Studies On Isolation And Molecular Epidemiology Of Mycoplasma Gallisepticum In Hubei Province

Mycoplasma gallisepticum (M gallisepticum) can cause severe chronic respiratory disease in farm flocks. M. gallisepticum infection is common in our country. It could lead to egg production decreased of layer and slaughter delayed of broiler. Once chickens suffered co-infection with Newcastle virus, Infectious brochitis or Escherichia, which could lead higher motality and incidence. Isolates, culture and serological identification for the detection of M. gallisepticum are mainly used currently in China. But these asssys are laborious and time consuming, it can not satisfy rapid application. However, little is known about epidemiology, drug resistance and pathogenic of Mycoplasma in chicken farms of Hubei Province. To aim directly at aboved-metioned, 16S-23S rRNA intergenic spacer region (ISR) was elected as a tool to investigate M gallisepticum in Hubei Province.The 16S-23S rRNA ISR is a nucleotide sequence with high conservatism and discontinuity. It could satisfy rapid detection. Moreover, it may be a useful molecular epidemiology tool and provide thereunder etiology assay of M. gallisepticum.In this study, facing the shortage of the present methods used in detecting, vacuity of epidemiology and etiology assay of CRD in poultry. The routine lab technology and Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used. And a PCR detection techonlogy based on the 16S-23S rRNA ISR was established. Moreover, the research of molecular epidemiology and etiology was advanced.1,Isolates, identification and resistance of M. gallisepticum with routine technology,A total of 172 field samples were collected from 47 farms in this region and 77 isolates were obtained by cultivation. These isolates were examined by routine microbiological, biochemical test and metabolism inhibition assays. We found that 57 isolates which metabolism inhibition titer was higher than 1:80. Then 16S rRNA PCR was used. The length of 16S rRNA PCR fragments was 186bp. So they were identified as M. gallisepticum. The positive rate of routine technology is 33.1%. In addition, we found that 20 isolates were not M. gallisepticum.The resistance test of some strains of M. gallisepticum showed that, sensitivity of clinical strains to antibiotics have area characteristic and majority strains were sensitive to drugs, such as Tiamulin, Tylosinum, Kitasamycin. (The minimum inhibition concentraion)2,16S-23S rRNA ISR PCR and molecular epidemiology of M. gallisepticumA PCR detection technology based on the 16S-23S rRNA intergenic spacer regions (ISR) gene fragments was established. A total of 172 samples were detected. We found that 70 isolates including 57 isolates were detected through routine technology which special fragment length was 850bp The positive rate is 40.7%. In addition, we found 20 isolates were M. gallinarum. Therefore, the PCR detection technology based on ISR have higher specificity than routine technology. Then PCR-RFLP technology was used at the same time. All isolates were divided into two groups through Rsaâ… (R1 and R2). Epidemiological investigation suggested that the R1 and R2 groups of M. gallisepticum appeared not to affect chickens at the same farm simultaneously. The R2 genotype of M. gallisepticum was more prevalent, evidentially by more farms and more samples were infected with this genotype of M. gallisepticum. The R2 group of M. gallisepticum appeared to be prevalent at one location and then changed to the R1 type of M. gallisepticum in the same city. This was the first report on genetic variants of M. gallisepticum in China Further analysis including sequenced, alignment and evolution analyzing of 16S-23S rRNA ISR revealed that there were three subgroups of M. gallisepticum with different genotypes. Some isolates were closely related to the reference strain of S6, some others to the F, and no single isolates to the reference strain of R.3,The assay of etiology about M. gallisepticum4-week-old chick were infected artificially with four strains of M. gallisepticum in which XA41 and ML21 strains were isolated in Hubei Province, which closely related to the reference strain of S6 and F in phylogenetic analysis respective, and S6 and F strains were reference strains. Provocative infection was made by New castle disease attenuated vaccine(â…£) eighe days later. Chicks infected with XA41 and S6 were sick, typical respiratory tract symptoms occurred. Pathoanatomical results, Scanning electron microscopy (SEM) and pathological section showed that respiratory were damage in serious degree. Serological reaction was positive. Mean Lesion Score of air sac were 3.0(S6),2.8(XA41)XA41. The virulense of XA41 and S6 for SPF embryoes were 80%. The chicken infected F and ML21 strain did not show any clinical symptom. Pathoanatomical results, SEM and pathological section showed that respitatory tract have not any damage. Mean Lesion Score of air sac were zero(F and ML21). Serological reaction was positive. The pathogenicity of F and ML21 strains for SPF embryoes were 40%. The study on etiology of XA41 and ML21 showed that the fomer was virulent strain that same with S6, and the latter was low virulent strain that same with F. Therefore, base on the study on etiology,16S-23S rRNA ISR could be infered as a useful molecular tool which can discrimination pathogenicity of different genotype of M. gallisepticum.