Construction Of K88ac~+ ETEC Attenuated Strain And Its Preliminary Application In Vivo Of Mouse

Piglet diarrhea is one of the important reasons of global pig industry losses. The major pathogen of the disease is enterotoxigenic Escherichia coli (ETEC), and its virulence factors mainly including adhesion factors and enterotoxin. Adhesin, also known as colonization factor, is the primary condition of pathogenic E. coli to settle reproduction in the gut, while enterotoxin is a kind of exotoxin, which was released by toxigenic E. coli during its growth and reproduction, and it can lead to water and electrolyte flowing to the intestinal lumen, causing diarrhea of the body. Enterotoxins of ETEC including heat stable enterotoxin (ST) and heat labile enterotoxin (LT). In this study, ETEC K88ac+ (C83902) was selected as experimental subject, which can express K88ac fimbriae and secrete LT and ST2. We successfully constructed attenuated ETEC by point mutations of LT virulence gene and knockout of ST2 gene, and achieved significant effects in the initial application in mice, the main process is as follows.LT gene of wild ETEC K88ac+strains were mutated by Red homologous recombination knockout technique. We designed specific primers according to published LT gene sequences, and then mutated the 63-bit of A subunit of LT gene encoding Ser (TCT) to a codon encoding Lys (AAA) to obtain mutant strains K88ac+LTS63K. Then, we knocked out ST2 gene of K88ac+ ETEC LT (S63K) strain, replaced with Gm resistance gene, both ends of which carryed dif sites. Bacteria can use their own Xer enzyme to excise Gm resistance gene, it was identified correctly by sequencing and named as K88ac+ ETEC LT (S63K) ΔST2. Rabbit ileum ligature experiment was conducted for detecting enterotoxin of K88ac+ ETEC LT (S63K) ΔST2, and we established K88ac+ ETEC (C83902), DH5α and CAYE as control groups. The results showed that the ratio of intestinal fluid volume and intestinal length is less than or equal to 0.9 in the groups that K88ac+ ETEC LT (S63K) AST2, DH5α sterile culture supernatant and CAYE medium were injected, while the ratio was 1.27 by injection of K88ac+ETEC (C83902) sterile culture supernatant. It indicated that K88ac+ ETEC LT (S63K) AST2 is an attenuated enterotoxigenic E. coli strain.The 8 weeks aged clean grade Balb/c mice were killed by cervical dislocation, we collected their ileum and cut into 5mm length segments, followed by washing 3 times with sterile PBS, soaking into bacterial fluid of K88ac+ ETEC(C83902), K88ac+ ETEC LT(S63K)ΔST2, DH5a and sterile PBS seperately, and incubated for 1h at 37℃. Then scrapped the ileal mucosa and coated glass slides, observed the bacteria under microscope after stained by methylene blue, the results showed that we can see the dyed E.coli only in the groups of K88ac+ ETEC (C83902) and K88ac+ETEC LT(S63K)ST2, while there is no dyed E.coli can be seen in the groups of DH5a and sterile PBS.109 CFU of K88ac+ ETEC LT (S63K) △ST2 were filled to the stomach of Balb/c mice, while K88ac+ ETEC (C83902) was set as the control group, the results showed that the mice inoculated with the attenuated K88ac+ E.coli didn’t cause any clinical symptoms, but trembling, loss of appetite, death and other symptoms were observed in control group, after inspection we can see lots of yellow fluid and intestinal adhesion. These indicated that Escherichia coli may adhere to the mucosa surface of murine ileum by k88ac fimbrials, and K88ac+ ETEC could cause BALB/c mice dying due to the enterotoxic released by it, but the attenuated K88ac+ ETEC LT (S63K)AST2 could not induce any clinical symtoms even inoculated at a high oral dose because of the point mutation and knockout of enterotoxic genes.The 8 weeks old Balb/c mices were inoculated with K88ac+ LT(S63K)AST2 via gavage, and 24h,48h,72h,96h,120h,144h and 168h post inoculation, gavage by K88ac wild strain. The results indicated that except for one mice appearing slight symptoms in the 168h post inoculation group, no other clinical symptoms observed. These results indicated that during one week after inoculation of the attenuated K88ac+ Escherichia coli can effectively prevent infection of K88ac+ enterotoxigenic Escherichia coli wild strain. We detected mouse feces by PCR method and found that the attenuated K88ac+ E.coli can keep in mouse intestine for 23 days, which reflected that the K88ac+ ETEC LT (S63K) △ST2 strain has the potential to be a kind of vaccine delivery vehicle.