Isolation,Biological Characteristics And Inactivated Vaccine Preliminary Developing Of Enterotoxin Escherichia Coli In Some Areas Of Anhui Province

Piglet yellow-white diarrhea occurs mostly before and after the birth and weaning of piglets,which is a disease difficult to cure in large-scale pig farms at present.Through clinical investigation,it was found that yellow and white diarrhea of piglets is one of the most frequent swine diseases in large-scale pig farms.The clinical manifestations of the infected piglets are anorexia,emaciation,dilated yellow diarrhea or viscous white diarrhea,even death.The main pathogen is Enterotoxigenic Escherichia coli(ETEC).The main pathogenic factors of ETEC are two kinds of enterotoxins,heat stable enterotoxin(ST)and heat labile enterotoxin(LT),which can cause diarrhea and even death of piglets,and seriously affect the healthy development of pig industry.Because of the high resistance and pathogenicity of ETEC strains,its threat to the breeding field deserves attention.This study investigated the epidemic situation of yellow and white dysentery in piglets of large-scale pig farms in some areas of Anhui Province,analyzed the biological characteristics of ETEC,including pathogenic factors,drug resistance and serotypes,and prepared inactivated vaccine to study the immune effect of mice,so as to achieve effective prevention and treatment of ETEC in large-scale pig farms in some areas of Anhui Province.1.Isolation and identification of ETEC and serotype analysis of piglets in some areas of Anhui ProvinceFour hundread and seventy-nine fresh feces and rectal swabs were collected from 17 large-scale pig farms in 6 areas of Anhui Province.110 ETEC strains were isolated by microbial marking,single colony morphology identification,identification of 16S rRNA and specific virulence genes by PCR.6 strains contained both ST and LT genes,97 ETEC strains contained ST gene only and 7 contained LT gene only.The serotypes of 110 ETEC strains were identified by PCR and serum agglutination tests.The results showed that 52 ETEC strains were typed,and the typing rate was 47.3%.13 serotypes were identified,including O8,O20,O128,O15,O25,O114,O3,O9,O137 and O141.In addition,O8,O20 and O128 were the dominant serotypes,accounting for 26.9%(14/52),21.2%(11/52)and 11.5%(6/52),respectively.The dominant serotypes existed in six regions.2.Antibiotic resistance and toxicity of ETEC in piglets in some areas of Anhui provinceThe drug resistance of 110 strains of ETEC to erythromycin,doxycycline,amoxicillin,trimethoprim/sulfamethoxazole and tetracycline was analyzed by 13 kinds of drug-sensitive paper.The results showed that the resistance rates of the 110 strains to erythromycin,doxycycline,amoxicillin,trimethoprim/sulfamethoxazole and tetracycline were 94.5%(104/110),100%(110/110),91.8%(101/110),94.5%(104/110)and 97.3%(107/110)respectively.All resistance rates were higher than 90%.Multidrug resistance is widely distributed,and 38.1%(42/110)of the strains with 10 or more multidrug resistance are concentrated in the central and southern areas of Anhui Province.51.8%of ETEC isolates were sensitive to cefotaxime(57/110),47.3%(52/110)of ETEC isolates were sensitive to Enrofloxacin and 90%(99/110)were sensitive to polymyxin B.The virulence of 31 dominant serotypes of ETEC strains was tested in ICR clean grade mice of four weeks old.Among them,14 were O8 serotype strains,11 were O20 serotype strains,6 were O128 serotype strains,and the intraperitoneal injection dose was 5×107CFU/mice.The results showed that 7 08 isolates leaded to 6 mice dead in each group,which were identified as virulent strains.8 of the O20 serotypes were virulent(6 mice died),4 of the O 128 strains were virulent(6 mice died).O8 strains FXYD4-1 was selected for half lethal dose(LD50)and minimum lethal dose(MLD)test.The LDso and MLD of FXYD4-1 was 5.1×106CFU/rat and 3×107 CFU/rat,respectively.3.Immune effects of inactivated vaccine of ETEC for piglets in some areas of Anhui province on miceThe monovalent inactivated vaccines were prepared with strains with different virulence of dominant serotype,including a virulent strain FXYD4-1 and an attenuated strain QJZX2 of O8 serotype,a virulent strain FDDS1 and an attenuated strain FXYQ1 of O20 serotype,a virulent strain QJXW2-2 and an attenuated strain YQBYZ2-4 of O128 serotype.The immune dose was 2×108 CFU/mice.The mice of 4-week-old immunized two weeks after the first immunization and challenged one week after the second immunization.The challenge strains were selected from dominant serotype virulent strains with a challenge dose of 5 x 107 CFU/mouse.The results showed that the protective rate of inactivated vaccine FXYD 4-1 to mice was 66.7%(2/6);while the protective rate of inactivated vaccine FXYQ1 to mice was 83.3%(1/6);the protective effect of inactivated vaccine FXYD4-1 and FXYQ1 to mice infect with serotype O128 strains was both 100%.So FXYD4-1 and FXYQ1 were mixed to make bivalent inactivated vaccine.The mice were immunized by intraperitoneal immunization with a dose of 2×108 CFU per mouse.The results showed that the protective effect of bivalent inactivated vaccine on mice was more than 50%(3/6).The titer of antibody before and after immunization showed that the first immunization still had certain protective effect,while the antibody after second immunization was better than that of first immunization.