PedaicGastoemeralogy ond Nutrtion Review Pathophysiology of Infectious Diarrhea: Changes in Intestinal Structure and Function E. V. O'Loughlin, *R. B. Scott, and *D. G. Gall Department of Pediatrics, University of Sydney, Sydney, New South Wales, Australia; and *Department of Pediatrics, University of Calgary, Calgary, Alberta Infectious diarrhea continues to be a major health problem throughout the world, particularly in de- veloping countries. A recent review by the World Health Organization of community-based survé lance studies in various third world countries esti ‘mated that in 1982 approximately 1 billion episodes of diarrhea occurred in children under 5 years of age. These studies excluded China (1). These re- sulted in approximately 5 million deaths and con- tributed to another major health problem in these countries, that of chronic malnutrition (2,3,4,5). In- fection is also a major cause of morbidity and mor tality in adults Diarrhea can be defined as increased frequency andior fluidity of bowel movements, i.e., passage of stools containing excessive water (6). A number of factors are known to contribute to excessive fluid and electrolyte losses, including () active secretion of fluid and electrolytes, (ji) decreased digestion and absorption of nutrients, and (ii) abnormal tran sit due to aberrations of intestinal motility. Other factors that may be involved in inducing diarrhea during infection such as abnormal intestinal blood flow, altered neural modulation of absorption and secretion, and alterations in endocrine and para- crine secretion will not be discussed further due to a paucity of experimental data. In the following re~ view, the authors, rather than presenting an exten- sive review of all pathogenic organisms, will elabo- rate on the mechanisms of diarrhea production and use pathogens as illustrative examples (Table 1). ‘Adaress correspondence and reprint requests to De. EV. O'Loughlin ut Department of Paediatrics & Child Health, Unk versity Teaching Unit, The Chilren's Hospital, P-0. Box 34, Camperdown, NSW 2680 Australia “Acoepted fo publication atthe office ofthe Founding Fitor INCREASED SECRETION OF FLUID ‘AND ELECTROLYTES Enterotoy and Chemical Secretagogues Cholera A wide variety of pathogenic bacteria elaborate enterotoxins and cytotoxins capable of stimulating intestinal secretion. Cholera isa classical prototype of enterotoxigenic diarrhea. While Vibrio cholerae ‘was first recognized as a cause of diarrhea in 1884 by Robert Koch, the role of enterotoxin was not, discovered until 1959 (7). V. cholerae colonizes the upper small intestine without inducing morphologic alterations in the mucosa (8-10). Following adher- cence to the mucosa, the organism elaborates a heat- labile toxin (LT) that, after binding to the apical ‘membrane of the mucosal cell, induces secretion of fluid and electrolytes. However, active transport processes for nonelectrolytes such as glucose are ‘unimpaired (11-13), Studies in isolated ileal mucosa from rabbits and humans performed under short- circuited conditions in Ussing chambers revealed that luminal administration of cholera toxin induces electrogenic chloride secretion by a cyclic AMP- ‘dependent process (14,15). Additional studies dem- ‘onstrated that cholera toxin induced adenylate cy- clase activity and increased intracellular concentra- tions of cyclic AMP (16-19) in animal models and also in biopsy specimens taken from humans with acute cholera (20). While considered primarily an affliction of the small intestine (11,21,22), recent studies in affected humans (23) demonstrated co- lonic involvement. The effect on the colon is pri- marily antiabsorptive. Evaluation of the cell regulation of cholera-6 E. V. O'LOUGHLIN ET AL. TABLE 1. Pathophysiological classification af infectious diarrhea Increased seretion| {) Enerotoxing Vibrio cholerae Enlerotongenie Escherichia col Salmonelia sp Clostridium dificil ip Chemical secretagogues ‘Entamoeba hsttyicn Gi) Insane repuation ‘Arachidonic acid metabolites ‘Salmoneta sp Ciowridium dificile Mist cell mediators Thichinle spiralis Decreased digestion and absorption “@ Disorgunzed epithelial renewal Rotavirus ‘Transmisible gastroenteritis virus (@ Epithelial struction Brush border damage aleropathogenic E. coll Giardia murte Yersinia enterocolitica Cytotoxin prodvction ‘Shigella. Epithelial invasion "Varsinia enterocolitica Helminths Disordered transit, ‘Teansnssible gastroenteritis vis Salmonella sp Shigella sp. Vibrio cholerae losin dificie Enterotoxigenie 6. col Thehinella spiralis induced intestinal seeretion has provided us with important insights into the normal physiology of in- testinal secretion. Cholera toxin (MW of 84,000) consists of five B subunits (MW of 11,500 each) arranged in a circular fashion, whose primary func~ tion isto bind the toxin to ganglioside receptors in the apical membrane, and an A subunit (MW 28,000) (Fig. 1). The A’ subunit translocates across the cell membrane and in the process is split into A, (MW 20,500) and A> (MW 7,500) (24). Ay is the active component. It stimulates adenylate cyclase 3g to the regulatory unit, G, protein, and this G, protein in an irreversibly activated in turm stimulates a catalytic component to produce cyclic AMP {recently reviewed by Spie- gel et al. 25)). ‘The cAMP effect on intestinal transport is be- ved to occur directly at the apical membrane This membrane contains cyclic AMP-lependent protein kinases, phosphatases, and several sub- 4 Pediat Ganoentrl Nut, Vo 12, No 11991 $l “al FIG. 1. Diagrammatic representation ofthe steps involved in the acivation of enloride secretion by cholera toxin. Chloride feeretlon and inhibition of neutral NaCl absorption aro de Ploted as If occurring Inthe same cell. See the text Tor & Sescrition. strate proteins (26-28). Activation of cAMP is as sociated with phosphorylation of several microvit- lus membrane proteins (29). Phosphorylation of proteins in the CI~ channel is thought to be the basis for Cl~ secretion (30). Heat-Stable Toxin (ST) Enterotoxigenic £. col is one of the commonest ‘causes of diarrheal disease, particularly in develop- ing countries (431-33). Enterotoxigenic E. coli elaborates a choleravlike LT but also produces a heat-stable toxin (ST). ST, unlike cholera toxin, does not bind to gangliosides of the microvilli of ‘mucosal cells and exhibits a very rapid onset of action. After binding to the receptor on the entero- eyte apical membrane, ST inhibits electroneutral Na*Cl~ absorption and induces Cl~ secretion. In- testinal fuid production parallels elevated intrace!- lular cyclic GMP and mucosal guanylate cyclase ac- tivities and can be mimicked by the cyclic GMP analogue dibutyl-cyclic GMP (db-cyclic-GMP) (33- 37). The secretion effect is not as potent as seen in cholera-induced secretion (38). The difference has been attributed to the decreased levels of guanylate cyclase activity in erypt enterocytes (39), sincePATHOPHYSIOLOGY OF INFECTIOUS DIARRHEA 7 crypt cells have been postulated as the site of active CI secretion (40), ‘The precise mechanism whereby guanylate cy: clase/eyelic GMP activation stimulates intestinal se~ cretion has not been determined. Guanylate cyclase complex is largely localized to the brush border (4D. A cyclic GMP-activated phosphorylation! dephosphorylation system has been identified in the apical membrane of a number of species (42,43), and in the rabbit a number of brush border mem= brane proteins have been phosphorylated following activation of this system (29). However, a role of cyclic GMP phosphorylation in the regulation of electrolyte transport has not been defined. Cyclic GMP-mediated secretion appears to occur primary in the small intestine, At least in rats (44) and rab- bits (45), ST has no effect on colonic transport. This is thought to be due to the absence of the cyclic GMP phosphorylation system (45). Miscellaneous Enterotoxin-Producing Bacteria Annumber of other bacterial pathogens are known to produce enterotoxins. Other pathogenic factors, including cytotoxin production and tissue invasion causing epithelial destruction and stimulation of lo- cal production of inflammatory mediators such as prostaglandins, may also be involved, making it i ficult to determine the exact role of toxin in symp- tom production. Some strains of Salmonella are capable of stim- ulating fluid secretion in ligated rabbit ileal loops (46,47), suggesting a possible role for enterotoxin. While initial studies failed to isolate enterotoxin, from crude culture filtrate (47), subsequent studies did reveal enterotoxigenic extracts in the bacterial cell wall (48,49), Isolation of toxin was amplified ‘when cultures were grown in the presence of mito- ‘mycin C (50), a manipulation thought to induce lysis of the bacterial cell wall, releasing fragments into the cell culture medium (51). Further evidence sup- porting enterotoxin production comes from the ob- servation of Chinese hamster ovary cell elongation by culture extracts. This effect was inhibited by monospecific cholera antitoxin or by adding mixed gangliosides to the cell culture (50,52). These exper imental observations suggest that Salmonella pro- duces a cholera-like enterotoxin. Other organisms such as Campylobacter jejuni (53-57) and Aeromonas hydrophila (58,59) produce cholera-like toxins, but their role in symptom pro- duction is less clear. Chemical Secretagogues ‘Another novel mechanism for intestinal secretion induced by a microorganism has been reported in studies on amebic dysentery (60). Cell-free extracts Of the parasite grown in axenic culture stimulate colonic secretion. When added to the serosal side of, the tissue in vitro under voltage clamp conditions, the amebic extracts stimulate calcium-dependent electrogenic CI secretion. The alterations in elec- trical parameters and ion transport can be inhibited by the serotonin antagonist, bufotenine, and par- tially inhibited by antibody to serotonin. The ame- bic lysates were found to contain high levels of se- rotonin, Thus, local serotonin production by the parasite may be involved in the pathogenesis of di- arthea production. Other neurohormones have been described in amebic lysates, including sub- stance P, neurotoxin, and acetylcholine (61). Like serotoxin, these agents stimulate CI~ secretion in ‘mammalian intestine. IMMUNE REGULATION OF INTESTINAL TRANSPORT Local iminune regulation of intestinal transport may occur by release of inflammatory mediators that act as intestinal secretagogues. Examples in- clude prostaglandins, mast cell mediators (hist: mine, serotonin, and leukotrienes), phagocytic cell factors (chemotactic peptide), and immune cell products (platelet activating factor, proteases, and hydrogen peroxide) Prostaglandins Prostaglandins of the E and F series induce intes- tinal secretion of fluid and electrolytes, a process part mediated via cyclic AMP (62). Studies in a va riety of animal models of Salmonella typhimurium have implicated a role for local prostaglandin pro- ‘duction in the intestinal fluid and electrolyte losses. In rabbits (47,63) and rhesus monkeys (64), invasive strains of Salmonella typhimurium induce mucosal destruction with severe inflammation in association with fluid and electrolyte secretion. Mucosal levels of cyclic AMP and adenylate cyclase are increased (63), suggesting a cyclic AMP-mediated secretory response. Indomethacin inhibits the intestinal se- cretion without altering the degree of mucosal in- flammation (65-67). Furthermore, inhibition of the polymorphonuclear neutrophil response with prior J Pedi Gastoetrl Nutr Yo. 2, No. 1 19818 E. V. O'LOUGHLIN ET AL nitrogen mustard treatment reduces the inflamma- tory response and intestinal uid secretion indepen- dently of any effect on intestinal morphology or CI~ secretory capacity (68). The findings indicate that cyclic AMP-mediated intestinal secretion induced by inflammatory mediators plays a role in some forms of invasive enteritis (69). This mechanism, however, does not provide a universal explanation for diarrhea production in all invasive infections. Other invasive organisms, such as Yersinia entero- colitica (10,71) and Treponema hyodysenteriae (72), 2 pathogen in pigs, both induce invasive disease ‘with widespread mucosal inflammation without in- ducing intestinal fluid and electrolyte secretion. Prostaglandins have also been implicated in the secretion induced by cholera toxin (65,67). Both as- pirin and indomethacin inhibit cholera toxin- induced secretion, an effect that appears to be in- dependent of cyclic AMP-mediated secretion (73,74). Indomethacin does not inhibit the cholera toxin increase in tissue cyclic AMP levels, and the later effects of cholera toxin and prostaglandin E on mucosal cyclic AMP are additive (19,67). The find ings suggest that indomethacin and aspirin antago- nize the effect of cholera toxin via a mechanism that, is independent of cyclic AMP-mediated Cl~ secre- ion, Similarly, prostaglandin inhibitors reduce in- testinal secretion induced by E. coli ST (75) but the role, if any, of prostaglandin in ST-induced secre- tion remains to be determined Colitis and diarrhea secondary to Clostridium aif- {ficile infection appear to result from two toxins (76). Toxin A, an enterotoxin, causes fluid secretion ‘when injected into rabbit ileal loops and toxin Bis a potent cytotoxin. Toxin A induces a severe enteri- tis, striking infiltration of the lamina propria with neutrophils, and increased production of pros- taglandins E, and leukotriene By. The secretory ef- fect appears to be mediated via products of the i flammatory cells since toxin A by itself does not induce secretion in Ty, cells (a colon Ca cel line) (77). Another possible origin of these secretagogues is from cells in the lamina propria. Under nonin- flammatory conditions, the subepithelium produces 80-90% of the arachidonic acid metabolites in intes- tinal mucosa in concentrations high enough to mod- ulate epithelial transport (78,79). However, itis not known whether these cells are responsible for the production of inflammatory mediators and secreta- gogues during pathological states such as infection. It remains to be determined if these toxins are si nificant in human C. difficile infection, Pedi Garoentrl Nut, Vo 12 No 1, 1991 Mast Cell Mediators Altered intestinal fluid and electrolyte secretion occurs during intestinal parasitic infestation. In vivo perfusion studies of rats infected with Nippo- strongylus brasiliensis (80) oF Trichinella spiralis (81) demonstrated net secretion. This effect appears 10 be due to intestinal anaphylaxis. Larval antigenic proteins elicited electrogenic CI~ secretion in j Jjunum from guinea pigs previously infected with Trichinella spiralis when studied in vitro under short-circuited conditions (82). However, a similar Fesponse was elicited in noninfected guinea pigs passively immunized with serum-containing anti- tichinella homocytotrophie antibodies of the IgE or IgG subclasses, but not in controls. The changes in electrical parameters and CI~ secretion were found to correlate with mast cell degranulation and hista- ‘mine and prostaglandin release (83). However, ‘other mast cell mediators such as substance P, va- soactive intestinal protein (VIP), leukotrienes, and serotonin are also capable of inducing intestinal se- cretion and conceivably may play a role in diarrhea production. It is also conceivable that mast cell products could modulate the enterie nervous sys- em and thereby alter transport. ‘The interaction between the immune system and the enterocyte during infectious gastroenteritis is an area of great interest. Little research has been done in this field particularly with respect to invasive bacterial infections. Understanding the interaction between immune cells and the enteroeyte will pro- vide new insights into mechanisms of host defense and hopefully will provide future opportunities for therapy, DECREASED DIGESTION AND ABSORPTION Abnormal digestion and absorption of nutrients particularly of dietary carbohydrates can produce diarrhea. Maldigestion and malabsorption occur in infectious insults that alter enterocyte differentia tion and that destroy the epithelial surface. Disorganized Epithelial Renewal: Viral Gastroenteritis Human rotavirus, initially recognized in the duo- denal mucosa of infants and young children with acute gastroenteritis (84,85), is now recognized as the commonest cause of gastroenteritis (86). Infor- mation on the pathophysiological studies of diar-PATHOPHYSIOLOGY OF INFECTIOUS DIARRHEA 9 thea in viral enteritis is derived primarily from pig- let models of transmissible gastroenteritis virus (TGE). Clinical studies of human rotavirus infection (85) and observations in piglets infected with human rotavirus (87) indicate a similar pattern of disease. Piglets infected with TGE develop diarrhea within 2440 h of infection. Initial studies suggested little morphological damage in the jejunum 40 h postinfection (88). Subsequent studies, however, in proximal and midjejunum and ileum demonstrated a range of morphological abnormalities throughout the small intestine (88,89). At 40 h, the mucosal lesion varied from mild partial to severe villus atro- phy and crypt hyperplasia. This was associated with autoradiographic evidence of accelerated turn- over of epithelial cells and a shortened enterocyte life span (88,90). Immune fluorescence microscopy identified TGE virus in villus epithelium in the up- per intestine within 12 h of infection (90). Antigen was consistently detected in all small intestinal re sions for 24 h aller infection. However, at 24 to 40 h postinfection, when diarthea was most severe, few virus particles remained in the epithelium. Electron microscopic studies demonstrated viral particles in the villus enterocytes but not crypt cells. up to 24 h postinfection. At 40 h, viral particles were not apparent and villus surface enterocytes had irregular sparse microvilli, apical nuclei, and fewer organelles in the apical cytoplasm, features ‘more in keeping with crypt enterocytes. ‘Mucosal enzyme activities in tissue from infected piglets also demonstrated a pattern of abnormalities, Consistent with an epithelium composed of imma- ture cryptslike enterocytes (91,92). Mucosal disac- ccharidase activities, alkaline phosphatase, and so- dium-potassium adenosine triphosphatase (Na, K*-ATPase), which are present in high concentra tions in mature enterocytes (91), were depressed in ‘mucosal homogenates and isolated villus cells from. all regions of the small intestine (93,94). Further more, thymidine kinase, a marker of proliferating crypt cells, was found to be markedly increased in villus enterocytes. These latter findings are in keep- ing with the morphological studies and suggest f ure of normal enterocyte maturation during migra- tion from the crypt to the villus. In vivo studies in infected piglets (88) using a marker perfusion technique demonstrated de- creased Na* and Cl~ absorption at the height of diarrhea. When studied in vitro under short- circuited conditions in Ussing chambers, infected tissue had decreased Na*Cl~ absorption (87,89, 94,95). Chloride secretory capacity was intact (04,95). militating against a role for cyclic AMP- mediated Cl~ secretion in this disease, Further- more, Na” efflux from isolated enterocytes is de- creased in TGE both inthe presence and absence of external slicose (89,95). Defective glucose absorp- tion appears to be the major transport abnormality In vivo glucose absorption is decreased in the jeju- rum in infected piglets (88). Studies in vitro dem- onstrated failure of glucose to stimulate Na* ab- sorption in both infected jejunum and ileum (87, 89,94,95) and markedly reduced 3-O-methyl-p- hicose absorption in the jejunum (96). Further- more, phlorizin, a potent inhibitor of glucose trans- port at the brush border, had no effect on 3-O- methyl-p-glucose flux, suggesting a lack of jejunal brush border receptors for glucose transport, a find- ing that was confirmed in jejunal brush border mem brane vesicles (97). The high-affinity brush border Deglucose transport system seen in control animals was absent in tissue from infected animals. Im- paired amino acid absorption has also been detected (98). These functional studies indicate that viral infection causes an inhibition of non-electrolyte- stimulated Na" Cl~ absorption and a marked im- pairment of glucose and amino acid-stimulated Na* absorption inthe small intestine of infected animals ‘A model for the pathophysiology of diarthea in viral enteritis is depicted in Fig. 2. Virus invades the mature villus enterocytes, causing increased des- quamation of villus cells. This leads to increased proliferation of crypt epithelium and migration rate. ‘This results in vill that are clothed with immature cxyptike enterocytes. This crypt-lke epithelium is deficient in brush border hydrolytic enzymes, brush border solute carriers, and basolateral Na”, K*- ATPase. These abnormalities lead to decreased electroneutral Na’ Cl” absorption, sodium- coupled solute absorption, and carbohydrate diges- tion and absorption, which contribute to the os- motic diarthea. Damage to the Absorptive Surface Brush Border Damage Damage to the absorptive surface occurs in a number of bacterial and parasitic infestations. This damage may result primarily from specific brush border damage or a more severe epithelial destruc- tion. Enteropathogenic E. coli are noninvasive Pediat Gastroenterol Net, Vo. 2, No.1 19110 E. V. O'LOUGHLIN ET AL. ie J FIG. 2. Pathophysolagie mechanisms of rtavirus-induoed diarrhea. Virus invades the mature enterocytes, which dae: quamate, to be replaced by immature erypt epithelium (Shaded). These cells ae ceiclont in brish border hysraytic ‘enzymes (not depicted) and solute (eg, glucose) coupled Na” absorption and neutral NaCl absorption, strains associated with diarthea, growth failure, and dehydration in infants (99). Diarthea is aggravated by the ingestion of oral nutrients, suggesting a mal- digestive or malabsorptive component. Morpholog- ical examination of jejunum from infected infants demonstrates moderate to severe atrophy, with in- ‘flammatory infiltrate and profuse adherent bacteria. Electron microscopic studies reveal bacteria adher~ cent to mucosal cells with loss of overlying glycoca- lyx and generalized shortening of microvilli (100- 103). In addition to the morphological abnor- malities, intestinal disaccharidase activities are de- creased, indicating a functional abnormality. A very similar appearance has been described in rabbits (RDEC-1) (104). This rabbit model closely parallels the human disease (105,106) and a Shi- gellaclike toxin has been identified in these strains (107,108). The ultrastructural findings in both the human and rabbit models suggest that abnormalities of digestive and absorptive function are due to beush border damage. Recent studies in the rabbit model of RDEC-1 indicate that this infection inhib- its electroneutral Na” CI” absorption in the cecum bbut does not induce CI~ secretion (109). Unfortu- nately, functional studies of small intestinal ion 4 Pediat Gatontrl Nutr, Vo. 12, No 1, 1991 transport and nutrient absorption do not appear to have been done. Brush border damage also occurs with Giardia lamblia infestation in humans. While studies of gestive and absorptive function are limited, some data are available from humans and observations from a mouse model of Giardia muris. Morpholog- ical examination of small intestine from infection Patients reveals a spectrum of change from normal mucosa to severe villous atrophy (110-116,120, 121). Abnormalities of lactose, sucrose, p-xylose, and triglyceride absorption, mucosal disaecharidase activities, and fecal fat excretion occur in a propor- tion of symptomatic individuals (112-114,117), ‘These parameters improve following successful therapy. Similar abnormalities are detected in a mouse ‘model of Giardia muris. Histological studies reveal at least mild villous atrophy in the jejunum (118) and crypt hyperplasia with increased turnover of epithe- lial cells (118,119). A pronounced disaccharidase deficiency is detected by 2 weeks postinoculation, corresponding to the maximal parasite load, Abnor- ‘mal disaccharidase activities are thought to be sec- ondary to direct brush border injury rather than fail- ure of enterocyte differentiation. Diffuse brush bor- der damage is also seen in a rabbit model of Yersinia enterocolitica and is discussed in more detail be- low. Cytotoxin Production Severe epithelial damage occurs in a number of invasive and cytotoxin-producing bacteria, e.g., Salmonella (64,66-68) and Shigella (120-126). Shi ella organisms induce a spectrum of disease in hi ‘mans and monkeys ranging from acute watery diar- thea to a fulminant dystenteric syndrome (120,121), This organism elaborates a toxin that disrupts the epithelium by inhibition of protein synthesis (122) However, in vivo perfusion studies of the small in- testine in monkeys infected with Shigella flesneri (121) revealed jejunal secretion of fluid and electro- Iytes. This occurs in the absence of morphological abnormalities, suggesting the possibility of a role for an enterotoxin, Shiga toxin has a similar struc- ture to cholera toxin (123). Some earlier studies sug gested that crude culture filtrates can induce secre tion with an associated increase in mucosal cyclic AMP (124,125). More recent studies, however, do not support a role for cyclic AMP-dependent Cl-PATHOPHYSIOLOGY OF INFECTIOUS DIARRHEA u secretion in the pathogenesis of diarrhea. Kandel and others (126) studied the effect of purified Shiga toxin on in vivo and in vitro electrolyte transport in rabbit jejunum. In ligated loops, toxin induced se- cretion that, when examined further in vitro, was due to inhibition of Na* CI~ absorption rather than stimulation of electrogenic CI” secretion. Toxin did not inhibit theophylline-induced CI” secretion, sug- gesting that a cyclic AMP-mediated mechanism was not involved. Further studies in isolated villus and crypt enterocytes were undertaken to localize toxin action, Villus cells had a greater content of Shiga toxin receptor, more binding sites, and were more sensitive to toxin-induced inhibition of protein syn- thesis. Thus, Shiga toxin exerts a preferential effect ‘on villus enterocytes and inhibits NaCI” absorp- tion. While this enterotoxic effect of Shiga toxin is not essential for the development of dysentery, it may mediate the watery diarthea seen in many pa- tients preceding the onset of bloody diarrhea. The impact on nutrient digestion and absorption has not been clearly defined in cytotoxin-producing infec- tions. Shiga toxin impairs in vitro absorption of glu- cose in rabbit ileum, which parallels the epithelial disruption induced by the toxin (126,127). Epithelial Invasion Recent studies in a reproducible animal model of Yersinia enterocolitica infection indicate that both epithelial destruction and brush border damage con- tribute to symptom production. New Zealand white rabbits infected with a human pathogenic strain of Yersinia enterocolitica develop weight loss and arthea (70). ‘The pathological injury, characterized by patchy microabscess formation, is present by day 3 throughout the small intestine, cecum, and proximal colon. By day 6, the injury is more severe in the ileocecal region and is associated with the partial villus atrophy and crypt hyperplasia. Mu- cosal disaccharidase activities are decreased in all regions of the small intestine but more severely so in the ileum (70). By day 14, enzyme activities have recovered in the jejunum but not the ileum. Elec- tron microscopy of infected small intestine demon. strates a marked reduction in microvillus brush bor der height and surface area (128) commensurate with the degree of disaccharidase impairment (Fig. 3). The major abnormality identified in intestinal transport is abnormal glucose absorption. lleum and jejunum from infected animals demonstrate de~ creased absorption of glucose and glucose-coupled sogium in both in vivo and in vitro studies (71), There was no evidence of active ion secretion. In the colon, despite morphological damage, there was evidence of enhanced absorption, suggesting co- Ionic salvage. The results of these studies indicate that small intestinal maldigestion and malabsorp- tion, particularly of carbohydrate, appear to be the major factor contributing to diarrhea production. ‘While lesions are detected at the light microscopic, level in small intestine, the most dramatic morpho- logical abnormality is seen at electron microscopy. ‘The diffuse atrophy of the brush border appears to explain the abnormalities of mucosal disaccharidase activities and glucose transport. Intestinal helminth infestations also induce se- vere morphological damage, brush border abnor- malities, and malabsorption (129), Interpretation of human studies is difficult, however, since human helminth infestation is often complicated by the presence of malnutrition, bacterial overgrowth, coinfection with other organisms, and tropical en- teropathy. Studies in animal models of Nippo- strongylus brasiliensus (80,130,131), Trichinella spiralis (81,132,133), and Hematospiroides dubius (134-136) indicate that infection induces small in- testinal morphological damage, villous atrophy, ‘crypt hyperplasia, intestinal brush border enzyme abnormalities, and diminished glucose and amino acid absorption. ‘Thus, the evidence indicates that a number of common infections and infestations interfere with normal digestion and absorption. This leads to 0s- motic loss of fluid and electrolytes, primarily in the small intestine, Diarrhea ensues if the ability of the colon to salvage fluid and electrolytes is over- whelmed, DISORDERED TRANSIT Intestinal motor function is intrinsic to the pro- cess of normal digestion and absorption. Activity of the circular and longitudinal muscle layers within the intestinal tract (a) triturates solids, (b) is coor- inated with fluid, electrolyte, and digestive secre tion through neural and hormonal reflexes, (c) mixes food with digestive secretions and brings lu- minal contents into contact with the absorptive ep- ithelium, (d) alters the surface area of intestine ex- posed to luminal contents, and (e) regulates the rateR E. V. O'LOUGHLIN ET AL. FG, 2. Electron micrograph of rabbit leum from (a) normal ang (b) Yersinia enterocoltca-intected animals, Yersinia entero- Ccoltce infection induced a cituse and marked recuction In microwlus height (> 16000) of intestinal transit and therefore the contact time during which mucosal absorption may occur (137,138), Diarrhea is a very common symptom of disturbed. ‘gastrointestinal function. In the mid-1970s, rapid advances in the understanding of epithelial trans- port led some authors to state that a causal link between changes in normal intestinal motor activity and diarrhea had not been established (139). How- ever, clinical observations indicate that fluid will not traverse the intestinal tract unless it is propelled by contractile activity. The diarrhea resulting from increased secretion or decreased absorption must, result in a change in motility. In fact, distention of the intestinal lumen with fluid will initiate peristaltic motor complexes (140) and an increased rate of transit, While alterations of intestinal motor activity are observed as a secondary response to disordered mucosal transport, causing accumulation of Muid and luminal distention, they may also occur inde~ pendently as a primary pathophysiologic mecha- nism of diarrhea (141). An increased rate of transit decreases the opportunity for digestion and absorp- tion, which leads to an increase in stool water loss and stool frequency. An increase in the rate of tran- sit can result from either an increase in propulsive activity or a decrease in contractions, impeding flow (140). Alterations of the normal patterns of intestinal motor activity are now well described in a variety of experimental animal models of bacterial (141-159), parasitic (160-171), and viral (172) infectious enteri- tis, and have been associated with alterations in the rate of transit and diarrhea. While the subject of normal intestinal myoelectric or motor activity and its control mechanisms is beyond the scope of this article, several excellent reviews provide a more detailed discussion (173,174). Mathias and his colleagues (141,148) have de- scribed two patterns of abnormal intestinal myo- clectrical activity in rabbit animal models of infec- tious bacterial enteritis—the migrating action po- tential complex (MAPC) and the repetitive burst action of potentials (RBAP). The MAPC was first ‘observed in ligated ileal loops of anesthetized rab- bits after laparotomy in response to instillation into the loop of live Vibrio cholerae, whole cell lysate of cholera, or purified enterotoxin (141). The MAPCPATHOPHYSIOLOGY OF INFECTIOUS DIARRHEA 2B was defined as an action potential discharge with a duration of 2.5 s or longer that propagated in an aboral direction over a distance of at least 2.5 em with a velocity of approximately 1 cm/s. The motor correlate of this activity was a propagating ring con- traction that was observed to propel intraluminal contents in an aboral direction. MAPCs were re~ stricted 10 the loop challenged and could not be reproduced by bolus injections of saline, which sug gested a localized control mechanism that was not just a response to enterotoxin-induced secretion and luminal distention. Tetrodotoxin (a blocker of, neural conduction), scopolamine hydrobromide (a muscarinic cholinergic antagonist of the smooth muscle receptor for acetylcholine), and trimetha- phan camsylate (an antagonist of nicotinic cholin- ergic receptors on ganglionic neurons within the en- teric nervous system) abolished the MAPC (142). Further studies subsequently demonstrated that cholera-exposed rabbit ileum exhibited MAPCs in vitro in the absence of central neural or hormonal influences (143), These findings are consistent with the hypothesis that a sensory-motor reflex arc within the enteric nervous system is capable of or- sanizing and maintaining the MAPC. How cholera toxin stimulates this reflex arc is not understood. ‘The secretory response to cholera toxin depends upon coordination of activity of the A and B sub- units. It appeared initially that binding of the B unit, lus GM, receptors could induce ithout stimulating fluid production (144). finding could not be reproduced by others (145). The mechanism(s) for the stimulation. of MAPC activity may still be associated with the cyclic nucleotide-dependent action of cholera toxin. It is also possible that cholera toxin-induced pros- taglandin synthesis is part of the mechanism stimu- lating MAPCs (146). Treatment with indomethacin, 2 prostaglandin synthetase inhibitor, abolishes the MAPC activity induced by cholera enterotoxin. MAPCs have also been described as the predomi- rant motor abnormality in the ligated ileal loop in response to enterotoxigenic E. coli and its heat- labile enterotoxin (147), and in the uninfected small intestine proximal to the ligated loop infected with invasive E. coli (148), Shigella (149), Salmonella ‘typhimurium (152), C. difficile (153), and Campylo- acter jejuni (155). ‘The mechanism of RBAP induction is unclear. Since this motor pattern oceurs in response to in vasive or cytotoxic enteritides, it is possible that inflammatory mediators released in response to lo- cal tissue injury regulate the response. Invasive E. coli (148) did not, but Shigella (149) did increase RBAP activity in the uninfected small bowel prox- imal to the ligated and infected ileal loop. The ap- pearance of RBAP in the proximal uninfected intes- tine is consistent with local circulation of absorbed toxin, release of a local mediator as a result of tissue invasion and inflammation, or the activation of a neural reflex (149). A reflex response induced by loop distention is unlikely since fluid accumulation was not observed, and loops infused with saline did not manifest alterations in motor activity. ‘Many of the bacteria that stimulate RBAP activ- ity appear to release a factor(s) that mediates the response. The florid cytotoxicity and alterations in myoelectric activity (MAPCs and RBAPs) seen in. the rabbit ileal loop in response to Shigella dysen- teriae are not induced by Shigella enterotoxin. However, they can be induced by a biologically ac- tive substance(s) that is present in a cell-free lysate of Shigella from which the enterotoxin has been removed (156). C. difficile produces a heat-labile enterotoxin that induces tissue culture assay posi= tivity, and a cytotoxin that causes in vivo mucosal injury. However, itis neither the cytotoxin nor en- terotoxin but an unidentified heat-labile sub- stance(s) in the crude culture filtrate that induces MAPCs and RBAPs. A high-molecular-weight fi tration product of the crude culture filtrate induced mucosal damage and significantly more RBAP ac- tivity than any other agent studied (153). The RBAP. activity induced by Campylobacter jejuni appears to be the result of a heat-stable substance with cy= totoxic activity (155). Certain strains of E. coli also produce a heat-stable enterotoxin that induces both, RBAP activity and a secretory effect, the latter by activating guanylate cyclase and increasing intra cellular cyclic GMP (137). The induction of RBAP activity appears to depend upon a mechanism other than the activation of the guanylate cyclase system, since cyclic 8-bromo-GMP induces secretion in the absence of RBAP activity In general, the MAPC is the predominant myo- electric and motor abnormality seen in association with secretory diartheas produced by noninvasive organisms and their enterotoxins, while the RBAP. is the predominant motor abnormality associated with the invasive or cytotoxic bacterial enteritidies that typically cause less diartheal output and more abdominal pain (155). It has also been suggested that the MAPC, which is a propulsive motor com- plex, is a motor defense mechanism that facilitates J Pedi Gasvoemrol Nutr, Val 12, No.1, 191“4 E. V, O'LOUGHLIN ET AL. clearance of offending toxins from the gastrointes- tinal tract (141), and that the RBAP, which is more frequently stationary, enhances the virulence of a bacterium by promoting stasis and facilitating pro- liferation of mucosal attachment and invasion (149), Determination of the specific role of the MAPC and RBAP in the pathogenesis of diarrheal illness is hampered by the fact that the studies have been, performed in isolated loops of anesthetized animals after laparotomy—conditions known to inhibit or abolish the normal patterns of motility seen in the conscious animal. Studies of the effect of cholera toxin on intestinal motor activity in conscious dogs indicate that cholera toxin significantly decreases the migrating motility complex (MMC) cyclic pe- riod (increased frequency of MCs), increases the percent of the MMC cycle occupied by irregular contractile or phase II activity, and in the fed state acts to decrease the propagation distance of phasic contractile activity (158). MAPCs were not ob: served; however, this might be a species difference. Scott et al. (159) studied the effect of an invasive Yersinia enterocolitica enteritis on ileal motor ac- tivity in conscious rabbits. They found a significant decrease in cycle period of the MMC (increased frequency of MMCS), increase in the duration of the period of continuous contractile activity or phase IIL of the MMC, and a significant increase in the rate of aboral transit. MAPC- and RBAP.like myo- electric complexes were present in control record- ings, and actually decreased in frequency in re- sponse to infection. Additional studies are needed in conscious animals and in humans before we as- cribe significance to the role of the MAPC and BAP in the pathogenesis of bacterial diarrhea. Parasitic Enteritis Alterations in intestinal motor activity have been described in association with a variety of parasitic infestations and appear to be part of an adaptive response contributing to expulsion of the organisms (161-165). Infection with coccidiosis (Bimeria mag- na) caused significant changes in the pattern of je- Junoieal motility between 4 and 12 days postinfec~ tion in conscious rabbits (160). There was an initial disruption and replacement of the MMC by a series of spike bursts occurring at 0.5-2 min intervals This was followed by a strong inhibition of spike activity from the 8th to the 12th days postinfection and a return to normal control levels by 4 days. Primary infection with the nematode parasite J Pediatr Gastroenterol Nur Vl 12, No.1, 1981 Trichinella spiralis is associated with an increased rate of transit in mice (161,162) and rats (163). The changes in intestinal myoelectric activity associated with both primary and secondary infection with this, organism have been studied in the rat (163,164) and in the dog (165). In the rat, primary infestation re- sulted in a significant decrease in slow wave fre- quency and myoelectric spike activity, and a signif- icant increase in the number of MAPCs and the interval between MMCs between 2 and 12 days postinfection, A secondary infection induced simi- lar changes within 15 min of challenge, presumably the result of an intestinal anaphylactic reaction in sensitized intestine. In dogs, primary infection caused similar changes with a decrease in amplitude and disruption of slow wave pattern, a general de- crease in myoelecttie spike activity, an increase in the interval between MMCs, and most notably the appearance of MAPC-like aborally propagating spike potential complexes. Trichinella spiralis infection not only induces changes in myoelectric and motor activity, but al- ters the mass, the contractility, or responsiveness of the smooth muscle in the intestinal tract. There is, «significant hypertrophy (166) and a differential re- sponse of longitudinal and circular muscle to the inflammation associated with infection (167,168). Longitudinal muscle exhibited a shift in the length tension relationship, with an increase in the maxi- ‘mum tension developed in response to stimulation with carbachol, while cireular muscle showed no shift in the lengthtension relationship but a de- crease in the maximum tension developed in re- sponse to carbachol. ‘Nippostrongylus brasiliensis, a nematode, is an enteric parasite of rodents. In rats, infection is as- sociated with diarrhea and an increased rate of in- testinal transit (169). It is not known what changes this infection causes in intestinal myoelectric and motor activity. However, the inflammatory re- sponse is associated with an increased maximum contractile response to several agonists including carbachol and a change in the muscles’ caleium- handling properties (170,171), Viral Gastroenteritis ‘The intestinal myoeleetric and motor abnormali- ties induced by a viral gastroenteritis have been studied in conscious piglets infected with a trans- missible gastroenteritis virus (coronavirus) (172). Infection induced a decrease in bursts of MAPC-PATHOPHYSIOLOGY OF INFECTIOUS DIARRHEA Is like spike activity and disrupted the periodicity of the MMC (Fig. 4). The authors suggested that dis- ruption of normal propulsive jejunal motor activity contributed to the diarthea and vomiting seen inthis disease. ‘There is a growing body of literature that suggests that during infectious enteritis, disturbances of nor- mal intestinal myoelectric and motor activity coex= ist with disturbances in transport and contribute t0 the clinical symptom of diarrhea. The literature also suggests that differences in the nature of the motor patterns induced by infection depend upon whether the pathogen releases a secretagogue, causes epi- thetial damage and inflammation due to tissue inva~ sion or eytotoxic injury, or initiates an immune re~ sponse such as anaphylaxis. However, there is a reed for a great deal of additional research. While Mathias and colleagues have clearly indicated the possible role of altered motility in the pathogenesis. of infectious diarrhea, much of their work has been confined to alterations of myoelectric and motor ac~ tivity in the ileum of rabbits under anaesthesia after laparatomy—procedures known to inhibit normal motility. The motor response of the intestine may be far different in a conscious animal. Furthermore, there are marked species differences in normal mo- tility, and it is reasonable to expect species variabil- ity in abnormal patterns induced by infection. The ‘mechanisms mediating alterations in motility may also vary between species. The effect of infection fon other regions of the gut needs to be studied, Pathogens exhibit differences in the preferred site of enteric colonization, and motility changes may vary depending upon the region of gut affected. In addition, alterations in motility are not necessarily confined to the region of gut exposed to the inva sive, eytotoxie, or enterotoxic effects of an infec- tious agent. While increased secretion and accumu- lation of intraluminal fluid may stimulate alterations in motility, it appears that in some cases alterations : ‘mmol memoir 2 ih etnrnepneatatenmte epee FIG, 4. Representative tractings of jeiunal myoslectrical activity trom 9-day-o piglets in (a) control and (b) a piglet 2 days attr Infection with transmiseibio gastoentertis virus (TOE). Electrodes Positioned § cm apart beginsing 20 em dlstal to tho ligament of Trst. The control recording demonstrates a migrating motilty complox. The tracing fom the TGE-infected piglet ‘demonstrates disruption of the migrating molly complex wth fallure of propagation ofthe intanbe spike activity charactaristc ‘of phase 8 (Reproduced with permission from vel 172)16 E. V. O'LOUGHLIN ET AL. in motility can be independent phenomena. The ‘mechanisms require further elaboration. ‘The term "motility" embraces two concepts: al- ‘erations in the pattern of movement of the wall of the intestinal tract, and changes in the rate of tran sit. The science of fluid dynamics explores the in- terrelationship of these two parameters (173). In vitro techniques have been developed to observe the myoelectric and motor activity of isolated seg- ‘ments of gut and correlate them with the oral and aboral changes in pressure and flow that they gen- crate (174). Ultimately, it will be necessary to un- derstand these relationships in vivo, and studies will have to be performed in humans to determine whether the pathophysiology of infectious enteritis, in animal models accurately predicts the pathophys- iology of the human disease state ‘CONCLUSION “The experimental evidence outlined implicates @ numberof mechanisms in infection induced intest- nal fluid and electrolyte losses. The concept of in- testinal seeretion as final common pathway of di- arthea production has given way to @ broader view that incorporates other pathophysiological mecha- nisms, including maldigestion and malabsorption of nutrients and abnormal intestinal mol. Some of these pathologic events such as inereased secretion, altered brush border architecture, and disturbed motility may be host defense mechanisms rather than occurring as a direct result of the infecting or- ganisms, Diarrhea may be the payoff for the host's attempt t0 "flush out” the offending organism or prevent attachment and invasion of epithelial sur faces. Future strategies for the control of darzhealdis- case will be directed towards preventive measures Such as improved standards of hygiene, clean water supplies, and the development of vaccines. How- ever, itis unlikely that acute diarhea wil be com- pletely eradicated. Many patients, particularly chil Gren, wl require active therapy such as oral rehy- dration therapy and the use of pharmacological antigiartheal agents. 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