Molecular characterization of opportunistic pathogens from avian Mycoplasma cases in Punjab

Abstract

Respiratory tract infections are of great importance in poultry industry, causing heavy economic losses. Mycoplasma gallisepticum and Mycoplasma synoviae are the most pathogenic organisms of the respiratory tract. Other respiratory tract infections includes both viral pathogens (Newcastle disease virus, Infectious bronchitis virus, avian influenza virus) and bacterial pathogens (Salmonella pullorum, Escherichia coli, Avibacterium paragallinarum, etc) cause disease independently and in association with each other. The study was designed to check the possible role of Mycoplasma infections in disseminating other respiratory pathogens. Further, the different diagnostic techniques including serum plate agglutination (SPA) test, cultural isolation and polymerase chain reaction (PCR) were applied are compared for their capabilities for the identification of the pathogens. Serum Plate Agglutination (SPA) test was used for serological screening test for Mycoplasma species. Samples including oral/ nasal swabs, lungs trachea and air sac swabs were collected from sero-positive and sero-negative flocks. Cultural isolation was on Frey’s Modified medium for Mycoplasma isolation, embryonated eggs for viral isolation and blood agar for other bacterial isolation. Polymerase chain reaction and Reverse transcriptase-polymerase chain reaction was optimized for the molecular identification of bacterial and viral pathogens, respectively. Multiplex PCR was also optimized for the simultaneous detection of respiratory tract pathogens of both bacterial and viral pathogens including Mycoplasma gallisepticum, Mycoplasma synoviae, Newcastle disease virus, Avian influenza virus and Infectious bronchitis virus using specific primers. To resolve further variation among opportunistic pathogenic species, the PCR products were sequenced and phylogenetic analysis was carried out. In the present study 34 flocks showing respiratory distress were visited for serological screening of Mycoplasma involvement in respiratory distress cases. Out of 34 flocks visited 27 (79.1%) were serologically positive. Based on PCR based diagnosis, irrespective of serological status the highest involvement of bacterial pathogens recorded was MG (31.8%), followed by E. coli (20.7%), MS (7.9%) and Av. paragallinarum (5.3%). Moreover, in case viral pathogens recovery from respiratory distress cases was recorded maximum in NDV (24.9%) then IBV (4.3%) and AIV (1.5%). The multiplex PCR was efficiently optimized for the simultaneous detection of respiratory tract infections including Mycoplasma gallisepticum, Mycoplasma synoviae, Newcastle disease virus, Avian influenza virus and Infectious bronchitis virus. Mycoplasma gallisepticum amplified 720bp PCR product, while Mycoplasma synoviae, yielded 270bp product. In case of viral pathogens Newcastle disease virus was identified by amplifying 320bp product, Avian influenza virus, 1050bp PCR product and Infectious bronchitis virus yielded 1720bp band. DNA sequences of Mycoplasma gallisepticum, Mycoplasma synoviae and Newcastle disease virus was submitted to GenBank as Mycoplasma gallisepticum (lp- gene) strain ABSuafMG2011 partial sequence, (GenBank accession no. JN114112). For Mycoplasma synoviae (16SrRNA gene) strain ABSfsdMS2011 partial sequence, (GenBank accession no. JN638722). While for Newcastle disease virus (Fusion gene) stains ABSuafND2011 partial sequence, (GenBank accession no. JN160608) and strain ABSfsdND2011 partial sequence (GenBank accession no. JN377950) In conclusion, the incidence of respiratory tract pathogens in sero-positive flocks for Mycoplasma was found higher as compared to sero-negative flocks. The true prevalence of the Mycoplasma infections is reflected by combining PCR results with SPA test. The present study also documented the involvement of indigenous strains of MG, MS and NDV in the respiratory distress cases. Multiplex PCR was successfully optimized for the simultaneous and early detection of respiratory tract infections.