Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal–mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.     

人源产肠毒素大肠杆菌疫苗的研发进展

腹泻是全球范围内引起5岁以下幼童死亡的第二大病因,而产肠毒素大肠杆菌(ETEC)是引起腹泻的最常见病原菌,其产生的细菌定植因子(CFs)和肠毒素是关键的毒力因子。CFs介导细菌黏附宿主小肠上皮细胞并完成定植,产生热敏肠毒素(LT)和热稳定肠毒素(ST)破坏宿主上皮细胞内的体液平衡,使体液和电介质过量分泌从而导致腹泻。预防ETEC腹泻的首选方法是使用能激发宿主产生抗黏附素免疫力和抗肠毒素免疫力的疫苗,阻断ETEC黏附和定植并中和肠毒素。目前一种名为Dukoral~的霍乱疫苗因能刺激机体产生抗热敏毒素免疫,已经被一些国家批准用于短期保护和预防旅行者腹泻。新型试验性ETEC候选疫苗正在研发中,旨在提供保护期长、反应谱广的抗ETEC感染免疫保护力。本文针对疫苗研发的关键问题和研究现状作一综述,并对未来的研究作出展望。     

A Chimeric protein of CFA/I,CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti‐CF and anti‐enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti‐CF and antitoxin immunogenicity was then assessed. To achieve high‐level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti‐CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion.

     

苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

[目的]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原菌,本研究通过调查苏北地区规模化猪场ETEC的流行情况,分析其生物学特性,研制具有免疫保护效果的优势血清型菌株的灭活疫苗,以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3-30日龄的仔猪新鲜粪样、肛拭子及小肠组织样,分离出ETEC,对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定;最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份,通过PCR鉴定及测序得到141株ETEC;血清凝集试验鉴定出85株菌的O抗原血清型,其中08、0101和0128为优势血清型,占定型菌株的61.2%(52/85),其他血清型包括09、03、020、0148、0149等;分析141株ETEC对14种常见抗生素的耐药情况,得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药,耐药率均高达80%以上;对恩诺沙星敏感性较高,敏感率达50.4%(71/141);对多粘菌素B和头孢噻肟中介耐药,占比分别为66%(93/141)和51.8%(73/141);多重耐药现象严重,其中10重耐药的菌株占比最大,为19%(27/141);小鼠攻毒试验测得08血清型强毒株YC-6的半数致死量(median lethal dose,LD50)为1.4×10^7 CFU/只,最低致死量(minimum lethal dose,MLD)为3×10^7 CFU/只;08血清型强毒株YC-6和0101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%,因此利用08血清型强毒株YC-6和0101血清型强毒株LYG-3研制二价灭活疫苗,结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查,得出其优势血清型,并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗,给临床ETEC的监测和防控提供参考。     

Shift in Phenotypic Characteristics of Enterotoxigenic Escherichia coli (ETEC) Isolated from Diarrheal Patients in Bangladesh

Background

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of bacterial diarrhea. Over the last decade, from 1996 to 2012, changes in the virulence antigen properties of ETEC such as heat labile (LT) and heat stable (ST) toxins, colonization factors (CFs), and ‘O’-serogroups have been observed. The aim of this prospective study was to compare changes in antigenic profiles of ETEC strains isolated from a 2% surveillance system at the icddr,b hospital in Dhaka, Bangladesh between 2007–2012 and an earlier time period of 1996–1998 conducted at the same surveillance site.

Methodology

In the surveillance system every 50^th patient attending the hospital was screened for major enteric pathogens including ETEC, Vibrio cholerae, Shigella spp. and Salmonella spp. from January 2007 to December 2012.

Principal Findings

Of the 15,152 diarrheal specimens tested between 2007–2012, the overall rate of ETEC isolation was 11%; of these, 43% were LT/ST, 27% LT and 30% ST positive. Isolation rate of ST-ETEC (p<0.009) and LT/ST ETEC (p<0.011) during 2007–2012 period differed significantly compared to those seen between 1996–1998. In comparison to the 1996–1998 period, difference in CF profile of ETEC isolates during 2007–2012 was observed particularly for strains expressing CS7 (12.4%), CS14 (9.5%) and CS17 (10.0%). The predominant CF types were CS5+CS6, CFA/I, CS7, CS17, CS1+CS3, CS6 and CS14. The most common serogroups among the CF positive ETEC isolates were O115, O114, O6, O25 and O8. A strong association was found between CFs and ‘O’ serogroups i.e. between CS5+CS6 and (O115 and O126); CS7 and (O114), CFA/I and (O78 and O126), CS17 and (O8 and O167) and CS1/CS2+CS3 and (O6).

Conclusion

The analyses show a shift in prevalence of antigenic types of ETEC over the study period; the information is important in designing effective ETEC vaccines with broad protective coverage.     

Temperature-regulated expression of the tac/lacI system for overproduction of a fungal xylanase in Escherichia coli

 Temperature-regulated expression of recombinant proteins in the tac promoter (P_tac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the P_tac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42°C were about 4.5 times higher than those of the cells grown at 23°C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl β-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated P_tac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacI ^q, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the λP_L system, the P_tac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best λP_L-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated P_tac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated P_tac system can be used for overproduction of some non-toxic recombinant proteins. Received: 27 June 1995/Received revision: 13 September 1995/Accepted: 30 September 1995     

Oral immunization with a lettuce-derived <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile toxin B subunit induces neutralizing antibodies in mice

Transgenic plants serve as attractive systems for the production and delivery of subunit vaccines, thus expression of an enterotoxigenic Escherichia coli (ETEC) antigen in an edible plant may lead to the development of a viable oral vaccine against cholera and ETEC diarrhea. In this study, expression of the heat labile toxin B subunit (LTB) from ETEC was performed in lettuce, and its immunological characterization was investigated. A total of 27 independent transgenic lines were established following Agrobacterium-mediated transformation. Selected lettuce lines were subjected to GM1-ELISA to confirm the proper quaternary structure of the LTB protein. Levels of accumulation of the pentameric LTB reached up to 0.05% of the total soluble protein (TSP) in T1 and T2 progenies of these lines. Oral immunization of Balb/c mice was conducted using three weekly doses of lettuce-derived LTB. This elicited specific and significant antibody responses in both serum and intestinal tissues. Moreover, mice immunized with lettuce-derived LTB showed diminished intestinal fluid accumulation following challenge with the cholera toxin. This study demonstrated that this plant-based vaccine may contribute to immunization practices against diarrheal diseases.     

Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains

Background

Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.

Methods

The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.

Results

The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.

Conclusions

This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics of E. coli infections.     

A multi-epitope fusion antigen candidate vaccine for Enterotoxigenic Escherichia coli is protective against strain B7A colonization in a rabbit model

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children’s and travelers’ diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.     

Identification of Novel Components Influencing Colonization Factor Antigen I Expression in Enterotoxigenic Escherichia coli

Colonization factors (CFs) mediate early adhesion of Enterotoxigenic Escherichia coli (ETEC) in the small intestine. Environmental signals including bile, glucose, and contact with epithelial cells have previously been shown to modulate CF expression in a strain dependent manner. To identify novel components modulating CF surface expression, 20 components relevant to the intestinal environment were selected for evaluation. These included mucin, bicarbonate, norepinephrine, lincomycin, carbon sources, and cations. Effects of individual components on surface expression of the archetype CF, CFA/I, were screened using a fractional factorial Hadamard matrix incorporating 24 growth conditions. As most CFs agglutinate erythrocytes, surface expression was evaluated by mannose resistant hemagglutination. Seven components, including porcine gastric mucin, lincomycin, glutamine, and glucose were found to induce CFA/I surface expression in vitro in a minimal media while five others were inhibitory, including leucine and 1,10-phenanthroline. To further explore the effect of components positively influencing CFA/I surface expression, a response surface methodology (RSM) was designed incorporating 36 growth conditions. The optimum concentration for each component was identified, thereby generating a novel culture media, SP1, for CFA/I expression. CFs closely related to CFA/I, including CS4 and CS14 were similarly induced in SP1 media. Other epidemiologically relevant CFs were also induced when compared to the level obtained in minimal media. These results indicate that although CF surface expression is complex and highly variable among strains, the CF response can be predicted for closely related strains. A novel culture media inducing CFs in the CF5a group was successfully identified. In addition, mucin was found to positively influence CF expression in strains expressing either CFA/I or CS1 and CS3, and may function as a common environmental cue.     

Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) causes an acute cholera-like diarrhoea in both humans and animals. We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface. Longus is composed of a repeating subunit of 22 kDa and its NH₂-terminal amino acid sequence revealed homology with the toxin-coregulated pilus of Vibrio cholerae, the bundle-forming pilus of enteropathogenic E. coli and type IV pilins of some Gram-negative bacterial pathogens. The longus structural gene (IgA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E. coli K-12. The presence of IngA was restricted to human ETEC strains. In contrast to other ETEC pili, IngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed. Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC. Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC. Longus could be significant in the immuno-prophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat-stable toxin only.     

Enterotoxigenic Escherichia coli in children with acute diarrhoea and controls in Teresina/PI, Brazil: distribution of enterotoxin and colonization factor genes

Aims: To investigate the distribution of the genes that encode enterotoxins and the colonization factors (CF) types as well as the antibiotic susceptibility profile of enterotoxigenic Escherichia coli (ETEC) isolated from children from the Brazilian Northeast. Methods and Results: We conducted a 3·5‐year prospective study that involved 250 children with and 150 without diarrhoea, aged 1–60 months, from low‐income families in Teresina/Brazilian Northeast. All samples were assayed for E. coli, enterotoxin and CF genes and antimicrobial susceptibility by microbiological methods and PCR. ETEC strains were isolated from 9·2% children with and 4·0% without diarrhoea. Infection was more common in children aged 6–24 months in rainy months. elt⁺/CFA/IV⁺ and elt⁺/CS14⁺ were the most frequent genotypes. Susceptibility to nalidixic acid, ciprofloxacin and gentamicin and resistance to ampicillin, cephalothin and sulfamethoxazole–trimethoprim were common. Conclusions: elt ⁺ isolates and ETEC strains harbouring genes encoding CFA/IV and CS/14 were the most common ETEC found in Brazilian Northeast. Significance and Impact of the Study: Our data, the first generated for north‐eastern Brazilian children, may be important for the development of an effective vaccine and for facilitation of an empirical choice of antibiotic treatment or prophylaxis for traveller’s diarrhoea in the area studied.     

Increased number of intestinal villous M cells in levamisole -pretreated weaned pigs experimentally infected with F4ac+ enterotoxigenic Escherichia coli strain

Immunoprophylaxis of porcine postweaning colibacillosis (PWC) caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae is an unsolved problem. Just as ETEC strains can exploit intestinal microfold (M) cells as the entry portal for infection, their high transcytotic ability make them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. We have developed a model of parenteral/oral immunization of 4-weeks-old pigs with either levamisole or vaccine candidate F4ac⁺ non-ETEC strain to study their effects on de novo differentiation of antigen-sampling M cells. Identification, localization and morphometric quantification of cytokeratin 18 positive M cells in the ileal mucosa of 6-weeks-old pigs revealed that they were: 1) exclusively located within villous epithelial layer, 2) significantly numerous (P< 0.01) in levamisole pretreated/challenged pigs, and 3) only slightly, but not significantly numerous in vaccinated/challenged pigs compared with non-pretreated/challenged control pigs. The fact that levamisole may affect the M cells frequency by increasing their numbers, makes it an interesting adjuvant to study development of an effective M cell-targeted vaccine against porcine PWC.Key words: M cells, levamisole, E. coli vaccine, weaned pigs.     

An adhesive factor found in strains ofEscherichia coli belonging to the traditional infantile enteropathogenic serotypes

Escherichia coli strains isolated from outbreaks of diarrheal disease were tested for the presence of adhesive factors. Fifty-one of these strains belonged to traditional infantile entero-pathogenic serotypes (EPEC) and 17 belonged to other serotypes. None of these strains were enterotoxigenic and none possessed colonization factors CFA/I or CFA/II, which have been described among strains of enterotoxigenicE. coli (ETEC). EnterotoxigenicE. coli strains from patients with diarrhea and strains which were neither EPEC nor ETEC from subjects without diarrhea were also examined. By means of a tissue culture technique using HEp-2 cells, a new adhesive factor was found to occur with greater frequency in EPEC strains. The adhesive factor was found less frequently in the other groups ofE. coli studied. It was distinct from type 1 pili and was not inhibited by the presence ofD-mannose.     

Protection of mice against enterotoxigenic E. coli by immunization with a polyvalent enterotoxin comprising a combination of LTB, STa, and STb

Currently available enterotoxigenic Escherichia coli (ETEC) vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). However, the induction of antitoxic responses against heat-stable enterotoxin a (STa) and b (STb) has merit as these two poorly immunogenic toxins are frequently associated with ETEC strains. In this study, we genetically constructed a trivalent enterotoxin fusion protein (STa–LTB–STb, abbreviated to SLS) in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. Mutagenesis at one disulfide-bridge-forming cysteine in STa led to a dramatic reduction in the STa toxicity of SLS; however, the fusion peptide retained the STb-associated toxicity. Immunization of mice with SLS protein elicited significant antibody responses to LTB, STa, and STb. Significantly, the mice antisera were able to neutralize the biological activity of both STa and STb. In the experiment to assess the protective effect of SLS immunization, the mortality of mice receiving SLS was significantly lower than their control cohorts (P < 0.01) after intraperitoneal challenge with ETEC. These results show that the trivalent fusion enterotoxin SLS has the potential to serve as a useful toxin-based vaccine against ETEC-induced diarrheal disease via a single immunogen.     

Molecular Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Diarrheal Patients in Korea during 2003–2011

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of infectious diarrhea in developing countries. In order to characterize the molecular features of human ETEC isolates from Korea, we investigated the profiles of enterotoxin and colonization factor (CF) genes by polymerase chain reaction (PCR) and performed multilocus sequence typing (MLST) with a total of 291 ETEC strains. The specimens comprised 258 domestic strains isolated from patients who had diarrhea and were from widely separated geographic regions in Korea and 33 inflow strains isolated from travelers visiting other Asian countries. Heat-stable toxin (STh)-possessing ETEC strains were more frequent than heat-labile toxin (LT)-possessing ETEC strains in the domestic isolates, while the detection rates of both enterotoxin genes were similar in the inflow isolates. The profile of CF genes of domestic isolates was similar to that of inflow isolates and the major CF types of the strains were CS3-CS21-CS1/PCF071 and CS2-CS3-CS21. Most of these 2 CF types were detected in ETEC strains that possess both lt and sth genes. The major MLSTST types of domestic isolates were ST171 and ST955. Moreover, the 2 major CF types were usually found concomitantly with the 2 major MLST STs, ST171 and ST955. In conclusion, our genotyping results may provide useful information for guiding the development of geographically specific vaccines against human ETEC isolates.     

A tale of two bacterial enteropathogens and one multivalent vaccine

Shigella and enterotoxigenic Escherichia coli (ETEC) are among the top four enteric pathogens that cause diarrheal illness in young children in developing countries and are major etiologic agents of travellers' diarrhoea. A single vaccine that could target both of these pathogens would have significant public health impact. In this review, we highlight the many pivotal contributions of Phillippe Sansonetti to the identification of molecular mechanisms of pathogenesis of Shigella that paved the way for the development of rationally designed, novel vaccines candidates. The CVD developed a series of live attenuated Shigella vaccine strains based on the most prevalent serotypes associated with disease. Shigella vaccine strains were engineered to express critical ETEC antigens to form a broadly protective Shigella‐ETEC multivalent vaccine.     

From scratch to value: engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> wild type cells to the production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and other fine chemicals derived from chorismate

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (l-phenylalanine, l-tryptophan, l-tyrosine) have been constructed. The largest demand is for l-phenylalanine (l-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides l-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for l-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, l-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed l-phenylalanine titers of up to 38 g/l of l-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.     

Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp)

Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.     

Histomorphometric evaluation of intestinal cellular immune responses in pigs immunized with live oral F4ac+ non-enterotoxigenic E. coli vaccine against postweaning colibacillosis

Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of porcine postweaning colibacillosis (PWC). Among fimbriae of porcine ETEC strains the best studied family of fimbriae are the members of F4 adhesins, existing in at least three variants: ab, ac, ad. Active immunization against porcine PWC is difficult due to: i) ETEC strains are only one of the essential predisposing factors, ii) the success of vaccinal antigen uptake depends on the presence of enterocyte receptors for F4 adhesins, iii) the intestinal immune system may react with tolerance or hypersensitivity to the same antigens depending on the dose and form of the vaccinal immunogen, and iv) kinetics of the specific immune responses may be different in the case of F4 (earlier) and the other ETEC adhesins, particularly F18 (later). The aim of this study was to test the effectiveness of a live attenuated F4ac⁺ non-ETEC vaccine against porcine PWC by analyzing quantitative differences in the small intestinal lymphoid and myeloid cell subsets of immunized (with or without levamisole given as an adjuvant) vs control non-immunized pigs. Four week-old pigs were intragastrically immunized with a vaccine candidate F4ac ⁺ non-ETEC strain 2407 at day 0, challenged 7 days later with a virulent F4ac⁺ strain ETEC 11-800/1/94, euthanatized at day 13 and sampled for immunohistology. Non-immunized pigs received saline at day 0 and were processed as the principals. Immunophenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidinbiotin complex method and corresponding morphometric data were analyzed using software program Lucia G for digital image analyses. Monoclonal antibodies reactive with surface molecules on porcine immune cells such as CD3, CD45RA, CD45RC, CD21 and SWC3 enabled clear insight into distribution patterns and amount of these cells within the gut-associated lymphoid tissues (GALT) examined. The numbers of jejunal and ileal cell subsets tested were significantly increased (at P<0.5 or lower) in both principal groups (vaccinated or levamisole primed-vaccinated) of pigs, compared to those recorded in the control non-vaccinated pigs. Based on the histomorphometric quantification of porcine intestinal immune cells from the GALT compartments tested, it is possible to differentiate the responses of pigs immunized by an experimental mucosal vaccine from those of non-immunized pigs.Key words: adjuvanted/nonadjuvanted E. coli vaccine, intestinal immune cells, pigs.