Screening Of Dominant Serotypes Of Escherichia Coli Causing Calf Diarrhea And Preparation Of Vaccine In Some Areas Of Hebei Province

As the scale of dairy farms in my country continues to expand and the farming density continues to increase,a variety of diseases have also occurred immediately under the influence of poor breeding and management conditions and untimely immunization,which threatens the health of dairy cows and creates a huge economy loss.Currently,diarrhea is the most common symptoms in calves.Many diseases can cause diarrhea in calves.Single pathogen or mixed infection of multiple pathogens can aggravate the condition of calves and cause serious calf death.Therefore,it is of great significance to determine the pathogen causing calf diarrhea and develop a vaccine for it to prevent and treat calf diarrhea.In this study,fresh feces from calves with diarrhea about 1 month old were collected from dairy farms in 7 regions of Hebei Province,using E.coli phoA gene,bovine viral diarrhea-mucosal virus E0 gene,bovine coronavirus N gene,and bovine rotavirus VP7 Gene,Cryptosporidium 18S rRNA gene primers for PCR detection of common pathogens of calf diarrhea.Isolation and identification of E.coli from the initial detection of E.coli positive materials,isolation and culture of bacteria,Gram staining,biochemical identification and PCR amplification of 16S rRNA gene;the isolation and identification of E.coli with 11 types of E.coli O factor typing serum Screening of dominant serotypes of Escherichia coli;Using specific primers of fimbriae to carry out PCR amplification,sequence determination and analysis of fimbriae genes of dominant serotype strains to identify the fimbriae antigen they carry;Also evaluated the pathogenicity of facultative fimbriae Escherichia coli through the mouse challenge test.Using the predominant serotype O101:F41:K99 strains of Escherichia coli that cause diarrhea in calves from dairy farms in some areas of Hebei Province,we have developed a whole-cell oil emulsion inactivated vaccine and F41-K99 natural fimbriae protein oil emulsion vaccine;Whole cell oil emulsion inactivated vaccine antigen is selected to inactivate E.coli with formaldehyde solution;prepare E.coli F41-K99 natural fimbriae protein oil emulsion vaccine fimbriae protein,use 60℃water bath lysis method and diafiltration method to remove fimbriae F41 And K99 is crude and pure;both the inactivated E.coli cell protein and the purified fimbriae protein are uniformly mixed in proportion with white oil,Span-80 and Tween-80 as adjuvants,and emulsified to make a vaccine;The two vaccines prepared were tested in terms of physical properties and sterility;mice and cattle were immunized with the two vaccines to test the safety of the vaccine,and a blank control group was set up.In the serum of days and 28 days,the antibody level in the serum was detected by the indirect ELISA method of E.coli cells and fimbriae;and 28 days after vaccination,the mice were challenged with 10-fold lethal dose,and 100-fold lethal dose to analyze the protection of the vaccines.The results showed that 48 cases of Escherichia coli,39 cases of bovine coronavirus,9 cases of bovine rotavirus,32 cases of bovine viral diarrhea mucosal virus,12 cases of cryptosporidium bovine were isolated and detected from 153 bovine diarrhea stool samples.Among them,no pathogen was isolated in 13 cases.After isolation,culture,biochemical tests and sequencing analysis,it was determined that 48 strains were E.coli;the result of E.coli O-factor type serotype identification found that the predominant serotype of E.coli with calf diarrhea in 4 regions in Hebei Province was O101.The results of PCR sequencing of fimbriae identified six strains of Escherichia coli with K99 fimbriae,seven strains of Escherichia coli with F41 fimbriae,and one strain was facultative K99-F41 fimbriae Escherichia.The mouse pathogenicity test results show that the minimum lethal dose of the isolate to mice is 1.5×1010 CFU/mL.Preparation of E.coli whole cell oil emulsion inactivated vaccine.The E.coli antigen was added to formaldehyde with a final concentration of 0.3%,and the cells were all inactivated after 24 hours.Crude extraction can obtain pure E.coli F41 and K99 fimbriae proteins.The inactivated Escherichia coli and crude fimbriae protein were mixed with adjuvants in proportions to prepare vaccines.After physical properties,sterility,and safety tests,both vaccines met the vaccine standards,and mice and cattle were vaccinated After the vaccine,there were no abnormalities.The indirect ELISA method was used to detect that the serum titers were as high as 1:6400 and 1:3200 after the mice were vaccinated with the whole-cell inactivated vaccine and the F41-K99 natural fimbriae protein vaccine;The highest can reach 1:102400 and 1:25600 respectively.The mouse challenge protection test showed that at 10 times the lethal dose of mice,the protection rate of the two vaccines on mice reached 100%;at 100 times the lethal dose of mice,the protection rate of the two vaccines on mice Respectively reached 84%and 67%.In summary,this study investigated the prevalence of calf diarrhea pathogens in dairy farms in some areas of Hebei Province,which can provide a theoretical basis for the prevention and treatment of calf diarrhea;the analysis of the dominant serotypes of E.coli lays a foundation for the study of E.coli disease;The developed whole-cell inactivated vaccine and facultative fimbriae vaccine have good development and application prospects as local candidate vaccines for calf diarrhea colibacillosis.