| In recent years,the respiratory diseases of sheep have become more and more frequently,and the pathogen of the disease is very complicated.Extraintestinal pathogenic E.coli is considered to be a major cause of infection in tissues and organs outside the intestines,including neonatal meningitis,urinary tract infections,and pneumonia.There are more and more reports of respiratory diseases caused by Extraintestinal Escherichia coli infection which becomes to be a threate to animal husbandry production and public health security in last several years.At the end of 2016,the lamb which suffered from cough,panting,muzzle,and respiratory tract symptoms from the Shihezi,Xinjiang,finally died out due to respiratory failure.The bacteria were isolated and identified from the lungs of infected lambs by routine methods combined with 16S rRNA sequence analysis to determine the bacterial pathogen and its characteristics in this study.Meanwhile pathogenicity of the isolated bacterias were tested by mouse and lamb.Drug sensitivity were analyzed.Besides drug resistance genes and virulence genes were detected by PCR.The lung tissue examination were observed.The ability to adhere and invade alveolar epithelial cells and biofilm formation were detected,to explore the biological characteristics of the pathogen.1.Isolation and identification of pathogenic Escherichia coli from sheep lung:Total10 strains of extraintestinal pathogenic E.coli were isolated and identified by biochemical identification,16S rRNA sequencing analysis,murine pathogenicity assays and specific virulence gene assays from lambs infected with respiratory tract at different times.The ExPEC XJ-07 which the median lethal dose was 109.24.24 CFU/mL caused capillary alveolar capillary hyperemia in lamb lungs with unclear boundaries,bronchial hyperemia,peripheral lymphocyte infiltration and hyperplasia.The isolate carried 8 virulence factors containing ibeA,ibeB,ibeC,iutA,fyuA,ireA,hlyD,afa and 7 resistance genes which were strA,str B,aadA1/aadA2,Bla(TEM1),Bla(OXA1),TetA and TetB.The strains are multidrug resistant to norfloxacin,enrofloxacin,ciprofloxacin,ofloxacin,cotrimoxazole,azithromycin,nornocycline,clindamycin,penicillin,amoxicillin,cefazolin,cephradine,streptomycin,gentamycin,doxycycline,midamycin,and florfenicol.2.Artificial infection test of pathogenic Escherichia coli from sheep lung:In order to verify the pathogenicity of E.coli isolated in sheep.Seven healthy lambs aged about 2 months were infected by the ExPEC XJ-07 through 5 ways of infection in this experiment.The clinical symptoms and the pathological changes of the main organs were observed.The routine blood analysis was performed,and isolated and identified bacteria from organs at the same time.The results showed that the best infections were oral?2×10100 CFU/m L?and intramuscular injection?2×109 CFU/mL?.At first two groups of lambs show symptoms of mild diarrhea at 9 hours after infection,and gradually disappeared.Symptoms of respiratory tract infections were the main symptoms as cough,gasping,rapid breathing,a large amount of secretions from the nasal cavity,and mental depression after 16 hours.The total number of white blood cells and neutrophils and body temperature was higher than the control group.Sheep's lungs exhibit varying degrees of bleeding and degeneration,and spleen and brain have bleeding.Myocardial fibers break,bronchial epithelial cells in the lung fall off,and inflammatory cells infiltrate were observed in experimental group.The same bacteria were isolated and identified from the liver,lung,spleen,brain and heart of the necropsy in lamb.Finally digestive tract infections and intramuscular injections successfully replicated the pathogenesis of ExPEC on lambs.3.Study on the biofilm formation ability of pathogenic Escherichia coli from sheep lung:10 ExPEC strains were cultured in the 96-well microplate and tube to test the ability of biofilms formation at 24 hours.The OD57070 of 10 ExPEC strains were detected at37?for 6 h,12 h,24 h,36 h,48 h,60 h,72 h,and the biofilm morphology were observed at different time.BF-positive strains were cultured in 96-well plates to draw the growth curve.The amount of bacterial nucleic acid and polysaccharide produced at 12h and 24h was observed by PI and FITC-ConA.The results showed that 4 strains of bacteria showed positive BF production,of which XJ-01 strain was moderately positive,XJ-03,XJ-05 and XJ-07 were weakly positive strains;These strains had the highest amount of BF formation about 60 h;The XJ-01 BF formation ability was slightly stronger than XJ-07 at 12h and 24h after observation with LSCM.The above results suggest that extra-intestinal pathogenic E.coli derived from sheep lung can produce BF to varying degrees,but the ability to produce BF is weak.4.Effect of pathogenic E.coli XJ-10 from sheep lung on mouse alveolar epithelium:To investigate the effect of Ex PEC XJ-10 on TC-1 in mouse alveolar epithelial cells,mouse alveolar epithelium TC-1 cell were cultured as monolayers and then inoculated with ExPEC XJ-10,ATCC13883 and ATCCA25922 at the ratio of 10:1.After inoculated bacteria into TC-1 about 30 min,60 min,90 min,and 120 min the cells were lysed,and then viable bacteria were quantified by plating aliquots of serial dilutions on LB agar.In the invasion,the operation steps are the same as the invasion test except time,which inoculated bacteria at2 h,6 h,and 12 h.Giemsa was used to observe the adhesion of three bacteria on the surface of TC-1 cells.The result shown that the numbers of ATCC13883 and ATCC25922 adherence to TC-1 cells were less than that of XJ-10,and the difference is significant at different time?p<0.05?.The numbers of K13883 invasive to TC-1 cells were less than that of XJ-10,and the difference is extremely significant at different time?P<0.01?.It was found that E.coli ATCC25922 had no invasiveness to TC-1 cells.Giemsa showed that XJ-10 had a large number of bacteria on the cell surface and gap,and the XJ-10 had stronger adhesion to TC-1cells than ATCC13883 and ATCCA25922. |
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