TRANSACTIONSOF THE ROYAL SOCIETY OFTROPICAL MEDICINE AND HYGIENE (1993) 87, SUPPLEMENT 3,17-21 s3117

Diarrhoeal disease:current concepts and future challenges

Pathogenesis of giardiasis

M. J. G. Farthing Department of Gastroenterology, St Bartholomew’s Hospital, West Smithfield, London, WCIA 7BE, UK

Abstract Giardiasis is the most common small intestinal protozoa1 infection and is found worldwide. The mechanisms by which Giardia duodenalis (=G. lamblia) produces chronic diarrhoea and malabsorption have still not been clearly defined. Many infections are associated with mild to moderate mucosal damage which, in animal models of infection, have functional correlates. Possible mechanisms include direct physical injury, release of parasite products such as proteinases or lectin, and mucosal inflammation associated with T cell activation and cytokine release. Other possible mechanisms of malabsorption include associated bacterial overgrowth and bile salt deconjugation, bile salt uptake by the parasite with depletion of intraluminal bile salts, and inhibition of pancreatic hydrolytic enzymes. Thus, there is no single mechanism to explain the diarrhoea and malabsorption caused by Giardia, which currently should be regarded as a multifactorial process.

Introduction Giardiasis is the most common protozoa1 infection of

the intestinal tract and is found worldwide throughout temperate and tropical locations (FARTHING, 1989; ADAM, 1991). Prevalence varies between 2% and 5% in the industrialized world and up to 20-30% in the develo- ping world. Although the clinical importance of this parasite has been debated since the beginning of this cen- tury, there is now indisputable evidence that Giardia duodenalis (=G. lamblia) can cause acute and chronic diarrhoea with intestinal malabsorption and may be re- suonsible for retarded growth and develonment in children (FARTHING et ;Z., 1986a; SULLIVAN et al., 1991). The maior clinical imuact of this narasite is in in- fantsand children during the first 3 yeais of life, in the undernourished, and in the immunocompromised.

Giardia cysts, which can be transmitted by direct per- son-to-person contact, water and food, are the infective form of the parasite. Following excystation, which is triggered by exposure to low pH and pancreatic hydro- lytic enzymes, trophozoites attach to the small intestinal epithelium, multiply by binary fission and thus effect colonization, predominantly in the proximal small intes- tine. The parasite completes its life cycle by encysting before leaving the host and once again entering the out- side environment. Encystation has been completed in vitro in the presence of increased concentrations of bile and high pH. Cysts can survive in the environment in cold moist conditions for weeks and possibly months.

Clinical manifestations of giardiasis Any hypothesis put forward to explain the pathogen-

esis of aiardiasis must be able to account for the clinical diversity of this infection, with respect to both its intesti- nal and extraintestinal manifestations. It should be able to take into account the broad spectrum of illness from asymptomatic carriage to severe chronic diarrhoea and malabsorption, the growth failure observed in some children and other well established, but less common, as- sociations such as lymphoid nodular hyperplasia, pro- tein-losing enteronathv and various other allergic and in- flammatory phenomena. Giardia accounts for-up to 7% of cases of acute diarrhoea and ll-45% of those with chronic diarrhoea (FARTHING, in press). At least 50% of symptomatic patients have biochemical evidence of car- bohydrate, fat and micronutrient malabsorption. A var- ietv of mechanisms has been described to explain these phenomena, which focus on (i) the intestinal mucosa and (ii) the intestinal lumen (KATELARIS & FARTHING, ig92).

Mucosal factors in the pathogenesis of diarrhoea Morphology

A variety of structural and functional abnormalities of

the small intestinal mucosa has been reported in humans and in animal models of giardiasis. In humans, Giardia can produce the complete repertoire of abnormalities of villous architecture, ranging from entirely normal light microscopical appearances, through partial to sub-total villous atrophy (YARDLEY et al., 1964; HOSKINS et al., 1967; ALP & HISLOP, 1969; AMENT & RUBIN, 1972; WRIGHT et al., 1977; DUNCOMBE et al., 1978; HART- LONG et al., 1979; ROSEKRANS et al., 1981; OBERHUBER & STOLTE, 1990). However, the majority of individuals have either normal or relatively mild villous shortening usually associated with an increase in crypt depth. Ex- perimental infections in gerbils, mice and rats can pro- duce similar abnormalities of mucosal architecture, al- though as in humans the abnormalities can often be mild (FAUBERT & BELOSEVIC, 1990). The gerbil provides a particularly good model to study small intestinal struc- ture and function, as weanling gerbils develop diarrhoea and have significant morphologTca1 abnormalities by day 6 of infection. with reduction in villous heirrht in the duodenum and increase in crypt depth in the diodenum, jejunum and ileum (BURET et al., 1992). In the ileum there is a small but significant increase in villous height. These early changes in villus and crypt morphology occur in the absence of any inflammatory infiltrate in the lamina propria and without an increase in the numbers of intraepithelial lymphocytes.

In human giardiasis, even when villous architecture appears normal bv hght microsconv, ultrastructural changes such as shorter&g and disru$on of the micro- villi are nresent (TAKANO & YARDLEY. 1965: HOSKINS et al., 1’967; M~RECKI & PARKER, 1967). The gerbil model has confirmed the significance of these observa- tions, demonstrating a marked reduction in microvillus membrane surface area in both jejunum and ileum, al- though these abnormalities were tr’ansient (BURET etal., 1992). This decrease in the height of microvilli was found uniformly and not specificilly related to sites of trophozoite attachment (BURET et al., 1992). From human studies there is evidence to suggest that the extent of the mucosal abnormality relates to the severity of the diarrhoea (WRIGHT et al., 1977; DUNCOMBE et al., 1978).

Disaccharidase activity In human giardiasis, morphological abnormalities have

been associated with reduction in lactase, sucrase and maltase activities in the microvillus membrane (DUN- COMBE et al., 1978; HARTONG et al., 1979). Similar ob- servations have been reported in experimental infection in mice, gerbils and rats (GILLON et-al., 1982; KHANNA et al.. 1988: BELOSEVIC et al.. 1989: BURET et al.. 1990: CEV~LLOS & FARTHING, 1992). Reduction in disacchari: dase activities are maximal when diarrhoea and villous morphological abnormalities are most pronounced.

S3118

Functional disorders Several studies in animal models suggest that there are

functional consequences of these structural abnormalities and the reduction in disaccharidase activity. In gerbils, basal transport of sodium and chloride ions was not dif- ferent from that in non-infected controls, but glucose- stimulated sodium absorption was significantly reduced in the ieiunum but not the ileum of infected gerbils in ex- periments using stripped small intestinal mucosa mounted in Ussing: chambers (BURET et al.. 1992). Per- fusion studies in iivo in animals have also shown im- paired water, sodium and chloride absorption in re- sponse to glucose, although basal transport was similar to that in controls (BURET et al., 1992). In a neonatal rat model of infection, basal transport of water, sodium and chloride ions was impaired, with some animals actually in a net secretory state for sodium and chloride ions. Per- fusion of a lactose-containing solution enhanced these transport abnormalities (CEVALLOS & FARTHING, 1992). Studies with brush border membrane vesicles from in- fected mice indicated that there is impairment of glucose and amino acid transport (SAMRA et al., 1987, 1988).

Mechanisms of mucosal inju y Structural and functional abnormalities in the small in-

testine can be detected in humans and experimentally in- fected animals, and it seems likely that these contribute, at least in part, to the diarrhoea and malabsorption asso- ciated with this infection. In experimental models, the abnormalities can be detected early in infection and in humans there appears to be some relationship between the extent of the structural abnormalities and the severity of diarrhoea (WRIGHT et al., 1977; DUNCOMBE et al., 1978). The mechanisms by which these abnormalities occur, however, are far from clear. Although there have been few reports of epithelial invasion (BRANDBORG et al., 1967; SAHA & GHOSH, 1977), Giardia is essentially a luminal enteropathogen and thus invasive episodes must be regarded as exceptional. Giardia trophozoites, how- ever, do attach to the epithelium and have been shown by electron microscopy to disrupt and distort microvilli at the site where the ventral adhesive disc interfaces with the microvillus membrane (ERLANDSEN & CHASE, 1974). Ventral disc imprints are particularly marked in the murine model, although they have also been reported in human infection. It seems unlikely, however, that the localized attachment sites can account for the widespread changes in microvillus membrane surface area observed in the small intestine (BURET et al.. 1992).

There is some evidence to sugiest that Giardia itself produces, and possibly releasesycytopathic substances into the intestinal lumen (SAMRA et al.. 1988’1. As vet no parasite product has been identified to’account for these changes in the intestine. Giardia does, however, contain a number of thiol proteinases which might attack surface glycoproteins and disrupt microvillus membrane inte- grity (HARE et al., 1989; PARENTI, 1989; NORTH et al., 1990). In addition, Giardia has been shown to express a surface mannose-binding lectin (FARTHING et al., 1986b) which is cleaved from a cytoplasmic precursor molecule by trypsin (LEV et al., 1986) and can mediate attachment to enterocytes (INGE et al., 1988). Dietary plant lectins can directly damage intestinal epithelial cells and pro- duce microvillus membrane abnormalities very similar to those seen in giardiasis (LORENZSONN & OLSEN, 1982; DOBBINS et al., 1986). It remains to be established whether any of these parasite products are injurious to host enterocytes.

An alternative explanation has been put forward to ex- plain the reduction in disaccharidase activity and the as- sociated impairment of carbohydrate and electrolyte ab- sorption. The increase in crypt depth seen in human infections and animal models is generally associated with increased crypt cell production rate and more rapid mi- gration of enterocytes along the villus. In other condi- tions where this occurs, such as coeliac disease, it results

in the villus becoming populated by relatively immature enterocytes with reduced digestive and absorptive capa- cities. Increased proliferation has been confirmed in the gerbil model (BURET et al., 1992) but, using thymidine kinase activity as a marker of maturity, there was no evi- dence in the jejunum or ileum that the cells repopulating the villus were less mature than those in non-infected control animals. It seems likely therefore that the struc- tural and functional abnormalities observed in the micro- villus membrane relate to direct injury rather than an- other secondary mechanism which is causing crypt cell proliferation.

In human giardiasis there is a variable immune re- sponse within the mucosa, although infection is often as- sociated with an increase in the numbers of lamina pro- pria lymphocytes and intraepithelial lymphocytes (WRIGHT & TOMKINS, 1977; DUNCOMBE et al., 1978; ROSEKRANS et al., 1981; GILLON, 1985). These inflam- matory changes have been more difficult to reproduce in experimental models. However, there is compelling evi- dence that T cell activation alone can produce villous atrophy. The enteropathy occurring in intestinal graft- versus-host disease and rejection of transplanted intesti- nal allografts is characterized by villous atrophy, crypt cell hyperplasia and a lymphocytic infiltrate. Using human foetal small intestinal explants it has been possible partly to characterize the mechanisms involved, by activating T cells with either pokeweed mitogen or anti-CD3 antibody; both approaches produced villous atrophy, crypt cell hyperplasia and increased interleukin 2 production, confirming T cell activation (MACDONALD & SPENCER, 1988). Further support for this hypothesis has been obtained from studies of experimental G. murk infection in athymic nuinu mice (ROBERTS-THOMSON & MITCHELL, 1978). Despite prolonged infection! the al- teration of villusicrypt cell ratio is less severe in nuinu mice than in immunocompetent controls. When Iympho- cytes from the spleens of immunologically intact mice were iniected into athvmic infected mice. histoloaical ab- normalities in the small intestine became m&e pro- nounced. However, reduction in the villusicrypt cell ratio did occur in the immunocompromised mice before reconstitution, and thus it seems likely that T cell-inde- pendent mechanisms are also involved. In addition, im- munosuppression in mice results in more profound ef- fects on disaccharidase activities in conventional animals, indicating that epithelial damage is not solely dependent on immune function (KHANNA et al., 1988). Further- more, although intraepithelial lymphocytes are fre- quently increased in number in giardiasis, and have been incriminated in contributing to immune mediated dam- age in experimental G. muris infection., intraepithelial lymphocyte numbers increased after villus shortening and the decrease in brush border disaccharidases had al- ready occurred (GILLON et al., 1982). Whether Giardia lectin can act as a mitogen and directly activate T cells re- mains to be established. However, challenge of mice (previously infected with G. muris) with G. muris tropho- zoite extract resulted in a rapid decrease in disacchari- dase activity, which might have been immune mediated (DANIELS & BELOSEVIC, 1992). The effect was most marked in the genetically susceptible C3H/HeN mice.

Luminal factors in the pathogenesis of diarrhoea Bacterial overgrowth

There is some evidence to suggest that symptomatic giardiasis is associated with increased numbers of aerobic and/or anaerobic bacteria in the proximal small intestine. In one studv from India. 8 of 17 natients 147%) with stea- torrhoea had more than lo4 aerobic bacteria cultured from duodenal fluid, whereas none from a giardiasis con- trol group without steatorrhoea had bacterial overgrowth (TANDON et al., 1977). Three of the natients with stea- torrhoea also had anaerobes present: TOMKINS et al. (1978) found increased numbers of aerobic bacteria in 9 of 14’symptomatic overland travellers (64%) with giar-

s3/19

diasis returning to the UK, 2 of whom also had an- aerobes present. Bacterial overgrowth can produce archi- tectural abnormalities in the small intestine similar to those seen in giardiasis, and thus it may have a role in producing mucosal injury.

Bile salt deconjugation The removal of the glycine or taurine conjugate from

bile salts reduces their solubility in aqueous solution and thus reduces their efficacy in micelle formation within the intestinal lumen. In addition, free bile salts are mem- branotoxic and can cause intestinal secretion, thus poten- tially contributing to the pathogenesis of diarrhoea: TAN- DON et al. (1977) found evidence of bile salt deconiu- gation in all‘of their Indian patients with bacterial over- growth and in 40% of giardiasis controls without malab- sorption, but other studies have not confirmed this (HALLIDAY et al., 1988). Giardia does not itself have the ability to deconjugate bile sale (SMITH et al., 1981; HAL- LIDAY et aZ., 1988).

periments with animal models indicate that this is at least in part genetically determined. However, there is in- creasing evidence to suggest that Giardia isolates differ in genotype and phenotype (NASH et al., 1985; ANDREWS et al., 1989; MELONI et al., 1989; CARNABY et al., 1991; KORMAN et al., 1992). There is also evidence to indicate that Giardia isolates may interact differently with their host with respect to their ability to colonize the host and nroduce clinical disease (AGGARWAL & NASH. 1987: DASH et al., 1987; CEVAL~OS & FARTHING, 199i; UDE- ZULU et al., 1992).

Bile salt uptake by Giardia Bile and bile salts appear to make important contribu-

tions to the life cycle of the parasite. Mammalian bile at low concentration has been shown to stimulate parasite growth and reduce generation time, and thus may be an rmportant colonization factor for this parasite (FARTHING et al.. 1983. 1985: KEISTER. 1983). The effect can be re- produced iartially by the’addition of conjugated bile salts alone, which appear to increase uptake of choleste- rol and membrane phospholipid that the parasite is un- able to svnthesise de nova (FARTHING et al., 1985). More recently-it has been shown that parasites grown’in bile are larger than those grown in a bile free medium, and that the presence of bile alters antigenic expression by the parasite (KATELARIS et al., 1991a). High concentra- tions of bile salts trigger parasite encystation.

During the course of bile stimulation experiments it became apparent that the parasite was consuming conju- gated bile salts (FARTHING et al., 1985). Further studies showed that this was relatively specific for Giardia (HAL- LIDAY et al., 1988) and that uptake appeared to be occur- ring by a carrier-mediated., active transport process. The metabolic advantage of bile salt uptake for the parasite has not been defined, although bile salts do appear to be fully internalized into the cytoplasm and not sequestered in the surface membrane. Theoretically, consumption of host bile salts during chronic diarrhoea could deplete the bile salt pool and thus contribute to fat malabsorption by impairing micellar solubilization of ingested fats and de- creasing the effectiveness of pancreatic lipase, the action of which is bile salt-dependent.

Inhibition of hydrolytic enzymes Intraluminal concentrations of trypsin, chymotrypsin

and lipase have been shown to be reduced in symptomatic patients with giardiasis (GUPTA & MEHTA, 1973; CHAWLA et al., 1975; OKADA et al., 1983). There is no evidence that this is due to a failure of pancreatic exocrine secretion but it could be related to the more recent obser- vation that live Giardia trophozoites and trophozoite soni- cates inhibit trvnsin activitv and linolvsis in vitro (SMITH et al., 1981; KATELARIS et hZ., 199ib;‘SEow etpl:,‘l993). The mechanism by which the parasite inhibits these enzyme activities has not been established but could be re- lated to the direct interaction between a parasite product, such as its own proteinases, and the host enzymes. How- ever, the pancreas has a large functional reserve and the magnitude of the reduction observed in clinical studies is itself unlikely to account for malabsorption; however, it could contribute to the cascade of abnormalities that together impair the absorptive mechanisms of the gut and contribute to diarrhoea and malabsorption.

Phenotypic and genotypic variation in isolates Host susceptibility to Giardia appears to vary and ex-

Sequelae of giardiasis Nutritional insufficiency and growth failure have been

associated with giardiasis, although the mechanisms have not been studied in detail. In the early stages of infection re- duced food intake is likely to be a major contributor, al- though as the illness progresses and malabsorption and stea- torrhoea become more apparent loss of energy substrates through the gut will compound the problem. However, these mechanisms remain speculative since energy balance studies have not been performed in chronic giardiasis.

Protein losing enteropathy has been described in sev- eral case reports (KORMAN et al., 1990; SHERMAN & LIEBMAN, 1980) but from a recent survey of children in The Gambia it appears that this is uncommon and rarely of a degree that has a major clinical impact (SULLIVAN et al., 1992). Loss of protein through the gut can occur as a result of a breach in mucosal integrity following inflam- mation and/or direct damage associated with enterocyte loss. The precise mechanisms of protein losing entero- pathy in giardiasis have not been defined.

Occasionally allergic and other inflammatory phe- nomena have been described in criardiasis. Although im- mediate-type hypersensitivity is a relatively common as- sociation with helminthic infections. it is rare with protozoa. However, urticaria, arthralgia and other aller- gic phenomena have been described. Increased serum immunoglobulin (Ig) E concentrations have been re- ported, but in one study where this was investigated in more detail there was no evidence that the IgE was Giar- dia-specific, suggesting that the intestinal damage in- duced by the parasite merely facilitated parenteral sen- sitization with food or other luminal antigens (FARTHING et al., 1984). Lymphoid nodular hyperplasia has been as- sociated with both chronic giardiasis and immune defi- ciency. Several studies have examined the prevalence of giardiasis in patients with hypogammaglobulinaemia and found it to occur in 29-71% of cases (AJDUKIEWICZ et al., 1972; WEBSTER et al., 1977; NAGURA et al., 1979). However, in one study from India 25 patients were de- scribed, all of whom had giardiasis and lymphoid nodu- lar hyperplasia but none had immunoglobulin deficiency (WARD et al., 1983). Thus, the relationship between lymphoid nodular hyperplasia, hypogammaglobulin- aemia and giardiasis remains unclear, although it appears that any 2 can occur in combination without any direct implication for pathogenesis. There is no clear indication as to the pathogenesis of lymphoid nodular hyperplasia, although several studies have shown a predominance of B cells producing IgM within the mucosa and lymphoid noduies, suggesting that there might be immune ‘over- activity’ against a luminal antigen, possibly with failure to switch from IgM to IgA production within the intestine.

References Adam, R. D. (1991). The biology of Giardia spp. Microbiological

Reviews, 55,706732. Aggarwal, A. & Nash, T. E. (1987). Comparison of two anti-

genitally distinct Gzardia lamblia isolates in gerbils. American Journal of Tropical Medicine and Hygiene, 36,325332.

Ajdeukiewicz, A. B., Youngs, G. R. & Bouchier, I. A. D. (1972). Nodular lymphoid hyperplasia and hypogammaglo- bulinaemia. Gut, 13,589-595.

Alp, M. H. & Hislop, I. G. (1969). The effect of Giardia Zumblia infestation on the gastrointestinal tract. Australasian Annals of Medicine, 18,232-237.

S3120

Ament, M. E. & Rubin, C. E. (1972). Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes. Gastroenterology, 62,2 16-226.

Andrews, R. H., Adams, M., Boreham, I’. F. L., Mayrhofer, G. & Meloni, B. P. (1989). Giardiuintestinalis: electrophoretic evidence for a species complex. InternationalJournal of Para- sitology, 19, 183-190.

Belosevic. M.. Faubert. G. M. & MacLean. I. D. 11989‘1. Di- saccharidase activity h the small intestine of gerbils (M&ones unguiculatus) during primary and challenge infections with Giardia lamblia. Gut, 30,1213-1219.

Bradborg, L. L., Tankersley, C. B., Gottlieb, S., Barancik, M. & Sartor, V. E. (1967). Histological demonstration of mucosal invasion by Giardia lamblia in man. Gastroenterology, 52, 143- 150.

Buret, A., Gall, D. G. & Olson, M. E. (1990). Effects of murine giardiasis on growth, intestmal morphology and disacchari- dase activity. Journal of Parasitology, 76,403-409.

Buret, A., Hardin, J. A., Olson, M. E. & Gall, D. G. (1992). Pathophysiology of small intestinal malabsorption in gerbils infected with Giardia lamblia. Gastroenterology, 103,506-5 13.

Carnaby, S., McHugh, T. D. & Farthing, M. J. G. (1991). DNA fingerprinting of Giardia lamblia with the Ml3 bacte- riophage genome. Gut, 32, A596-A597.

Cevallos, A. M. & Farthing, M. J. G. (1992). Differences in functional mucosal damage between Giardia lamblia isolates. Gut, 33, S44.

Chawla, L. S., Sehgal, A. K., Broor, S. L., Verma, R. S. & Chhuttani, I’. N. (1975). Tryptic activity in the duodenal as- pirate following a standard test meal in giardiasis. ScandinavianJournal of Gastroenterology, 10,445-447.

Daniels, C. W. & Belosevic, M. (1992). Disaccharidase activity in the small intestine of susceptible and resistant mice after primary and challenge infections with Giardia muris. American Journal of Tropical Medicine and Hygiene, 46,382-390.

Dobbins, J. W., Laurenson, J. I’.! Gorelick, F. S. & Banwell, J. G (1986). Phytohaemagglutmin from red kidney bean (Phaseolus vulgaris) inhibits sodium and chloride absorption intherabbitileum. Gastroenterology,90,1907-1913.

Duncombe, V. M., Bolin, T. D., Davis, A. E., Cummins? A. G. & Crouch,, R. L. (1978). Histopathology in giardiasis: a correlation with diarrhoea. Australian and New Zealand Jour- nal of Medicine, 8,392-396.

Erlandsen, S. L. & Chase, D. G. (1974). Morphological alter- ations m the microvillus border of villous epithelial cells produced by intestinal micro-organisms. American Journal of Clinical Nutrition, 27,1277-1286.

Farthing, M. J. G. (1989). Host-parasite interactions in human giardiasis. QuarterlyJournal of Medicine, 70, 191-204.

Farthing, M. J. G. (in press). Giardiasis as a disease. In: Giardiat From Molecules to Disease and Beyond, Reynoldson, J. A., Thompson, R. C. A. & Lymbery, A. J. (editors). Walling- ford, UK: CAB International.

Farthing, M. J. G., Varon, S. R. & Keusch, G. T. (1983). Mammalian bile promotes growth of Giardia Zumbliu in axenic culture. Transactions of the Royal Society of Tropical Medicine and Hygiene, 77,467-469.

Farthing, M. J. G., Chong, S. & Walker-Smith, J. A. (1984). Acute allergic phenomena in giardiasis. Lancet, ii, 1428.

Farthing, M. J. G., Keusch, G. T. & Carey, M. C. (1985). Ef- fects of bile and bile salts on growth and membrane lipid uptake by Giardia lamblia: possible implications for pa- thogenesis of intestinal disease. Journal of Clinical Investigation, 76,1727-1732.

Farthina, M. I. G.. Mata. L.. Urrutia, I. I. & Kronmal, R. A. (1986;). Natural history of Giardia’mfection of infants and children in rural Guatemala and its imoact on ohvsical growth. AmericanJournal of Clinical Nutritidn, 43,393’-463.

Farthing, M. J. G., Pereira, M. E. A. & Keusch, G. T. (198613). Description and characterization of a surface lectin from Giar- dia lamblia. Infection and Immunity, 51,661-667.

Faubert, G. M. & Belosevic, M. (1990). Animal models for Giardia duodenalis type organisms. In,: Giardiasis, Meyer, E. &.($oditor). Amsterdam: Elsevier Science Publications, pp.

Gillon? J. (1985). Clinical studies in adults presenting with giar- cl!:;; ,to a gastromtestmal unit. Scottzsh Medzcal Journal, 30,

Gillon, J., Al Thamery, D. & Ferguson, A. (1982). Features of small intestinal pathology (epithelial cell kinetics, intraepithe- lial lymphocytes, disaccharidases) in a primary Giardia murk infection. Gut, 23,498-506.

Gupta, R. K. & Mehta, S. (1973). Giardiasis in children: a study of pancreatic functions. Indian Journal of Medical Re- search, 61,743-748.

Halliday, C. E. W., Inge, I’. M. G. & Farthing, M. J. G. (1988). Giardia-bile sale interactions in vitro and in vivo. Transactions of the Royal Society of Tropical Medicine and Hy- giene, 82,428-432.

Hare, D. F., Jarroll, E. L. & Lindmark, D. G. (1989). Giardia lambha: characterization of proteinase activity in tropho- zoites. ExperimentalParasitology, 68, 168-175.

Hartong, W. A., Gourley, W. K. & Arvanitakis, C. (1979). Giardiasis: clinical spectrum and functional-structural abnor- malities of the small intestinal mucosa. Gastroenterology, 77, 61-69.

Hoskins, L. C., Winawer, S. Y., Broitman, S. A., Gottlieb, L. S. & Zamcheck, N. (1967). Clinical giardiasis and intestinal malabsorption. Gastroenterology, 53,265-279.

Inge, I’. M. G., Edson, C. M. & Farthing, M. J. G. (1988). At- tachment of Giardia lamblia to mammalian intestinal cells. Gut, 29,795-801.

Katelaris, I’. H. & Farthing, M. J. G. (1992). Diarrhoea and malabsorption in giardiasis: multifactorial process. Gut, 33, 295-297.

Katelaris, I’. H., McHugh, T. D., Carnaby, S., Cavellos, A. M., Char, S. & Farthing, M. J. G. (1991a). Bile modulates genotypic and phenotypic characteristics of Giardia lambha. Gut, 32, A1260.

Katelaris, I’. H., Seow, F. & Ngu, M. C. (1991b). The effect of Giardia lamblta trophozoites on lipoylsis in vitro. Parasitology, 103,35-39.

Keister, D. B. (1983). Axenic culture of Giardia lambha in TYI- S-33 medium supplemented with bile. Transactions of the Royal Society of Tropical Medicine and Hygiene, 77,487-488.

Khanna, R., Vinayak, V. K., Mehta, S., Kumkum & Nain, C. K. (1988). Giardia lamblia infection in immunosuppressed animals causes severe alterations to brush border membrane enzymes. Digestive Diseases and Sciences, 33, 1147-l 152.

Korman, S. H.,, Bar-Oz, B., Mandelberg, A. & Matoth, I. (1990). Giardiasis with protein-losing enteropathy: diagnosis by faecal ort-antitrypsin determination. Journal of Pediatric Gastroenterology and Nutrition, 10,249-252.

Korman, S. H., LeBlancq, S. M., Deckelbaum, R J. & Van Der Ploeg, L. H. T. (1992). Investigation of human giardiasis by karybtype analysis. Journal of Clinical Investigation, 89, 1725-1733.

Lev, B., Ward, H., Keusch, G. T. & Pereira, M. E. A. (1986). Lectin activation in Giardia Zumblia by host protease: a novel host-parasite interaction. Science, 232,71-73.

Lorenzsonn, V. & Olsen, W. A. (1982). In vivo responses of rat intestinal epithelium to intraluminal dietary lectins. Gastroen- terology, 82, 838-848.

MacDonald, T. T. & Spencer, J. (1988). Evidence that acti- vated mucosal T cells play a role in the pathogenesis of enteropathy in human small intestine. Journal of Experimental Medicine, 167,1341-1349.

Meloni, B. P., Lymbery, A. J. & Thompson, R. C. A. (1989). Characterisation of Gzardia isolates using a non-radiolabelled DNA probe and correlation with the results of isoenzyme ana- lysis. American Journal of Tropical Medicine and Hygiene, 40, 629-637.

Morecki, R. & Parker, J. G (1967). Ultrasound studies of the human Giardia lumblia and subjacent jejunal mucosa in a sub- ject with steatorrhea. Gastroenterologv, 52, 151-164.

Nagura, H., Kohler, P. F. & Brown, W. R. (1979). Immunocy- tochemical characterization of the lymphocytes in nodular 14vom$p&oj!, hyperplasia of the bowel. Laboratory Investigation,

Nash, T. E., McCutchan, T., Keister, D., Dame, J. B., Con- rad, J. D. & Gillin, F. D. (1985). Restriction-endonuclease analysis of DNA from 15 Giardia isolates from humans and animals3oumal of Infectious Diseases, 152, 64-73.

Nash, T. E., Herrington, D. A., Losonsky, G. A. & Levine, M. M. (1987). Experimental human infections with Giardia lamblia. Journal of Infectious Diseases, 156,974-984.

North, M. J., Mottram, J. C. & Coombs, G. H. (1990). Cys- teine proteinases of parasitic protozoa. Parasitology Today, 6, 270-275.

Oberhuber, G. & Stolte, M. (1990). Giardiasis: analysis of histo- logical changes in biopsy specimens of 80 patients. Journal of Clinical Pathology, 43,641-643.

Okada, M., Fuchigami, T., Ri, S,., Kohrogi, N. & Omae, T. (1983). The PTPABA pancreatic function test in giardiasis. Postgraduate MedicalJournal, 59, 79-82.

Parenti, D. M. (1989). Characterization of a thiol proteinase in Giardia lamblia. Journal of Infectious Diseases, 160, 1076 1080.

Roberts-Thomson, I. C. & Mitchell, G. F. (1978). Giardiasis in mice. I. Prolonged infections in certain mouse strains and hy-

S3121

pothymic (nude) mice. Gastroenterology, X,42-46. Rosekrans, I’. C. M., Lindeman, J. & Meijer, C. J. L. M.

(1981). Quantitative histological and immunohistochemical findings in jejunal biopsy specimens in giardiasis. Vichows Archiv (Pathologic unddnatomie), 393, 145-151.

Saha, T. K. & Ghosh, T. K. (1977). Invasion of small intestinal mucosa by Giardia lamblia. Gastroenterology, ‘72,402-405.

Samra, H. K., Garg, U. C., Ganguly, N. K. & Mahajan, R. C. (1987). Effect of different Giardia lamblia inocula on glucose and amino acids transnort in the intestinal brush border mem- brane vesicles of infected mice. Annals of Tropical Medicine and Parasitology, 81,367-372.

Samra, H. K., Ganguly, N. K., Garg, U. C., Goyal, J. & Maha- jan, R. C. (1988). Effect of excretory-secretory products of Giardia lamblia on glucose and phenylalanine transport in the small intestine of Swiss albino mice. Biochemistry Inrerna- tional, 17,801-812.

Seow, F., Katelaris, I’. H. & Ngu, M. (1993). The effect of Giardia lamblia trophozoites on trypsin, chymotrypsin and amylase in vitro.

Sherman, I’. & Liebman, W. M. (1980). Apparent protein-los- ing enteropathy associated with giardiasis. American Journal of Diseases in Childhood, 134,893-894.

Smith, I’. D., Horsburgh, C. R. & Brown, W. R. (1981). In vitro studies on bile acid deconjugation and lipolysis inhibition by Giardia lamblia. Digestive Diseases and Sciences, 26, 700- 704.

Sullivan, I’. B., Marsh, M. N., Phillips, M. B., Dewit, O., Neale, G., Cevallos, A. M., Yamson, I’. & Farthing, M. J. G. (1991). Prevalence and treatment of giardiasis in chronic diar- rhoea and malnutrition. Archives of Disease in Childhood. 60. I I 304-306.

Sullivan, P. B., Lunn, P. G., Northrop-Clewes, C. A. & Far- thing, M. J. G. (1992). Parasitic infection of the gut and protein-losing enteropathy. 3ournal of Paediatric Gastroentero- lopv and Nutrition. 15.404407.

Tak&o, J. & Yardley,‘J. H. (1965). Jejunal lesions in patients with giardiasis and malabsorption. An electron microscopic

study. Bulletin ofJohns Hopkins Hospital, 116413-429. Tandon, B. N., Tandon, R. K., Satpathy, B. K. & Shriniwas.

(1977). Mechanism of malabsorption in giardiasis: a study of bacterial flora and bile salt deconjugation in upper jejunum. Gut, 18, 176-181.

Tomkins, A. M., Drasar, B. S., Bradley, A. K. & Williamson, W. A. (1978). Bacterial colonization of jejunal mucosa in giar- diasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72,33-36.

Udezulu, I. A., Visvesvara, G. S., Moss, D. M. & Leitch, G. T. (1992). Isolation of two Giardia lamblia (WB strain) clones with distinct surface protein and antigenic profiles and differ- ing infectivity and virulence. Infection and Immunity, 60, 2274-2280.

Ward, H., Jalan, K. N., Maitra, T. K., Agarwal, S. K. & Ma- halanabis, D. (1983). Small intestinal nodular lymphoid hyperplasia in patients with giardiasis and normal serum im- munoglobulins. Gur, 24, 120-126.

Ward, H. D., Lev, B. I., Kane, A. U., Keusch, G. T. & Per- eira, M. E. A. (1987). Identification and characterisation of taglin, a mannose-6-phosphate binding, trypsin-activated lec- tin from Giardia lamblia. Biochemistry, 26,8669-8675.

Webster, A. D. B., Kenwright, S., Ballard, J., Shiner, M., Slavin, G., Levi, A. J., Loewi, G. & Asherson, G. L. (1977). Nodular lymphoid hyperplasia of the bowel in primary hypo- gammaglobulinaemia: study of in vivo and in vitro lymphocyte function. Gut, 18,364-372.

Wright, S. G. & Tomkins, A. M. (1977). Quantification of the lymphoid infiltrate in jejunal epithelium in giardiasis. Clinical and Experimental Immunology, 29,408-412.

Wright, S. G., Tomkins, A. M. & Ridley, D. S. (1977). Giar- diasis: clinical and therapeutic aspects. Gut, 18, 343-350.

Yardley, J. H., Takano, J. & Hendrix, T. R. (1964). Epithelial and other mucosal lesions of the jejunum in giardiasis. Jejunal biopsy studies. Bulletin of Johns Hopkins Hospital, 115, 389- 406.