Mycoplasma gallisepticum (MG) is one of the smallest self replicating infectious agent, it lacks cell wall so cell membrane is the outer most boundary. Its cell membrane is made up of sterols which not only gave it rigidity but also make it fastidious to grow. MG is the causative agent for chronic respiratory disease (CRD). Isolation identification and serodiagnosis are routinely used in laboratories. However among the serodiagnosis rapid serum agglutination (RSA) test is mainly used for early detection for its sensitivity, rapidity and cost effectiveness.
Keeping in VIew the importance of RSA antigen test a study was conducted to prepare
standardize RSA antigen from local isolate and compare its sensitivity with a commercial RSA antigen. Molecular characterized local isolate of MG procured from University Diagnostic Lab University of Veterinary And Animal Sciences, Lahore, was grown in 11 different media formulations to identify a media with better antigenic yield. Affects of different in process quality control parameters; bacterial concentration (0.75%, 1 %,
1.25% PCV), diluents (normal saline, PBS, HBSS) inactivants (heat, formalin) and preservatives (thiomersal sodium, sodium azide, phenol) were studied in terms of their influence on the sensitivity of prepared RSA antigens. These prepared antigens from local isolate were further compared with a commercial RSA antigen. The results of antigenic yield were statistically analyzed through One Way A OVA test. All the
media formulations support the growth of MG isolate except Frey's media with 7.5% fetal
bovine serum and 7.5% chicken serum that revealed no growth or packed cell volume. The
maximum bacterial growth in terms of packed cell volume was obtained from frey's media with 12% horse serum. So this media was further use in the production of antigens. Sensitivity of RSA antigens with different bacterial concentrations in terms of packed cell volume, preservatives, inactivants was more using HBSS followed by PBS and normal saline.
However difference in increasing the sensitivity of RSA antigen in terms of agglutination with
known serum was statistically found significant. Formalin inactivated RSA antigens were
somewhat more sensitive as compared to the heat inactivated using different diluents and
bacterial concentrations. Sensitivity of RSA antigens in different diluents, inactivants and
preservatives was a little bit more with 1.25% pev followed by 1.00% pev however it was least with 0.75% Thiomersal sodium added RSA antigens were more sensitive followed by phenol and sodium azide. PBS and HBSS based, formalin inactivated and preservative (thiomersal sodium, phenol, sodium azide) added antigens having bacterial concentration of 1.25% and 1 % (peV) given agglutination at 1 :30 dilution of known positive MG serum. The sensitivity of all these antigens was equal to the commercial RSA antigen. All the prepared RSA antigens and commercial anitigen showed no agglutination with known negative serum. Sensitivity of most of the prepared and a commercial antigen was comparable. The findings of this study will be helpful for further recommendations of local RSA MG antigen used in the diagnosis of chronic respiratory disease.