Colonization factor antigens of enterotoxigenic Escherichia coli : with special reference to monoclonal antibodies and methods for epidemiological studies / Yolanda Lopez-Vidal.

Tese (doutorado)-- University of Göteborg, 1990.

Enterotoxigenic Escherichia coli (ETEC) cause diarrhoea worldwide but are especially common in developing countries. To cause disease, ETEC must be able to colonize the small intestine and produce heat-stable enterotoxin (ST) or heat-labile toxin (LT) or both. The attachment of ETEC is mediated by different fimbrial antigens: colonization factor antigen I (CFA/I), CFA/II, CFA/IV (PCF8775) and probably several others yet to be defined. The aims of this study included: (1) to define the role of PCF8775 and its subcomponents in the ability of ETEC to colonize the intestine and to cause diarrhea as well as to induce protective immunity in rabbits; (2) to produce and characterize monoclonal antibodies (MAbs) against the different CFAs and to develop simple methods for identification of CFA-carrying bacteria based on these MAbs; (3) to assess the incidence of CFAs and their subcomponents, i.e. the coli surface-associated antigens CS1-CS6, as well as the enterotoxins of ETEC isolated from children in a highly endemic area. Studies in a nonligated intestine rabbit model (RITARD) showed that ETEC strains carrying CFA/IV or the CS6 subcomponent alone are capable of colonizing the intestine and of inducing protective immunity against diarrhea caused by CFA/IV-positive bateria. A nitrocellulose replica method has proved to be useful for identification of individual CFA-positive colonies in stool cultures, e.g. from animals infected with CFA-expressing bacteria as well as of CFA-positive or negative mutants. MAbs were raised against CFA/I and different CS-factors of CFA/II and CFA/IV and characterized with regard to isotype and CFA specificity. Selected MAbs were used for development of CFA detection methods: a CFA inhibition-ELISA that allows very sensitive and specific identification of CFAs was developed. By this method, it was shown that the amount of CFA/CS factors expressed on the surface by different ETEC strains varied considerably. A prospective epidemiological study was conducted in children below 5 years in Mexico to determine the relative distribution of ETEC with different CFA/CS factors. ETEC infection occured with a monthly incidence of 3 episodes per 10 children (during the ETEC seasonal peak). The illness-to-infection rate diminished progressively with age, suggesting the development of natural immunity against ETEC diarrhea. Strains carrying CFA/IV, CFA/I and CFA/II were found in 23%, 18% and 5% of the ETEC isolates respectively. The combination of CS1+CS3 was seen on 67% of the C-FA/II-positive strains whereas CS4+CS6 and CS5 CS6 strains were equally prevalent (42% and 43% respectively) among the CFA/IV-positive isolates. Children with ETEC isolated from the stool were more likely to have diarrhea than ETEC-free age-matched controls. The risk of having diarrhea associated with a strain carrying CFA/I, CFA/II or CFA/IV vs CFA-negative ETEC strains was the same. The results of this study documented the importance of PCF8775 as a colonizing factor and as a protective antigen. The generation of specific MAbs enabled the development of improved methods for detection and characterization of CFA/I, CFA/II and CFA/IV on ETEC bacteria. The use of these methods has identified CFA/IV as the predominant colonization factor on ETEC isolated from Mexican children.