| Mycoplasma is abundant in nature and there were at least 28 species of mycoplasma that could infect poultry.Most species of mycoplasma were not pathogenic under normal conditions,only a few species of mycoplasma can cause avian disease.Pathogenic mycoplasma infection alone or in combination can not only lead to chronic respiratory disease,arthritis,airbag inflammation and so on in poultry,but also lead to poor growth and development of poultry,reduced production performance,and produced very serious economic losses.In this study,two different kinds of mycoplasma were isolated from peacock with dyspnea and commercial broiler with paralysis.At the same time,mycoplasma etiological identification,diagnostic method establishment and whole genome sequencing were carried out.The report was shown below.1.Isolation,identification,biological characteristics and phylogenetic analysis of 16 S rRNA gene of mycoplasma(1)Mycoplasma from peacockA new mycoplasma was isolated from peacock with respiratory symptoms by routine method.Morphological identification,physiological and biochemical experiments and 16 S rRNA gene sequencing of mycoplasma were carried out to confirm that the isolated pathogen was a completely new mycoplasma,we named it Peacock20181011.This isolate belongs to the Mycoplasma genus of the Mycoplasma family,but was a different species from Mycoplasma gallisepticum(MG)and Mycoplasma synoviae(MS),which were common in poultry.Peacock20181011 grows faster.After 2 to 3 times of pure cultures,the color of the culture medium changed from red to orange after 12 to 20 hours liquid culture,while other mycoplasmas such as MG and MS generally do not change color until 48 to 72 hours culture.The results of homology comparison analysis showed that the similarity between Peacock20181011 and the MGC classical model strain NCTC10183(LR214950)was 99.8%,while the similarity comparison with the classical poultry strains MG?MS was only 73.9% and 88%.Comprehensive comparison shows that this strain was a new MGC strain in China.The pathogenicity study showed that Peacock201810111 did not cause SPF chicken lesions and did not kill chicken embryo,but it could cause chicken embryo growth retardation and claw curl,and had certain pathogenicity of chicken embryo.The experimental results of minimum inhibitory concentration(minimum inhibitory concentration experiment,MIC)showed that Peacock20181011 were sensitive to 6 different drugs,such as KAN,FFC,et al,which provided a basis for clinical treatment against the disease.(2)Mycoplasma from broilerA mycoplasma was isolated from the paralyzed joint fluid of broiler by the routine method of mycoplasma isolation and identification.The results of comparison of nucleotide homology for 16 S rRNA gene showed that WF2018 was highly homologous to MS model strains with a similarity of 98.5%.However,the similarities with MGC,MG,Mycoplasma gallinarum and Mycoplasma meleagridis(MM)were 92.8%,77.4%,89.5% and 90.1%,respectively.These results proved that the newly isolated strain was identified as MS and named WF2018.The inoculation test of chicken embryo showed that WF2018 could kill SPF chicken embryo,cause slow development of chicken embryo,and the pathogenicity was more serious than MGC.Meanwhile,the MIC result was the same with MGC.2.PCR and LAMP(1)PCRA pair of PCR primers was designed for the highly conserved region of MGC 16 S rRNA gene with species specificity by Primer 5 software.The temperature optimization,specificity and sensitivity of PCR reaction were evaluated.The results showed that the optimal reaction temperature was 52 ?,the minimum detection dose was 1.07 pg/?L,and there were no cross reactions with MG,MS,A.laidlawii,and so on.PCR had better specificity and sensitivity,and was suitable for the rapid diagnosis of clinical MGC.(2)LAMPWe designed LAMP primers for the species-specific high-conservative segment of Mycoplasma synoviae VLHA by Primer Explorer V5 software,and optimize the reaction conditions.The results showed that the optimum temperature of the LAMP reaction was 63?,the amplification time was 60 min,and the lowest detection dose was 807 fg/?L.Importantly,LAMP was negative reactions with MG,MGC and A.laidlawii,and could be specific for MS diagnostics.It provides a method for MS specific diagnosis.3.Genome sequencing and functional analysisThe whole genome of Peacock20181011 and WF2018 were sequenced by using the second and third generation sequencing technology,and the genomic components and functions were analyzed.(1)Peacock20181011The total genome length of Peacock20181011 was 1 183 913 bp with 28.7%(G+C);898 genes were predicted;the genome included 46 non coding RNAs,including 3 copies of 5S rRNA,4 copies of 16 S rRNA and 4 copies of 23 S rRNA respectively.There were some differences in genome among Peacock20181011,MGC model strain NCTC10183 and South African epidemic strain B2096 8B.We annotated the genome of Peacock20181011 with COG and GO databases,and found that there were many functional genes;at the same time,we compared with KEGG databases,and predicted its metabolic pathway,and found that Peacock20181011 had a relatively complete glycolysis/gluconeogenesis pathway.In addition,it was predicted that there were 20 virulence factor genes and 2 tetracycline resistance genes.(2)WF2018The total genome length of WF2018 was 809 346 bp with 28.36%(G + C);743 genes were predicted;The genome included 48 non coding RNAs,including 3 copies of 5S rRNA,2 copies of 16 S rRNA and 2 copies of 23 S rRNA respectively.The genomic information of this strain was similar to that of MS model strain NCTC10124 and epidemic strain HN01 in China.The data base of COG,GO and KEGG were compared and analyzed.It was found that there were some differences in protein function classification and some metabolic pathways between WF2018 and GX11-T.In addition,it was predicted that there were 67 virulence factor genes,54 genes are highly similar to VLHA,and there were 8 antibiotic resistance genes,319 and 631 were related to fluoroquinolone antibiotic resistance,707 was related to rifamycin resistance. |
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