBV and ILTV was 8.03%.The rate of mixed infection of three pathogens was 1.21%.It was found that the disease occurs all the year round,and there is no seasonal disease.However,the detection rate of mixed infection in December is the highest.The co-infection rate of IBV and ILTV,H9N2 AIV and ILTV,H9N2 AIV and IBV was 21.52%,10.13%,4.22%,respectively,and the mixed infection rate of the three viruses was 3.38%.According to the characteristics of host distribution,the detection rate of three diseases in broilers is higher than that in laying hens,especially for the detection rate of IBV,as high as 54.07%.According to regional distribution,the rate of pathogens in Jiangsu,Shandong and Hebei is higher,of course,it may also be related to the uneven sampling number in each province.H9 AIV,IBV and ILTV are still prevailing in poultry industry in China,which pose a heavy load on the prevention and control.Epidemiological surveillance should be done to point out the direction of disease prevention.The detection method established in this study has the nature of quick detection and good specificity,provide a powerful technology for clinical diagnosis and epidemiological investigation. |
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