Virulence factors in Escherichia coli isolated from piglets with neonatal and post-weaning diarrhea in Japan

A total of 567 strains of Escherichia coli were isolated from piglets with neonatal diarrhea (ND) or post-weaning diarrhea (PWD) in Japan. They were investigated for enterotoxigenicity and possession of adhesins and O antigens. There were clear differences between the strains of ND origin and those of PWD origin in the occurrence of enterotoxigenic E. coli (ETEC) strains, type of enterotoxin and frequency of adhesins: ETEC was found in 77 (25.7%) of 300 strains of ND origin and in 137 (51.3%) of 267 strains of PWD origin. ETEC strains producing heat-labile enterotoxin (LT) or heat-stable enterotoxin (STa), alone or in combination were evenly distributed among the strains of PWD origin. In contrast most of the ETEC strains of ND origin produced LT alone. Adhesins appeared in 42 (54.5%) of 77 ETEC strains of ND origin and in 36 (26.3%) of 137 ETEC strains of PWD origin. Adhesins were less common in ETEC strains of PWD origin than in those of ND origin. Some K99-positive ETEC strains of PWD origin produced both LT and STa. There was a similarity in the distribution of O antigens, particularly O149 and O157, between the strains of ND origin and those of PWD origin.     

Enterotoxigenic Escherichia coli in the jejunum of piglets with neonatal diarrhea

Thirty-two Escherichia coli colonies were taken from the primary step of cultivation of the jejunal contents of each of 10 dead piglets which had suffered from diarrhea. The organisms of each colony were examined for the presence of adhesion fimbria (F4 (K88) and F5 (K99)), production of heat-stable and heat-labile enterotoxin and of colicins.The presence of heat-labile enterotoxin in the intestinal content of the necropsied pigs was also tested, and results correlated with enterotoxin production of the isolated E. coli strains. In all but 3 pigs, 50–80 % of the E. coli strains were found to produce one or both of the enterotoxins and to possess the F4 of the F5 antigen. All bacteria producing both heat-labile and heat-stable enterotoxin proved to belong toi O group 149 and to possess the F4 antigen. Strains from 1 pig belonged to O group 64 and possessed the F5 antigen; these bacteria produced heat-stable enterotoxin only. Most of the enterotoxin-producing E. coli also produced colicins.After each subcultivation, the strains produced less heat-labile enterotoxin, some becoming negative when assayed.     

Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E. coli isolates from pigs with diarrhoea

The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E. coli strains isolated from pigs with postweaning diarrhoea. Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes. Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods. It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant. The most common EAST1-positive E. coli serotype was O149:K91 and these strains were mostly LTI/STII-positive. A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA(+) isolates tested possessed the F4 antigen. Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis.     

Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs.

A novel common colonization factor was detected in enterotoxigenic strains of Escherichia coli isolated from intestinal contents of piglets affected with postweaning diarrhoea. This factor was antigenically distinct from the previously described K88, K99, F41, 987P, CFAI, CFAII and Att25 fimbrial antigens. E. coli strains possessing this factor adhered to the pig intestinal brush borders and one strain, used in experimental infection in weanlings, colonized the intestinal epithelium and induced diarrhoea. Examination of 212 toxigenic strains of E. coli isolated from weanlings revealed the presence of the novel common colonization factor in 83 strains, belonging to serogroups O25, O108, O138, O141, O147 and O157. The antigen K88 was detected in 47 strains belonging to serogroups O8, O141, O147 and O149.     

Enterotoxin plasmids in bovine and porcine enterotoxigenic Escherichia coli of O groups 9, 20, 64 and 101.

Fourteen strains of Escherichia coli of serogroups characteristic of porcine class 2 enterotoxigenic E. coli isolated from pigs or calves were selected for genetic studies. The strains were examined for their ability to cotransfer a number of plasmid-mediated properties during conjugation with E. coli K-12. These properties were antibiotic resistance, and the production of heat-stable enterotoxin, the K99 antigen and colicin and the ability to ferment raffinose. Distinction was made between the two types of heat-stable enterotoxin, STa and STb. All 14 strains were antibiotic resistant and 11 of them cotransferred antibiotic resistance and heat-stable enterotoxin. One strain which transferred heat-stable enterotoxin also transferred the raffinose gene. Among six K99-positive strains which transferred heat-stable enterotoxin, five always cotransferred K99. Three strains had 100% cotransfer of colicin as well as heat-stable enterotoxin and K99. Drug resistance determinants were cotransferred at high frequency with heat-stable enterotoxin for six of eight multiple drug resistant enterotoxigenic E. coli. A 100% cotransfer of combinations of heat-stable enterotoxin, K99, colicin and antibiotic resistance was often associated with a single plasmid band on agarose gel electrophoresis. For some strains, the genes for STa and STb were on the same plasmid and for others they were on separate plasmids. The enterotoxin plasmids ranged in size from 5.2 to 85 Mdal. Heterogeneity in molecular size occurred among enterotoxin plasmids in E. coli of the same serogroup and recovered from the same animal host species.     

Development of intestinal antibodies against Escherichia coli antigens in piglets with experimental neonatal E. coli diarrhoea

Intestinal immune responses to Escherichia coli antigens were studied in conventionally reared piglets orally infected on the first day of life with a virulent enterotoxigenic E. coli (O149: K88). During the first week of life intestinal antibodies were produced against the homologous lipopolysaccharide (LPS) as well as against the K88 antigen and the heat-labile enterotoxin (LT). On Day 7, anti-LPS antibodies of the IgA and IgG classes were detected in most piglets, whereas anti-K88 antibodies of the IgG and IgM classes predominated; antibodies against the enterotoxin were usually of the IgG class. In 21-day-old piglets antibodies of all immunoglobulin classes had usually been produced. In most cases, the levels of intestinal antibodies were substantially higher on Day 21 compared to Day 7, but the levels varied considerably both between and within litters. The intestinal immune responses did not correlate with the severity of clinical symptoms. One-, 7- and 21-day-old piglets reared in a specific-pathogen-free (SPF) herd lacked significant intestinal antibodies to the antigens examined. The oral challenge did not stimulate systemic immune responses. After colostral intake, all piglets had high antibody levels in the circulation. These levels decreased continuously during the 3-week study period. The possibility that high amounts of antibodies in colostrum could interfere with this early intestinal antibody formation should be considered when planning vaccination programmes against E. coli diarrhoea in piglets.     

Prevalence of fimbrial antigens and enterotoxins in nonclassical serogroups of Escherichia coli isolated from newborn pigs with diarrhea

Ninety-nine nonclassical serogroups isolated from newborn pigs with neonatal diarrhea were tested for fimbrial antigens F4(K88), F5(K99), F6(987P), F41, and F165, and for heat-labile enterotoxin, heat-stable enterotoxin a (STa), heat-stable enterotoxin b, verocytotoxin, and cytolethal-distending toxin. Thirty-two strains, belonging mostly to serogroups O64:K"V142,", O9:K103, and O20:K101, were F5-positive and usually produced STa, although 5 strains produced only heat-stable enterotoxin b. Fifteen strains, belonging mostly to serogroups O64:K"V142" and O20:K101, were F41 positive and usually produced STa. Twenty-three stains, belonging mostly to serogroups O64:K"V142," O9:K103, and O20:K101, were F6-positive and usually produced STa. Several strains produced more than one fimbrial antigen, either F5 and F41, F5 and F6, F6 and F41, F6 and F165, or F5, F6, F41, and F165. None of the strains produced heat-labile enterotoxin, verocytotoxin, or cytolethal-distending toxin. The indirect immunofluorescence test was much more sensitive than was the slide agglutination test for the detection of each of the fimbrial antigens F5, F6, F41, and F165 on strains grown in vitro. The production of F6 by certain strains for which only a low proportion of cells were F6-positive in vitro, as demonstrated by immunofluorescence, was confirmed by experimental infection of newborn pigs.     

Attachment of E. coli-bearing K88 antigen to equine brush-border membranes

Equine small intestinal brush-border membranes, from 40 adult horses were tested in vitro for the presence of receptors for the Escherichia coli adhesive antigens K88ab, K88ac and K99. Only K88-positive strains of E. coli adhered strongly to horse brush-border membranes. In contrast, a K88-negative mutant strain J2, 2 K99-positive strains and 3 E. coli strains isolated from foals failed to adhere to horse brush-border membranes. Purified K88ac pili when reacted with equine brush-border membranes inhibited to a great extent the adhesion of K88-positive E. coli. Similarly, K88-positive E. coli previously reacted with K88 antibody, did not attach to equine brush-border membranes. Oral inoculation of 4 newborn foals with strains of K88-positive enterotoxigenic E. coli, producing either heat-stable or heat-labile enterotoxin, caused diarrhoea in 1 animal.     

Experimental colibacillosis in gnotobiotic piglets exposed to 3 enterotoxigenic serotypes

Three strains of enterotoxigenic Escherichia coli (ETEC) (064:KSNT, K88ac; 020:KSNT, K88ac and 08:K85ab, K99) originally cultured from outbreaks of diarrhoea in piglets a few hours old, were administered orally to gnotobiotic piglets. There was a marked age-related difference in the clinical response to infection between the 3 strains although they all produced heat-stable toxin. All 3 strains produced severe clinical signs of depression, anorexia, vomiting, diarrhoea, followed by dehydration and death in one-day-old piglets. In piglets infected at 3 days of age the two K88+ ETEC caused diarrhoea and death but the K99+ ETEC induced moderate diarrhoea only. In piglets infected at 7 days of age, the 064 strain produced severe diarrhoea and death, and 020 strain caused mild diarrhoea in 3 of 6 piglets with one death while the 08 strain caused no illness. Pathological changes in the intestinal tract associated with these infections were minimal, or absent. Immunofluorescent staining with homologous hyperimmune sera demonstrated adherence of the 3 ETEC strains to the brush border of small intestinal epithelial cells. Fluorescing organisms were observed in all infected piglets irrespective of the severity of clinical signs but the degree and extent of colonisation varied with the age of the piglets and the infecting strain. This may explain the difference in clinical response between the 3 strains.     

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.     

Fimbriae and enterotoxins associated with Escherichia coli serogroups isolated from pigs with colibacillosis

A comprehensive study of 223 Escherichia coli isolates from pigs with colibacillosis included determination of O serogroups, detection of heat-labile enterotoxin, heat-stable enterotoxin (STa and STb), and identification of K88, K99, 987-P, F-41, and type 1 fimbriae. The incidence of the various E coli types among isolates of pigs of different ages was also determined. Escherichia coli bearing K88 fimbriae accounted for 48% of all isolates studied, were most often of serogroup O157, O149, or O8, and usually produced labile toxin alone or in combination with STa or STb. These E coli were commonly isolated from pigs in each age group studied (0 to 5 days, 6 to 10 days, 11 to 24 days, and greater than 24 days). Escherichia coli bearing 987-P accounted for 30% of the isolates, were most often of serogroup O141 or O20, and usually produced STa. Escherichia coli bearing K99 accounted for 13% of the isolates, usually were of serogroup O101 or O8, and almost always produced STa. Escherichia coli bearing 987-P or K99 were most often isolated from pigs less than 6 days of age. Fimbriae F-41, when identified, were usually on E coli of serotype O101:K99. Although infrequently found, type 1 fimbriae were on E coli of most of the serogroups identified in this study.     

The Significance of Proliferation and Enterotoxin Production by Escherichia coli in the Intestine of Gnotobiotic Pigs

The significance of enterotoxin production and proliferative ability of Escherichia coli in the intestinal tract as related to porcine enteric colibacillosis was studied in 68 gnotobiotic pigs.

The animals were monocontaminated at seven to ten days of age with eight selected strains of E. coli. The strains were two naturally occurring porcine enteropathogens — P155 (0149:K91;K88a,c:H10) and P307 (08:K87;K88a,b:H19), two nonenteropathogenic strains — P104 (0139:K82:H1) and F11 (018-ab:K?:H14), and four enterotoxigenic derivatives of the above strains — P104(P155), P104(P307), F11(P155) and F11(P307). The response of the animals was evaluated on the basis of clinical observations and necropsy lesions 22 hours after exposure to the organisms. E.coli counts were determined at seven different levels of the intestinal tract. Cell free extracts of the intestinal contents were examined for enterotoxic activity by the ligated pig intestine loop test.

All of the strains possessing the enterotoxin plasmid produced enterotoxin in the pig's intestine and were capable of causing diarrhea. The nonenteropathogenic E. coli failed to do so. The strains possessing the P155 enterotoxin plasmid were more virulent than the corresponding derivatives with the P307 enterotoxin plasmid. Strains P155, P307 and P104(P155) proliferated in the upper small intestine at a greater rate and were more virulent than the other strains. The numbers attained in the upper small intestine by the other enterotoxigenic derivatives were comparable to those of their nonenteropathogenic parent strains.

It was considered that enterotoxin produced by E. coli was the essential factor for causing a diarrheic response in gnotobiotic pigs. The virulence of each of the tested strains appeared to be governed by the degree of enterotoxicity associated with a particular enterotoxin plasmid, the numbers attained by these organisms in the upper small intestine, (but not in the lower small intestine or in the colon), and by other undetermined factors.

     

Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea

The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea. Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected. E. coli. strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII). F107 fimbriae were demonstrated in association with E. coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c. Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains. The genetic determinant of SLT-IIv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c. Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv. Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes. Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes. In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates. The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates. In these latter strauns enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.     

Detection with nonradioactively labeled probes of the toxin genes of different E. coli pathotypes from swine]

We tested hemolytic E. coli from 86 pigs with edema disease or colidiarrhea. They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes. By slide-agglutination we detected 38 cases with E. coli O149:K88, 28 with E. coli O139:82B and 20 with E. coli O141. E. coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes. Edema disease E. coli O139:82B reacted with the SLTII probe. E. coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns. All the E. coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII. No strains isolated from pigs with edema disease possessed any of these enterotoxin genes. Gene probe technique confirmed the serological method as useful tool for diagnosing E. coli O149:K88 and O139:82B as ETEC or VTEC, respectively. On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E. coli of serogroup O141.     

Colicin resistance in relation to pathogenicity factors in strains of Escherichia coli isolated from the intestinal tract of piglets

Three hundred and fifteen E. coli strains isolated from the intestine of piglets were examined for K-antigens 88 and 99, enterotoxin production and colicin resistance. Of these strains 308 belonged to one of 3 following different groups: Group 1: 0149, K88, producing heat-labile (LT) and heat-stable (ST) enterotoxins, group 2: 064, K99, producing ST, and group 3: variable O-antigens, no K-antigens or enterotoxin production.Almost 100 % of the E. coli strains were found to be resistant to colicins E₁, E₃, Ia, H and D+X. Resistance to colicins E₂, B+M, V and K+X were found in 91.7 %, 43.8 %, 49.8 % and 62.2 % respectively.E. coli strains in group 1 were always (resistant to colicin E₂, while about 87 % of the other strains were resistant to this colicin. E. coli strains in group 2 were more often resistant to colicin B+M, V and K+X (65 %, 94 %, 83 %) than strains in group 1 (37 %, 24 %, 64 %) and strains in group 3 (37 %, 52 %, 46 %).E. coli strains in group 2 showed a high degree of multiresistance, 45.1 % of the strains being resistant to all of the 9 colicins. About 10 % of the other strains were resistant to all of the 9 colicins.E. coli strains harbouring the enteropathogenicity factors K99 antigen and ST production, showed a higher degree of colicin resistance than both the E. coli strains with K88 antigen and ST and LT production, and the E. coli strains lacking enteropathogenicity factors.     

Characteristics of verotoxigenic Escherichia coli from pigs.

Porcine verotoxigenic Escherichia coli were characterized with respect to frequency of occurrence, serogroup, and association with disease, weaning, and selected properties of the bacterium. Of 668 strains of E. coli from southern Ontario pigs with enteric disease, 32 (4.8%) produced verotoxin at 10(3)-10(7) cytotoxic doses per mL of culture supernatant. Of 22 isolates which belonged to O serogroups 138, 139 and 141, 15 produced verotoxin. Among other enterotoxigenic types of E. coli, two of 57 isolates of O157:K"V17" and two of 96 isolates of O149:K91 were verotoxigenic. The remaining 13 verotoxigenic E. coli belonged to O groups 2, 107, 120, 121 and 130. An additional 21 verotoxigenic E. coli belonging to O groups 138, 139 and 141 and three to O157:K"V17" were identified in a collection of 47 E. coli recovered from weaned pigs with enteric disease. Verotoxigenic E. coli were associated with postweaning diarrhea, bloody stools, sudden death and edema disease. They were isolated at similar frequencies (14%) from healthy weaned pigs, and from weaned pigs with enteric disease. Isolation rates from neonates were low and significantly different from rates in weaned pigs. Neutralizing antibody to verotoxin was not detected in the sera of 45 pigs, which included pigs from herds with a history of edema disease. Verotoxin was not associated with production of colicin, hemolysin, or enterotoxins or with any of 23 biochemical properties of the organisms. The serological data indicate that porcine verotoxigenic E. coli are not a common source of verotoxigenic E. coli for humans. Porcine verotoxin may play a role in postweaning diarrhea and absence of detectable neutralizing antibody in serum may be an important aspect of pathogenesis.     

Serological, enterotoxin-producing and biochemical properties of Escherichia coli isolated from piglets with neonatal diarrhea in Norway

Altogether 235 strains of Escherichia coli isolated from jejunal content of piglets with neonatal diarrhea were examined for serological, enterotoxin-producing and certain biochemical properties. Of 198 strains examined, 84 (42 %) belonged to O-group 149, while 14 (7 %) strains belonged to each of the O-group s 8 and 64. Seventy strains (30%) could not be grouped with the sera used. The remaining strains were distributed among the following O-groups with only a few strains in each group: 2,6,9,32,45,98 and 141. Eighty out of 84 E. coli strains of O-group 149 possessed the K88 antigen and produced heat labile enterotoxin (LT). Besides, LT production was demonstrated in 3 out of 14 strains of E. coli 08. K88 antigen was demonstrated in only 1 strain not belonging to O-group 149. Among strains of E. coli 064 12 out of 14 were K99 positive. This antigen was not demonstrated in E. coli strains of other O-groups. A close relationship was demonstrated between strains of E. coli 0149 possessing the K88 antigen and the ability to ferment both raffinose and adonitol. This ability was only detected in 2 other strains of E. coli not belonging to O-group 149.     

致犊牛腹泻肠毒素大肠杆菌多重PCR检测方法的建立

肠毒素大肠杆菌((Ent erot oxi geni c E.col i,ETEC)是引起犊牛腹泻的主要病原之一。本试验建立了多重PCR检测ETEC毒力因子F41菌毛、K99菌毛和STa、LT肠毒素相关基因的技术方法。试验对影响PCR扩增的dNTP、引物浓度以及退火温度等因素进行优化,确定了多重PCR的特异性和灵敏性。结果表明:所建立的多重PCR方法快速、特异、灵敏,在2.5h-3h内就可以完成,为致犊牛腹泻肠毒素大肠杆菌的快速准确检测提供另一种选择。     

The effectiveness of inactivated vaccines applied parenterally to sows to control Escherichia coli diarrhea in piglets in an industrial fattening farm

Different vaccines against Escherichia coli diarrhea of piglets were applied parenterally in pregnant sows at an industrial fattening farm. The following vaccines were used: vaccine No. 1 with non-complete Freund's adjuvant. Tween 80 and Arlacel A, comprising O149:K91,K88; O139:K82; O8:K87,K88; O141:K85,K88; and O64:K? E. coli serotypes; vaccine No. 2 with paraffin oil instead of Freund's adjuvant, comprising the same E. coli serotypes as the vaccine No. 1; stable specific vaccine with 10% aluminium hydroxide, based on E. coli serotypes most frequently isolated from piglets which died at the farm (O149:K91,K88; O8:K87,K88; O20:K17; O64:K?); Gletvax K88 (Wellcome) and NOBI-VAC LT-K88 (Intervet International). The number of piglets which died up to the moment of weaning in comparison to the number of born ones was considered as an indicator of acquired protection. It was found that the most effective in conferring protection against E. coli diarrhea were: vaccine No. 1 and NOBI-VAC. The differences in the mortality rate between piglets originating from sows vaccinated with these vaccines and those from unvaccinated ones were statistically significant (P < 0.05). No significant differences were noted between controls and animals vaccinated with the remaining vaccines.     

表达Ⅰ型耐热肠毒素的重组大肠杆菌诱发仔猪小肠炎症的分子机制研究

本试验旨在研究表达Ⅰ型耐热肠毒素(STa)的重组大肠杆菌诱发7日龄仔猪小肠炎症反应。选取24头7日龄健康仔猪,随机分为4个处理组:对照组(人工乳)、STa组(人工乳+2×10~9 CFU重组菌LMG194-pBAD-STa)、LMG194组(人工乳+2×10~9 CFU大肠杆菌LMG194)和K88组(人工乳+2×10~9 CFU大肠杆菌K88),每组6头。试验第5天进行攻毒,第7天屠宰取样,观察空肠组织形态学变化,并检测血清中炎性细胞因子含量及空肠免疫相关基因的相对表达量。结果显示,与对照组相比,STa与K88组仔猪空肠绒毛明显萎缩变短,有明显损伤甚至脱落;STa、LMG194及K88组血清中IL-10、IL-8和IL-6浓度均显著升高(P<0.05),LMG194和K88组血液和肠道iFABP浓度均显著降低(P<0.05),STa和K88组血清中TNF-α浓度显著降低、NF-κB浓度显著升高,LMG194组NF-κB浓度显著降低、TNF-α浓度显著升高(P<0.05);STa和LMG194组CCL2和CXCL9基因表达水平显著上调(P<0.05),VNN1基因表达水平显著下调(P<0.05),STa和LMG194组IFN-γ组基因水平分别显著上升和下降(P<0.05);K88组CXCL9和IFN-γ基因表达水平显著下调(P<0.05),VNN1基因表达水平显著上调(P>0.05),CCL2基因表达水平无明显差异(P>0.05)。以上结果表明,表达Ⅰ型耐热肠毒素STa的重组大肠杆菌几乎具有与K88一样的毒性,可导致7日龄仔猪小肠黏膜受损,诱发严重的炎症反应,导致仔猪腹泻,可作为仔猪腹泻模型的候选菌株。