Mamo, Badaso (Professor)Bekele, Legesse2018-06-292023-11-082018-06-292023-11-082015-06http://etd.aau.edu.et/handle/123456789/4792The study and entire laboratory works ware conducted from December 2014 to April 2015 in National Veterinary Institute, Bishoftu, Ethiopia. A total of 120(20 chickens from four farms and 40 backyard chickens from commercial farms and local markets respectively) were used for the study. The chickens were slaughtered at national veterinary institute postmortem room and tracheal, air sac and lung samples were collected from slaughtered chickens. Isolation and molecular characterization of Mycoplasma gallisepticum strains was done based on standard recommended methods. And the objective of this study is to identify local strains and to undertake molecular characterization of Mycoplasma gallisepticum circulating in the chicken population of farms in Bishoftu. This can help to device strategies in controlling the disease mainly through developing more effective vaccine which will replace the currently being imported Mycoplasma gallisepticum vaccines by some farmers. Then propagation was undertaken using appropriate culturing procedure. The mycoplasmal DNA was extracted and polymerase chain reaction was conducted for amplification of Mycoplasma gallisepticum Mgc2 gene. Amplified deoxyribonucleic acid fragments were analyzed by conventional 2% agarose gel electrophoresis incorporating appropriate size markers, followed by examination under Ultra violate light. The Polymerase chain reaction product for Mycoplasma gallisepticum was 185 bp. A number of 3 pooled (from 30 chickens) mycoplasma isolates were recovered from chickens in 2 farms and from back yard chickens in Bishoftu. Of these, 3 Mg isolates were identified using growth inhibition and rapid serum agglutination tests. Among six isolates (3 pooled and 3 lyophilized National veterinary institute isolates), (50%) or 3 samples were found strong positive and (50%) or 3 samples were weak positive to Mycoplasma gallisepticum as they gave 185 bp products, similar to the positive control when visualized electrophoretical analysis. Finally oil based Inactivated Mycoplasma gallisepticum vaccine was produced in suitable clean and secure accommodation, well separated from production facilities of National Veterinary institute. In this study, formaldehyde inactivated Montanide ISA70 based Mycoplasma gallisepticum vaccine from the Polymerase chain reaction confirmed positive from Samuel local isolate of National Veterinary Institute was prepared and evaluated in chickens. The amount of immune antigen per 0.5ml of the dose was 10 7 Colony forming units of the bacteria. At the age of 16 weeks, the chickens were randomly divided into three groups (A, B and C), each having twenty birds. Each bird of group B was vaccinated group of imported- live vaccine with 30μl dropped in the eye and each bird of group C was inoculated with 0.5 ml indigenous or trial vaccine subcutaneously at mid neck region and group A was used as a control. xiv After challenge test, among non-vaccinated chickens ( control or group A) 2 chickens were died (10 %), thicken and cloudy appearance of the air sac showed 18 (90% ), 2 chickens were not showed thickened and cloudy air sack ( 10% ). Although among vaccinated group (inactivated vaccine or group C) all chickens did not show clinical signs or post mortem changes (100 %). From attenuated imported live vaccine (group B) no clinical signs or post mortem changes was observed (100 %). It was concluded that oil based Mycoplasma gallisepticum vaccine induces protective level of anti Mycoplasma gallisepticum antibodies in chickens. Keywords: Adjuvant, Chickens, Inactivated vaccine, Mycoplasma gallisepticum, and polymerase chain reactionenAdjuvantChickensInactivated vaccineMycoplasma gallisepticumand polymerase chain reactionThesis