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The Identification and Establishment of the PCR Assay of Mycoplasma Gallisepticum Jinan Isolate

Author: ZhangLei
Tutor: ZhuRuiLiang
School: Shandong Agricultural University
Course: Veterinary
Keywords: Mycoplasma Gallisepticum Jinan isolate Identification Structural protein Multiplex RT-PCR Establishment
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 171
Quote: 1
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Abstract

Mycoplasma gallisepticum (MG) is a primary pathogen causing chronic respiratory disease in chicken, which is characterized by coughing, nasal discharge, rattling and air-saccaulitis. Usually chickens suffered co-infection with virus and bacteriumd, and led to forced-breathing and increased mortality. MG infection is common in flocks in our country, but mortality is low, which mainly led to high unhealthy chicken rate and egg production of layer dropped, feed conversion efficiency reduced and the period of onset to market prolonged, the cost of prevention added. At the same time, it could cause drug residua in poultry products. MG infection may result in economic losses in the poultry industry.One isolates was got from the infected or suspicious infected chickens came from a pheasantry of Shandong Jinan. On the basis of typical clone shape with frig egg and bacteria recovery test, 1 of mycoplama isolates was purified and was identified as Mycoplasma gallisepticum by colony morphology observation, red adsorption test colony, physiological and biochemical and serological appraisal. The isolate was named as JN. JN strain in liquid medium with tetrazolium and without glucose make medium color orange-red, their colonies in agar-solidified medium surface are typical of Fried egg shape, about 0.1~0.4 mm, colony can absorb 1% of red cell of chicken, MG antiserum can make JN strain metabolic inhibition.Through growing of the JN strain in different serum medium, it was easier in the pig serum medium, replacing expensive and less sources of horse serum. From the chicken annulus trachealis’spathogenic test of MG, JN strain can cause annulus trachealis cilia damage more, can make them dead quickly, pathogenicity larger than strains F. At the same time, SDS-PAGE map showed JN strain structural protein was different from F strains structural protein. So, we can conclude the correlation between the pathogenicity and structural proteins.Three primers were designed for mycoplasma gallisepticum (MG), mycoplasma synoviae (MS) and mycoplasma meleagridis (MM) and the size were MG 732bp,MS 208bp,MM 850bp. Through a single polymerase chain reaction (PCR), the corresponding segment can be got, under the optimized conditions, the multiple PCR established can also get three segments, the same as single PCR. Experiment showed that the multiple PCR can be used to detection of MS, MG and MM, convenient, high sensitivity and specificity.Aiming at chicken infective bronchitis virus (IBV), avian influenza virus (AIV), Newcastle disease virus (NDV), avian infectious laryngotracheitis virus (ILTV), four primers were designed and the sizes are IBV 1720bp, AIV 550bp, NDV 300bp, ILTV 647bp. And multiple RT-PCR was establisheded with MG, MS. Under the optimized conditions, it can simultaneously got corresponding segment clearly. Experiments indicated that the multiple RT-PCR can be used to identify and diagnose mixed infecting six kinds of respiratory infectious disease.Drug soaking, temperature fluctuation and fuming were used to treat Breeding eggs. The results demonstrated that three methods could drop the hatchery rate to some degree, but the vertical transmission infection of mycoplasma gallisepticum in chickens was cut down dramatically. Comprehensive index from hatchery rate, positive rate antibody against MG and average body weight of 35-day-old chicken, suggested that the temperature fluctuation method was more available for elimination of vertical transmission of MG in chickens.The research isolated and identified JN strain, and the molecular biological detection method was established. And more, the study laid a foundation for the research of genetic engineering vaccine and the comprehensive prevention and cure of the infection of MG in the future.

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