Isolation And Identification Of A Strain Of Mannheimia Haemolytica SYS-1 From Sheep And Its Pathogenicity Study In Mice

Mannheimia haemolytica is one of the most important and recognized pathogenic agents of pneumonia in sheep and other ruminants around the world.In wild North American bighorn sheep herds,pneumonia caused by the bacterium resulted in 90%mortality.It has also been reported that this bacteria-caused lung disease also exists in the flock in various areas of China,but it has not caused enough attention from relevant administrations.In this experiment,a M.haemolytica strain was isolated from lung tissue samples collected from a sheep farm in Yulin,Shanxi Province,named M.haemolytica SYS-1strain.The sample was inoculated on 5%sheep blood agar plates and incubated at 37°C for 24 h.Then a hemolytic,gray and translucent round colony was chosen for the further pure culture.And then the isolate was identified by gram staining,biochemical tests,16S r RNA gene sequencing and genetic evolution analysis.Moreover,its serotype,virulence factors,drug susceptibilities and the pathogenicity in mice were tested.The isolate strain was short rod-shaped gram-negative bacteria showing round dew-like colonies.The hemolytic ring can be seen in the case of light transmission;the fermentation results were fermented glucose,sorbitol,mannitol,with the production of acid rather than gas,without fermented mushroom sugar,aesin,mannose,lactose.Urease test,indole test,and citrate utilization test were all negative,while the touch enzyme and oxidase tests were positive.The biochemical results indicate that the isolates was M.haemolytica;16S r RNA sequencing results were compared with the known sequence BLAST in Gen Bank,from which 98.68%identity with the published M.haemolytica Denmark isolate(Registration number:NR＿114448.1)was found.Through mapping the genetic evolutionary tree,it has also found that,it was in the same clade as the Danish isolates,indicating the same source.Further,the serotype of M.haemolytica SYS-1 was identified as A1 by multiplex PCR,and its main virulence factor leukotoxin was detected(LKT).The results of drug susceptibility test indicated that the isolates showed sensitivity to enrofloxacin and florfenicol,but was moderately sensitive to ceftriaxone and showed resistance to gentamicin and erythromycin.After culturing the purified M.haemolytica mansoni SYS-1 strain in Martin broth medium at 6.25×10~8cfu/m L.This was administered in Kunming mice by oral and intraperitoneal injection.No mice of the oral group died within 3 days.After continued intraperitoneal injection,one mouse died within 24 hours.After necropsy and pathological section observation,pulmonary congestion,spleen with white necrosis,with liver congestion;one mouse died 3 days after autopsy and pathological section observation,with the large area of liver congestion and brain tissue congestion.In addition,there was no increase in body temperature of mice in each group.In conclusion,the combination of clinical body temperature changed,necropsy and pathological section results suggested that the death of mice may be due to the release of exotoxin.This experiment provides a preliminary scientific basis for the treatment and prevention and control of the bacterium through the isolation and identification of the clinical isolate Mannheimia haemolytica SYS-1 in sheep and the animal pathogenicity test study,and enriches the data for the epidemiological investigation of clinical lung disease in cattle and sheep.