Pathogenicity of porcine enterotoxigenic Escherichia coli that do not express K88, K99, F41, or 987P adhesins.

Three-week-old weaned and colostrum-deprived neonatal (less than 1 day old) pigs were inoculated to determine the pathogenicity of 2 enterotoxigenic Escherichia coli isolates that do not express K88, K99, F41, or 987P adhesins (strains 2134 and 2171). Strains 2134 and 2171 were isolated from pigs that had diarrhea after weaning attributable to enterotoxigenic E coli infection. We found that both strains of E coli adhered in the ileum and caused diarrhea in pigs of both age groups. In control experiments, adherent bacteria were not seen in the ileum of pigs less than 1 day old or 3 weeks old that were noninoculated or inoculated with a nonpathogenic strain of E coli. These control pigs did not develop diarrhea. Antisera raised against strains 2134 and 2171 and absorbed with the autologous strain, grown at 18 C, were used for bacterial-agglutination and colony-immunoblot assays. Both absorbed antisera reacted with strains 2134 and 2171, but not with strains that express K99, F41, or 987P adhesins. A cross-reaction was observed with 2 wild-type K88 strains, but not with a K12 strain that expresses K88 pili. Indirect immunofluorescence with these absorbed antisera revealed adherent bacteria in frozen sections of ileum from pigs infected with either strain. We concluded that these strains are pathogenic and express a common surface antigen that may be a novel adhesin in E coli strains that cause diarrhea in weaned pigs.     

Protection against enteric colibacillosis in pigs suckling orally vaccinated dams: evidence for pili as protective antigens

Pregnant gilts were vaccinated orally with Escherichia coli that produced pilus antigens K99 or 987P. The vaccines were live or dead enterotoxigenic E coli (ETEC) or a liver rough non-ETEC strain which has little ability to colonize pig intestine. Pigs born to the gilts were challenge exposed orally with K99+ or 987P+ ETEC, which did not produce heat-labile enterotoxin or flagella and which produced somatic and capsular antigens different from those of the vaccine strains. Control gilts had low titers of serum and colostral antibodies against pilus antigens, and their suckling pigs frequently had fatal diarrhea after challenge exposure. Serum antibody titers against pilus antigens of the vaccine strains increased in the gilts after vaccination with liver ETEC, and the colostral antibody titers of these gilts were higher than those of controls. Pigs suckling such vaccinated gilts were more resistant than controls to challenge strains were of different pilus types, and it could not be attributed to enterotoxin neutralization by colostrum. In contrast to the live ETEC vaccines given to the pregnant gilts, the liver rough non-ETEC and dead ETEC vaccines stimulated little or no production of antibody against pilu, and the pigs born of these vaccinated gilts remained highly susceptible to challenge exposure. The results support the hypothesis that pilu can be protective antigens in oral ETEC vaccines. It was indicated that in the system reported, protection depended on living bacteria for the production of pilus antigens in vivo or for the transport of pilus antigens across intestinal epithelium.     

Development of a novel live vaccine delivering enterotoxigenic Escherichia coli fimbrial antigens to prevent post-weaning diarrhea in piglets

The efficacy of a novel, live delivery vaccine was examined for protection against post-weaning diarrhea in pigs. An expression/secretion plasmid harboring genes encoding enterotoxigenic Escherichia coli K88ab, K88ac, FedA and FedF fimbriae was constructed and harbored in an attenuated Salmonella, which was used as the vaccine candidate. Groups A (n=3) and B (n=3) sows were orally immunized with the candidate vaccine and PBS as a control, respectively, at 8 and 11 weeks of pregnancy. All group piglets were challenged with two challenge strains at 5-week-old. All immunized sows had significantly increased IgG and IgA levels in both serum and colostrum to individual adhesins compared to the control (p ≤ 0.05). Immune response in Group A piglets were significantly increased (p ≤ 0.05). Furthermore, no clinical signs were observed in Group A piglets after the challenge and no challenge strains were detected in rectal swabs, while diarrhea was observed in 47.8% control piglets and challenge strains were isolated from all the diarrheic piglets. These results show that immune response of sucking piglets can maintain at higher levels through the milk of the immunized sows and vaccination of sows with the candidate may protect colibacillosis in weaned piglets.     

Iscom (immunostimulating complex) vaccines containing mono- or polyvalent pili of enterotoxigenic E. coli; immune response of rabbit and swine

Iscom (immunostimulating complex) vaccines were prepared to contain K88ab, K88ac, K99 and 987P pili (fimbriae) of enterotoxigenic E. coli bacteria as monovalent or quadrivalent preparations. The iscoms injected into rabbits and into pigs elicited similar or higher immune response in both animal species than the oil adjuvanted vaccine containing about 5 times more of the same pilus protein. It is concluded that inclusion of pili into iscoms results in immunogenic preparations likely worth pursuing for vaccine production against enterotoxic colibacillosis of newborn pigs. The iscoms did not induce local reaction at the injection sites in contrast to the oil adjuvanted vaccines.     

大肠杆菌K_(88ac)与LT(A~-B~ )抗原基因质粒在猪霍乱沙门氏菌弱毒株中的表达

应用细菌质粒转化技术,将大肠杆菌K_(?)与LT(A~-B~ )抗原基因重组质粒转入猪霍乱沙门氏菌弱毒菌苗株中.对获得的其中8个转化子进行鉴定的结果表明,转化的细菌仍保持沙门氏菌的形态、生化及抗原特性,同时可稳定地表达K(?)和LT-B两种抗原.用微量间接血凝试验、抗甘露糖豚鼠红细胞凝集试验(MRHA)、ELISA等对转化菌表达的K(?)抗原进行了测定,用间接免疫溶血试验对其表达的LT-B抗原进行了测定.结果,这两种抗原在转化的细菌中均可高效表达.电镜下观察,转化的细菌在其表面形成菌毛样结构.这种转化细菌表现出猪霍乱沙门氏菌与产肠毒素性大肠杆菌的两种抗原特性,为这种双价工程菌苗的研制提供了有价值的候选菌株.本研究结果还表明,猪霍乱沙门氏菌弱毒菌苗株可作为基因转化的有效受体菌.     

Preliminary characterization of Escherichia coli isolated from pigs with diarrhoea in Venezuela

The aetiology of neonatal porcine diarrhoea was studied in 15 different herds located in the north-western region of Venezuela. Of 56 strains of Escherichia coli analyzed, 16 (28.6%) were shown to produce heat-stable (STa) enterotoxin, as detected by infant mouse assay. Only four of these STa+ isolates also possessed the K88 pilus antigen, two were 987P+ and none possessed the K99 antigen, leaving 10 STa+ samples in which no pilus antigen was identified. Among the 40 STa negative samples were six K88+ specimens, one K99+, four 987P+, one which reacted as K88+ + K99+ and one K88+ + 987P+. Considering as pathogenic any strain showing at least one of the characters studied, pathogenic E. coli were detected with an overall frequency of 42.9%, being more prevalent during the second week of life. An electrophoretic analysis of the plasmid content of the field isolates of E. coli, revealed the presence of numerous species of extrachromosomal DNA, although no direction association could be made between a particular plasmid and any of the pathogenic characteristics identified. Results of Southern blot analysis indicate that the STa enterotoxin was preferentially encoded within an endemic plasmid of 4.9 Md. Other plasmids present in the E. coli isolates could be related to antibiotic resistance. With the exception of one strain, all E. coli isolates were resistant to more than one of the nine drugs tested; multiresistant E. coli were frequently isolated, including four strains which were resistant to seven antibiotics.     

产肠毒素性大肠杆菌K99菌毛蛋白抗原基因的克隆与表达

应用PCR从产肠毒素性大肠杆菌（ETEC）中扩增出不含信号肽序列的K99菌毛蛋白基因片段，克隆测序后，将该片段连接到E.coli表达载体pET28a( )中，转化E.coli表达菌株BL21（DE3），筛选得到可诱导表达K99抗原的工程菌株。经IPTG诱导，分离纯化K99重组蛋白，以其免疫新西兰大白兔，获得重组蛋白的兔抗血清；免疫印迹分析表明，此重组蛋白制备的抗血清能与标准的K99强毒株姓明显的抗原抗体反应。     

Susceptibility of Chinese Meishan and European large white pigs to enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, K99, or F41

Conventionally raised Chinese Meishan and European Large White pigs were intragastrically challenge exposed with 2.1 x 10(10) enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, F41, or F41 plus K99. In response to challenge exposure with the K88-positive (K88+) organisms, 96% of Large White pigs died within 48 hours, whereas none of the Meishan pigs died. Both breeds of pigs had similar susceptibility to strains bearing 987P or F41. Lastly, Meishan pigs were found to be more susceptible than Large White pigs to a strain expressing K99 and F41. In pigs with diarrhea, challenge-exposure strains intensively colonized the jejunum (10(8) to 10(10) bacteria/g of tissue) and, to less extent, the duodenum (except K88+ strain, which comprised 10(8)/g). In most cases, jejunal concentrations of the challenge-exposure strains were substantially lower in pigs that did not have diarrhea. Half the resistant Meishan pigs eliminated the K88+ strain from the intestines. Colostral antibody titer that agglutinated challenge-exposure strains did not differ between Meishan and Large White gilts. Results indicate that resistance of pigs to the K88+ strain did not extend to enterotoxigenic strains bearing other well-known factors. They indicate, in addition, that genetic resistance to K88+ strains described in pigs in Europe may exist in pigs in China.     

Prevalence of K88, K99, and 987P pili of Escherichia coli in neonatal pigs with enteric colibacillosis

One hundred nineteen live neonatal pigs with diarrhea less than or equal to 2 weeks old were euthanatized, and frozen sections of their ilea were submitted to an indirect fluorescent antibody technique to identify K88, K99, and 987P pili (also referred to as F-4, F-5, and F-6 pili, respectively) in Escherichia coli. Ten-centimeter ileal sections were used to determine numbers of lactose-fermenting bacteria. Of 52 pigs in which E coli pili were found, 14 had K88 (27%), 23 had K99 (44%), 13 had 987P (25%), and 2 had K88 and K99 simultaneously (4%). Numbers of lactose-fermenting bacteria were significantly (P less than or equal to 0.05) higher in pigs with piliated E coli than in pigs without piliated E coli. Results of this study indicated that piliated E coli are a major cause of enteric disease in neonatal swine in Michigan, and that in pigs less than or equal to 2 weeks of age, K99 was the most frequently encountered pilus antigen.     

Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins

Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.     

大肠埃希菌K88ac菌毛蛋白亚基因的原核表达及其免疫原性研究

从E.coli C83902中扩增出不含信号肽序列的K88ac菌毛蛋白亚基基因片段,将其克隆到表达载体pQE-30中,构建了原核表达载体pQE30-K88ac,并转入E.coli XL1-Blue中,经IPTG诱导后,重组蛋白以包涵体形式获得高效表达.Western blot结果显示,表达的蛋白能够被K88ac单抗识别,用纯化的蛋白免疫小鼠,能够抵抗1 MLD大肠埃希菌强毒株C83902的攻击,这表明构建的工程菌株XL1-Blue(pQE30-K88ac)可以作为预防幼畜大肠埃希菌性腹泻基因工程菌苗的候选株.     

Relationship between virulence and adherence of various enterotoxigenic Escherichia coli strains to isolated intestinal epithelial cells from Chinese Meishan and European large white pigs

In vitro adherence to intestinal epithelial cells by enterotoxigenic Escherichia coli strains bearing K88, K99, F41, or 987P adhesins and of their variants not bearing adhesins (K88-, K99-, or F41-) was investigated in European Large White and Chinese Meishan pigs. Possible relationship between adherence and virulence was also examined. The K88-positive (K88+) strain strongly adhered to intestinal epithelial cells from 26 of 28 Large White pigs. This strain had previously been found to be highly virulent for Large White pigs. The only surviving pig was of nonadherent phenotype, and cells from 4 dehydrated moribund pigs had strong adherence. By contrast, the same K88+ strain found previously to have little pathogenicity for Meishan pigs adhered with variable intensity to cells from 17 of 23 Meishan pigs; correlation was not evident between adherence and virulence. The K99+ F41+ strain of porcine origin and the F41+ strain generally adhered strongly to cells from 24 and 23 Meishan pigs, respectively, and to cells from 25 of 26 Large White pigs. Correlation was not found between adherence and virulence for the 2 strains. A K99+ F41+ strain of bovine origin adhered to cells from 20 of 22 Meishan and 22 of 23 Large White pigs, and a K99- F41+ variant adhered to cells from 19 of 23 Meishan and 23 of 24 Large White pigs. The adhesin-negative variants never adhered to intestinal epithelial cells. Strain 987 known not to readily produce 987P adhesin after in vitro growth never adhered to cells during the test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine monoclonal antibodies to the F5 (K99) pilus antigen of E. coli, produced by murine/bovine hybridomas

Lymph node cells from calves immunized with purified pilus antigen of K99+ enterotoxigenic E. coli (ETEC) were fused with mouse myeloma (NSO) cells, and with non-Ig producing mouse/calf hybridomas or with a bovine Ig-producing mouse/calf/calf secondary hybridoma. Lines secreting bovine monoclonal IgG1 specific for K99 pilus antigen in an ELISA were obtained in each case. The two lines derived from xenohybridoma fusion partners have been secreting anti-K99 bovine monoclonal antibody for over one year in continual passage. None of the antibodies cross-reacted with other pilus types including K88, CFAI, CFAII, 987P or CP; they all inhibited agglutination of horse RBC (which have a K99 receptor) in the presence of K99 antigen; they showed positive fluorescence in an indirect binding assay on K99+ ETEC and inhibited K99+ ETEC adhesion to piglet enterocytes. These antibodies have potential prophylactic and therapeutic use in control and treatment of diarrhoea.     

Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.     

Effects of weaning on diarrhea caused by enterotoxigenic Escherichia coli in three-week-old pigs

We attempted to determine whether weaning is required for induction of diarrhea in pigs with postweaning enterotoxigenic Escherichia coli infection. Three-week-old newly weaned pigs and their suckling littermates were inoculated with the K88+ enterotoxigenic E coli strain M1823B. Fourteen of 21 weaned and 12 of 20 suckling pigs were genetically resistant to intestinal adhesion by the K88+ strain of E coli; they remained healthy, and gained weight at similar rates. Both groups of K88-resistant pigs gained weight faster, and shed fewer bacteria of strain M1823B in their feces, than did their K88-susceptible counterparts. Diarrhea developed in K88-susceptible pigs in the weaned (6 of 7 pigs) and suckling (4 of 8 pigs) groups, and 1 of the 4 affected suckling pigs died from complications resulting from diarrhea. The incidences of diarrhea, weight gain rates, and the numbers of strain M1823B shed in feces of susceptible weaned and suckling pigs were not significantly (P greater than 0.05) different. Diarrhea scores of susceptible weaned pigs were significantly (P less than 0.02) higher than those of susceptible suckling pigs on the second day after inoculation. In this experimental model, it was concluded that weaning is not required for induction of diarrhea, but may modestly increase its severity.     

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.     

Evaluation of monoclonal antibodies to K88, K99, F41 and 987P fimbrial adhesins for the detection of porcine enterotoxigenic Escherichia coli in paraffin-wax tissue sections

A panel of monoclonal antibodies against fimbrial adhesins of porcine enterotoxigenic Escherichia coli were evaluated for the detection of enteric colibacillosis in paraffin-wax embedded sections of piglet small intestine. Using the immunoperoxidase technique, monoclonal antibodies were used to detect epitopes on the K99 adhesin and on the a and c regions of the K88 adhesin. However, monoclonal antibodies to the F41 and 987P adhesins failed to react in sections with organisms colonising the intestine of gnotobiotic piglets monoinfected with strains bearing those adhesins, whereas corresponding polyclonal antisera gave positive results. In contrast to apparent expression of all K99 organisms, only a proportion of organisms were identified by monoclonal or polyclonal antibodies as expressing K88. In some instances, failure of immunostaining was attributed to prolonged storage of tissue in formalin.     

Postweaning diarrhea in swine: effects of oxytetracycline on enterotoxigenic Escherichia coli infection

Investigators have found that oxytetracycline decreases the adhesion of K88+ Escherichia coli to intestinal epithelial cells in vitro. This occurs with oxytetracycline-sensitive E coli at drug concentrations less than those required to prevent growth and with E coli that are resistant to the drug. We conducted experiments to determine whether oxytetracycline alters the disease caused by an oxytetracycline-resistant K88+ enterotoxigenic strain of E coli. Oxytetracycline-treated pigs (inoculated with K88+ E coli) did not differ from nontreated pigs in the incidence or severity of diarrhea, nor in the shedding of K88+ E coli. However, during recovery, weight gain by treated pigs was slower than that of nontreated pigs. The control pigs were not inoculated with E coli, and they remained clinically normal. Oxytetracycline-treated controls gained weight faster than nontreated controls. Some controls were genetically resistant to K88+ E coli, others were susceptible. The K88-resistant oxytetracycline-treated controls gained weight faster than the K88-susceptible oxytetracycline-treated and non-treated controls.     

Evaluation of monoclonal antibodies to K88, K99, F41 and 987P fimbrial adhesins for the detection of porcine enterotoxigenic Escherichia coli in paraffin-wax tissue sections

A panel of monoclonal antibodies against fimbrial adhesins of porcine enterotoxigenic Escherichia coli were evaluated for the detection of enteric colibacillosis in paraffin-wax embedded sections of piglet small intestine. Using the immunoperoxidase technique, monoclonal antibodies were used to detect epitopes on the K99 adhesin and on the a and c regions of the K88 adhesin. However, monoclonal antibodies to the F41 and 987P adhesins failed to react in sections with organisms colonising the intestine of gnotobiotic piglets monoinfected with strains bearing those adhesins, whereas corresponding polyclonal antisera gave positive results. In contrast to apparent expression of all K99 organisms, only a proportion of organisms were identified by monoclonal or polyclonal antibodies as expressing K88. In some instances, failure of immunostaining was attributed to prolonged storage of tissue in formalin.     

Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies

Escherichia coli is one of the most important causes of postweaning diarrhea in pigs. This diarrhea is responsible for economic losses due to mortality, morbidity, decreased growth rate, and cost of medication. The E. coli causing postweaning diarrhea mostly carry the F4 (K88) or the F18 adhesin. Recently, an increase in incidence of outbreaks of severe E. coli-associated diarrhea has been observed worldwide. The factors contributing to the increased number of outbreaks of this more severe form of E. coli-associated diarrhea are not yet fully understood. These could include the emergence of more virulent E. coli clones, such as the 0149:LT:STa:STb:EAST1:F4ac, or recent changes in the management of pigs. Development of multiple bacterial resistance to a wide range of commonly used antibiotics and a recent increase in the prevalence and severity of the postweaning syndromes will necessitate the use of alternative measures for their control. New vaccination strategies include the oral immunization of piglets with live avirulent E. coli strains carrying the fimbrial adhesins or oral administration of purified F4 (K88) fimbriae. Other approaches to control this disease include supplementation of the feed with egg yolk antibodies from chickens immunized with F4 or F18 adhesins, breeding of F18- and F4-resistant animals, supplementation with zinc and/ or spray-dried plasma, dietary acidification, phage therapy, or the use of probiotics. To date, not a single strategy has proved to be totally effective and it is probable that the most successful approach on a particular farm will involve a combination of diet modification and other preventive measures.