Isolation,Identification Of Morganella Morganii From Bovine And Establishment,Appliacation Of Its PCR Detection Method

Morganella morganii belongs to Morganella,Enterobacteriaceae,which can cause infections in humans and other animals.Some studies pointed out that Morganella morganii has become a new pathogen of cattles,which can cause diarrhea in calves.From April 2016 to October 2017,three strains of Morganella morganii were isolated from nasal swabs of cattles with respiratory disease complex,indicating that Morganella morganii may also be a potential pathogen of Bovine Respiratory Disease Complex?BRDC?.Klebsiella pneumoniae is a common conditional pathogen causing respiratory tract infections in cattles,while Salmonella infections affects the occurrence and development of BRDC.Moreover,the reported PCR specific primers of Morganella morganii may also be amplified with Salmonella typhimurium predicting by Primer-BLAST.In order to eliminate the possible interference caused by Salmonella typhimurium to the PCR detection of Morganella morganii,the specific primers of Morganella morganii,Klebsiella pneumoniae and Salmonella typhimurium were synthesized referring to reference literature,a triple-PCR method for the detection of Morganella morganii,Klebsiella pneumoniae and Salmonella typhimurium was established and used for the detection of case samples,in order to provide reference for the diagnosis and epidemiological investigation of Morganella morganii and Klebsiella pneumoniae infection in cattle production.Test 1:Isolation and identification of Morganella morganii from bovineOn April 10 of 2016,April 21 of 2017 and October 11 of 2017,three batches of nasal swabs from cattles with BRDC were collected from some beef farms in Guang'an and Yibin,Sichuan Province,soon afterwards pathogenic isolation and identification were done.The 16SrRNA gene sequencing,construction of phylogenetic tree,biochemical test,drug sensitivity test and pathogenicity test were carried out.Three strains of Morganella morganii were obtained and tentatively named 16GA7,17GA61.2 and 17YB9.The biochemical characteristics of three strains were identical,while their sensitivity to drugs showed significant differences.16GA7 was sensitive to piperacillin,tetracycline and enrofloxacin,17GA61.2 was sensitive to carboxybenicillin,piperacillin,amikacin,enrofloxacin and florfenicol,17YB9 was sensitive to carboxybenicillin,kanamycin,tetracycline,minocycline,enrofloxacin and florfenicol.The pathogenicity of three strains were also different.Infection in mice could cause depression,holding together,abdominal breathing and viscous secretions in the eyes,mouth opening breathing occured in dying mice.Pulmonary hemorrhage was the main pathological change in dissection.Tissue sections showed that all three strains caused pulmonary hemorrhage,alveolar wall thickening,inflammatory cell infiltration;splenic congestion;liver cord disorder,hepatocyte swelling and necrosis;narrowing of renal capsular space.In addition,17GA61.2 could also cause disappearing of alveolar structure and necrosis in lung,increasing of multinuclear macrophages in spleen and white pulp structure blurred;17YB9 could cause vacuolar degeneration in spleen,liver and kidney in mice.Test 2:Establishment of triple PCR for detection of Morganella morganii,Klebsiella pneumoniae and Salmonella typhimuriumChose the 16S rRNA gene variable sequence of Morganella morganii,phoE gene of Klebsiella pneumoniae and fliC gene of Salmonella typhimurium as target genes,by optimizing the volume of primers,the number of cycles and annealing temperature,a triple PCR method was established for the detection of Morganella morganii,Klebsiella pneumoniae and Salmonella typhimurium,and its specificity,sensitivity and repeatability were evaluated.The reaction system was 30?L,including 15?L 2×Master Mix,7?L ddH₂O,1?L each of forward primer and reverse primer for Morganella morganii and Klebsiella pneumoniae,0.5?L each of forward primer and reverse primer for Salmonella typhimurium,1?L each of DNA of Morganella morganii,Klebsiella pneumoniae,and Salmonella typhimurium.The amplification procedure was 94?5 min;94?45 sec,59?1 min,72?1 min,35 circles;72?10 min.The PCR detection could not product bands while detecting Acinetobacter baumannii,Proteus mirabilis,Serratia nematodiphila,Salmonella enteritidis,Serratia marcescens and Escherichia coli.The detection limits for Morganella morganii,Klebsiella pneumoniae and Salmonella typhimurium were 1×10⁸CFU·mL^-1,1.32×10⁷CFU·mL^-1and 1.78×10⁷CFU·mL^-1.Repeatability results were well.Test 3:Application of triple PCR for detection of Morganella morganii,Klebsiella pneumoniae and Salmonella typhimuriumFrom December 2018 to January 2019,45 samples from BRDC infected cattles from some farms in Guang'an,Sichuan Province were collected and tested.Thirteen?28.89%?positive samples of Morganella morganii and eight?17.78%?positive samples of Klebsiella pneumoniae were detected by triple-PCR,among which three?6.66%?samples were infected by both Morganella morganii and Klebsiella pneumoniae.After isolation,culture and identification of bacteria,Morganella morganii were isolated from nine?20%?samples,Klebsiella pneumoniae were isolated from seven?15.56%?samples,one?2.22%?sample contained Morganella morganii and Klebsiella pneumoniae simultaneously.The coincidence rate between the results of triple PCR and bacteria isolation of Morganella morganii reached 91.11%,and the coincidence rate of Klebsiella pneumoniae was 97.78%.No Salmonella typhimurium positive samples were detected by both methods.Conclusion:1,Three strains of Morganella morganii 16GA7,17GA61.2 and 17YB9 were successfully isolated from some bovine nasal swabs infected with BRDC.They may be new pathogens of BRDC.2,Triple PCR detection methods of Morganella morganii,Klebsiella pneumoniae and Salmonella typhimurium were successfully constructed.The reaction system was 30?L,including 15?L 2×Master Mix,7?L ddH₂O,1?L each of forward primer and reverse primer for Morganella morganii and Klebsiella pneumoniae,0.5?L each of forward primer and reverse primer for Salmonella typhimurium,1?L each of DNA of Morganella morganii,Klebsiella pneumoniae,and Salmonella typhimurium.The amplification procedure was 94?5 min;94?45 sec,59?1 min,72?1 min,35 circles;72?10 min.3,The coincidence rates between results of the triple PCR and traditional bacterial isolation methods were 91.11%for Morganella morganii and 97.78%for Klebsiella pneumoniae.No Salmonella typhimurium was found in the samples.