In vivo emergence of enterotoxigenic Escherichia coli variants lacking genes for K99 fimbriae and heat-stable enterotoxin

Abstract

Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin. Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K9 and STaP. Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes. Variants were recovered from 10 of 13 suckling pigs that survived the infection. Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants. Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants. Variant colonies were detected more frequently among pigs in some litters than in others. The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K9 antibody in her colostrum. Variants also occurred in colostrum-deprived, artificially reared pigs. However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs. More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody. Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99-STaP+) and some lacked both genes (K99-STaP-), but none was of the K99+ STaP-type. Our results confirmed reports of gene loss from enterotoxigenic E. coli during infection. They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations. However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease. Fimbriae (pili) and enterotoxins are virulence factors of enterotoxigenic Escherichia coli (ETEC) (1,15). Fimbriae mediate adherence of E. coli to host intestinal epithelium, and enterotoxins stimulate intestinal secretion of water and electrolytes, resulting in diarrhea. Some ETEC strains of the K88 (F4) and K99 (F5) fimbrial antigen types lose these antigens (and, apparently, the plasmids carrying the genes encoding them) during experimental infections in mice (5). Loss of K88 antigen also occurs during infections in pigs, which are natural hosts for K88+ ETEC (17,18). A human ETEC strain (6) and an enteropathogenic E. coli strain (7) have been shown to lose genes encoding heat-labile enterotoxin and an adherence factor, respectively, during infection in humans.Loss of K88 in pigs is thought to be caused by antibody, because it was detected in immunized pigs but not in control pigs (18) and because it can be induced in vitro by growth in immune colostrum (8,17,18). It has been suggested that the antibodies involved in loss of K88 are not K88 specific but rather are directed against a novel antigen(s) and act by a novel mechanism which also impedes the spread ...

The role of pathogenic Escherichia coli in the etiology of veal calf hemorrhagic enteritis

“…Adhesion and colonization by K99-positive ETEC can be blocked by K99 antibody produced by the K99 pilus vaccines which are extensively used in cattle. However, K99 antibodies could lead to the emergence of K99-negative ETEC in vivo (Mainil et al, 1987 ). The enterotoxigenic aggregative adhering E. coli may be opportunistic organisms in some circumstances.…”

Genetic characterization and virulence of enterotoxigenic Escherichia coli mutants which have lost virulence genes in vivo

“…Nagy et al (16) reported that antibody selected for K99mutants in vitro but that these mutants revert to K99+ phenotype when subcultured, suggesting that point mutations had occurred which prevented K99 expression. The K99 DNA probes used here and by Mainil et al (11) would not have differentiated between the wild type and variants with point mutations affecting K99 expression, which might also occur in vivo.…”

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Loss of K99 and STaP genes from enterotoxigenic Escherichia coli 431 during infection occurred by either plasmid curing or plasmid deletion. These mutants expressed the F41 adhesin and colonized neonatal pigs, but only those mutants that retained STaP caused diarrhea with significant weight loss.In vitro and in vivo loss of enterotoxigenic Escherichia coli virulence genes has been reported to occur by plasmid curing (5,6,8,9,11,(16)(17)(18). Pigs infected with enterotoxigenic E. coli 431 (O101:K30:NM, K99+ F41+ STaP+) shed mutants which had lost both K99 and STaP or only K99 (11).

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“…In vitro and in vivo loss of enterotoxigenic Escherichia coli virulence genes has been reported to occur by plasmid curing (5,6,8,9,11,(16)(17)(18). Pigs infected with enterotoxigenic E. coli 431 (O101:K30:NM, K99+ F41+ STaP+) shed mutants which had lost both K99 and STaP or only K99 (11). Mutants which had lost STaP and retained K99 were not found.…”

Preface

“…Because E. coli is a highly flexible organism (acquiring new virulence characters or masking the ones that may be disadvantageous for survival) (Mainil et al, 1987), and because there are several kinds of infections (due to viruses and protozoa as described above) and conditions that may predispose the host to colonization by ETEC, thereby enhancing the chances for E. coli to utilize its pathogenic potential, the protection of pigs and calves from pathogenic E. coli is a constant challenge for farmers and veterinarians alike. As described in the previous sections, the knowledge on adhesins and receptors for colonization by different pathotypes of E. coli has been utilized quite extensively for diagnostic purposes (antifimbrial diagnostic sera and reagents) and for the prevention of diarrhoeal diseases (mainly in the form of killed maternal vaccines containing fimbrial antigens).…”