Efficacy of enteric-coated protease in preventing attachment of enterotoxigenic Escherichia coli and diarrheal disease in the RITARD model

Abstract

In this study, we report on a novel approach based on modification of the intestinal surface to prevent diarrhea caused by enterotoxigenic Escherichia coli (ETEC). The removable intestinal tie adult rabbit diarrhea (RITARD) model was used to test the efficacy of an enteric-coated protease preparation (Detach; Enzacor Technology Pty. Ltd.) in the prevention of bacterial attachment and diarrheal disease caused by colonization factor antigen I-positive (CFA/I+) E. coli H10407. Protease was administered orally to rabbits 18 h prior to challenge with 10" bacteria. Four groups of rabbits were inoculated with different ETEC strains which produced different combinations of adhesin and enterotoxin or with sterile phosphate-buffered saline. Occurrence of diarrhea during the subsequent 24-h incubation period was recorded. Oral administration of protease was successful in reducing diarrhea and diarrhea-induced death in six of seven (86%) rabbits infected with CFA/I+, heat-stable and heat-labile toxin-positive E. coli (H10407). Seven of eight (87%) rabbits not protected by protease treatment died or developed severe diarrhea. Quantitative analysis of bacterial cultures obtained from the small intestine of rabbits showed a significant (P < 0.001) 2,000-fold reduction in CFU per centimeter of intestine following treatment with protease. The efficacy of protease treatment was 99.5%, with very wide confidence limits (>0 to 99.9%). The data indicate that the use of protease to prevent ETEC diarrheal disease has considerable potential.

Long-term systemic and mucosal antibody responses measured in BALB/c mice following intranasal challenge with viable enterotoxigenicEscherichia coli

“…However, there are potential concerns in the use of these animal models to study ETEC infections, namely, the inability of ETEC to elicit an immune response in the animal, adhere to and colonize the animal gut, and induce symptoms consistent with diarrhea, in addition to the resistance of the animal to ETEC with age. The removable intestinal tie adult rabbit diarrhea (RITARD) model has been used in the study of ETEC-induced acute diarrheal disease (Mynott et al, 1991), but this procedure is surgically invasive and hence is very stressful to the animal.…”

Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment

“…Finally, the role of the HA/protease in detachment merits further consideration from the standpoint of vaccine development (a vibrio which adheres longer is more likely to stimulate an effective immune response) and for its role in the ecology of V. cholerae (a vibrio which is prevented from detaching is less likely to cause devastating epidemics). Further, a recent study (28) indicated that pretreatment of rabbits with an enteric-coated protease preparation prevented intestinal colonization by E. coli. The HA/protease might similarly be useful as a prophylactic agent in preventing colonization by V. cholerae.…”

Detection of attachment of enterotoxigenicEscherichia coli(ETEC) to human small intestinal cells by enzyme immunoassay

“…Brush border vesicles (BBV) were prepared from both rabbit (New Zealand White, 2.0 kg) and human jejunum. The jejunum was the pre-ferred site, as it had previously been shown in animal studies that this region was most heavily colonised by ETEC organisms and had the highest pilus receptor activity [31,32]. Specimens of human small intestine (approximately 2-5 cm in length) were obtained from adult patients and were provided by C.W.…”

“…Specific antisera against CFA were produced as previously described [32]. Antisera directed against human preparations were raised in paired rabbits contained in isolation unit facilities at Fairfield Infectious Diseases hospital (Fairfield, Victoria, Australia) to avoid any risk from viral/bacterial contaminants which could theoretically have been present.…”