Infant Rabbit Model Research Articles

Shiga toxin 2-induced intestinal pathology in infant rabbits is A-subunit dependent and responsive to the tyrosine kinase and potential ZAK inhibitor imatinib

Shiga toxin producing Escherichia coli (STEC) are a major cause of food-borne illness worldwide. However, a consensus regarding the role Shiga toxins play in the onset of diarrhea and hemorrhagic colitis (HC) is lacking. One of the obstacles to understanding the role of Shiga toxins to STEC-mediated intestinal pathology is a deficit in small animal models that perfectly mimic human disease. Infant rabbits have been previously used to study STEC and/or Shiga toxin-mediated intestinal inflammation and diarrhea. We demonstrate using infant rabbits that Shiga toxin-mediated intestinal damage requires A-subunit activity, and like the human colon, that of the infant rabbit expresses the Shiga toxin receptor Gb3. We also demonstrate that Shiga toxin treatment of the infant rabbit results in apoptosis and activation of p38 within colonic tissues. Finally we demonstrate that the infant rabbit model may be used to test candidate therapeutics against Shiga toxin-mediated intestinal damage. While the p38 inhibitor SB203580 and the ZAK inhibitor DHP-2 were ineffective at preventing Shiga toxin-mediated damage to the colon, pretreatment of infant rabbits with the drug imatinib resulted in a decrease of Shiga toxin-mediated heterophil infiltration of the colon. Therefore, we propose that this model may be useful in elucidating mechanisms by which Shiga toxins could contribute to intestinal damage in the human.

An Escherichia coli O157-Specific Engineered Pyocin Prevents and Ameliorates Infection by E. coli O157:H7 in an Animal Model of Diarrheal Disease

AvR2-V10.3 is an engineered R-type pyocin that specifically kills Escherichia coli O157, an enteric pathogen that is a major cause of food-borne diarrheal disease. New therapeutics to counteract E. coli O157 are needed, as currently available antibiotics can exacerbate the consequences of infection. We show here that orogastric administration of AvR2-V10.3 can prevent or ameliorate E. coli O157:H7-induced diarrhea and intestinal inflammation in an infant rabbit model of infection when the compound is administered either in a postexposure prophylactic regimen or after the onset of symptoms. Notably, administration of AvR2-V10.3 also reduces bacterial carriage and fecal shedding of this pathogen. Our findings support the further development of pathogen-specific R-type pyocins as a way to treat enteric infections.

Mechanism of Pressure-Overload Right Ventricular Hypertrophy in Infant Rabbits

Although pressure-overload right ventricular hypertrophy is a long-term risk in some congenital heart diseases such as tetralogy of Fallot, how it develops is unclear. The aim of this study was to investigate the mechanism of development of this right ventricular heart failure.Pulmonary artery banding in 10-day-old rabbits induced pressure-overload right ventricular hypertrophy as they grew. Comparisons were made with age-matched sham controls (n = 24 per group). In weekly serial echocardiography, the right ventricular contraction and diastolic function decreased from 3 weeks after surgery (P < 0.01), and the right ventricle became hypertrophic from 4 weeks after (P < 0.05). Pressure-overload increased cardiomyocyte apoptosis from 4 weeks postoperatively (TUNEL staining and Western blotting analysis, P < 0.05); and fibrosis occurred in the right ventricular cardiomyocytes at 8 weeks after operation (Masson's trichrome stain, P < 0.01). In our model, pressure-overload to the right ventricle caused the right ventricular disorder, hypertrophy, and fibrosis. Apoptosis of right ventricular cardiomyocytes was involved in progression. We have shown for the first time the mechanism whereby pressure-overload right ventricular hypertrophy develops in an infant rabbit model.

Back to the Future: Studying Cholera Pathogenesis Using Infant Rabbits

ABSTRACTCholera is a severe diarrheal disease, caused by Vibrio cholerae, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation of V. cholerae into 3-day-old rabbits pretreated with cimetidine led to lethal, watery diarrhea in virtually all rabbits. The appearance and chemical composition of the rabbit diarrheal fluid were comparable to those of the “rice-water stool” produced by cholera patients. As in humans, V. cholerae mutants that do not produce cholera toxin (CT) and toxin-coregulated pilus (TCP) did not induce cholera-like disease in rabbits. CT induced extensive exocytosis of mucin from intestinal goblet cells, and wild-type V. cholerae was predominantly found in close association with mucin. Large aggregates of mucin-embedded V. cholerae were observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influences V. cholerae’s association with the host intestine and its exit from the intestinal tract. Our model should facilitate identification and analyses of factors that may govern V. cholerae infection, survival, and transmission, such as mucin. In addition, our results using nontoxigenic V. cholerae suggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-V. cholerae vaccines.

Reactogenicity of live-attenuated Vibrio cholerae vaccines is dependent on flagellins

Cholera is a severe diarrheal disease caused by the motile Gram-negative rod Vibrio cholerae. Live-attenuated V. cholerae vaccines harboring deletions of the genes encoding cholera toxin have great promise for reducing the global burden of cholera. However, development of live vaccines has been hampered by the tendency of such strains to induce noncholeric reactogenic diarrhea in human subjects. The molecular bases of reactogenicity are unknown, but it has been speculated that reactogenic diarrhea is a response to V. cholerae's flagellum and/or the motility that it enables. Here, we used an infant rabbit model of reactogenicity to determine what V. cholerae factors trigger this response. We found that V. cholerae ctx mutants that produced flagellins induced diarrhea, regardless of whether the proteins were assembled into a flagellum or whether the flagellum was functional. In contrast, approximately 90% of rabbits infected with V. cholerae lacking all five flagellin-encoding genes did not develop diarrhea. Thus, flagellin production, independent of flagellum assembly or motility, is sufficient for reactogenicity. The intestinal colonization and intraintestinal localization of the nonreactogenic flagellin-deficient strain were indistinguishable from those of a flagellated motile strain; however, the flagellin-deficient strain stimulated fewer mRNA transcripts coding for proinflammatory cytokines in the intestine. Thus, reactogenic diarrhea may be a consequence of an innate host inflammatory response to V. cholerae flagellins. Our results suggest a simple genetic blueprint for engineering defined nonreactogenic live-attenuated V. cholerae vaccine strains.

Mitochondria-initiated apoptosis triggered by oxidative injury play a role in total parenteral nutrition–associated liver dysfunction in infant rabbit model

Purpose The aim of the study was to investigate oxidative injury and apoptosis as the mechanisms underlying total parenteral nutrition (TPN)–associated liver dysfunction. Methods Twenty New Zealand rabbits (2 weeks old) were divided into 2 groups as follows: 10 in the control group (maternal feed) and 10 in the TPN group. The rabbits in the TPN group received continuous PN infusion through a silastic catheter inserted in the right jugular vein. Results After 10 days of treatment, the serum levels of total bilirubin and bile acid were significantly higher in the TPN group than in the control group ( P < .01, respectively). The light microscopic findings in the TPN rabbits included inflammatory cell infiltration and hepatic steatosis. Electron microscopy showed change in the cytosolic vacuoles and rare microvilli in the microbile duct. Moreover, 10 days of treatment resulted in an inhibition of the superoxide dismutase (SOD) activity in hepatocytes, an increase of the malondialdehyde level, a significant increase in cytochrome c release from the mitochondria, a significant increase in caspase 3 activity, and increased apoptosis ( P < .01, individually). Conclusions Oxidative damage may be one of the essential mechanisms of TPN-associated liver dysfunction. Moreover, mitochondria-initiated apoptosis triggered by oxidative damage may play an important role in this process.

Probiotic acidified formula in an animal model reduces pulmonary and gastric bacterial load

Background/Purpose We previously reported that a diet acidified with citric acid effectively reinforces gastric acid protection against bacterial colonization and translocation. In this study, our objective was to examine a biologically acidified formula hypothesized to be more physiologic than formula acidified with free acid. This study was Institutional Animal Care and Use Committee (IACUC) approved and designed to determine whether this diet is better tolerated and equally effective to acidification with citric acid against gut colonization and subsequent bacterial translocation in a premature infant rabbit model. Methods A total of 89 rabbit pups born via cesarean delivery 1 day preterm were randomly assigned to 3 feeding groups: Pelargon Nestle at pH 4.55; NAN Nestle, a control diet at pH 7.0 with similar composition; and NAN Nestle acidified in the laboratory with citric acid at pH 4.55. Pups were gavage fed every 12 hours with Enterobacter cloacae challenges of 10 colony-forming units per milliliter of diet per feed and killed on day 3 of life. Lungs, liver, spleen, mesenteric lymph nodes, stomach, and cecum were cultured and quantitatively analyzed for target organism growth and statistically analyzed using χ 2 and Kruskal-Wallis tests. Results Pelargon, compared to acidified NAN and NAN, significantly reduced the incidence of gastric colonization (15/33 [45%], 21/27 [78%], and 25/29 [86%], respectively; P < .01) and pulmonary colonization (10/33 [30%], 19/27 [70%], 21/29 [72%]; P < .01). Comparing the bacterial logs of colonized groups, the same benefit is observed in the lungs (0.77 ± 1.22, 1.89 ± 1.41, 2.12 ± 1.47; P < .01). Gut colonization and bacterial translocation were equivalent between treatment groups (mesenteric lymph nodes: 10/33 [30%], 11/27 [40%], 8/29 [27%]; spleen: 10/33 [30%], 7/27 [26%], 8/29 [27%]; liver: 10/33 [30%], 6/27 [22%], 9/29 [31%]; cecum: 33/33 [100%], 27/27 [100%], 29/29 [100%]). Conclusion Biologically acidified formula demonstrated superior protection against pulmonary and gastric colonization compared to normal pH and diets acidified with free acid. Its effects may potentially reduce clinical pulmonary infection.

CadA negatively regulates Escherichia coli O157:H7 adherence and intestinal colonization.

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

Type 2 Secretion Promotes Enterohemorrhagic Escherichia coli Adherence and Intestinal Colonization

Enterohemorrhagic Escherichia coli (EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. We found that the Z2053 gene (designated adfO here), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfO mutant exhibited marked reductions in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, we found that the adfO mutation did not affect colonization but that the colonization of an etpC (T2SS) mutant was reduced approximately 5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, our findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.

Effects of Rigid Fixation on the Growing Neurocranium of Immature Rabbits

The improved intraoperative long-term skeletal stability achieved with rigid fixation techniques has led to their widespread popularity and application. However, experimental studies have revealed some drawbacks related to metallic implants and long-term results of clinical studies, especially in pediatric patients, has confirmed the results of experimental studies. Our aim in this experimental study using an infant rabbit model is to answer the following question: "Does short-term skeletal stability cause long-term growth inhibition?" Forty, 9-day-old New Zealand white albino rabbits were divided into four groups: 1) experimental, n=6: plated across the right coronal suture and two screws on each side of the left coronal suture; 2) re-operation, n=6: the same materials as group I were placed, and only the plate was removed at the end of 1 month; 3) sham, n=6: sham control with simulated surgery and two screws on each side of the left coronal suture; 4) control, n=2: no operation. The animals were killed 6 months after microplate application, and the skulls were evaluated both grossly and cephalometrically. Gross examination showed that the plates and the screws were covered by bony overgrowth and caused bony irregularity and regional bone degeneration. The parietal bones on the plated sides became striated and lost their concave shape. Cephalometric analysis demonstrated overt mastoid tip deviation toward, or shortening of cranionasal length on, the side with rigid fixation. We conclude from our study that rigid fixation during skeletal development causes growth retardation and should not be used in the growing child.

Radiation-Induced Craniofacial Bone Growth Inhibition: Efficacy of Cytoprotection following a Fractionated Dose Regimen

Severe craniofacial growth disturbances are noted in 66 to 100 percent of children with head and neck cancers who received radiotherapy during their growing years. The authors have previously demonstrated the prevention of radiation-induced craniofacial bone growth inhibition following single-dose orthovoltage radiation to the orbitozygomatic complex in an infant rabbit model through the administration of the cytoprotective agent amifostine (WR-2721) before radiation treatment. The purpose of this study was to investigate the efficacy of cytoprotection using a fractionated dose regimen that better approximates the clinical application of radiation therapy. Thirty 7-week-old male New Zealand rabbits were randomized into three groups (n = 10), each receiving six fractions of orthovoltage radiation to the right orbitozygomatic complex: group C, sham irradiation control; group F35, total dose of 35 Gy; and group F35A, total dose of 35 Gy with administration of amifostine 200 mg/kg intravenously 20 minutes before each fraction. Bone growth was evaluated up to skeletal maturity (age 21 weeks) with serial radiographs and computed tomography scans for cephalometric analysis, bone volume, and bone density measurements. Fractionated radiation resulted in significant (p < 0.05) bone growth inhibition compared with sham radiation in 16 of 21 cephalometric parameters measured and significantly (p < 0.05) reduced bone volume of the rabbit orbitozygomatic complex. Pretreatment with amifostine before each radiation fraction prevented growth deformities in four cephalometric parameters and significantly (p < 0.05) attenuated these effects in another seven parameters compared with radiated animals. Bone volumes were also significantly (p < 0.05) improved in F35A animals compared with F35 animals. This study establishes that fractionation of orthovoltage radiation does not prevent the development of growth disturbances of the rabbit craniofacial skeleton and also demonstrates that preirradiation administration of amifostine is highly effective in the prevention and attenuation of radiation-induced craniofacial bone growth inhibition.

Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli-induced diarrhea and intestinal inflammation in infant rabbits.

Enterohemorrhagic Escherichia coli (EHEC) is a group of food-borne pathogens that can cause diarrhea, colitis, and the hemolytic uremic syndrome (HUS). The importance of several of the proposed EHEC virulence factors lacks experimental verification in animal models. The limitations of current animal models led us to reexamine the infant rabbit model for the study of EHEC pathogenicity. Here, we report that intragastric inoculation of a Shiga toxin 2 (Stx2)-producing E. coli O157:H7 clinical isolate into infant rabbits led to severe diarrhea and intestinal inflammation but no signs of HUS. We constructed a set of isogenic derivatives of this isolate with deletions in several putative virulence genes, including stx(2), eae, tir, and ehxA, to investigate the contribution of individual virulence factors to EHEC pathogenicity. stx(2) increased the severity and duration of EHEC-induced diarrhea. Furthermore, although stx(2) had no role in EHEC intestinal colonization nor was it required for EHEC-induced inflammation, stx(2) altered how the host responded to EHEC infection by promoting heterophilic infiltration of the colonic epithelium and lamina propria. Intragastric inoculation of purified Stx2 also induced inflammation and diarrhea in this model. Diarrhea and intestinal inflammation were also dependent on EHEC colonization, as EHEC derivatives with deletions in eae and tir did not colonize, form attaching and effacing lesions, or develop clinical signs of disease. Our studies indicate that infant rabbits are a useful model for investigation of the intestinal stage of EHEC pathogenesis and suggest that Shiga toxin may play a critical role in causing diarrhea and inflammation in patients infected with EHEC.

After-cataract and secondary glaucoma in the aphakic infant rabbit

Purpose To study the association between after-cataract and secondary glaucoma after lensectomy and 5-fluorouracil treatment in an experimental infant rabbit model. Setting St Erik Eye Hospital, Karolinska Institute, Stockholm, Sweden. Methods Lensectomy was performed in both eyes of 16 3-week-old rabbits. One randomly selected eye in each rabbit was injected with 2.5 mg of 5-fluorouracil (5-FU) at surgery and 5.0 mg the day after surgery to reduce the formation of after-cataract. Axial length, corneal thickness, corneal diameter, and intraocular pressure were measured preoperatively and 4 times during the 6 months following surgery. Six months after surgery, the wet weight of the after-cataract was determined. Results In 16 aphakic eyes treated with 5-FU, no or a minimal amount (<0.001 g) of after-cataract developed. None of the eyes showed signs of secondary glaucoma. Of 16 aphakic eyes not treated with 5-FU, 10 developed considerable amounts of after-cataract (>0.10 g); 8 of these developed glaucoma. The other 6 eyes had no or minimal after-cataract and did not develop secondary glaucoma. The relationship between after-cataract and secondary glaucoma was statistically significant. Conclusion A significant relationship between the amount of after-cataract and the development of secondary glaucoma was found in aphakic infant rabbit eyes.

Construction and characterization of recombinant Vibrio cholera strains carrying heterologous genes encoding non-01 antigen or cholera enterotoxin

In an attempt to study the effect of heterologous genes on the virulence of Vibrio cholerae01 and non-01, rfbgenes encoding biosynthesis of non-01 antigens were introduced by homologous recombination into the chromosome of V. cholerae01 strain 569B (serotype Inaba, biotype classical). Recombinant strains were obtained which were not agglutinated with the diagnostic cholera 01 antiserum and were not sensitive to the cholera diagnostic bacteriophage, but produced as much cholera toxin as 569B and were highly virulent in the infant rabbit intra-intestinal injection model. These data indicate that the rfbgenes from the studied V. choleraenon-01 did not alter the virulence phenotype of V. cholerae01. In contrast, cloned ctxABgenes from V. cholerae01 encoding cholera toxin introduced into a non-pathogenic strain lead to efficient secretion of cholera toxin but to only low virulence in the infant rabbit model.

Prevalence of a Vibrio cholerae 18-kDa antigen in vibrios

An 18-kDa protein that occurs in Vibrio cholerae has been described as an in vivo and low-iron regulated outer membrane antigen. Monoclonal antibodies which recognized this antigen were protective as passive vaccines in the infant rabbit model of cholera disease. In this study, those monoclonal antibodies were used in three immunological assays for surveillance of various bacteria for the 18-kDa antigen. ELISA, and Western blot assays gave variable results with bacteria or outer membrane preparations. The biodot assay was the most sensitive test, detecting the 18-kDa antigen in 29 of 29 V. cholerae strains, independent of biotype or serotype. A few other Gram-negative bacteria and V. parahaemolyicus reacted weakly with our antibodies and antiserum.

BIOAVAILABILITY AND CSF PENETRATION OF CIPROFLOXACIN IN EXPERMENTAL HAEMOPHILUS INFLUENZAE MENINGITIS

Ciprofloxacin (C) is a new quinolone antimicrobial with a broad spectrum of activity and oral bioavailability that may be useful for the therapy of systemic infections in children. Pharmacokinetics, CSF penetration and efficacy of this agent were studied in an infant rabbit model of bacteremic Haemophilus (Hib) meningitis induced by intranasal inoculation. C concentrations were determined by bioassay. Uninfected rabbits were given 25 or 50 mg/kg orally and intravenously.Serum concentrations (conc.) peaked 1-3 h after oral dosing and bioavailability (AUC po/AUC iv) was 40.5%. Rabbits with Hib meningitis were given C 50 mg/kg po q12h. Peak serum cone. (3.3±1.5) and (3.0 h) were similar to uninfected animals, but CSF cone, were higher (mean 0.14 ug/ml, penetration 7.8±1.6%). Serum cone. 12 h after an oral dose (0.16 ug/ml) were still 10X the Hib MIC. CSF cultures showed 2500 cfu/ml before and 16 cfu/ml 2h after treatment. CSF cultures at 6 h were sterile. C warrants further study in the treatment of systemic Hib infections.

Streptococcus faecium M 74 in Control of Diarrhoea Induced by a Human Enterotoxigenic Escherichia coli Strain in an Infant Rabbit Model

Streptococcus faecium strain M 74 was evaluated as a prophylacticum for enterotoxigenic Escherichia coli (ETEC) diarrhoea with human isolates of E. coli with CFA/I and CFA/II surface fimbrial haemagglutinins (adhesins) in a rabbit model. Young rabbits (3 to 4 days old) were given S. faecium organisms (5 X 10(9)) 15 min before (group A), 6 h before (group B) and 12 h after (group C) challenge with ETEC organisms. Only 4 out of 26 rabbits in group A, 6 out of 21 in group B and 7 out of 23 in group C developed diarrhoea. In conclusion, this S. faecium strain M 74 seems efficiently to protect animals from ETEC diarrhoea when given as a prophylactic agent at a high dose. This animal model seems useful for comparative studies on new preventive methods for ETEC diarrhoea such as testing probiotics and antiadhesive drugs.

A new principle for prevention of diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) possessing colonization factor antigen (CFA/I).

The infant rabbit model, developed to study human ETEC with CFA/I (Evans et al., 1975), was used to evaluate agarose (Sepharose) gel substituted with a hydrophobic ligand for prevention of diarrhoeal symptoms. In a group of rabbits given 0.02 g of palmitoyl Sepharose 1 h after an oral infection with 10(9) ETEC organisms with CFA/I antigen, only 2/11 animals developed diarrhoea, while in control groups given the unsubstituted Sepharose (hydrophilic) gel or ETEC organisms alone, all animals developed diarrhoea. In groups of rabbit given the hydrophobic gel 6 h after the ETEC organisms, only 2/6 animals showed a short period of diarrhoea. The "hydrophobic principle" to prevent ETEC infections will be studied in other animal models and in human volunteers.

Dissociation and Recombination of the Subunits of the Cholera Enterotoxin (Choleragen)

Abstract By gel filtration under dissociating conditions in acid urea, the cholera enterotoxin (choleragen) is separated into two fractions, A and B. Fraction A, which elutes first from columns of Bio-Gel P-60, is unique to the toxin and is not found in the spontaneously formed enterotoxoid (choleragenoid) after similar treatment. Neither A nor B alone has significant toxicity in either the infant rabbit model or adult rabbit skin tests. However, after neutralization and dialysis of mixtures of A and B (regardless of whether the latter is derived from choleragen or choleragenoid) the toxin activity in both systems is restored as is the characteristic electrophoretic mobility of the original toxin. Antiserum against the holotoxin precipitates both fragments but A and B give reactions of non-identity. Antiserum against choleragenoid does not precipitate A. Peptide mapping indicates that choleragen contains a number of peptides not present in choleragenoid. The A fragment can further be dissociated into two parts by treatment with thiol reagents. It now seems evident that the cholera enterotoxin consists of two major kinds of non-covalently associated subunits. Of these, the B type, comprising choleragenoid, is likely to be responsible for interaction with GM1 ganglioside-like host cell receptors and the A fragment for biologic activity, i.e., the activation of adenyl cyclase.

Toxigenic Escherichia coli: a cause of infantile diarrhea in Chicago.

Approximately 1000 children per year are admitted to Cook County Hospital with acute diarrhea. The etiologic agent cannot be found in the majority of cases. Six hundred strains of Escherichia coli were isolated from the stools of 29 children with diarrhea not yielding salmonella and shigella. Classic serotypes of enteropathogenic Esch. coli (EPEC) were found in nine of 29 cases. Esch. coli enterotoxin was assayed in an infant rabbit model; strains causing fluid accumulation and distention of the small bowel were found in 24 of 29 cases. This reaction could be induced by sterile filtrates as well as viable cultures. Two or three enterotoxin-positive strains were often present in the same stool specimen. Simple serotyping for EPEC identified pathogens in 31 per cent of cases of diarrhea whereas the animal model incriminated pathogenic strains in 83 per cent of cases.