Study On The Biological Characteristics,Genome Of Mannheimia Glucosida,and Establishment Of A Species-Specific PCR

Mannheimia glucosida belongs to the genus Mannheimia of Pasteurella and is a gram-negative coccobacillus.Recent studies have found that,as an important respiratory pathogen,it can infect bovine?sheep and antelopes and cause pneumonia.In addition,the infection of M.glucosida is associated with the occurrence of ovine mastitis.At present,there are no reports of M.glucosida infection in animals in China.In 2020,during an etiological study of pneumoinae in a sheep farm in Sichuan Province,we isolated 5 strains of M.glucosida.In order to further analyze its biological characteristics,this study conducted research on its biochemical characteristics,main virulence factors,pathogenicity to mice,and antimicrobial susceptibility.Meanwhile,in order to further analyze the molecular biological characteristics of the pathogen,the whole genome of a strain of M.glucosida was sequenced,and a preliminary bioinformatics analysis was performed.Afterwards,a PCR method for specific detection of M.glucosida was developed.The methods and results are described briefly as follows:1.Isolation and identification of M.glucosida from sheep and its biological characteristicsIn this study,deep nasal swabs were collected from 30 sheep with respiratory diseases,and the bacteria were isolated.The Mannheimia genus specific PCR,combined with the esculin hydrolysis experiment,was used to detect M.glucosida.Subsequently,the biochemical characteristics,virulence genes,pathogenicity to mice,antimicrobial sensitivity,and lktA gene subtypes of the isolats were determined.The following results were obtained.(1)By using Mannheimia-specific PCR and esculin hydrolysis test,5 strains of M.glucosida were identified and named G1-G5.This is the first report of M.glucosida infection in sheep in China.(2)Biochemical characteristics analysis found that the 5 isolates were all positive for?-glucosidase and negative for ornithine decarboxylase.This results enriched the biochemical properties of M.glucosida.(3)The detection of virulence genes found that all isolates carry adh,a virulence gene related to host cell adhesion and plpD,a virulence gene related to bacterial escape from host cell phagocytosis,suggesting that all strains have strong adhesion and colonization capabilities.(4)The pathogenicity results for mice showed that the median lethal dose(LD₅₀)of the G1 strain was 1.35×10⁶CFU/mL,indicating that the strain is virulent.The DNA of M.glucosida can be detected in various organs and tissues of mice after artificial infection.Histopathological analysis found that this strain can cause damage to multiple organs of mice,indicating that this strain has a wide range of organ tropism in mice.(5)The antimicrobial susceptibility test showed that the isolates were resistant to streptomycin,reaching 100%,this result can be used to guide the medication plan of the farm.(6)The lktA allelic typing showed that the 5 isolates belonged to the allelic lktA4,which was consistent with previous reports.However,two new allele subtypes were identified in this study,which was named lktA4.7 and lktA4.8.2.M.glucosida whole genome sequencing and establishment of species-specific PCR methodIn order to further understand the molecular biological characteristics of M.glucosida,this study sequenced the entire genome of G1 isolates,and conducted preliminary bioinformatics analysis of coding genes,virulence genes,andantimicrobial resistance genes.Furthermore,the bglA gene encoding?-glucosidase was selected as the target gene,and a species-specific PCR method for the detection of M.glucosida was established.(1)The full-length genome of M.glucosida G1 strain is 2 363 903 bp with a GC content of 41.25%.The number of coding genes is 2 264 and the number of non-coding genes is 84.Through the prediction of virulence genes,it was found that there are 96 virulence genes,of which the most virulence genes involved in endotoxin action(29),indicating that after the lysis of this strain,lipopolysaccharides play a pathogenic effect.Through the prediction of antimicrobial resistance genes,it is found that there are 46 drug resistance genes;indicating that this strain has a wide range of drug resistance genes.(2)The three housekeeping genes of 16S rRNA,infB and sod A were used to construct a phylogenetic tree.Phylogenetic analysis showed that 16S rRNA and infB have high homology among the species of genus Mannheimia,while sodA was highly conserved in same species and different from other species,suggesting sod A is a good target for the phlogenetic analysis of genus Mannheimia.(3)The established bglA-based PCR method for has good specificity and high sensitivity.Its detection limit is 27.8 copies/mL for bacterial genome and 56.7 CFU/mL for bacterial cultrue.This methods could detect M.glucosida in 18 out of 80clinical samples with a detection rate of 22.5%(18/80),indicating that this method can be used for the detection of M.glucosida in clinical samples.This results also suggested that the pathogen is prevelant in sheep and is worthy of further study.In summary,this study was the first to isolate M.glucosida from domestic sheep,and conducted a preliminary analysis of their main biological characteristics and the whole genome sequence.At the same time,a species-specific PCR method for the detection of M.glucosida was established.The results of this study provided a theoretical basis for the in-depth study of the biological characteristics of M.glucosida,and also provide a useful tool for identification and detection of the pathogen and rapid diagnosis and epidemiological investigation of the infection.