In first part of present study 380 samples were collected from clinically suspected cases of layers suffering from respiratory diseases in and around Lahore. Samples were subjected for mycoplasma isolation by using Frey's medium. Plates with positive growth revealed characteristic colonies on 8th day post inoculation that reached maximum in size and growth at 15th day post inoculation. Out of 380 samples, 104 (27.36%) samples were positive on culture. Isolates were identified through growth inhibition test (GIT) by using hyper immune sera raised in rabbits. Isolates were further confirmed by PCR. Similarly, tracheal swabs and tissue samples of lungs and trachea collected under refrigeration were also subjected for DNA analysis. Out of 380 samples 264 (69.5%) were positive on PCR analysis. By comparing two diagnostic techniques it was found that PCR was more sensitive and reliable technique for screening of Mycoplasma gallisepticum.
In second part of study experiment isolates were analyzed for protein profile of Mycoplasma by standardization of two techniques Sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and western blotting. These techniques help to find out any antigenic variation in prevailing strain of mycoplasma. During our study five bands of protein were detected with molecular size of 32.35 kDa, 43.65 kDa, 52.48 kDa, 64.56 kDa and 70.8 kDa. These proteins were extracted from whole cell of Mycoplasma gallisepticum isolates. On comparing the molecular sizes it was found that isolated species showed low antigenic variation, analyzed by SDS-PAGE. Western blot was used to determine the specific protein of Mycoplasma gallisepticum with the use of specific polyclonal antibody raised in rabbit. The positive reaction site was shown on nitrocellulose membrane confirming target species of CRD.
During third part of present study, it was concluded that aerosol route of infection causes early disease, followed by intra tracheal and per-oral route respectively. The severity of infection was found more in aerosol and intra tracheal routes of inoculation than per-oral route which was found to be very mild. The general gross lesions observed in the above two groups were hemorrhages in trachea with mucous plug. There was air sacculitis, hemorrhages in the lungs, salpingitis and putrefied eggs in the ovary. On histopathological examination lesions were found in trachea, lungs and oviduct.
Re-isolation was carried out to confirm antigen in experimentally inoculated birds. Paraffin embedded sections of trachea, lungs and oviduct were processed for immunohistochemical examination in order to confirm the antigen of Mycoplasma gallisepticum within tissue. A positive immunochemical reaction was found in lungs and oviduct. Which represents that antigen was same as inoculated during study of pathogenesis.