Tropical sprue in gnotobiotic rats?
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Animals↗
Biomedical subjects
F A Klipstein
Publications and source records attributed to F A Klipstein.
Enterotoxigenicity of colonising coliform bacteria in tropical sprue and blind-loop syndrome.
The enterotoxigenicity of strains of Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli, which represented the predominant coliform species isolated from the jejunum of 12 patients with tropical sprue and 5 with the blind-loop syndrome, was quantitatively assessed in terms of the ability of toxin preparations to induce water secretion as assayed by in-vivo perfusion in the rat jejunum. All 12 patients with sprue harboured 1 or more highly toxigenic strains--14 of the 16 strains isolated from this group produced heat-labile and/or heat-stable toxins which were as potent as toxins derived from strains isolated from persons with acute diarrhoea and documented as toxigenic. None of the 9 strains isolated from patients with the blind-loop syndrome produced potent toxins. This difference between the coliform bacteria in sprue and the blind-loop syndrome probably accounts, at least partly, for the different intestinal response in these two disorders to contamination by these organisms.
Acute Disease↗
Enterotoxigenicity of enteropathogenic serotypes of Escherichia coli isolated from infants with epidemic diarrhea.
Enteropathogenic serotypes of Escherichia coli which have been incriminated by epidemiological evidence as responsible for epidemics of acute diarrhea in infants are often found to be nontoxigenic when tested by conventional systems such as Y1-adrenal, Chinese hamster ovary, and suckling mouse assays. Twelve such strains, representing four different enteropathogenic serotypes, were examined for their capacity to elaborate toxic materials which alter water transport. Ultrafiltration fractions prepared to contain either a high-molecular-weight, heatlabile or a low-molecular-weight, heat-stable form of toxin from each strain were perfused through rat jejuna in graded concentrations ranging from 100 mug to 0.1 ng/ml. Ten of the twelve enteropathogenic strains produced one or both toxin forms that induced water secretion at concentrations of 1 to 10 ng/ml. Values in this range are considered indicative of clinically significant enterotoxigenicity in this assay system, and toxins from well-documented toxigenic strains examined in this study were active at these same concentrations. Similar preparations from ten control strains from healthy persons were either inactive or evoked water secretion only at concentrations of 10 to 100 mug/ml. These observations suggest that enteropathogenic serotypes of E. coli isolated from epidemics of infantile diarrhea produce diarrhea by elaborating potent heat-labile and heat-stable toxin forms which alter water transport but which are inactive in conventional assay systems. The manner in which these toxins differ either quantitatively or qualitatively from those which stimulate the conventional test systems is unknown.
Animals↗
Immunological relationship of different preparations of coliform enterotoxins.
Antisera raised in rabbits to ultrafiltrate toxin preparations containing either the heat-labile (LT) toxin form obtained from whole cell lysates or broth filtrates or the heat-stable (ST) toxin form prepared from broth filtrates from nontoxigenic and toxigenic strains of Escherichia coli and Klebsiella were examined for their ability to neutralize the secretory effect on water transport of these toxins in the rat jejunum as determined by the in vivo marker perfusion technique. Antisera to the heat-labile toxin derived from whole cell lysate preparations from nontoxigenic strains had no neutralizing effect. Antisera to both types of LT preparation from both toxigenic strains neutralized, with several exceptions, all of the homologous and heterologous LT toxins as well as a heat-labile toxin preparation derived from sequential ultrafiltration of cell-free whole cell lysates which had a defined molecular weight of between 30,000 and 100,000. These antisera also neutralized homologous and heterologous ST preparations obtained from broth filtrates, but they had no neutralizing effect on low-molecular-weight, ST toxin material obtained during the sequential ultrafiltration of cell lysates. Antisera to ST prepared from broth filtrates had no neutralizing capacity against either LT or ST toxin preparations. These observations (i) indicate that the immunological relationship of E. coli and Klebsiella LT and ST toxins extends to antisera raised against LT prepared by several different methods, (ii) raise the possibility that, based on the response to antisera to LT, there may be several immunologically heterogeneous forms of low-molecular-weight ST toxin, and (c) confirm the lack of immunogenicity of ST.
Antibodies, Bacterial↗
Reversal of jejunal water secretion by glucose in rats exposed to coliform enterotoxins.
Glucose absorption and glucose-facilitated water transport were assessed in rats exposed to semipurified preparations of the heat-labile (LT) and heat-stable (ST) enterotoxins of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae by in vivo jejunal perfusion of these toxins alone and with varying amounts of glucose. Progressive increases in the glucose concentration of from 12 to 56 mM resulted in incremental rises in water absorption from perfusates containing each of these toxins, with similar delta water values to those for the electrolyte perfusion solution without toxins. Water secretion was reversed to absorption by the addition of 12 mM glucose when secretion was mild, but 24 mM was required to achieve this in the presence of severe secretion; absolute values for water transport did not return to normal in any instance. Glucose absorption from solutions containing 24 and 56 mM glucose was severely reduced by all of the ST toxins and marginally reduced by some of the LT toxins; the effect of LT was not attributable to the toxin itself, however, but to another unidentified factor present in LT, but not ST, preparations from both toxigenic and nontoxigenic strains. These observations indicate that glucose-facilitated water transport remains intact in intestinal tissue exposed to various coliform enterotoxins and that this occurs despite the presence of impaired glucose absorption in some instances. They provide a rational basis for treatment with oral glucose-electrolyte solutions of persons whose diarrhea is caused by toxigenic coliform bacteria.
Animals↗
Factors responsible for weight loss in tropical sprue.
The respective roles of reduced dietary intake and malabsorption in the pathogenesis of weight loss in persons with chronic tropical sprue have been evaluated . Dietary intake was found to be significantly (P less than 0.001) less in a group of 45 patients with tropical sprue, all of whom had anorexia due to deficiency of folate and/or vitamin B12, than in a group of 51 healthy Puerto Ricans. Weight loss was equally prominent in those patients with tropical sprue who had normal absorption of fat and protein as in those who had excessive fecal loss and reduced absorption of these nutrients. Treatment of five sprue patients with folic acid or vitamin B12 for 2 weeks resulted in improved appetite and increased in dietary intake with weight gain in the absence of significant improvement in intestinal absorption. Treatment with oral tetracycline for a similar period of time in five other patients was not associated with vitamin repletion, return of appetite or weight gain. These observations indicate that reduced dietary intake resulting from anorexia caused by vitamin deficiency is a significant, and sometimes the most important, factor in the pathogenesis of weight loss in persons with chronic tropical sprue.
Anorexia↗
Relative enterotoxigenicity of coliform bacteria.
The enterotoxigenicity of 12 strains of coliform bacteria (Enterobacter cloacae, Klebsiella pneumoniae, and Escherichia coli) isolated from the gastrointestinal (GI) tract of persons with either acute diarrhea or tropical sprue and of 13 strains of the same species isolated from urine (GU) cultures was determined. Fractions of heat-labile (LT) and heat-stable (ST) toxins of each strain were separated by ultrafiltration, and the effect of graded concentrations (range, 100 microgram-10 pg/ml) on water transport was assessed by in vivo perfusion in the rat jejunum. Enterotoxigenic activity was defined as inducement of net secretion of water. All 12 of the GI strains and six of 13 GU strains elaborated enterotoxins, but there was a millionfold quantitative difference in the potency of the toxins produced. All of the GI strains produced one or both forms of toxin, which had a minimal effective concentration of as low as 0.1-10 ng/ml, whereas the GU strains produced toxins of weak or, rarely, of intermediate potency.
Animals↗
Immunological interrelationships between cholera toxin and the heat-labile and heat-stable enterotoxins of coliform bacteria.
Cholera toxin (CT) and the heat-labile (LT) toxin of Escherichia coli are known to share antigenic properties. The present study examined the immunological relationship of CT and the LT and heat-stable (ST) toxins of E. coli, Klebsiella pneumoniae, and Enterobacter cloacae. The neutralizing capacity of equine CT antiserum and of antiserum raised in rabbits to the LT toxin of the three species of coliform bacteria was evaluated by determining their capacity to inhibit the action of purified CT and semipurified ultrafiltration preparations of the coliform LT and ST toxins in inducing water secretion as assayed by the in vivo marker perfusion technique in the rat jejunum. One milliliter of antiserum to CT and to E. coli and Klebsiella LT completely neutralized the secretory action of each of these three toxins; effective serial dilutions of CT antiserum extended to 1 to 4, whereas those of the antisera to LT were limited to 1 to 2 in most instances. One milliliter of antiserum to E. cloacae LT partially neutralized each of the three coliform LT toxins; serial dilutions were inactive. Antiserum to E. cloacae LT did not neutralize CT. Antiserum to CT and to each of the three coliform LT toxins also had a weak neutralizing effect on the ST toxins of E. coli and Klebsiella, but they did not affect E. cloacae ST. Adsorption of the antiserum to CT and to each of the three LT toxins by incubation with a heat-inactivated preparation of either the homologous or a heterologous LT toxin completely abolished the neutralizing capacity of the antisera towards both LT and ST. These observations indicate that the immunological interrelationship of CT and E. coli LT extends to the LT toxins of Klebsiella and E. cloacae and, further, that these immunological properties are shared to a lesser extent by the ST toxins of E. coli and Klebsiella.
Antigen-Antibody Reactions↗
Effect of folate deficiency of the intestinal mucosa on jejunal transport in the rat.
The role of folate deficiency in the pathogenesis of the abnormalities of structure and transport of the small intestine that are often present in certain disorders such as alcoholism is unclear. One reason for this is that the folate status of the intestinal mucosa has never been ascertained in humans. Previous investigations have shown that a deficient diet supplemented with sulfa does produce folate deficiency of the intestinal mucosa in weanling rats and that this is associated with diarrhea and structural abnormalities of the jejunum. In the present study, we used then in vivo marker perfusion technique to examine the jejunal transport of water, sodium, xylose, glucose, and L-leucine in such folate-deficient rats. Supplementation of the normal diet with sulfa did not produce folate deficiency of the intestinal mucosa, but it was associated with a moderate reduction in water absorption; this was attributed to cecal enlargement in these rats. Rats placed on a folate-deficient diet plus sulfa developed markedly depressed serum and jejunal folate concentrations, net secretion of water and sodium, and persistent diarrhea; absorption of xylose, glucose, and L-leucine remained normal, however. Folate repletion of these rats by water and sodium transport to absorption ano cessation of diarrhea. These observations indicate that folate deficiency of the intestinal mucosa alters the transport of water and electrolytes but not that of such solutes as xylose, glucose, and L-leucine. They suggest that when transport abnormalities of these solutes occur in folate-deficient alcoholics, this in not due to the folate deficiency but rather to other pathogenic factors such as ethanol.
Animals↗
Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin.
The enterotoxic material in cell-free growth preparations of Klebsiella pneumoniae serotype 5 was purified by sequential ultrafiltration and gel filtration (GF) procedures and the fractions were assayed for enterotoxic activity by determining their ability to induce in vivo net water secretion in the rat jejunum. Whole-cell lysates were inactive. Anaerobic broth culture conditions yielded a 10-fold increase in toxin production over aerobic conditions. Enterotoxic activity was absent in the UM-10 retentate of the broth filtrate but present in both the retentate and filtrate of the UM-2 membrane. GF of the two UM-2 ultrafiltration fractions through a Sephadex G-25 column yielded an active eluate, whose potency was increased by 10- or 200-fold, in or adjacent to the void volume. When subsequently passed through a G-50 column, these pools eluted at a Kav of between 0.4 and 0.6 and were further increased in potency by two- or fivefold. A second equally potent fraction was also recovered in the void volume of the G-50 eluate of the UM-2 filtrate; this may represent a polymer. Progressive purification by GF was associated with an increased protein and decreased carbohydrate content of the most active fractions. The most active G-50 eluate of the UM-2 retentate had a minimal effective enterotoxic dose of 5 mug/ml and that of the filtrate was less than 0.1 mug/ml. Heating the active GF eluates to 100 C for 30 min did not abolish enterotoxic activity and lowering the pH to 1 or incubation with either Pronase or trypsin had no effect on activity. These observations indicate that K. pneumoniae heat-stable enterotoxin is probably a single toxin with an apparent molecular weight in the range of 5,000. The elution characteristics during GF as well as the chemical composition of the most purified enterotoxin fractions indicate that the toxin is not associated with endotoxin.
Animals↗
Partial purification and properties of Enterobacter cloacae heat-stable enterotoxin.
Cell free preparations of the whole-cell lysate and ultrafiltration (UF) fractions of broth cultures of a strain of Enterobacter cloacae, isolated from a Puerto Rican with tropical sprue, were assayed for their ability to induce in vivo net water secretion in the rat jejunum. The whole-cell lysate and UM-10 retentate of broth cultures were inactive. The UM-2 retentate and filtrate were active at a concentration of 100 mug/ml or more; the toxigenic activity was entirely retained, and increased to 1 mug/ml, by a UM-05 membrane; washing this retentate yielded a fraction with an activity of 10 ng/ml. Stationary aerobic culture conditions yielded the most active UF fractions when ammonium sulfate was used as the precipitating agent, whereas anaerobic culture conditions produced the most active fractions in broth cultures precipitated by acetone. Passage of the active acetone-precipitated UF fractions through a Sephadex G-25 column yielded eluate pools with enhanced toxigenic activity in, or adjacent to, the void volume, but maximum activity of the ammonium sulfate-precipitated UM-05 retentate eluated at a Kav of 0.38 to 0.52. Neither of the most active gel filtration elution fractions of the UM-05 retentates contained detectable carbohydrate, suggesting that the toxin is not associated with endotoxin. Toxigenic activity was unaltered by exposure to a temperature of 100C for 30 min, lowering the pH to 1, or incubation with either Pronase or trypsin. These observations indicate that the strain of E. cloacae under study elaborates a heat-stable enterotoxin htat has approximately the same molecular weight and shares many of the characteristics of the heat-stable enterotoxin produced by some strains of Escherichia coli and Klebsiella pneumoniae.
Aerobiosis↗
Assay of Escherichia coli enterotoxins by in vivo perfusion in the rat jejunum.
Assay of Escherichia coli enterotoxins by in vitro perfusion in rats was evaluated by examining the effects of variously prepared fractions of heat-labile (LT) and heat-stable (ST) toxins on water transport in this system. The assay was found to respond equally well, in a dose-related manner, to both LT and ST; it was sufficiently sensitive to detect the toxigenic effect of concentrations as small as 1 ng/ml. With the assay, it was found that LT is produced in cultures grown under aerobic, but not anaerobic, conditions; in contrast, ST is elaborated in stationary aerobic and anaerobic broth cultures but not in those grown under agitated aerobic conditions. Both toxins can be precipitated by either ammonium sulfate or acetone. The two toxin forms were completely separated from each other by sequential ultrafiltration. LT alone (thermolabile after exposure to 100 degrees C for 30 min) was retained by a PM-30 membrane, and ST alone was present in UM-2 retentates; ST was retained more effectively by a UM-05 membrane, with a 1,000-fold increase in activity over that of the UM-2 retentate. Washed ultrafiltration retentates containing either LT or ST derived from the proper culture conditions all induced water secretion at concentrations of 100 ng or less per ml. These results indicate that in vivo perfusion in rats is a sensitive, duplicable assay for both the LT and ST forms of E. coli enterotoxin.
Animals↗
In memoriam: Gordon Hamilton Fairley 1930-1975.
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Allergy and Immunology↗
Contamination of the small intestine by enterotoxigenic coliform bacteria among the rural population of Haiti.
Coliform bacteria were isolated by either aerobic or anaerobic culture techniques from aspirates of the proximal small intestine of 4 of 5 Haitians with tropical sprue, but not from any of 10 well nourished Haitians who had milder gastrointestinal complaints and abnormalities. Klebsiella (Klebsiella pneumoniae and Klebsiella ozaenae) was cultured from the jejunal aspirates of 2 sprue patients and Escherichiae coli from the other 2. Fifteen colonies of coliform bacteria cultured from each aspirate were specifically identified by their biotype. In three instances, every colony in each aspirate was the same; In three instances, every colony in each aspirate was the same; in the fourth aspirate, two biotypes of E. coli were present, one of which grew under both aerobic and anaerobic culture conditions and another which grew only under anaerobic conditions on initial isolation. A randomly selected strain of each of the five coliform biotypes isolated was examined for enterotoxigenicity by determining the effect of variously prepared cell-free preparations on water transport in the rat jejunum using standard marker perfusion techniques. Every strain tested was toxigenic; one produced only a heat-stable toxin, one produced a heat-labile toxin only, and three elaborated both forms of enterotoxin. These observations indicate that most Haitians with tropical sprue have colonization of the proximal small intestine by a specific strain of enterotoxigenic coliform bacteria, but such is not the case among Haitians who have milder intestinal abnormalities.
Animals↗
Enterotoxigenic intestinal bacteria in tropical sprue. III. Preliminary characterization of Klebsiella pneumoniae enterotoxin.
Cell-free broth filtrates of a strain of Klebsiella pneumoniae serotype 5 retained their capacity to induce fluid secretion in the rabbit ileal loop model after heating (100 C for 30 min), acid treatment (pH 4.4), and dialysis through viscose tubing. Sequential passage of an acetone precipitate of the broth filtrate through Diaflo membranes yielded an active fraction in the UM 2, but not the UM 10, retentate; this observation indicates that the molecular weight of the enterotoxin is in the range of 1,000-10,000. Klebsiella pneumoniae enterotoxin thus resembles the heat-stable enterotoxin of Escherichia coli in a number of respects.
Animals↗
Enterotoxigenic intestinal bacteria in tropical sprue. IV. Effect of linoleic acid on growth interrelationships of Lactobacillus acidophilus and Klebsiella pneumoniae.
The factors responsible for colonization of the small intestine by enterotoxigenic coliform bacteria in Puerto Ricans with tropical sprue are unknown, but epidemiological observations have suggested that they may be related to an increased dietary intake of long-chain unsaturated fatty acids, particularly linoleic acid, which is known to exert an inhibitory effect on the growth of gram-positive organisms that normally comprise the flora of the small intestine. We have examined, by using a glucose-limited continuous-culture system, what effect this fatty acid exerts on the growth relationships of enteric gram-positive and coliform bacteria. In this system, colonization by an invading strain of Klebsiella pneumoniae was prevented by the presence of an established culture of Lactobacillus acidophilus, principally by virtue of a lowered pH of the medium that was incompatible with Klebsiella growth. However, when the population density of L. acidophilus was reduced by the presence of a sufficient concentration of linoleic acid, the invading K. pneumoniae successfully colonized the system and, once established, suppressed the growth of L. acidophilus. These observations indicate that, under the conditions of our chemostat, gram-positive enteric bacteria suppress coliform growth and that this effect is reversible by the presence of linoleic acid. It remains to be established, however, what pertinence these in vitro observations have to conditions within the intestinal tract of persons living in the tropics.
Enterotoxins↗
Effect of Klebsiella pneumoniae enterotoxin on intestinal transport in the rat.
The effects on intestinal transport of either a semipurified preparation of enterotoxin elaborated by Klebsiella pneumoniae or similaryly prepared control material were tested by marker perfusion studies in the small intestine of rats. At a concentration of 2 mg/ml, the enterotoxin produced net secretion of water, Na, and Cl in both jejunal and ileal segments; HCO3 transport was not affected. Net secretion was evident within 30 min after intorduction of the toxin and was maximal after 90 min. The addition of 56 mM glucose to the enterotoxin-containing perfusion fluid resulted in reversal of water and Na transport to net absorption in both intestinal segments. The enterotoxin also produced a significant depression of xylose absorption in both the jejunum and ileum but did not affect the absorption of either glucose or L-leucine. Intestinal structure was not altered after perfusion of the toxin but insillation of approximately one-quarter of the total perfusion dose into a ligated jejunal loop for 18 h produced fluid secretion and structural abnormalities. These observations confirm the fact that other species of coliform bacteria in addition to tescherichia coli are capable of elaborating an enterotoxin. Such species commonly contaminate the small intestine of persons with tropical sprue and it is suggested that chronic exposure of the intestinal mucosa to the enterotoxin elaborated by these bacteria may be a factor in the pathogenesis of intestinal abnormalities in thid disorder.
Animals↗