Genome Sequencing And Comparative Genomics Analysis Of Mycoplasma Anatis And Mycoplasma Columbinum

Mycoplasma anatis is a kind of opportunistic pathogenic Mycoplasma, which can cause infectious sinusitis of duck, especially high susceptibility to ducklings. It can cause infraorbital sinus, nasal cavity, trachea and air sac lesions, characterized by infraorbital sinus swelling. Recovered ducks growth slowly and development retardation, impact on the economic benefits directly. Mycoplasma columbinum was first isolated in1978, which can be isolated from Throat swabs of not only apparently healthy pigeons, but also those pigeons showing signs of mild respiratory disease. The etiological status of M. columbinum is still hasn't confirmed yet. There are some researchers propos that M. columbinum has a direct relation with respiratory disease, but report infrequently in recent years.With the development of genome swquencing technology and widely used of comparative genomics analysis, the microbiologics research goes in a newly and rapid development stage. Based on the softwares and datebases, the two sequenced genomes were analyzed integrally and comparatively, which shows the information of the genomic structure, the evolutionary position, the virulence factor by this study. And the virulence of different M. anatis strains were compared by experimental infection. The main task as follows:1. Genome sequence and analysis of M. anatis1340M. anatis1340was found to be928,687-bp chromosome with a G+C content of26.64%and contained88.74%coding sequences. A total of778protein-coding genes were predicted with an average length of1059bp. The genome includes4rRNA loci and33tRNA genes. Based on the genome functional annotation, the classification of genes, the gene components of metabolitc pathway, protein features and transport associated gene in1340genome were analyzed and summed up.2. Genome sequence and analysis of M. columbinum SF7The genome of M. columbinum was composed of748,210bp with an average GC content of26.95%and a coding percentage of89.08. There were612putative ORFs with average length of1,089bp,2rRNA operons, and30tRNA genes. After the genome annotation and gene classification, the gene components of metabolitc pathway, protein features as well as estriction and modification genes in SF7genome were analyzed and generalized.3. Phylogenetic analysis and comparative genomics analysis of M. anatis1340and M. columbinum SF71340and SF7were completed phylogenetic analysis and comparative genomics analysis by using other21different genomes of Mycoplasmataceae, which were complete genome sequenced, as the reference genome. It is revealed that1340and SF7were not quite similar to Mycoplasma gallisepticum which was an avian pathogen involved in respiratory disease in chickens and turkey. They were in low identity, and located at two big different evolutionary branchs of the23strains in total. There are some rapid evolutionary genes which are mutant frequently by ka/ks analysis. It means there is evolutionary selection in1340and SF7genome. The SSR analysis results of the7Mycoplasma genomes include M. anatis1340and M.columbinum SF7genomes suggest that the content of Single nucleotide G and C could be the auxiliary criterion to determine the evolutionary relationship. And, the difference of Mono-SSR between Mycoplasma strains associated with the host specificity. The result shows that1340and SF7has fewer virulence associated genes than M. gallisepticum str. Rlow, the number of virulence associated genes in1340and SF7was only56.2%and37.5%of Rlow separately. It is very interesting that the vlhA gene family, which is very important to pathogenic and immune evasion in M. gallisepticum, is missing in1340and SF7. These results can explain the pathological characteristics of M. anatis and M. columbinum why they lack of the ability dispersing to deeper respiratory tract.4. Experiment infection with Mycoplasma anatisIn order to compare the difference of pathogenicity between M. anatis str.1340and M. anatis str. SJQ, the infective experiment was carried out. The result of average body weight analysis indicates that M. anatis SJQ group weight gain fastest and M. anatis1340plus E. coli group is slowest, which meant M. anatis could be more pathogenic when co-infected with Escherichia coli than infected with M. anatis only. Based on the result of pathogenic analysis of M. anatis1340genome, several virulence factors were choosen which were amplified by PCR using M. anatis1340genome and M. anatis SJQ genome as the template separately. Then the products were sequenced and BLAST to identify the indentity between products in M. anatis1340and M. anatis SJQ genome. It showed that each pair of products from the two genomes was highly identical to each other, the average indentity over97%. It is suggest that there no significant difference of pathogenicity between M. anatis1340and M. anatis SJQ.