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1.
Many strains of enterotoxigenic Escherichia coli (ETEC) that cause diarrhoea in young piglets secrete a heat-labile enterotoxin (LTp) that binds to specific glycoconjugates on porcine intestinal epithelial cells. Binding of LTp to an appropriate glycoconjugate facilitates the uptake and trafficking of the toxin into the cell, where it stimulates intracellular changes that promote fluid secretion and diarrhoea. The objective of the current study was to identify the LTp-binding glycoconjugates on porcine intestinal epithelial cells, the natural target cells for LTp. We found that LTp binds, in an age-correlated manner, to an acidic glycosphingolipid (GSL) that co-migrated with GM1 on thin-layer chromatography (TLC), a small acidic GSL that appears to be a sulphatide, a neutral GSL that co-migrated with neolactotetraglycosylceramide (nLc4) on TLC, and two glycoproteins (36 and 205 kDa). Of these potential LTp receptors, the GM1-co-migrating GSL was detected most intensely in young animals, while the other four LTp-binding glycoconjugates were detected most intensely in older pigs (54 weeks). Since ETEC primarily cause disease in young piglets, the GM1-co-migrating GSL is the most likely candidate for a functional LTp receptor.  相似文献   

2.
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.  相似文献   

3.
Enterotoxigenic Escherichia coli was isolated from a 3-day-old foal with diarrhea. The isolate was distinguished from nonpathogenic E coli by determining the presence of pili and enterotoxin production. A standard slide agglutination test was performed, using pooled antisera that contained antibodies against K99 and F41 pilus antigens, K87 capsular antigen, and 0101 somatic antigen. Agglutination of the antisera occurred in the presence of the isolate. Piliation was verified by use of negative-contrast electron microscopy. Further, the isolate produced a heat-labile enterotoxin-like antigen that cross-reacted with a reagent containing formalin-treated, heat-killed Staphylococcus aureus (cowan 1 strain) bearing anti-cholera antibodies. On the basis of the aforementioned procedures and the absence of other identifiable enteric pathogens, we believe that E coli was responsible for causing diarrhea in the foal.  相似文献   

4.
We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests. K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method. LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies. It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type. There was 100% correlation between the slide agglutination and ELISA tests. Of the 103 strains tested 68.9% produced LT or STa or both toxins. LT-positive strains were the most common ones in both groups of piglets. All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study. Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E. coli strains isolated from piglets.  相似文献   

5.
During 1972 to 1974, 112 Escherichia coli strains isolated from diarrheic piglets were recieved from different parts of the Province of Quebec, Canada. Fifty-six strains elicited a positive gut loop response in three week old piglets and were then considered as Moon's class 1 enteropathogens, while four of the 56 remaining strains reacted only in ten day old piglets and were classified as class 2 enteropathogens. Forty-eight strains produced both a heat-labile and a heat-stable enterotoxin, while 12 isolates which included the four class 2 enteropathogens produced only a heat-stable enterotoxin. Fifty-one enterotoxigenic strains could be serogrouped using OK antisera against E. coli strains commonly associated with colibacillosis in piglets. The most common serogroups encountered were O157: K "V17"; 88a,c, O149:K91; 88a.c. O157:K"V1c, O149:K91; 88a.c, O157:K"V17"; 88 a,c or a,b and O45:K"E5"; 88a,c. No significant difference was observed in the fermentation patterns, antibiotic susceptibility, colicin production, production of a filterable hemolysin and transferable tetracycline resistance between the enterotoxigenic and the nonenterotoxigenic strains.  相似文献   

6.
Specific, double-sandwich ELISAs for beta and epsilon toxins were developed by coating wells of microplates with specific sheep antitoxin IgG and using specific rabbit antitoxin IgG as detecting antibodies. The assay for beta toxin detected a minimum level of 8 ng/ml of purified toxin. The assay for epsilon toxin was capable of detecting 2 ng/ml of purified toxin. When applied to detect the toxin in intestinal contents using 50% fetal bovine serum as diluent the lowest amounts detected were about at least 30 ng/ml for beta toxin and 4 ng/ml for epsilon toxin. Clear differences in ELISA readings of both assays have been found between culture filtrates from toxin and non-toxin producing strains. These results suggested that both assays described in this study could detect their respective toxin in buffers, culture supernatants or in intestinal contents.  相似文献   

7.
已构建的能表达大肠杆菌K88ac-ST1-LTB融合蛋白的工程菌株BL21（DE3）（pXKST3LT5)及其表达产物经动物试验证实没有毒性反应。用从IPTG诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原，免疫小鼠，结果免疫小鼠至少能抵抗2MLD的大肠杆菌强毒株C83902（K88ac,ST^ ,L^ )的攻击，用提取的包涵体免疫家兔后，采集的血清能够中和天然ST1的毒性，这表明构建的工程菌株BL21（DE3）（pXKST3LT5)可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株。  相似文献   

8.
Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is economically one of the most important diseases for the swine industry. Porcine ETEC strains typically express K88 or F18 fimbria and heat-labile (LT) and/or heat-stable (STa, STb) enterotoxins. However, recent studies indicate that EAST1 toxin, adhesin involved in diffuse adherence (AIDA-I) and porcine attaching and effacing-associated factor (paa) may also be expressed by ETEC strains associated with diarrhea. To better understand the virulence factors of E. coli strains that cause PWD, we applied PCR to screen for K88, F18, F41, 987P and K99 fimbrial genes; LT, STa, STb, Stx2e and EAST1 toxic genes; and AIDA-I, paa and EAE adhesin genes in E. coli strains recently isolated from young pigs with PWD in the US. Of 304 E. coli isolates from diarrheic pigs submitted for testing, 175 (57.6%) strains possessed fimbrial genes: K88 (64.6%), F18 (34.3%), F41 (0.57%), K99 (0.57%), 987P (0); toxin genes: LT (57.7%), STb (72.6%), STa (27.4%), STx2e (17.4%), EAST1 (35%); and adhesin genes: AIDA-I (26.9%), paa (60%), EAE (1.1%). All toxin genes except the EAST1 toxin gene, were almost exclusively associated with K88+ or F18+ isolates, and most of these isolates carried multiple toxin genes. The non-fimbrial adhesin paa was found present in over half of the K88+ isolates. A total of 129 (42%) isolates carried no fimbrial genes, including 66 (21.7%) isolates that did not have any of the above virulence genes. These results suggest a broad array of virulence genes associated with PWD in pigs.  相似文献   

9.
1125 and 1146 E. coli strains isolated from suckling and weaned piglets with diarrhea, respectively, and 724 strains from healthy piglets were tested for the presence of fibriae and production of enterotoxins. The fimbriae were determined by hemagglutination and slide agglutination tests, enterotoxins—by the use of ileal loop test in piglets (LT and STb enterotoxins) and suckling mouse assay (STa enterotoxin). It was found that 72.8 and 53.0% strains, isolated from diseased suckling and weaned piglets, respectively, possessed specific fimbrial hemagglutinins, in most cases with K88 antigen. Additionally, 987P fimbriae were detected in 14.0 and 0.7% strains isolated from piglets with diarrhea. Only 5 strains (0.7%) recovered from healthy piglets had specific fimbriae, usually with undetermined antigenic structure. F1 fimbriae (called common or unspecific) were found in strains isolated both from diseased (15.2 and 16.3% strains, respectively) and healthy piglets (27.1% strains). It was noted that the strains isolated from suckling and weaned piglets with diarrhea in most cases were enterotoxigenic (90.5 and 69.1% strains, respectively) and most frequently produced heat-labile toxin LT alone or with STb. 18.5% of enterotoxigenic strains isolated from healthy piglets produced STa toxin.  相似文献   

10.
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.  相似文献   

11.
Rectal swabs collected from piglets with diarrhoea from commercial pig farms were examined for the presence of enterotoxigenic Escherichia coli (ETEC) using DNA hybridisation methods. The probes specifically detected genes for the K88 and K99 fimbrial antigens and the heat-labile and heat-stable enterotoxins. DNA hybridisation methods detected more ETEC than could be detected by either enzyme-linked immunosorbent assay (ELISA) or slide agglutination methods, and also offered the opportunity to test for fimbrial antigens and toxins concurrently. The DNA hybridization method was shown to be applicable to ETEC detection in mixed growths cultured directly from rectal swabs to filters. The method eliminates the need for toxin tests using animals and enables very large numbers of samples to be investigated. The use of toxin probes has revealed large numbers of ETEC with uncharacterized fimbrial antigens.  相似文献   

12.
E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.  相似文献   

13.
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.  相似文献   

14.
Pregnant gilts were vaccinated with two doses of alhydrogel adsorbed fimbrial antigens of Escherichia coli (K88ab, K88ac, K99 and 987P) supplemented with beta toxoid of Clostridium perfringens type C. Their piglets, and piglets of nonvaccinated gilts, were subsequently orogastrically challenged with one or other of the four fimbrial types of enteropathogenic E coli. Some of the vaccinated animals were reinjected with a single dose of the vaccine during second gestation and their piglets, and piglets of non-vaccinated sows, were challenged the same way as were litters of gilts. Blood serum and colostra were examined for antibodies to the four fimbrial antigens of E coli and for antitoxin to beta toxin of C perfringens type C. It was found that: (1) a highly significant reduction in mortality and morbidity was achieved in vaccinated litters against all four challenge strains of E coli; (2) excretion of K88ab and K88ac but not of K99 and 987P challenge strains was significantly reduced; (3) revaccination of sows by a single dose of the vaccine during second gestation conferred complete protection against mortality and highly significant protection against morbidity; (4) no correlation was noted between colostral or seroagglutinins to fimbrial antigens of E coli and mortality rates in litters challenged with homologous fimbrial types of E coli, but good correlation was found between colostral precipitins to K88 antigens and mortality rates in litters; (5) antitoxin value in 97 per cent of colostrum of vaccinated sows was 10 iu equivalent of C perfringens type C toxin or more per ml of colostrum.  相似文献   

15.
The role of Escherichia coli as a causative agent of diarrhea in cats was investigated. Isolates of E coli from healthy and diarrheic cats were serotyped and investigated for their biochemical characters, production of cytotoxin activity on Vero cells, heat-labile enterotoxin, heat-stable enterotoxin, and hemagglutination of erythrocytes from other animal species. None of 48 investigated strains produced heat-labile enterotoxin or heat-stable enterotoxin, nor did they agglutinate erythrocytes. Most strains were hemolytic and belonged to O-serotypes 2 and 6. Cytotoxin activity on Vero cells was significantly more common and produced in greater amounts among E coli strains isolated from diarrheic cats, and was neutralized by anti-Shiga-like toxin I serum.  相似文献   

16.
Live, avirulent Escherichia coli vaccine strains were constructed and tested for efficacy in preventing colibacillosis in 4-week-old pigs. Either or both of 2 plasmids were inserted into avirulent E coli strain G58-1 (0101:NM). These plasmids were pPMC4, which encodes for LTb subunits of heat-labile enterotoxin, and pDHF1, which encodes for K88ac fimbriae. Litter- and weight-matched pigs were removed from sows when they were 10 days old and vaccinated orally with the constructed strains or with G58-1 (negative control vaccine) when they were 2 weeks old and 5 days later. All pigs were challenge-inoculated with virulent E coli strain 3030-2 (O157:K88, LT+, STb+) 2 weeks after the first vaccination. Only 1 pig vaccinated with G58-1/pPMC4/pDHF1 developed diarrhea and none died following challenge inoculation. Seventeen of 31 control pigs developed diarrhea and 11 died. Of 18 pigs vaccinated with G58-1/pDHF1 then challenge-inoculated with the virulent strain, 5 developed diarrhea and 2 died. Fifteen of 18 litter- and weight-matched controls developed diarrhea and 8 died. When compared with G58-1 (negative control), G58-1/pPMC4 afforded no protection to pigs challenge-inoculated with 3030-2.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.  相似文献   

20.
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.  相似文献   

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