A Multiplex PCR assay for simultaneous detection of common respiratory pathogens of poultry in Andhra Pradesh

Respiratory pathogens detection in poultry by Multiplex PCR

Abstract

The present study was undertaken to screen the common respiratory pathogens like avian mycoplasmas (Mycoplasma gallisepticum and Mycoplasma synoviae), Infectious laryngotracheitis virus (ILTV) and  Infectious bronchitis virus(IBV) in poultry farms of Andhra Pradesh by developing suitable diagnostic tool i.e. Multiplex PCR assay for rapid detection and differentiation of these pathogens. Mixed respiratory infections are common in commercial poultry due to intensive rearing in large numbers. Among the respiratory pathogens avian mycoplasmas, IBV and ILTV are the major etiology causing significant economic losses to poultry industry. To reduce the losses, early detection and differentiation of common respiratory pathogens, the at a time detection and differentiation of common respiratory pathogens by application of multiplex PCR assays have been regularly used in poultry. In our study we standardized a multiplex PCR assay by targeting individual gene for each pathogen and used this test for rapid screening of above agents. 228 pooled cloacal swabs, 228 pooled nasal swabs, 228 pooled tracheal swabs samples  from the ailing birds and 152 pooled tracheal tissues, 152 pooled  lung tissues and 76 pooled oviduct  samples   from  the dead birds were collected from the suspected 19 poultry farms in A.P. as  labelled accordingly.  For extraction of Nucleic acid the Trizol method was used. All nineteen pooled clinical samples and were subjected for PCR individually. A Multiplex PCR test  was developed for in  a single tube detection of all common four  pathogens by choosing individual genes i.e., N gene for IBV, ICP4 gene for ILTV, 16S r RNA gene for MG and vlhA gene for MS. The results revealed that 10 farms (MG), 7 farms (MS), 5 farms (MG AND MS) and 2 farms were found to be positive for ILTV and none of them found to be positive for IBV. Among the 17 positive farms ,60 cloacal swabs were positive for MG,120 tracheal swabs,96 nasal swabs, 72 tracheal swabs, 64 lung tissues were found were found  positive for MG,MS and 8 oviduct samples were found positive for MS. Among the 2 positive farms, twenty-four tracheal swabs and sixteen tracheal tissues were found positive for ILTV.