Biological Characterization and Comparative Diagnostics of Mycoplasma gallisepticum Isolates

Abstract

Mycoplasma gallisepticum infections are of great economic importance for commercial poultry. Strategies to maintain M. gallisepticum free flock, include biosecurity practices to minimize likelihood of exposure, use of antibiotics in case of infected flocks to culminate spread of infection and reduce production losses or to use vaccines where exposure to infectious agent cannot be controlled. Continuous monitoring of commercial flocks is imperative for early control of disease. Highly infectious nature of organism is responsible for rapid spread of infection within a flock. Once the flock gets infected, management of infectious agent to suppress clinical infection is arduous for poultry farmer. The present study was undertaken to determine sero-surveillance, biological and molecular characterization of M. gallisepticum as well as to evaluate the effectiveness of lab diagnostic tests and vaccines used for the control of this disease in commercial and backyard poultry. The serological study was designed to assess status of anti-mycoplasma antibodies in poultry, along with molecular detection of suspected and infected flocks. Attempts were made to recover field isolate from infected flocks. To evaluate seroprevalence of M. gallisepticum among commercial poultry, unvaccinated layer and breeder farms were investigated from year 2015-2018. Serum plate agglutination (SPA) test and Enzyme linked immunosorbent assay (ELISA) were performed to detect and quantify antibodies. Molecular prevalence of disease was assessed from 2016-2019 through polymerase chain reaction (PCR). Using modified Frey’s media, isolation of M. galllisepticum from PCR positive field samples was done. High cumulative seropositivity was detected in backyard poultry (59.80%) than in commercial poultry (41.93%). Molecular detection rate of M. galllisepticum from selected study areas was 11.93% and isolation rate was 10.5%. To unveil the biological behaviour of local M. gallisepticum isolates, pathogenesis of an isolate designated as Pak MG1 (ARL-1963) was studied. Experiment was setup to judge pathogenicity of isolate by determining level of tracheal lesions and air-sacculitis caused in experimental birds. In addition, dissemination of infection to internal body organs was also evaluated. It was found that PakMG1 (ARL-1963) was localized to upper respiratory tract and cause mild air sac lesions. No dissemination to internal body organs was noticed. xviii Molecular characterization of 03 Pakistani M. gallisepticum isolates i.e. Pak MG1(ARL 1963), Pak MG2 (ARL- 2020) and Pak MG3 (ARL- 2668) was carried out by gene target sequence analysis of 03 surface protein genes and 01 lipoprotein gene ((gapA, lp, pvp A, mgc2). The study provided an understanding about genetic relatedness of local isolates, Pak MG1(ARL-1963), and Pak MG3 (ARL- 2668) were more closely related to each other and distinct from Pak MG2 (ARL- 2020). For cheap availability of rapid serological test to local farmers, a study was designed to develop SPA antigen using a local M. gallisepticum isolate (Pak MG1). Developed SPA antigen was tested for sensitivity, specificity and evaluated for shelf life. A comparison study was conducted using in house SPA antigen and commercially available antigen. It was found that in house SPA antigen can be a good alternative of commercially available imported antigen with 100% sensitivity and specificity. Early and sensitive molecular detection of M. gallisepticum has been of foremost importance to initiate therapeutic management of disease. Validation of insulated isothermal PCR (iiPCR) was conducted in comparison with real-time PCR (qPCR) and conventional PCR (con-PCR). Analytical and diagnostic performance of assay was evaluated and compared with qPCR as gold standard. Detection limit of iiPCR was found comparable with that of qPCR. It was found that iiPCR can be a good alternative to qPCR. To evaluate efficacy of commercially used M. gallisepticum vaccine, a longitudinal study was conducted on vaccinated breeder farms. The study was based on serological and molecular investigation of 08 breeder farms located in Islamabad, Khyber Pakhtunkhwa and Punjab region of Pakistan. For determination of antibody titers induced by vaccination, post vaccination baseline of live and killed M. gallisepticum vaccines was developed. High antibody titers with refernce to vaccination titers, were detected from all the farms included in the study from 2017-2019. This provided an evidence of incompatibility of M. gallisepticum vaccine used by farmers to the circulating strains in Pakistan.