| Mycoplasma ovipneumoniae (MO) is the etiological agent of atypical pneumonia ofsheep and goats. This disease is characterized by cough, nose running, progressiveemaciation and hyperplastic interstitial pneumonia. The disease is prevalent worldwide andcauses huge economic loss to sheep and goat industry. In China, MO was first isolatedfrom sheep by Hu et al(1982), and then MO infections were reported in several provinces,including Gansu, Liaoning, Sichuan, Yunnan, Jiangsu and Xinjiang, posing serious threatto sheep and goat industry in China.HSP70is a highly conserved protein among species of Mycoplasma. It is amolecular chaperone and could be used as an adjuvant. Classification and identification ofmycoplasmas can be achieved based on HSP70sequence. However, it is commonlyobserved that the results of HSP70-based phylogeny and16S rRNA-based phylogeny arediscrepancy, suggesting that hsp70can provide different view of phylogeny. Therefore,hsp70is useful for molecular phylogenetic analysis. Despite of its conservation, hsp70genes show diversity to a certain extent between species (within same species). Althoughthe whole genome sequence of strain MO SC01and HSP70gene sequence of referencestrain Y98are available, there was no report concerning the genetic diversity of clinicallyisolated strains.In present study, we analyzed the genetic diversity of MO HSP70genes fromclinically isolated strains, elaborated the molecular characters of HSP70protein andutilized it to diagnostic technology.1. Genetic diversity of MO HSP70geneThe complete open reading frames (ORF) of HSP70were cloned and sequencedfrom Y98standard strain as well as from14clinically isolated strains. The Y98HSP70ORF is1815bp in length, coding a protein of604amino acids. In contrast to Y98, all14clinically isolated strains HSP70ORF is1818bp, coding for605amino acids with aGlutamine (Gln) insert at position593. The variation between Y98and clinical isolatessuggests that it is valuable to analyze the genetic diversity of HSP70in MO. Sequence analysis of hsp70from Y98and clinical isolates showed34.16%GCcontent on average,96.1%-100%nucleotide identity and97.7%-100%amino acid identity.Bioinformatics analysis of the predicted protein demonstrated the existence of atransmembrane region and absence of signal peptide. Peptides33-144,198-347and467-600are potential antigenic determinants. There are124variable nucleotides and19deduced variable amino acids. These sites distributed randomly along the whole gene. Thenucleotide mutation rates are7%within1-600bp,7.7%within601-1200bp and5.8%within1201-1818(1815) bp. Two short regions, from1-300bp and1400-1818(1815) bp,are highly conserved in MO but variable in other Mycoplasma species, could serve aspotential molecular targets for diagnosis.Phylogenetic analysis between16Mycoplasma species based on HSP70revealed thatMO has the closest evolutionary relationship with Mycoplasma hyopneumoniae and alsoclose with Mycoplasma conjunctivaem, but far away from other species that infect sheepand goats. Difference between phylogenetic trees based on HSP70and16S rRNA wasobserved, which is in agreement with the research in Mycoplasma capricolum. Withinspecies, phylogenetic analysis based on HSP70divided Y98standard strain and clinicallyisolated strains into two clusters. Fourteen clinically isolated strains were further clusteredinto two groups. Interestingly, all5strains from Lezhi Goat Farm were clustered togetherinto Cluster1A, all4strain from Zigong Goat Farm were clustered together into Cluster1B. Five strains from Jianyang Goat Farm fall into either Cluster1A or Cluster1B.Although there is no clear link between hsp70sequences and origin, strains from LezhiGoat Farm and Zigong Goat Farm demonstrated tendency of regional distribution. whichneeded to be proven in the future.2Establishment of a PCR assay for the detection of MO based on HSP70Previous investigations have indicated that nucleotides and protein sequences of MOexhibit high heterogeneity. The genome GC content is extremely low and the distributionof nucleotide is uneven. In addition, little is known about the feature of its genes andgenetic diversity. All above make it difficult to choose suitable target genes and to designprimers for PCR method. McAuliffe L developed the only method for detection of MO byPCR which targets16s rRNA. In present study, HSP70genes from15MO strains were sequenced and the sequences were used to design primers. Primer pair amplifying127-261bp of HSP70ORF was selected, as this region is relatively conserved for all strainsand specific for MO. By using this primer, we established a sensitive and specific PCRassay. All35strains from clinically positive goats were identified without non-specificdetection of any other common pathogens. As low as4.4×10-3ng DNA template could bedetected successfully by our method, which is10-fold more sensitive than McAuliffe’smethod. The PCR method established in this study will be useful for detection of MOinfection.3Establishment of real-time quantification PCR assay for the detection of MO basedon HSP70Real-time quantification PCR assay was also developed based on HSP70for detectionof MO. The primer concentration, template concentration, annealing temperature and cyclenumber were optimized. PCR product of HSP70present only one peak on melting curvewith melting temperature of80.2℃±0.5℃. Non-specific amplification and primer dimerswere not observed. The amplification efficiency was103.51%with a linear range from7×108to7×103copies and a correlation coefficient of0.99985. The standard equationwas y=-3.551x+33.052. The lowest detection limit was7copies. Under the optimalcondition,35strains of clinically positive MO were all identified, but the unrelatedpathogens, such as M. mycoides subsp. mycoides. LC, M. mycoides subsp. Capri, M.bovis,M.agalactiae, M.arginini, Mannheimia haemolytica, Escherichia coil013andStaphylococcus aureus SW07were all negative. This assay is106-fold and105-fold moresensitive than McAuliffe’s method and our conventional methods, respectively. As it isspecific, sensitive and quantificational, this method is valuable for MO infection diagnosis. |
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