Comparison Of PCR Methods For Detection Of Mannheimia Haemolytica And Investigation Of Mannheimia Infection In Goats From Sichuan Province

Mannheimia is a member of the Pasteurellaceae family,which has six named species,mostly opportunistic pathogens,mainly causing pneumonia,sepsis and mastitis in ruminants.Among the species,M.haemolytica is the main pathogen causing bovine respiratory disease complex(BRDC),but also can cause ovine pneumonia,mastitis and acute sepsis in newborn lambs,which is currently the most important pathogen in Mannheimia spp.For M.haemolytica,a variety of PCR methods have been successively established targeting different genes,but there is no comparative analysis on the specificity,sensitivity and detection ability of clinical samples for each method,which is not conducive to providing methodological reference and guidance for the epidemiological investigation,diagnosis and pathogen detection and identification of clinical M.haemolytica.Therefore,in this study,according to the established PCR methods for the detection of M.haemolytica at home and abroad,the primers of each method were analyzed by bioinformatics,and on this basis,the specificity and sensitivity of each method and the detection of clinical samples were compared and analyzed in order to provide a reliable basis for the selection of PCR methods for M.haemolytica.In addition,in view of the lack of research data on the infection of Mannheimia other than M.haemolytica in China,in this study,goat nasal swab samples were collected from some areas of Sichuan Province to detect and isolate the pathogens of M.haemolytica,M.glucosida and M.granulomatis,and the sod A gene of some Mannheimia strains isolated was also analyzed for genetic evolution to provide a basis for the prevention and control of Mannheimia infection.1.comparative study of PCR methods for M.haemolyticaSix PCR methods established for six target genes of M.haemolytica lktD,gcp,rpt2,phssa,16 Sr RNA and art J-lkt C were used in this study(lktD-PCR,gcp-PCR,rpt2-PCR,phssa-PCR,16 Sr RNA-PCR and art J-lkt C-PCR).First,bioinformatics analysis was performed for each primer.On this basis,the specificity and sensitivity of each PCR method were compared,and the detection rates of nasal swab samples and the detection ability of mock-infected lung tissue samples by each method were compared and analyzed.The results are as follows:(1)Bioinformatic analysis revealed that the primer sequences targeting four target genes,gcp,phssa,art J-lkt C,and 16 Sr RNA,were only highly homologous to M.haemolytica target genes and had no homology with other pathogens.Primers for lktD-PCR showed homology with several other Mannheimia species except for their ability to completely match the M.haemolytica target gene fragment,suggesting that they are not very specific.The primers of rpt2-PCR matched the target fragments of different M.haemolytica target genes with different lengths,which may be related to the fact that this gene fragment is prone to variation.(2)The specificity comparison study revealed that lktD-PCR,gcp-PCR and phssa-PCR were positive for M.haemolytica,M.glucosides,and M.ruminanlis in the genus Mannheimia,which were Mannheimia specific PCRs;although 16 Sr RNA-PCR,art J-lkt CPCR,and rpt2-PCR were specific for M.haemolytica species,they could only amplify some M.haemolytica strains and were not versatile,and the combination of these three methods could detect all M.haemolytica in this study.(3)Through sensitivity comparison studies,it was found that lktD-PCR had the highest sensitivity among the three Mannheimia "genus-specific" PCR methods and could be used as a preliminary test for clinical Mannheimia infection.The sensitivity of 16 Sr RNA-PCR was the highest among the three M.haemolytica "species-specific" PCR methods.(4)Comparison of the detection rates of nasal swab samples revealed that the highest detection rate of lktD-PCR was 55.56%.Through the detection and comparison of simulated infection samples of M.haemolytica in lung tissues,it was found that each PCR method could amplify specific bands,which could be directly used for the detection of clinical lung tissue samples.2.Investigation of Mannheimia capricolum infection in some areas of Sichuan ProvinceA total of 231 goat nasal swab samples were collected from five regions of Sichuan Province in this study,and various Mannheimia infections were detected by specific PCR methods,while Mannheimia was isolated and identified,and the sod A gene of the isolated strains was analyzed for genetic evolution.The results are as follows:(1)231 goat nasal swab samples were tested.The total infection rate of Mannheimia was 61.47%,the infection rate of M.haemolytica was 49.35%,the infection rate of M.glucosida was 3.46%,and the detected samples of M.granulomatis were 0.These results indicate that Mannheimia is widespread in large-scale goat farms in Sichuan,with M.haemolytica as the most important pathogen,but the situation of M.glucosida infection also exists.(2)Through strain isolation and identification,a total of 89 Mannheimia strains were isolated,including 85 M.haemolytica,2 M.glucosida,and 2 M.ruminalis.(3)Genetic evolution analysis of the sod A gene of the isolated strains revealed that M.haemolytica was closely genetically related to M.glucosida,but distant from M.ruminalis,M.varigena,and M.granulomatis.In conclusion,this study compared the specificity,sensitivity and clinical sample detection of commonly used PCR methods for M.haemolytica,providing a reference for the selection of PCR methods for M.haemolytica in clinical use.Through the investigation of Mannheimia infection in some areas of Sichuan Province,the prevention and control of Mannheimia provides useful data.