| Mycoplasma hyopneumoniae causes porcine enzootic pneumonia in Tibetan pigs,a chronic respiratory disease occurring worldwide in the pig industry.It is also one of the pathogens causing porcine respiratory disease complex syndrome(PRDC)in Tibetan pigs.Due to few information of porcine enzootic pneumonia can be found for Tibetan pigs,according to the research experience of mycoplasma hyopneumonia of slaughter pigs,the Mycoplasma hyopneumoniae enzyme-linked immunosorbent assay(ELISA)antibody(MP-Ab)quantitative detection kit was employed to check the serum positive rate,to investigate the epidemiology and dynamics of mycoplasma hyopneumonia in Tibetan pigs.Mycoplasma hyopneumoniae was Confirmed from lung’s lesions of slaughter Tibetan pigs by observation of pathological section,the separation,identification,purification,culture expansion of pathogenic microorganisms.Antibiotic sensitivity and resistance of chemical drugs were carried out.Mycoplasma hyopneumoniae 7448 strains was employed to Primers designed,Analysis of 16 S rRNA,according to the M.h specific conserved gene the preparation of P36 were detected by PCR M.h to determine the pathogen,The standard J strains,7448 strains,7422 strains,232 strains were employed as the reference sequence for whole genome sequencing in order to understand the differences in genomics and proteomics.In order to accurately understanding the etiology,revealing the basic mechanisms of disease.On the basis of success isolation and purification of the freeze-dried bacteria,provide a basis for the development of Tibet pig mycoplasma pneumonia vaccine and effective prevention and control of Tibet pig mycoplasma pneumonia.1 Prevalence and phylogenetic analysis of Mycoplasma hyopneumoniae from Tibetan pigsMycoplasma hyopneumoniae was detected by PCR(polymerase chain reaction),gel electrophoresis and imaging,and 16 S rRNA sequencing.Inflammatory lung lesions were observed in 29 of 100 suspected cases.Invasion of focal lymphocytes was confirmed by Paraffin sectioning and HE staining of lung sections.Bronchoalveolar lavage fluid(BALF)was assayed by PCR.The prevalence of Mycoplasma hyopneumoniae in Tibetan Pig herds(via ELISA)was 72.6% in 3 counties(Mainling,Nyingchi,and Gongbo’gyamda)during the winter of 2014.The difference in prevalence between six growing stages was statistically significant(P<0.05).Anti-Mycoplasma hyopneumoniae antibody was detected in 83.48% and 79.17% of Growing pigs and piglets,respectively.10% of pigs tested positive for M.hyopneumoniae via PCR and gel electrophoresis of BALF.The presence of Mycoplasma hyopneumoniae in serum was higher in piglets and growing than other groups.2 Isolation,purification,staining,identification and expansion culture of Mycoplasma Hyopneumoniae from Tibetan PigsM.hyo was isolated from consolidated lungs of Tibetan pigs by liquid culture and agar plate colony picking for purification.Bacterial liquid stained with Wright’s stain and Giemsa stain.Mycoplasma hyopneumoniae strain 7448(Gen Bank:NC＿007332)was employed as the standard(reference)strain.PCR was used to amplify the 16 s rRNA gene and the species-specific gene P36 of M.hyo from the bacteria isolated from pig lungs.The PCR products were sequenced by the shotgun method.Genetic similarity was assessed by DNA sequence comparison;the gene sequences were >98% similar to those of database reference strains of M.hyo.3 Antibacterial test of mycoplasma pneumonia in vitroThe Mycoplasma Hyopneumoniae from Tibetan Pigs were highly sensitive to Two terpene class,macrolides,tetracycline and quinolones,the MIC of the four kinds of drugs were lower than 0.05μg / mL.The MIC of Lincosamides was 0.13 ～ 0.3 μg/mL,medium sensitive.Moreover,concering sensitivity or resistance to the quinoxaline and ampicillin,MIC≥16 μg/mL;the MIC of berberine higher than the general chemical,MIC≥32 μg/mL;for nano silver,MIC was 0.5～1 μg/mL.For the vaccine strain 168(attenuated live vaccine strain),RM48 strain(attenuated live vaccine strain)and J strain(non-virulence strain),the sensitivity of the same antimicrobial drugs were lower than the Mycoplasma Hyopneumoniae from Tibetan Pigs,especially J strains of erythromycin thiocyanate resistance is common.4 Macrolide resistance selection in Tibetan pigs with a high load of Mycoplasma hyopneumoniaeCurrently,tylosin tartrate is the first-line treatment for M.hyopneumoniae infections in China.However,the efficacy of tylosin tartrate and resistance to this treatment in M.hyopneumoniae infections of Tibetan pigs are unknown.Here,we examined the prevalence of M.hyopneumoniae infection in Tibetan pigs at three intensive farms in Tibet,China.In addition,we investigated the efficacy of tylosin tartrate treatment for porcine enzootic pneumonia by monitoring M.hyopneumoniae DNA eradication dynamics and macrolide resistance.Eighty-two out of 450(18.2%)Tibetan pigs tested positive for only M.hyopneumoniae,and most of these animals(85.1%)had symptoms and signs of pneumonia.The elimination of M.hyopneumoniae DNA was substantially faster in Tibetan pigs with lower pre-treatment M.hyopneumoniae load,and the total eradication rate was 97.4%(75/77).Two Tibetan pigs tested positive for M.hyopneumoniae that contained macrolide resistance-determining mutations in the 23 S rRNA gene.Our study is the first to confirm that the G2059 A mutation is closely related to M.hyopneumoniae resistance to Tylosin tartrate(a16-membered macrolide).Our results indicate that the pre-treatment M.hyopneumoniae load may be an effective predictor of macrolide treatment efficacy(and possibly other antimicrobial agents)and macrolide resistance.Moreover,our results suggest that Danofloxacin mesylate can be used as an alternative drug for the treatment of macrolide-resistant M.hyopneumoniae infection acquired during intensive farming.5 Identification,genome sequencing and bioinformatics analysis of M.hyopneumoniae in Tibet pigs.Using Illumina HiSeq 4000 sequencing platform,320 Mb raw data was collected for sample 2B.Then we constructed the scaffolds of sample 2B and filled the gap by PCR.The genome size of sample 2B of Mycoplasma hyopneumonia Tibet1 strain was 909,064 bp,and the number of contig was 237,the number of scaffold was 57,GC content was28.53%.When genome analysis results of sample was exported,we found that the genome of Mycoplasma hyopneumonia Tibet1 strain contained 1,565 genes,the total length of genes was 610,647 bp,which makes up 67.17% of genome,the number of tandem repeat sequence was 91,the total length of tandem repeat sequence was 3,402 bp,which makes up 0.3742% of genome,the number of minisatellite DNA was 50,the number of microsatellite DNA was 15,the number of tRNA was 21,the number of rRNA was 3.6 Comparative genomic analysis of virulent strain(Mycoplasma hyopneumonia Tibet1)and attenuated strain of Mycoplasma hyopneumoniae(vaccine strains).Annotations of the Virulence Factors of Pathogenic Bacteria(VFDB)database revealed virulence factors of the Mh TB1 strain,including glyceraldehyde-3-phosphate dehydrogenase(MG＿301),the molecular chaperone Dna K(CT396),an oligopeptide ABC transporter,a permease component(oppF),an ATP-dependent protease(clpE),a putative prolipoprotein p65(p65),pyruvate dehydrogenase(lipoamide)e1-beta chain(pdhB),elongation factor Tu(tuf),lipoyltransferase and lipoate-protein ligase family protein(lplA1),phosphopyruvate hydratase(eno),membrane nuclease,lipoprotein(nuc),and hemolysin(hlyA).Comparative genomic analyses revealed genome characteristics of Mh TB1 related to high-altitude hypoxia(plateau hypoxia)and unique virulence factors. |
