| Pathogenic Escherichia coli (E. coli) are an important kind of pathogen for causing diarrhea in piglets that result in severe diarrhea, dehydration and seriously affect the survival rate of piglet. Due to the continuous emergence of resistant strains of E. coli, the effect of drug treatment is getting worse. Therefore, the development of effective prevention of piglet diarrhea in E. coli vaccine has important practical implications for the prevention of occurrence of the disease.The epidemic states of pathogenic E. coli are complex. The distribution and frequency of virulence factors can vary considerably from region to region and over time in a given region. The purpose of this study was to investigate the distribution of virulence gene in porcine pathogenic E. coli, providing evidence for antigen selection of vaccine. Based on the epidemic character, explore the feasible for preparing oral vaccine delivery vector using attenuated E. coli.A total of 381 E. coli strains, obtained from 290 fecal samples from pigs on 38 farms, were tested for fimbriae (K88, K99,987P, F41, F18, F17), non-fimbrial adhesins (AIDA-I, paa, CS31A, eae, saa), enterotoxin (LT-Ⅰ, LT-Ⅱ, STa, STb, EAST1), Shiga toxin (Stxl, Stx2, Stx2e), pathogenicity islands (HPI, LEE), a-haemolysin (hlyA), afa8 gene cluster (afaD, afaE) and sepA genes by PCR. Out of the 381 isolates,206 carried at least one virulence gene. Of the 206 virulence positive isolates, the virulence factor genes detected were EAST1 (n= 120), irp2 (n= 59), paa (n= 50), STb (n= 41), AIDA-1 (n= 34), LT-I (n=23), ler (n= 11), hlyA (n= 9), K88 (n= 8), eae (n= 8), STa (n=7), sepA (n= 6), F18 (n= 5), afaD (n=3), afaE(n=3) K99 (n= 2), and Stx2e (n= 1), with most of the isolates carrying several virulence genes. These results demonstrate that EAST1 (58%), irp2 (29%), AIDA-I (16.5%), paa (24%) and STb (20%) are prevalent virulence factors expressed by E. coli strains, and a relative minority of isolates contained the colonization factors, which located at no more that half of the strains.Pathogenic E. coli induced diarrhea of piglets mainly infected the piglets from digestive tract. Therefore, immunization of piglets with oral vaccine is more suitable for preventing this disease. In this study, STa gene of wildtype E. coli 0142 was deleted by G-DOC and X. Red recombinant system for constructing the attenuated E. coli O142:△ STa. In addition, pseudogene yaiT was selected as the inserting site for forgein gene for constructing the double selection platform based on Kil and mKate2 genes. Further, ER-A and ER-B used as oral vaccine were constructed and analyzed for toxicity test, survival properties in imitative gastrointestinal environments, safety test, bacterial growth test, heredity stability test, fimbrial growth test and colonization ability in intestinal tracts. The results of survival properties in imitative gastrointestinal environments showed that ER-A and ER-B could keep high survival rate in gastric fluids of PH range from 2.5 to 3.5. In the intestinal fluid environment, ER-A and ER-B showed a trend of growth. ER-A and ER-B also showed a good tolerance to bile, which was able to propagate well in 0.05% of bile. The safety test showed that the mice were not caused to death orally fed with 109CFU and 1010CFU of ER-A and ER-B, indicating that ER-A and ER-B were safe to animals. Growing the E. coli strains in LB medium showed that the deletion of STa gene and the insertion of LT192-STa13 or LT192-STb fusion gene into the genome did not substantially affect bacterial growth. The structural stability assay showed that ER-A and ER-B had good stability properties, with no loss of the LT192-STa13 or LT192-STb fusion gene. Electron microscopy showed that the deletion of the STa gene (0142:△STa) and insertion of the LT192-STa13 or LT192-STb fusion gene did not affect the fimbriae expression. The result of the colonization assay was that ER-A and ER-B were able to colonize the intestinal tract of immunized mice.BALB/c mice were immunized via oral with ER-A and ER-B. The results showed that anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P < 0.05) IgG antibodies were detected in the serum of immunized mice in ER-A group (A group), ER-B group (B group), and mixture group (M group) on the days 7 to 21. Anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P< 0.05) IgG antibodies were almost detected in the lac feminium, spleens, mesenteric lymph nodes and intestinal mucus samples on day 42. Anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P< 0.05) IgA antibodies were detected in the fecal of immunized mice on the days 7 to 28. Anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P< 0.05) IgA antibodies were almost detected in the lac feminium, spleens, mesenteric lymph nodes and intestinal mucus samples on day 42. The results of lymphocyte proliferation and mesenteric lymphocyte proliferation showed that recombinant proteins and enterotoxins stimulated the potent production of specific T-lymphocytes. The results of flow cytometry showed a marked shift towards Th2-mediated immunity in mesenteric lymph node cells, indicating that oral immunization cannot stimulate system Th immune response, but stimulated local mucosal immune response in mice. Results of in vitro and in vivo tests showed that serum, spleens, mesenteric lymph nodes and intestinal mucus samples collected from immunized mice demonstrated obvious inhibition to enterotoxins.Piglets and sows were further immunized via oral with ER-A and ER-B. The result of the colonization assay was that ER-A and ER-B were able to colonize the intestinal tract of immunized piglets and sows. Results of ELISA showed that anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P< 0.05) IgG antibodies were detected in the serum of immunized piglets in ER-A group (A group), ER-B group (B group), and mixture group (M group) on the days 7 to 28. Anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P< 0.05) IgG antibodies were almost detected in the lac feminium, spleens, mesenteric lymph nodes and intestinal mucus samples on day 42. Anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P< 0.05) IgA antibodies were detected in the fecal of immunized pigs on the days 14 to 28. Anti-LTA (P< 0.05), anti-LTB (P< 0.05), anti-STa (P< 0.05), anti-STb (P< 0.05) and anti-F41 (P< 0.05) IgA antibodies were almost detected in the lac feminium, spleens, mesenteric lymph nodes and intestinal mucus samples on day 42. The results of lymphocyte proliferation showed that recombinant proteins and enterotoxins stimulated the potent production of specific T-lymphocytes. The results of flow cytometry showed a marked shift towards Th2-mediated immunity in mesenteric lymph node cells, indicating that oral immunization cannot stimulate system Th immune response, but stimulated local mucosal immune response in piglets. Results of in vitro and in vivo tests showed that serum, spleens, mesenteric lymph nodes and intestinal mucus samples collected from immunized piglet demonstrated obvious inhibition to enterotoxins. The intestinal villus depth, intestinal crypt depth and ratio of intestinal villus depth and crypt depth of immunized piglets were significant difference from that of control piglets after being challenged (P< 0.05).In this study, virulence factors of porcine pathogenic E. coli were investigated, indicating the main virulence gene of epidemic strains were EAST1, irp2, AIDA-I, paa, and STb. However, only minority of the strains harbor the colonization factors. These results provide evidence for selecting the antigen of vaccine. The immune effect of mice and pigs showed that oral vaccine induce humoral immunity and celluar immunity. The oral vaccine wereprotective for pigs, and exhibiting well prospect of attenuated E. coli for antigen delivery vector. |
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