Influence of bovine intestinal fluid on the expression of K99 pili by Escherichia coli

A study was conducted to determine whether intestinal fluid collected from various portions of bovine intestine differed in its effect on production of K99 pili by Escherichia coli. The small and large intestines of 7 calves, euthanatized 4 hours after a final feeding of milk, were divided into 6 to 9 segments from which intraluminal fluids were collected. Depending on the amount of fluid collected, up to 20 E coli strains that express K99 pili were grown on media prepared from the content of each specimen and then were tested for K99 pilus expression. In general, intestinal fluid from the most proximal small intestinal segments were more suppressive to K99 pilus expression than was fluid from more distal segments of small intestine. Only about 20% of the E coli test strains expressed K99 pili when grown on medium prepared from proximal small intestinal segmental fluid, whereas greater than 90% did when grown on medium prepared from distal small intestinal segmental fluid. Fluid from the large intestine varied considerably from calf to calf in its effect on K99 pilus expression. A correlation was found between K99 pilus expression and pH of the intestinal fluid, with the lower pH values (characteristic of proximal intestinal segmental fluid) being suppressive. The correlation between K99 pilus production and the pH of the medium was verified, using defined laboratory media adjusted to various pH values. Strains of E coli grown in medium at or below pH 5.5 failed to express K99 pili, whereas the same strains when grown in medium at or above pH 6.5 expressed K99 pili in abundance.     

Evaluation of a monoclonal antibody to the K99 fimbrial adhesin produced by Escherichia coli enterotoxigenic for calves, lambs and piglets

All the K99+ Escherichia coli grown at 37 degrees C stained strongly with a peroxidase labelled K99 monoclonal antibody using a direct immunoperoxidase staining procedure. There was no reaction when these bacteria were cultured at 18 degrees C or when K99- E coli were grown at either temperature. The binding of the monoclonal antibody to K99 antigen was inhibited by OK antisera to heterologous K99+ E coli but OK antisera to E coli producing adhesins other than K99 were without effect. Using the slide agglutination test the reactions of the monoclonal antibody were identical to those of a polyclonal antiserum to K99 when both were used in parallel to examine 100 K99+ E coli from at least 10 somatic O groups and 1308 K99+ E coli from at least 82 different somatic O groups submitted for routine serological typing in England or the, USA. The monoclonal antibody reacted with K99+ E coli in cryostat sections of the ileum from a piglet infected with E coli strain B44 (O9: K30, K99, F41) but there was no reaction with similar material from piglets infected by E coli strains 1751 (O101: F41), X177/81 (O9: K103, 987P) or Abbotstown (O149: K91, K88ac).     

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.     

The nucleotide sequence of the genes, fanE and fanF of Escherichia coli K99 fimbriae.

K99 fimbriae of enterotoxigenic Escherichia coli consist of eight different subunits. A major subunit called fimbrillin forms fimbrial structure and a minor subunit called adhesin localizes at the tip of fimbriae and recognizes host receptor ganglioside. Within this eight gene cluster, fanE and fanF have not yet been sequenced. In this study, fanE and fanF genes were sequenced by analyzing several DNA fragments produced by endonuclease or exonuclease digestion. The fanE gene encoded 227 amino acids containing 20 amino acids of signal peptide starting from GTG (valine) and showed a homology to fanA-fanB. The fanF gene encoded 271 amino acids containing 20 amino acids of signal peptide starting from ATG (methionine) and showed homologies to the fanD gene, fimbrillin gene of F41, adhesin gene of P fimbriae (papG) and adhesin gene of Type 1 fimbriae (fimH). E and F subunits had fifteen and fourteen hydrophobic domains, respectively, which periodically appeared possibly forming a hydrophobic region.     

The role of K antigens of enteropathogenic Escherichia coli in colonization of the small intestine of calves.

The colonizing and proliferating abilities of enterotoxigenic acapsular or K99- mutants of bovine enteropathogenic Escherichia coli strains were compared with those of their capsulated and K99+ parent strains in the small intestine of infected colostrum-fed calves. Calves infected with the enteropathogenic E. coli parent strains developed profuse diarrhea and severe dehydration. None of the calves which received the acapsular mutant developed diarrhea and one of three calves inoculated with the K99- enterotoxigenic mutant developed moderate diarrhea. The parent enteropathogenic E. coli strains colonized the middle and lower small intestine; in these areas a layer of specific immunofluorescence against the enteropathogenic E. coli covered most villi and 80% of the organisms were associated with the intestinal wall. The acapsular mutant strain failed to colonize the small intestine and fluorescent bacteria were not observed in any area of the small intestine. The K99- mutant proliferated to a lesser extent than did the K99+ parent strain in all areas of the small intestine but moderately colonized the lower small intestine where fluorescent bacteria were observed to cover parts of the intestinal villi.     

鸡大肠杆菌活的非可培养状态发生相关基因克隆与分析

本研究旨在从分子水平探究细菌活的非可培养状态(VBNC)的发生机制。应用冰乙酸和4℃联合诱导条件,使鸡大肠杆菌进入VBNC状态,并利用mRNA差异显示技术(DDRT-PCR)获得VBNC相关基因。结果表明,从VBNC大肠杆菌中筛选得到的3个差异片段与大肠杆菌23S核糖体RNA基因序列具有较高的核苷酸同源性,分别为98%、98%和99%,而氨基酸的同源性也均在97%以上,表明这3个序列是大肠杆菌23S核糖体rRNA基因的部分序列,同时也是与大肠杆菌VBNC状态发生密切相关的基因。由此推知,当正常大肠杆菌在未暴露任何压力下时,其转录水平较低,特别是23S rRNA的某一(些)基因不显示或受到强烈抑制。当进入VBNC状态后,面临生存压力时,这一(些)基因转录水平明显强于正常状态,而核糖体作为蛋白质合成的主要结构与场所,其某些基因也将积极参与新蛋白质的生物合成。     

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.     

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.     

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。     

The studies on the aetiology of diarrhoea in neonatal calves and determination of virulence gene markers of Escherichia coli strains by multiplex PCR

The purpose of this study was to determine aetiological agents of diarrhoea in neonatal calves and to investigate virulence gene markers of Escherichia coli strains isolated from calves by multiplex polymerase chain reaction (PCR). Eighty-two diarrhoeic calves and 18 healthy calves were used as subjects. Faeces were taken from the rectums of all the calves and were subjected to bacterial culture. Antigen enzyme-linked immunosorbent assay (ELISA) was performed to detect rotavirus, coronavirus and E. coli K99 in faeces of all the calves. A multiplex PCR was used to characterize E. coli strains in all the calves. Escherichia coli was isolated from 37 faeces samples, Enterococcus ssp. was isolated from 22 faeces samples and Salmonella was isolated from one faeces sample in diarrhoeic calves. Furthermore, only E. coli was isolated from all 18 faeces samples of healthy calves. Of the 37 E. coli isolated from diarrhoeic calves, K99 (18.9%), F41 (18.9%), heat-stable enterotoxin a (STa) (18.9%), Shiga toxin 1 (Stx1; 13.5%) and Shiga toxin 2 (Stx2; 5.4%) and intimin (8.1%) genes were identified by multiplex PCR. Of the 18 E. coli isolated from healthy calves, K99 (16.6%) and intimin (55.5%) genes were identified by PCR. A total of 15 rotavirus, 11 coronavirus and 11 E. coli K99 were detected in diarrhoeic calves by the antigen ELISA. As a result, this study shows that rotavirus, coronavirus, E. coli and Enterococcus ssp. were determined to play a role in the aetiology of diarrhoea in the neonatal calves. K99, F41, STa, Stx1 and Stx2 were found as the most common virulence gene markers of E. coli strains isolated from calves with diarrhoea. Multiplex PCR may be useful for characterization of E. coli isolated from calves.     

Prevalence of four enterotoxin (STaP, STaH, STb, and LT) and four adhesin subunit (K99, K88, 987P, and F41) genes among Escherichia coli isolates from cattle

Colony hybridizations with DNA probes for 3 heat-stable (STaP, STaH, and STb) enterotoxins and 1 heat-labile (LT) enterotoxin and for 4 adhesins (K99, F41, K88, 987P) were performed on 870 Escherichia coli isolates to determine pathotypes prevalent among enterotoxigenic E coli (ETEC) isolated from cattle in Belgium. One hundred thirty-two E coli isolates (15.2%) hybridized with probes STaP, K99, and/or F41. The 5 other probes were not hybridized by E coli isolates. Therefore, only STaP enterotoxin and K99 and F41 adhesins were virulence factors of ETEC isolated from cattle. Two major pathotypes accounted for 95% of the ETEC: STaP+K99+F41+ (67.4%) and STaP+K99+ (27.3%). The last 5% of probe-positive isolates had STaP+, STaP+F41+, or K99+F41+ minor pathotypes. Of 12 American ETEC isolates also assayed, 7 were positive with STb and/or 987P probes (pathotypes STaP+STb+, STaP+ 987P+, or STaP+STb+987P+) and may be porcine- rather than bovine-specific enteropathogens. The remaining 5 American ETEC isolates belonged to 3 minor pathotypes (STaP+, STaP+F41+, and K99+F41+) also found among Belgian E coli isolates. Such isolates may be derivatives of STaP+K99+F41+ or STaP+K99+ ETEC after in vivo or in vitro loss of virulence genes and/or non-ETEC isolates, which have acquired virulence genes by in vivo transfer.     

产肠毒素大肠杆菌双重PCR检测方法的建立

为了快速检测和鉴定产肠毒素大肠杆菌菌毛(K88和K99)基因,本研究设计合成了针对K88、K99的2对特异性引物,对扩增条件进行优化,建立了检测K88和K99的双重PCR方法。该方法对K88、K99基因的扩增产物大小分别为237和314 bp;最终确定dNTP终浓度0.4 mmol/L,K88、K99的引物终浓度均为25 μmol/L,退火温度为52℃。试验结果表明,该方法具有良好的灵敏性和特异性。用所建立的双重PCR方法对实验室分离的23株大肠杆菌进行检测,结果显示,K88单重PCR阳性2株,K99单重PCR阳性3株,K88和K99双重PCR阳性5株。本研究建立的双重PCR检测方法为致幼畜腹泻产肠毒素大肠杆菌的快速准确检测提供了方法。     

Fimbriae in Escherichia coli isolated from the small intestine of piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively.Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated.Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.     

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.     

Protection of the nursing pig against experimentally induced enteric colibacillosis by vaccination of dam with fimbrial antigens of E coli (K88, K99 and 987P)

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.     

Prevalence of enterotoxigenic Escherichia coli in calves in Scotland and northern England

Eighty-eight of 1529 (5.7 per cent) Escherichia coli isolates from diarrhoeic and clinically normal calves in Scotland and northern England were found to possess the K99 pilus antigen (K99+). There was complete correlation between possession of K99 antigen, heat stable enterotoxin production and ability to dilate intestinal loops. The diagnosis of calf enterotoxigenic E coli infections may therefore be based on the detection of K99 antigen alone. Enterotoxigenic E coli was isolated from 23 of 306 (7.5 per cent) diarrhoeic calves from eight of 70 (11.4 per cent) farms and was not isolated from clinically normal calves. Infected calves were between one and three days old. A survey by an enzyme-linked immunosorbent assay found 3.0 per cent and 3.9 per cent of sera from calves and cows respectively to contain antibodies to K99 antigen. The prevalence of other enteropathogenic organisms in calf faeces is also discussed.     

Development of vaccines for bacterial diseases using recombinant DNA technology

A vaccine was prepared using recombinant DNA techniques to prevent fatal enterotoxigenic Escherichia coli diarrhea in swine. The product, which is a subunit vaccine, was prepared by mechanical and chemical removal of pilus adhesins from the surface of genetically engineered strains of E. coli. The vaccine contains the pilus adhesins K88, K99, and 987P plus an adjuvant. The genes responsible for production of K88 and K99 were separately cloned into the multicopy vector pBR322. K88 was found to be encoded on a 7.6-kilobase HindIII-EcoRI fragment, and K99 was found to be encoded on a 7.15-kilobase BamHI fragment. Strains containing the recombinant plasmid for K99 produced up to ten times more K99 than strains containing the wild-type plasmid. Vaccination of pregnant pigs with the vaccine led to production of pilus-adhesin-specific antibodies that were transferred to the piglets in colostrum and milk. Pilus-adhesin-specific antibodies neutralized the adhesiveness of the pili on enterotoxigenic E. coli, thus preventing attachment, colonization, and disease. Mortality of pigs in litters from vaccinated pigs due to experimentally induced enterotoxigenic E. coli diarrhea was reduced 10-to-20-fold (depending upon the challenge strain), and the incidence, severity, and duration of diarrhea were also reduced.     

Enzyme-linked immunosorbent assay, using monoclonal antibody, to detect enterotoxic Escherichia coli K99 antigen in feces of dairy calves

A modified, double-antibody, enzyme-linked immunosorbent assay (ELISA) was developed to detect the K99 pilus antigen of enterotoxic Escherichia coli (ETEC) in feces of calves. Extremely high positive to negative ratios (greater than 200) were obtained by using monoclonal antisera as the primary antibody. Strong positive reactions were obtained with strains of E coli known to produce the K99 antigen; however, non-enteropathogenic E coli (strains not producing the K99 antigen), Salmonella, Proteus, Klebsiella, Pseudomonas, Staphylococcus, Streptococcus, and rotavirus produced negative results. Seventy-five fecal samples, 8 from healthy calves and 67 from calves with neonatal calf diarrhea were examined with the K99 ELISA for the presence of ETEC. Rotavirus test and fecal culture results were available on feces from calves with diarrhea and were used with the K99 ELISA results to determine the specific cause of the disease. Enterotoxic E coli was the predominant agent detected in the feces of 29 diarrheal calves less than 5 days of age. Mixed infections of rotavirus and ETEC were also common in these calves, but rotavirus infections alone were not detected. In 38 calves greater than or equal to 5 days, rotavirus was detected without ETEC. Of these calves, only 2 produced positive tests with the K99 ELISA. Salmonella sp and Proteus sp were detected from 5 of 67 calves with diarrhea.     

Isolation by phage display of recombinant antibodies able to block adherence of Escherichia coli mediated by the K99 colonisation factor

K99 fimbriae are important for intestinal colonisation by bovine strains of enterotoxigenic Escherichia coli. The mode of action of this colonisation factor is well understood and specific immune responses are protective. K99 was therefore chosen for this study as a model to test if antibodies with anti-adhesion activity could be isolated from recombinant libraries using phage display techniques. Potentially, this strategy could be used to understand better the action of bacterial colonisation factors and aid the design of therapies (e.g. vaccines, purified protein products or bacteria bearing colonisation-blocking antibodies) to inhibit bacterial adherence. The major fimbrial subunit from K99, FanC, was purified from a clinical E. coli isolate. The protein was coated to plastic immunotubes and used as a target for selection of antibodies from the Tomlinson I and J libraries of single chain (scFv) antibodies. Clones able to recognise K99 were isolated by iterative rounds of binding, elution and amplification. scFv antibodies chosen from the resulting panel were purified and their specificity confirmed by ELISA. Pre-incubation of several scFvs with bacteria expressing K99 fimbriae inhibited the agglutination of erythrocytes. Further investigation by microscopy confirmed that when E. coli expressing K99 were exposed to scFv antibodies, the binding of bacteria to erythrocytes was blocked with high efficiency. The study showed that recombinant antibodies were able to block the action of a bacterial colonisation factor and hence that phage display techniques might be applied to the identification of less well-characterised virulence factors and the analysis of their structure and function.     

Protection of lambs against enteric colibacillosis by vaccination of ewes

Pregnant ewes were vaccinated twice, seven weeks and three weeks before lambing, with a multivalent formalin-killed Escherichia coli vaccine containing an added K99, F41 antigen preparation. Lambs born to vaccinated and unvaccinated ewes were exposed to oral infection with E coli B44 (09:K30, K99, F41). All 10 lambs from vaccinated ewes were protected whereas all 10 control lambs developed severe diarrhoea and five died or were killed in extremis. In the following year, previously immunised ewes were given a single dose of the vaccine two weeks before lambing. Eleven of their 12 lambs were protected against a similar challenge, which caused the death of six of eight control lambs and severe diarrhoea in the two survivors. Higher levels of antibody to the K99, F41 preparation were detected by enzyme-linked immunosorbent assay in the serum and colostrum from vaccinated ewes and in the serum of their lambs when compared with similar samples from control ewes and lambs.