SEROLOGICAL AND MOLECULAR CHARACTERIZATION OF MYCOPLASMA GALLISEPTICUM FROM LAYER POULTRY IN NORTHERN PART OF BANGLADESH A DISSERTATION

Abstract

Poultry industry targets the production of animal protein of excellent quality with low costs and helps in national economy for generating jobs and foreign exchange credits for the balance of trade. But outbreaks of different infectious diseases are one of the major constraints of poultry farming. Mycoplasma gallisepticum organism is one of the most infectious organism and economically significant throughout the world. The present research was performed with the objective of serological and molecular characterization of Mycoplasma gallisepticum organism from field samples in order to get pure culture of the etiological agent. The epidemic behavior of the etiological agents were studied based on age and breed of birds, seasons and location where the birds were rearing. The incidence rate of infections were recorded as per information collected from farmers by using a set of questionnaire, symptoms of affected birds and post mortem lesions. In association with the epidemiological investigation, the overall incidence rate was 11.54%. For detection of specific antibody of Mycoplasma gallisepticum, 920 sera were tested from ten selected farms based on location, age groups, seasons, breeds and flock size, by SPA test and found 526 sera were positive. The overall incidence was 57.17%. The highest incidence rate (61.96%) was found at Dinajpur and the lowest (51.09%) at Rangpur in comparison to other districts. The highest incidence (64.78%) was found in above 40 wks followed by 59.30% in 21-40wks, 56.52% in 9-20wks and 48.26% in 0-8wks respectively. The prevalence was found 61.96% in winter and 52.39% in summer season, 59.78% in Sonali and 56.52% in Isa brown breed, 61.96% in large and 51.44% in small flock. No significant difference was observed as their location and breed but significant was in age and season variation. Furthermore 526 SPA positive sera were tested by iELISA test and 164 (31.18%) sera were positive. 80 representative sera were tested by HI from iELISA positive sera and found that 15 (18.75%) sera were positive. A total of 156 different organs of trachea, lungs and air sacs were cultured for isolation of etiological agent of Mycoplasmosis and the positive cases were 5.77%. The highest rate (13.46%) of organism was found in tracheal swabs followed by 1.92% in air sac and 1.92% in lung. For molecular characterization of Mycoplasma gallisepticum, 48 different organs were tested by direct PCR without culture and overall positive was 12.5% (6 bands). Among them 25%, 6.25% and 6.25% were found from tracheal swabs, air sacs and lungs respectively and 0.64% (1 band) from culture positive isolates. The DNA sequencing of the present study showed 99 to 100% per cent similarity with the sequence of the duck isolate from South Africa, chicken isolate of Spain and USA as demonstrated by blast (NCBI). It is recommended that the samples from live birds are sufficient enough to identify the organism and tracheal swab samples gave more isolations than from lung and air sac by molecular technique. It is justified that PCR is a serviceable tool in the accurate diagnosis of Mycoplasma infections, not only for its sensitivity but also for its high specificity. This technique overcome culture method as it depends on the direct detection of the micro organism’s DNA without the need for cultivation. PCR has allowed the study of microbial genes, directly amplified from samples. It is a sensitive, easy, rapid and inexpensive technique and the most important advantage is eliminating the need for isolation of Mycoplasma gallisepticum.