DISACCHARIDASE DEFICIENCIES IN GERBILS (MERIONES...

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DISACCHARIDASE DEFICIENCIES IN

GERBILS (MERIONES UNGUICULATUS)

IMMUNE TO GIARDIA LAMBLIA

by

SHAWN RASHEED MOHAMMED

A Thesis submitted to the Faculty of Graduate Studies and Research in

partial fui fi Il ment of the requirements for the degree of

Institute of Parasitology

McGill University, Montreal Quebec, Canada

Master of Science

<CShawn Mohammed August 1994

Nome SüawtJ R..AS..tf~"b MDHAtnIlÎ~~ . Dissertation Abstracts Intemational i~ arranged by broad, general sublect categories Pleo.e select the one sublect which most neor\y describes the content of your dis~rtahon Enter the correspondlrlg four-digit code ln the spaces provided

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SHORT TITLIl~:

DISACCHARIDASE DEFICIENCIES 1'N GIARDIA SIS

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TABLE OF CONTENTS

Title Page . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 • • • • i

Table of Contents .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . il

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Abrégé ................................... , .... , ... .. vii

Acknowledgements ... . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . ix

Thesis Office Statement ................................... x

Statement of Contribution .................................. xii

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII

List of Tables .. , ...................................... xv

List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X vi

GENERAL INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Taxonomy ....................................... 3

Life Cycle and Morphology ............................ 4

ln Vitro Cultivation of Giardia Trophozoites ... . . . . . . . . . . . . . .. 6

Animal Models for Human Giardiasis ...................... 8

Humoral Immune Responses ............................ 10

Cellular Immune Responses ............................ 13

Antigens of G. lamblia ............................... 16

Pathology ....................................... 18

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Disaccharidase Deficiencies in Giardiasis .... . . . . . . . . . . . . . . . . 21

MANUSCRIPT 1. DISACCHARIDASE

DEFICIENCIES IN MONGOLIAN GERBILS (MERlON ES

UNGUICULA TUS) PROTECTED AGAINST GIARDIA LAMBLIA ....... .. 24

ABSTRACT . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . 25

INTRODUCTION ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 26

MATERIALS AND METHODS ....................... .. 29

Parasites .................................... 29

Animals ...... . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . 29

Preparation of the G. lamblia

and E. histolytica Trophozoite Extracts . . . . . . . . . . . . . . . . . 30

• Quantification of Trophozoites in the Gerbil Small Intestine .... 30

Measurement of Intestinal Disaccharidases ............ . . . 31

Preparation of Intestinal Homogenate . . . . . . . . . . . . . . 31

Assay for Intestinal Disaccharidases . . . . . . . . . . . . ... . 31

Reagents .............................., 32

Collection of G. lamblia Excretory/Secretory Products ....... 32

Measurement of Protein Concentration ................. 33

Statistical Analysis . . . . . . . . . . . . . . . . .. ;. . . . . . . . . . . . 33

RESULTS ....................................... 34

Primary Infection with Live G. lamblia Trophozoites ........ 34

Challenge wlth Live G. lamblia Trophozoites .... . . . . . . . . . 36

• Challenge with the Soluble Extract of G. lamblia Trophozoites ...... 36

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Challenge with the Soluble Extract of E. histolytic:a Trophozoites ... 39

Challenge with G. lamblia Excretory/Secretory Products .. .... 41

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 43

REFERENCES .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 49

CONNECTING STATEMENT ............................... 52

MANUSCRIPT 2. IDENTIFICATION OF A FRACTION OF

GIARDIA LAMBLIA TROPHOZOITE EXTRACT ASSOCIATED

WITH DISACCHARIDASE DEFICIENCIES IN MONGOLIAN GERBILS ... 53

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 54

INTRODUCTION .............................. .... 55

MATERIALS AND METHODS ......................... 58

• Parasites .................................... 58

Anirnals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Preparation of the erude Extract of G. lamblia Trophozoites . . . . 59

Column Chromatography .......................... 59

Sodium Dodecyl Sulfate-

Polyacrylamide Gel Electrophoresis (SDS-PAGE) ........... 60

Measurement of Intestinal Disaccharidases . . . . . . . . . . . . . . . 60

Preparation of Intestinal Homogenate . . . . . . . . . . . . . . 60

Assay for Intestinal Disaccharidases . . . . . . . . . . . . . . . 61

Reagents ..........................,.... 61

Measurement of Protein Concentration ................. 62

• Statistical Analysis .............................. 62

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RESULTS ................... , ................... 63

Fractionation of the Soluble Extract of G. lamblia Trophozoites ..... 63

Challenge with the Fractions of the G. lamblia Soluble Extract. ..... 63

Fractionation of FI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Challenge with Fractions Fla and Flb ................. 68

SOS-PAGE .................................. 71

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... 73

REFERENCES ............ . . . . . . . . . . . . . . ......... 1 78

GENERAL DISCUSSION .................................. 81

REFERENCES ........................................ 86

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ABSTRACT

Studies using Mongolian gerbils found that during a primary infection with

Giardia lamblia trophozoites, disaccharidase activities were decreased from day 10

post-infection (p.i.) untiJ weIl past elimination of the parasite. However, during a

challenge infection, enzyme deficiencies were short-lived. A challenge with a soluble

extract of G. lamblia trophozoites also resulted in reductions in disaccharidase activity.

The degree of these reductions in ~nzyme activity was dependent on the extract dose.

Gel filtration of the trophozoite eTUde extract resulted in fractions FI, F2, and F3.

However, only a challenge with FI led to disaccharidase deficiencies. Further

separation of FI resulted in fractions Fla and Flb. Impairments of enzyme activity

were obtained only in gerbils challenged with Flb. Protein analysis of Flb revealed

several high and low molecular weight bands. When gerbils previously exposed to G.

lamblia were challenged with an extract of Entamoeba histolytica trophozoites,

disaccharidase activities remained comparable to controls. Moreover, enzyme levels

in gerbils challenged with excretory/secretory G. lamblia products were affected i:t a

manner which was inconsistent with the live parasitic chaI.lenge. Results suggest that

the disaccharidase deficiencies in giardiasis are parasite-specifie and are induced by a

heat-stable constituent(s) of fraction Flb, possibly through an immune resiJÜnse to an

antigenic component of this parasite fraction.

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ABREGE

Des études effectuées chez des gerboises mongoliennes ont montré que lors

d'une infection primaire avec des trophozoites de Giardia lamblia, l'activité des

disaccharidases diminue à partir du IDe jour après mfection jusqu'au-delà de

l'élimination du parasite. Cependant, lors d'une infection secondaire, la diminution de

l'activité enzymatique a été de courte durée. Une deuxième exposition, cette fois avec

un extrait soluble de trophozoites de G. lamblia, a résulté aussi en une réduction de

l'activité des disaccharidases. Le taux de réduction est lié à la dose de l'extrait. Les

fractions FI, F2 et F3 sont obtenues par filtration sur gel de l'extrait non-purifié de

• trophozoites. Cependant, seulement l'inoculation avec la fraction FI a induit une

déficience de l'activité des disaccharidases. Une séparation supplémentaire de la

fraction FI, a mené à deux autres fractions, soit Fla et Flb. La diminution de

l'activité enzymatique est observée seulement chez les gerboises inoculées avec la

fraction F lb. L'analyse protéique de la fraction Flb a révélé plusieurs bandes de poids

moléculaires variés. Lorsque les gerboises, précédemment infectées avec G. lamblia,

sont inoculées avec un extrait soluble de trophozoites de Entamoeba hisrolytica,

l'activité des disaccharidases est demeurée semblable à celle des gerboises témoines.

De plus, le niveau enzymatique était différent dans les gerboises inoculées avec les

produits secrémnt/excrétant de G. lamblia comparativement à celles infectées avec le

parasite vivant. Ces résultats suggèrent que la diminution de l'activité des

• disaccharidases dans les giardioses est spécifique à ce parasite. De plus, cette

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diminution est induite par un (des) composant(s), stable(s) à la chaleur, contenu~ dans

la fraction Flb, et est possiblement causée par une réponse immune à un ou des

composants antigéniques contenus dans cette fraction .

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ACKNOWLEDGEMENTS

A number of people were of assistance to me during my stay at the Institute.

Firstly, 1 would Iike to thank my research/thesis supervisor, Dr. Gaétan Faubert, for

his support and guidance throughout my graduate studies. Special thanks also go to my

advisors: Dr. Kris Chadee, for his advice and encouragement, and Dr. Dick MacLean.

ln addition, 1 would like to extend my appreciation to Dr. Elias Georges, Dr. James

Smith, Dr. Marilyn Scott and Dr. Charles Tanner for the help they provided.

Many thanks go to Darren Campbell, Dr. Nasreen Bughio, Dr. Wayne Butscher,

Vivian Lewis, Nancy Laporte, Norma Bautista-L6pez and Kis Djamiat', 1 for their

assistance, friendship and moral support during the many hours spent together in the

laboratory.

1 am grateful to animal-care technicians Susan Frappier, Brenda Lepitzki,

Martha Robinson and Joanne Tansey for the kind treatment of my many gerbils over

the years. My appreciation also goes out to Dr. George Lubega, Kathy Keller, Dr.

Bakela Nare, Dr. Carl Lowenberger, Dr. Dwayne Lepitzki, Elida Campos, Sil-king

Tse, Christiane Trudeau and Rosanne Séguin for their assistance in various ways. The

secretarial help of Mary LaDuke and Shirley Mongeau is sincerely acknowledged.

1 would like to express my gratitude to the Institute of Parasitology of McGill

University and Dr. Faubert for providing me with financial help.

Finally, 1 thank my parents and brother, Stephan, for their constant love, moral

support and encouragement.

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THESIS OFFICE STATEMENT

The following is cited in accordance with the regulations of the Faculty of

Graduate Studies and Research, McGiIl University:

"Candidates have the option, subject to the approval of their Department, of

including, as part of their thesis, copies of the text of a paper(s) submitted for

publication, or the clearly-duplicated text of a published paper(s), provided that these

copies are bound as an integral part of the thesis. If this option is chosen, connecting

texts, providing logical bridges between the different papers, are mandatory.

"The thesis must still conform to ail other requirements of the "Guidelines

Concerning Thesis Preparation Il and should be in a Iiterary form that is more than a

Mere collection of manuscripts published or to be published. The thesis must include,

as separate chapters or sections: (1) a Table of Contents, (2) a general abstract in

English and French, (3) an introduction which clearly states the rationale and objectives

of the study, (4) a comprehensive general review of the background Iiterature to the

subject of the thesis, when this review is appropriate, and (5) a final ove rail conclusion

and/or summary. Additional material (procedural and design data, as weil as

descriptions of equipment used) must be provided whcre appropriate and in sufficient

detail (eg. in appendices) to a110w a clear and precise judgement to be made of the

importance and originality of the research rcported in the thesis.

"In the case of manuscripts co-authored by the candidate and others, the

candidate is required to make an explicit statement in the thesis of who contributed

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to such work and to what extent; supervisors must attest to the accuracy of such

claims at the Ph.D. Oral Defense. Since the task of the examiners is made more

difticuJt in these cases, it is in the candidate's interest to make perfectly clear the

responsibilities of the different authors of co-authored papers."

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STATEMENT OF CONTRIBUTION

The experimental work reported herein (Manuscript 1 and Manuscript 2) was done

by Shawn R. Mohammed.

This thesis was written by Shawn R. Mohammed.

Dr. G. M. Faubert acted as researchlthesis supervisor.

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LIST OF FIGURES

MANUSCRIPT 1:

Figure 1. Disaccharidase activities over time in gerbils

with a primary infection of live G. lamblia trophozoites .... 35

Figure 2. Disaccharidase activities over time

in gerbils challenged with live G. lamblia trophozoites . . . . . 37

Figure 3. Disaccharidase activities in gerbils

challenged orally with varying amounts of the

soluble extract (antigen) of G. lamblia trophozoites 50

days after a primary infection with 1 x lO6 live trophozoites .... 38

MANUSCRIPT 2:

Figure 1. Chromatograph of the soluble

extract of sonicated G. lamblia trophozoites ........... 64


challenged with different fractions of

the soluble extract of G. lamblia trophozoites ........ .. 65


challenged with varying amounts of fraction

FI of the soluble extract of G. lamblia trophozoites ....... 67

Figure 4. Chromatograph of fraction FI of

the soluble extract of sonicated G. lamblia trophozoites .. .. 69


challenged with fractions Fla and Flb ...........•... 70

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• Figure 6. SOS-PAGE of the crude extract of G. lamblia

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tfophozoites and partially purified fractions of the extract ... 72

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LIST OF TABLES

MANUSCRIPT 1:

Table 1. Disaccharidase activities in gerbils

previously exposed to G. lamblia and challenged

with a soluble extract of E. histolytica trophozoites . . . . . . . . 40

'fable 2. Disaccharidase activities in gerbils

previously exposed to G. lamblia on day 4 post-

challenge with in vitro-released excretorylsecretory products ...... 42

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LIST OF ABBREVIATIONS

Ald Aldolase

BD Blue Dextran 2000

BSA Bovine serum albumin

ELISA Enzyme-linked immunosorbent assay

Fig. Figure

g Gravit y

• GVHR Graft-versus-host reaction

Hel Hydrochloride

HEPES N-2-hydroxyethy11,iperazine-N'-2-ethanesulfonic acid

HR High resolution

IEL Intraepithelial lymphocyte

IFN-ex/{J Interferon-ex/ {J

Ig Immunoglobulin

kDa Kilodaltons

MW Molecular weight

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n Number in sample

NK Natural killer

O.D. Optical density

Oval Ovalbumin

PBS Phosphate-buffered saline

p.c. Post-challenge

p.L Post-infection

RnA Ribonuclease A

• SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

S.E. Standard error

SEM Scanning electron microscopy

sIgA Secretory IgA

Tel. T cytotoxic/suppressor

Th T helper

TYI-S-33 Trypticase yeast extract iron seru!1l #33

U Unit

VSP Variant surface protein

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GENERAL INTRODUCTION

Giardia lamblia is a single-celled parasite capable of infecting the

gastrointestinal tract of humans, a~ '~ei1 a~ ')ther animais. This organism is endemic

throughout the world, with the highest prevalence occurring in the tropics and sub

tropics (Wolfe, 1992). The flagellated trophozoite form of the parasite colonizcs the

host's small intestine by attaching to the epithelium of the mucosal villi via its ventral

dise. Trophozoites may then become encysted within a protective wall and the resultant

cysts subsequently pass from the host in the feces. The cysts are transmitted throllgh

contaminated food or water, or direct person-to-person contact. Althollgh not fatal, the

morbidity associated with giardiasis can be considerable, especially in children. The

symptoms of giardiasis vary from none to chronic diarrhea with malabsorption. The

reason for the spectrum of clinical manifestations is not weil understood, but may

include differences in host factors, as weil as the pathogenicity of different G. lamblia

strains (Adam, 1991). Individuals may eliminate the parasite after a variable length of

time without therapy. However, a number of drugs, such as quinacrine, metronidazole,

and furazolidone, are effective in the treatment of infected patients (Wolfe, 1992).

Disaccharidase deficiencies have been a commonly reported manifestation of

giardiasis (Jennings et al., 1976), contributing to the mal absorption of nutrients seen

in this disease. The aim of this researeh was to examine the process by which G.

lamblia causes disaccharidase deficiencies in the small intestine of Mongolian gerbils

and, ultimately, to assist in exploring mechanisms for inhibiting the enzyme deficiencies

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and preventing the Jarger probJem of malabsorption. This, in turn, may help to further

define the relationship between the parasite and the host intestinal mueosa, and lead to

the development of strategies for interrupting the )ife cycle of G. lamblia. The short

term objectives of this project were to: (a) characterize the effects on disaccharidase

activity following primary and challenge inoculations with the live parasite and erude

extract of the trophozoites; (b) attempt to purify the fraction of the parasite responsible

for the decreases in disaccharidase activity; (c) determine the specificity of the effects

on disaccharidase activity in immune animais; and (d) examine the effects of G. lamblia

excretory/secretory products on disaccharidase activity.

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REVIEW OF LITERA TURE

Taxonomy

The tirst reported observation of G. lamblia is normally attributed to van

Leeuwenhoek, who described it in 1681. While van Leeuwenhoek undoubtedly saw

sorne sort of motile protozoan, the identification as Giardia has been a matter of debate.

It was next reported in 1859 by Lambl, and subsequently a large number of diffcrent

giardias were described (Ackers, 1980). Unfortunately there is much confusion

regarding the nomenclature of these protozoa. Both Giardia and Lamblia are used as

genus names. Giardia is the name used in the Western World and Lamblia is usually

used in Eastern European countries (Ackers, 1980). The number of species within the

genus Giardia has been a subject of controversy, with at least 40 having been

described. Many of these species are indistinguishable from each other on

morphological grounds (Faubert, 1988). Filice (1952) concluded that the only stable

morphological feature of the trophozoite is the median body, and he used the shape of

this structure to div ide the genus into three groups. They are G. agilis, which is found

in tadpoles and frogs; G. duodenalis (which includes G. intestinalis/G. lamblia), which

infects humans and other mammals; and G. muris, which is found in rodenls and birds.

In North America, G. lamblia is used to refer to the human parasite, whereas G.

intestinalis is often used in Europe. Studies, including work on cross transmission

using laboratory animais (Grant and Woo, 1978), average dimensions of trophozoites

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(Solovjev, 1975), and isoenzyme profiles (Bertram et al., 1983), are being used to

further eharaeterize Giardia inter- and intra-strain differences.

G. lamblia is plaeed 10 the foHowing taxonomie classification:

Phylum: Protozoa (Honigberg, 1964)

Subphylum: Sarcomastigophora (Honigberg and Balamuth, 1963)

Superclass: Mastigophora (Diesing, 1866)

Class: Zoomastigophora (Calkins, 1909)

Order: Diplomonadida (Wenyon, 1926)

Family: Hexamitidae (Kent, 1880)

Subfamily: Octomitinae (Von Prowazek, 1904)

Lire Cycle and Morphology

There are two stages in the life cycle of G. lamblia: the trophozoite, which is

the vegetative form, and the cyst, which is the infective form. After cysts are ingested,

gastric acidity induces excystment in the stomach. The trophozoites then move into the

small intestine and divide by binary fission, resulting in mature trophozoites. These

trophozoites attach to the epithelium of the gut by 10eans of their sucking dise and

evade enzymatic degradation by unknown mechanisms. Further down the smaU

intestine, encystation oecllrs on exposure to bile salts. During this proeess, the flagella

are 10st and a hyaline wall is secreted. Cysts are eventually excreted and are infective

to the next host after a latent period of 3 to 20 days (Shandera, 1990) .

The trophozoite of G. lamblia measures approximately 12-15 I-tffi in length and

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5

6~8 #Lm in width, and resembles a pear cut in half lengthwise. There arc two nuclei in

the anterior half of the organism. It exhibits bilateral symmetry and has four pairs of

flagella extending from basal granules at the anterior pole of the !)uelei (Fee\y ('t al.,

1984). The dorsal surface is convex and may have s1lght depressions rctaled to

underlying cytoplasmic vacuoles. Its ventral surface is concave and contains a structure

caUed the sucking disc, which is refined for adherence to surfaces. Transmission

electron micrographs show a microtubular network that seems to be associated with the

attachment disc, and a series of vesicles that line up along the dorsal surface and may

be associated with nutrition of the orgamsm (Stevens, 1982). The median body, a

structure unique to the genus Giardia, is located in the posterior half of the organism

and is described as claw~hammer in shape in G. lamblia trophozoites. This structure

is composed of microtubules and is of unknown function. The cytoplasm is known to

contain axonemes, glycogen particles, free ribosomes, rough endoplasmic reticulum and

vacuoles, but no mitochondria. In addition, a structure identified as a golgi apparatus

has been reported in in vitro encysting trophozoites (Reiner et al., 1990).

The cyst is oval in shape and is approximately 8-12 #Lm in size. The cyst wall

is approximately 0.3 #Lm thick and is composed of fibrillar elements (Sheffield and

Bjorvatn, 1977). The eyst eontains two to four nuclel, basal bodies and axoncmcs of

flagella, axostyles associated with caudal flagella, rough endoplasmic reticulum,

cytoplasmic masses, vacuoles, and fragmented pieces of the sueking dise and median

bodies (Feely et al., 1984). in addition, each cyst usually eontains two trophozoites.

The chemical nature of the cyst wall is not completely understood. Although chitin has

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6

been suggested as a major component of the cyst wall (Ward et al., 1985), studies by

Jarron and coneagues (1989) have found that N-acetylgalactosamine is the primary

amino sugar of the cyst wall. They did not detect N-acetylglucosamine, the primary

amino sugar of chitin. The cyst wall is resistant to water and changes in temperature,

and the cyst can survive in the environ ment for over 20 days when it is free of fecaI

debris (Faubert et al., 1986).

ln Vitro Cultivation of Giardia Trophozoites

A method for the long term cultivation of G. lamblia trophozoites in vitro was

developed in 1960, when Karapetyan was able to culture the trophozoites for seven

months, in the presence of the yeast-like fungus Candida guillermondi. The complex

culture medium included serum, chick fibroblasts, tryptic meat digest, and Hank's or

Earle's balanced salt solutions. Subsequently, Karapetyan (1962) modified the method

and maintained G. lamblia for five months by replacing C. guillermondi with

Saccharomyces cerevisiae. However, he was unable to axenize the culture of Giardia.

In 1970, Meyer reported axenically culturing Giardia trophozoites isolated from

rabbits, chinchillas, and cats. The cultures were axenized by separating the

trophozoites from S. cerevisiae using a U-shaped culture tube. The yeast remained on

one side while the motile trophozoites moved to the opposite arm of the tube. Then,

in 1976, Meyer reported the establishment ofaxenic cultures of Giardia trophozoites

isolated from humans. The medium used, called HSP-l, contained human serum,

Hank's balanced salt solution, phytone peptone (a papaic digest), and cysteine.

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In 1980, Visvesvara adapted the Giardia trophozoites grown in HSP-l medium

to Diamond's TPS-l medium - a medium introduced by Diamond in 1968 to axenically

culture Entamoeba histolytica trophozoites. Due to difficullies in obtaining Panmede

(for the TPS-l medium), which supported growth, Gillin and Diamond (1980)

established axenic cultures of Giardia in Diamond's TYI-S-33 (trypticase, yeast extract,

iron, serum). This medium originally consisted of a nutrient broth, bovine serum, and

a vitamin-Tween 80 mixture. Later, mammalian bile was added to the medium because

it was shown to promote the growth of the trophozoites (Farthing et al., 1983; Keister,

1983). It has a1so been shown that the reducing agent L-cysteine is required for the

growth of the trophozoites, as weIl as for attachment of these trophozoites to the culture

vessel (Gillin and Reiner, 1982) .

It is of note that only the trophozoites of G. lamblia (duodenalis type) have been

successfully cultured in vitro. Although TYI-S-33 is now the standard medium used

for the axenic cultivation of Giardia trophozoites, it remains a complex and chemically

undefined medium. There are isolates of G. lamblia which will not grow in the

currently available culture media (Meloni and Thompson, 1987) and thercfore the

development of a defined medium would be helpful for studying the in vitro

requirements of the parasite. Gillin and colleagues (1986) have shown that biliary

lipids can support the in vitro growth of G. lamblia in the absence of serum. In

addition, Bifulco and Schaefer (1992) found that the serum in TYI-S-33 can be reptaced

by Ultroser Q, a characterized mixture, without affecting the growth of the parasite .

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Animal Models for Human Giardiasis

Systematic study of human giardiasis requires a satisfactory animal model.

Roberts-Thomson et al. (1976) developed a reproducible mouse model for giardiasis

using G. muris cysts. Unfortunately G. muris differs physiologicaBy and pathogenically

from G. lamblia, and it does not infect humans. Domestic animaIs such as mongrel

dogs (Hewlett et al., 1982), cats (Kirkpatrick and Greene, 1985), and kittens (WOO and

Paterson, 1986) have been tested for experimental infection by G. lamblia, but they

show only low levels of susceptibility - making it difficult to reproduce experiments.

Sehgal et al. (1976) reported that adult and weanling rats become infected at low

rates when inoculated with cysts of G. lamblia. A significantly higher infection rate

was observed once the parasite had been passaged through rats. Craft (1982) reported

an infection rate of 100% in weanling Sprague-Dawley rats following oral inoculation

with 150 G. lamblia cysts. Anand et al. (1980) studied the pathogenesis of

malabsorption during giardiasis, using adult Wistar albini> rats, and found a significant

fall in the transport of glucose and glycine in intestinal segments, but observed no

mucosal abnormalities. Contrary to these findings, Woo and Paterson (1986) were

unable to infect adult Wistar rats with G. duodenalis. These studies show that rats have

a poor susceptibility to infection and seem to lack pathology at the gut level, and

therefore are not very sui table hosts to study human giardiasis.

Vinayak et al. (1979) proposed a mouse model of G. lamblia infection using

weanling Swiss mice and reported a 100% infection rate. Hill et al. (1983) showed that

suckling CF-l mice could be infected through an oral inoculation with cultured Giardia

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trophozoites. Swiss mice between 2 to 4 weeks old have been used to study the

immune response to a Giardia infection (Kanwar et al., 1985; Kanwar et al., 1986;

Vasudev et al., 1982). Unfortunately, successful infections with G. lamblia can only

occur in weanling and young mice and therefore these studies on the immune response

to Giardia must take imo account the immunologically immature status of the animais

used. The age-dependent susceptibility of mice to G. lamblia limits their usefulness as

an animal model.

In 1983, Belosevic et al. demonstrated that adult Mongolian gerbils (Meriolle.(j

unguiculatus) are highly susceptible to infection with cysts or cultured tropholoites of

G. lamblia. A reproducible pattern of infection was observed and gerbils inoculated

with cultured Giardia trophozoites produced infections which were similar to those

observed following inoculation with cysts isolated from patients. Cyst release was

intermittent tiuoughout the infection and most gerbils eliminated the trophozoites from

the small intestine in six to seven weeks. The animais were protected against challenge

infections for up to eight months, following a primary infection. Faubert et al. (1983)

demonstrated the uniform susceptibility of gerbils to G. duodenalis-type organisms from

beavers and cats, as weil as to G. muris. Wang el al. (1986) studied the pathology in

gerbils infected with G. lamblia and concl uded that the gerbil-Giardia model paralleled

sorne of the clinical manifestations and histopathological changes seen in humans. So

the Mongolian gerbil seems to be a useful model for studying human giardiasis because

the animal can be infected wh en its immune system is fully competent and the gerbils

exhibit characteristics of the disease which are similar to those seen in humans.

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Humoral Immune Responses

There is much evidence to suggest that antibodies play an important role in

imrnunity to Giardia. It has been shown that infections with G. lamblia are more

corn mon arnong hypogammaglobuUnemic individuals (Hughes el al., 1971; Ament and

Rubin, 1972; Hermans et al., 1976). Thompson et al. (1977) found that the nurnber

of Ig-bearing plasma cells in the lamina propria is significantly higher in G. lamblia

infected individuals. Ridley and Ridley (1976) demonstrated the presence of anti

Giardia antibodies in the serum of infected humans. Il has been suggested that these

antibodies may play a role in the elimination of the parasite from the gut. Radulescu

and Meyer (1981) found that opsonization of trophozoites of G. lamblia with serum

obtained frorn rabbits irnmunized with antigens of the trophozoites resulted in a

significantly higher phagocyto~is of the parasite by peritoneal macrophages of rabbits.

Hill et al. (1984) found that human serum containing anti-Giardia antibodies killed up

to 98% of the trophozoites in vitro. Il was suggested that this killing was complement

mediated. In addition, the attachment of G. lamblia to intestinal epithelial ceUs can be

inhibited by trophozoite-specific antibodies (Inge el al., 1988).

Anti-Giardia IgG can he detected, using an enzyrne-Iinked immunosorbent assay

(ELISA), in more than 80% of patients with syrnptomatic infection and antibody titres

appear to rernain elevated for rnonths or even years after a primary infection (Goka et

al., 1986). Anti-Giardia IgG titres may be elevated in asymptomatic individuals in

endemic areas, indicating previous exposure to the parasite (Goka et al., 1986). These

litres are presumably maintained by repeated exposure lo Giardia antigens without

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production of symptomatic infections. The relationship between the presence of anti-

Giardia IgG and protective immunity h,as not been established (Farthing, 1990).

Studies suggest that IgG antibodÏl!s may hdp in the clearance of G. muris infections in

mice. For example, Heyworth (986), usung an immunofluorescence assay, identitied

both IgG and IgA antibodies on the surface of G. muri.\' trophowites isolated from

BALB/c mice, starting from day 10 of the infection. Other studies have demonstrated

the in vitro killing of Giardia trophozoites by specifie IgG 1 (Nash et al. 1 1988) and

IgG3 (Heyworth, 1992) monoclonal antibodies, in the presence of complement. Nash

and Aggarwal (1986) raised IgG monoclonal antibodies which reacted with a 170 kDa

surface antigen on WB, RS, and Isr strains. Two of these monoclonal antibodies were

cytotoxic for trophozoites which possessed this antigen. The mechanism of killing is

unknown, but was shown to be complement independent.

Using an immunodiffusion test, Jokipii and Jokipii (1982) found that total serum

IgM levels were slightly elevated in patients with recently acquired giardiasis. Anti

Giardia serum IgM has been detected in patients by indirect immunofluorescence and

byan ELISA (Gok.a et al., 1986). Anti-Giardia IgM titres appear to increase early in

infection and then decline within 3 weeks. Sharma and Mayrhofer (1988a) found a

transient IgM response in rats during primary and secondary infections with two isolates

of G. lamblia. Deguchi et al. (1987) demonstrated the capability of anti-G. lamblia

IgM to sensitize the parasite in vitro for complement Iysis by the classical pathway, as

weil as by a unique pathway that requires Cl and factor B, but not C2 and C4. Lysis

of trophozoites can be achieved by C5b to C8, without C9. In 1988, Butscher and

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Faubert produced five IgM monoclonal antibodies against G. muris trophozoites which

were able to kill these trophozoites in vitro, in the presence of complement. When

there was no source of complement, these monoclonal antibodies were capable of

agglutinating the trophozoites and impairing fIagellar movement. One of the

monoclonal antibodies, when injected intraperitoneally into mice, was found to reduce

the intestinal Giardia burden.

Roberts-Thomson and Anders (1981) have shown the presence of an ti-Giardia

serum IgA in human giardiasis. A more recent study in Indian and United Kingdom

patients suggests that only one third of patients with active infection have detectable

anti-Giardia IgA (Goka et al., 1989). However, raised titres were not found in local

control subjects, suggesting that the presence of specifie anti-Giardia IgA is indicative

of current infection (Farthing, 1990). Although there is little information on the role

of secretory IgA (sIgA) in human giardiasis, there is evidence to suggest that

individuals with sIgA deficiency are more susceptible to giardiasis (Zinneman and

Kaplan, 1972). ln a clinical study, mothers with giardiasis were found to have anti

Giardia sIgA antibodies in their milk (Nayak et al., 1987). Their breast-feeding

children had a lower incidence of giardiasis than infants born to uninfected mothers.

Experimental infections in mice with G. muris support the view that production of sIgA

is impor tant for eradication and prevention of infections (Snider and Underdown, 1986;

Heyworth el al., 1988). 80th sIgA and IgG antibodies have been demonstrated on the

surface of G. muris trophozoites (Snider and Underdown, 1986) and termination of the

infection was c10sely related to increased concentrations of anti-Giardia sIgA in

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intestinal fluid (Heyworth, 1986). A biliary IgA response has also been documented

in rats after inoculation of live G. lamblia trophozoites into the intestine (Loftness (JI

al., 1984). It was found that, in these rats, sIgA coated the surface of the trophozoites.

ineluding the flagella and dorsal and ventral surfaces. Studying rats infected with G.

duodenalis, Sharma and Mayrhofer (1988b) found IgA antibodies attached to 3 % of the

trophozoites on day 7 post-inoculation and to 70% of trophozoites on day 10. In

addition, jejunal biopsies from human patients have shown sIgA on the surface of G.

lamblia trophozoites (Briaud el al., 1981). AIso, studies in G. lamblia-infected rats

have suggested that sIgA antibodies can agglutinate the parasite and/or prevent

attachment of the trophozoites to intestinal epithelial eells (Inge et al.. 1988).

However, there are sorne findings which conflict with the view that sIgA is uniformly

associated with an ability to clear the infection. For example, specifie anti-Giardia

sIgA concentrations are normal in C3H/He mice, yet these mice develop ehronic

infections (denHollander et al., 1988).

Allergie manifestations only occasionally occur in giardiasis, in association with

inereased total serum IgE (Farthing et al., 1984). Farthing (1990) suggests that either

Giardia has little ability to elicit a type 1 hypersensitivity response or there is liule

systemic exposure of Giardia antigens. The latter explanation is consistent with the

biology of the parasite, Giardia being mainly a lumenal and non-invasive pathogen.

Cellular Immune Responses

An inflammatory response in the small intestinal mucosa often accompanies

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human infection with Giardia (Wright and Tomkins, 1977; Hartong et al., 1979).

There is an increase in lymphocyte nurnbers both within the lamina propria and in the

epithelium which, when associated with partial or sub-total villous atrophy, can be so

severe as to resemble untreated coeliac disease. Intraepithelial lymphocyte numbers

decrease when the infection resolves. However, there has been no detailed study of

lymphocyte phenotypes in human giardiasis (Farthing, 1990).

Cellular immune responses have been studied in detail during experimental

infection with G. muris in mice. As in sorne human infections, G. muris in mice

induces increased numbers of lymphocytes in the small intestinal epithelium, an event

which closely parallels the reduction in parasite numbers in the intestinal lumen (Gillon

et al., 1982; MacDonald and Ferguson, 1978). Intraepithelial lymphocytes are mainly

T ceUs, whereas in the lamina propria there is an increase in both Band T ceUs.

During a G. muri:;- infection, lymphocyte numbers in murine Peyer's patches have been

found to be more than twice the basal levels, but return to normal following resolution

of the infection. Approximately 30% of these Peyer's patch lymphocytes are T helper

(TJ ceUs and 6% are T cytotoxic/suppressor (Te/J ceUs (Carlson et al., 1986).

Hypothymic nude T cell-defi.:ient mice experience a markedly prolonged infection with

G. muris compared to immunocompetent strains (Roberts-Thomson and Mitchell, 1978;

Stevens et al., 1978). In these hypothymic mice the number of L3T4 + /Th ceUs are

profoundly reduced, whereas the numbers of Tels ceUs and macrophages are relatively

normal (Carlson et al., 1987). It is suggested, therefore, that Th cells are critically

important for the ability of mice to cIear G. muris infections. Th ceUs may be involved

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in switching B cell IgM to IgA production during infection (Clark and Holberton.

1986). The role of T ceUs in the antibody response to G. lamblia in humans remains

unclear. One limited study in humans by Gottstein and colleagues (1991) found that

Th cells from Giardia-infected individuals proliferate in vitro in response to G. lamblia

antigens. This proliferation was associated with CD4 + peripheral blood mononllc1ear

ceUs depleted of CD8+ cells, but not with periphera1 blood mononuclear cells depletcd

of CD4 + ceUs.

Congenitally mast cell-deficient mice (Wf/Wf) have prolonged experimental G.

muris infections lasting 8 weeks or more, compared with BALB/c mice which c1ear the

infection in 4 to 5 weeks (Erlich el al., 1983). These observations sllggest that mast

cells play a role in controUing infection. It is possible that degranulating mast cells

release mediators which are directly toxic to the parasite or that their release iflcreases

the access of other effector cells through changes in vascular permeability (Farthing,

1990).

Although lymphocytes and granulocytes do not exhibit spontaneous cytotoxicity

for G. lamblia, Smith et al. (1983) found that granulocytes are cytotoxic for

trophozoites in the presence of serum containing anti-G. lamblia antibodies.

Neutrophils from patients with giardiasis were shown to effe.ct antibody-dependent

cellular cytotoxicity against G. lamblia in vitro. Anti-Giardia IgG was found to be the

main antibody responsible for sensitization. These antibodies were not cytotoxic for

Giardia in the absence of granulocytes, even in the presence of complement.

Natural killer (NK) ceUs, however, are not likely to be invûlved in the expulsion

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of the parasite. Studies using NK cell-deficient beige mice found that they were able

to clear G. muris infections as quickly as immunocompetent C57BL/6J mice (Heyworth

et al., 1986).

Tissue macrophages have a critical role in the mucosal immune response since

they present antigens to T lymphocytes. In addition, there is evidence that macrophages

act as effector cells for the clearance of the parasite during experimental G. muris

infection. Tissue macrophages have been observed in contact with and engulfing G.

muris trophozoites (Owen et al., 1981) and rabbit peritoneal macrophages will engulf

opsonized G. lamblia trophozoites in vitro (Radulescu and Meyer, 1981). Mouse

peritoneal macrophages have been shown to kill G. muris trophozoites in vitro, a

process which can be enhanced by the addition of immune serum or milk containing

anti-Giardia IgG and IgA. Belosevic and Faubert (1986) found that macrophages from

the G. muris-resistanl BIO.A mice were more phagocytically active and more

chemotactically responsive during a G. muris infection, as compared to those from the

susceptible A/J mice. Peripheral bl00d monocytes have also been shown to exhibit

spontaneous cytotoxicity against G. lamblia (Smith et al., 1982a).

Antigens of G. lamblia

lt has become clear that isolates of G. lamblia, although morphologically

identical, are different both genotypically and phenotypically. There are both inter- and

intra- strain antigenic differences (Smith et al., 1982b; Aggarwal and Nash, 1988) .

These variations may explain the broad spectrum of clinical disease observed in

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giardiasis, as weIl as the absence of a reliable diagnostic test.

A variety of antigens have been detected, but little is known about their structure

and properties. Some of them have been shown to be glycoproteins with hydrophobie

dornains. Einfeld and Stibbs (1984) identtfied an 82 kDa surface glycoprotein in four

different G. lamblia isolates. Others have reported both 82 and 56 kDa surface

antigens common to P-l, Isr and WB strains of G. lamblia (Kumkum et al., 1988a).

Sorne patients with giardiasis developed an IgM response to the 82 kDa antigen, which

was associated with the resolution of the infection (Kumkum et al., 1988b). Char and

colleagues (1991) found that there was a 57 kDa antigen which was recognized by

serum IgG from Giardia patients. In addition, patients with non-persistent or

asymptomatic giardiasis have been shown to respond to a 56 kDa antigen with much

higher antibody titres th an individuals with persistent infections (Vinayak et al., 1989).

The immunization of Swiss mice with this 56 kDa antigen leads to resistance to G.

lamblia infections (Vinayak et al., 1992). Antibodies to a 170 kDa surface antigen in

G. lamblia are known to be cytotoxic to the parasite (Nash and Aggarwal, 1986). Each

G. lamblia isolate usually expresses one major variant surface protein (VSP) which

covers the entire trophozoite surface (Pimenta et al.! 1991). Thes.:! VSPs undergo

spontaneous variation in vitro and are likely a family of cysteine rich proteins which

can be secreted into the growth medium (Aggarwal el al., 1989). G. lamblia isolates

with different VSPs vary in their su sceptib ilit Y to intestinal proteases and this may lead

to differences in virulence (Nash et al., 1991).

Certain internaI antigens have also becn identified (32-170 kDa), sorne of which

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appear to be highly immunogenic and associated with the cytoskeleton (Torian et al.,

1984). One of these proteins is the tubulin-associated protein "giardin", which has a

rnolecular weight of approximately 30 kDa. A variety of low molecular weight proteins

have also been idf'ntified, although their precise location in the parasite is uncertain

(Farthing, 1990). Sorne Giardia antigens are excreted/secreted during in vitro growth.

These antigens have been found to range in size from 94 kDa to 225 kDa (Nash et al.,

1983).

Considerable attention has been devoted to cyst antigens, which in sorne

instances appear to be different from the major trophozoite antigens, although a 65 kDa

antigen is corn mon to both (Rosoff and Stibbs, 1986a). This 65 kDa antigen is resistant

to proteolytic degradation and to prolonged storage at 40 C and -200 C in 10% formalin

and distilled water (Rosoff and Stibbs, 1986b). A group of antigens ranging from 21

to 39 kDa have been shown to appear early during in vitro encystation, and 66, 78, 92

and 103 kDa antigens are observed later (Reiner et al., 1989). Cyst antigens of 66, 78,

94, 103 and 120 kOa have been recognized by secretory and serum IgG, IgM and IgA

antibodies from patients with giardiasis (Reiner and Gillin, 1992).

Pathology

Light microscopy of the slnall intestinal rnucosa frorn Giardia infected

individuals frequently reveals histological changes. These changes inc1ude varying

degrees of infiltration of polymorphonuciear leukocytes and lymphocytes into the

epithelium, accumulation of mononuc1ear leukocytes in the lamina propria, shortened

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villi (decreased ratio of villous to crypt cells), loss of the brush border, damage to

epithelial ceUs, and an increase in epithelial cell mitosis (Yardley (If ai., 1964: Takano

and Yardley, 1964; ~-Ioskms et al., 1967; Wright and Tomkins, 1977). These changes

range from minimal injury to extensive histological damage wilh total villous atrophy,

flattening of the epithelial cells, and dense mononuclear cell infiltration (Levinson and

Nastro, 1978). Studies by Saha and Ghosh (1977) and others have sllggested that G.

lamblia can invade the intestinal mllcosa. These researchers demonstrated the presence

of trophozoites within the mucosa. However, such reports of intestinal invasion by

Giardia are not universally accepted (Owen et aL, 1979).

Electron microscopy has also provided much information on histological changes

occurring in giardiasis. Scanning electron microscopy (SEM) of the intestinal mllcosa

from mice infected with G. muris reveals circlIlar indentations on the epithelial surface

where trophozoites had been attached (Owen et al., 1979). Using SEM, Erlandsen

(1974) observed that the trophozoites of G. muris in rats heavily infected with the

organism almost completely coyer the apical two thirds of the villi. Transmission

electron microscopy of the epithelium of jejunal mucosa from persons with giardiasis

shows that ultrastructural changes in epithelial ceUs accompany intlamed as weil as

noninflamed regions of the mucosa (Takano and Yardley, 1964). Thesc changes

include swelling of membrane-bound cytoplasmic sh uctures, distortion of nuclei, and

reduction of the height and number of epithelial cell microvilli. Studies of G. muris

infections in mice by Roberts-Thomson and colleagues (1976) found that mice which

were inoculated with high numb~rs of cysts had a greater impairment of weight gain

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20

and more severe alterations in small intestinal architecture than mice given lower cyst

dosages. However, these changes were reported to be transient, with normal mucosal

structure retuming foUowing elimination of the parasite.

Changes in intraepithelial lymphocyte (IEL) numbers have also been observed

in giardiasis. Miee infected with G. muris have been reported as having significantly

higher IEL counts than those normally observed, from 3 to 10 weeks post-infection

(Gillon et al., 1982). These increased IEL counts persist even after the parasite is

eliminated from the intestine. The IELs are mainly T cells and it has been proposed

that these T cells secrete lymphokines which can contribute to villous damage (Gillon

el al., 1982). In addition, IELs have been found to exhibit direct cytotoxicity during

G. lamblia infections in micc (Kanwar et al., 1986). However, a more recent study

suggested that the IELs involved in the decline phase of a G. lamblia infection are

mainly Tb' not Tc/., cells (Vinayak et al., 1991). The specifie mediators secreted by

IELs in a G. lamblia infection have yet to he examined.

Other a1terations that have been described in human giardiasis inelude fat and

vitamin B'2 malabsorption, indicating that intestinal dysfunetion can extend into the

ileum (Hoskins et al., 1967; Wright et al., 1977). Vitamin A deficieney has been

reported to be associated with G. lamblia infections in children (Mahalanabis et al.,

1979). In sorne cases, protein-Iosing enteropathy and redueed serum carotene levels

have been observed (Sherman, 1980). AIso, individuals with reduced gastric acidity

have been found to be more likely to develop protein-energy malnutrition during a G.

lamblia infection, as nutrients are more difficult to absorb in these patients (Slonim et

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al., 1976). It has been proposed that the induction of bacterial proliferation in the

smal1 intestine concurrent with a G. lamblia infection could result in the malabsorption

found in giardiasis (Tomkins et al .• 1978). However, this mechanism has not been

proven.

Based on the histological alterations observed in human and animal giardiasis.

a number of pathogenic mechanisms have been proposed as explanations for intestinal

dysfunction. These include the presentation of a mucosal barrier to the passage of

nutrients due to the presence of a high number of trophozoites, cellular in jury and

inflammatory reactions, and mucosal cell invasion by the trophozoites (Solomons,

1982) .

Disaccharidase Deticiencies in Giardiasis

The final stage of carbohydrate digestion occurs on the luminal surface of small

intestinal epithelial cells, which is known as the brush border or microvillar membrane.

In this membrane several glycosidases, which hydrolyze di- and oligo- saccharides

originating from ingested nutrients, are present. These glycosidases are referred to as

disaccharidases. The activity of disaccharidases is known to be modified by various

factors such as age, nutritional status, hormones, pancreatic secretions, and dietary

composition (Goda and Koldovsky, 1988). Diseases that damage the small intestinal

mucosa, such as giardiasis, also affect this enzymatic function (Banai et al., 1990).

Disaccharidase deficiencies have been consistently identified as one of various

abnormalities of small intestinal pathology in giardiasis (Jennings et al., 1976) .

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Quantification of brush border disaccharidase enzyme levels during human giardiasis

has shown that lactase, sucrase, and leucylnaphthylamidase are reduccd (Hartong et al.,

1979). MacDonald and Ferguson (1978) reported that, in chronic murine giardiasis,

tissue sucrase activity is reduced - as seen in the jejunum in the "mild" human disease.

Measurement of tissue disaccharidases in murine giardiasis showed the most profound

decreases to be in lactase levels, which reached a peak 2 weeks after infection, at which

time the activities of sucrase and maltase were also below normal (Ferguson et al.,

1980). By the fourth week after infection, activities for all enzymes examined were

similar to controi levels. In c1inical terms, it has been noted that the lactase deficiency

can lead to osmotic diarrhea after ingestion of milk (Ferguson et al., 1980). Studies

by Belosevic et al. (1989) found that G. lamblia induced a transient decrease in

disaccharidase activity during the acute phase of a primary infection in gerbils. There

was a 30% to 85% decrease in enzyme activity on days JO and 20 post-infection. This

decrease was related temporally to the highest trophozoite burden in the small intestine.

These authors also found that challenge inoculation of gerbils with G. lamblia

trophozoites resulted in a sharp and immediate (24 hours after challenge) decrease in

the activity of the enzymes. This reduction in enzyme activity was found to be

independent of the size of the challenge inoculum and it occurred in the absence of

trophozoites in the intestine. In addition, challenge of gerbils previously exposed to G.

lamblia with the soluble extract of the trophozoites also resulted in disaccharidase

deficiencies, comparable with that induced by a challenge with live trophozoites. These

authors concluded that the disaccharidase deficiency associated with the primary

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23

infection probably results from a direct effect of the parasite on the brush border of the

small intestine, while the deficiency in the secondary infection is probably due to the

local immune responses of the host. The suggestion that G. lamb/lU causes brush

border injury with consequent reduction in enzyme levels is supported by the finding

that elimination of the parasite reverses both the morphological and functional brush

border changes (Hartong et al., 1979). However, other resCc'lrchers have reported

decreases in brush border enzyme activity in the absence of microvillous damage

(Jennings et al., 1976). A more recent study by Daniels and Belosevic (1992)

examined disaccharidase activities in susceptible and resistant mice during G. muris

infections. They observed decreases in enzyme activity in susceptible C3H/HeN mice

after challenging with an antigen extract of the trophozoites. However, liule change

occurred in disaccharidase activity after a similar challenge in resistant C57Bl/6 mice.

Therefore these authors concluded that more severe disaccharidase deficiencies occur

in susceptible hosts following parasite antigen challenge.

[References for the General Introduction and Review of Literature follow the

General Discussion.]

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MANUSCRIPT 1

DISACCHARIDASE DEFICIENCIES IN

MONGOLIAN GERBILS (MERIONES

UNGUICULATUS) PROTECTED AGAINST

GIARDIA LAMBLIA

Shawn R. Mohammed and G. M. Faubert


McGill University

Macdonald Campus

21 , 111 Lakeshore Road

Ste. Anne de Bellevue, Quebec

Canada H9X 3V9

(submitted for publication)

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ABSTRACT

The activities of the disaccharidases lactase, maltase, sucrase and trehalase were

examined in gerbils during Giardia lamblia infections. In a primary infection with

trophozoites, the activities of al} 4 enzymes were reduced from day 10 post-infection

(p.i.) and remained at low levels well past the elimination phase of the infection.

However, during a challenge infection, the decreases in disaccharidase activity were

short-lived, with impairments only on days 2 and/or 4 post-challenge (p.c.). Sucrase

activity was not affected by a challenge infection. When 'll mg of a soluble extract

of G. lamblia trophozoites was used to challenge gerbils previously exposed to the live

parasite, the pattern and duration of enzyme deficiencies were comparable to the

challenge wÏth the live parasite. In addition, decreasing the extract dose used to

challenge the gerbils led to smaller disaccharidase deficiencies. Gerbils previously

exposed to G. lamblia were also challenged with a soluble extract of Entamoeba

histolytica trophozoites and this had no effect on the disaccharidase activities.

Therefore. the presence of the intact parasite was not necessary to induce reductions in

enzyme activitv in immune animals. As weil, the effects in the secondary infection are

parasite-specifi: and may involve the host's immune response to Giardia antigens.

Immune gerbils were further challenged with the in vitro-released excretory/secretory

products of G. lamblia. Under our experimental conditions, disaccharidase activities

were found to be affected by the~,e products in a manner which was inconsistent with

the live parasitic challenge and this merits further study.

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INTRODUCTION

Giardia lamblia is an anaerobic protozoan parasite of worldwide distribution

which is found in the small intestine of humans, as weil as other animals. It is the most

frequent cause of defined waterborne outbreaks of diarrhea in developed countries

(Craun and Jakubowski, 1987; Wilson et al., 1982). The parasite exists in two forms:

the vegetative, flage.llated trophozoite and the infective cyst. Individuals infected with

Giardia may remain asymptomatic carriers or may develop sorne of the clinical

manifestations of giardiasis, such as chronic diarrhea, nausea, abdominal cramps and

distension, and malabsorption (Adam, 1991) .

The intestinal epithelium is the site of interaction between Giardia and the host,

and a mucosal immune response follows. However, the precise nature of this immune

response remain~ unknown. Several abnormalities of the small intestine have been

consistently identified in human giardiasis and these include increased intraepithelial

lymphocyte counts (Wright and Tomkins, 1977), crypt hyperplasia with shortened villi,

increased lamina propria cellularity (Yardley et al., 1964), and disaccharidase

deficiencies (Jennings el al., 1976). The pathogenic mechanisms by WhlCh Giardia

causes these abnormalities of the gut remain undefined.

Intestinal brush border enzyme deficiencies can be clearly shown during

protozoan disease and are commonly reported abnormalities of the gut (Buret et al.,

1990). Quantification of brush border disaccharidase levels during human giardiasis by

Hartong el al. (1979) showed that lactase, sucrase, and leucylnaphthylamidase were

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reduced. Measurement of tissue disaccharidases in murine giardiasis showed the most

profound changes to be in lactase levels, which reacherl a peak two weeks after

infection, at which time the activities of sucrase and maltase wt'r~ also below normal

(Ferguson et al., 1980). In addition, in the gerbil animal model, decreases in enzyme

activity were observed not only in a primary G. lamblia infection, but also in challenge

infections (Belosevic et al., 1989). This reduction in enzyme activity in the immune

animaIs was found to be independent of the size of the challenge inoculum and it

occurred even in the absence of trophozoites in the intestine. 8elosevic and colleagues

also found that a challenge with a crude extract of the trophozoites resulted in lowered

disaccharidase activity, suggesting the involvement of the host's immune response in

disaccharidase deficiencies. A more recent study examined the variability in the

decreases in disaccharidase activity during G. muris infections in susceptible and

resistant mice. It was found that after challenging the immune mice with an extract of

the trophozoites, the susceptible mice showed more severe decreases in disaccharidase

activity than the resistant strain (Daniels and Belosevic, 1992). Studying the effects of

G. muris on the mouse mucosal ultrastructure, Buret el al. (1990) found that brush

border disaccharidase deficiencies are linked to a loss of microvillus surface area.

However, the precise mechanisms invoived have yet to be elucidated.

The disaccharidase deficiencies ohserved in giardiasis contribute to host

malabsorption of carbohydrates, with subsequent fluid accumulation in the intestinal

lumen and diarrhea. These deficiencies may involve the immune response to the

parasite and it has been clearly established that gerbils exhibit a long lasting immunity

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to challenge infection with G. lamblia (Lewis et al., 1987; Belosevic et al., 1983). We

therefore undertook a more extensive characterization of the effects on disaccharidase

activity in gerbils previously exposed to G. lamblia. In this study, our objectives were

to: (1) examine the activity of four disaccharidases at various times following a

challenge infection with live trophozoites; (2) measure enzyme levels following a

challenge inoculation with known amounts of the soluble extract of G. lamblia

trophozoites; (3) determine the specificity of the effects that the soluble extract of G.

lamblia trophozoites has on the disaccharidases by challenging gerbils previously

exposed to G. lamblia with the soluble extract of Entamoeba histolytica trophozoites;

and (4) examine the effects of the excretorylsecretory products of G. lamblia

trophozoi tes , released in vitro, on disaccharidase activity .

• 29

MATERIALS AND METHODS

Parasites

G. lamblia trophozoites, WB strain (American Type Culture Collection no.

30957), originally isolated from a patient with chronic symptomatic giardiasis (Smith

et al., 1982) were used in this study. Trophozoites were maintained axenically in filter-

sterilized TYI-S-33 (trypticase, yeast extract, iron, and serum) medium adapted for

a.lamblia (Gillin and Diamond, 1979), with 10% adult bovine serum (Sigma Chemical

Co., St. Louis, U.S.A.), and supplemented with 100 units/mL of penicillin (Sigma) and

100 ILg/mL of dihydrostreptomycin sulphate salt (Sigma), in the absence of bovine bile.

• They were passaged twice weekly.

Pathogenic E. histolytica trophozoites, strain HM 1 :IMSS, originally isolated by

L. Diamond (National Institutes of Health, Bethesda, U.S.A.) and cultured in Dr. K.

Chadee' s laboratory (Institute of Parasitology, McGill University, Montreal, Canada)

were used for the amebic study. Trophozoites were maintained axenicaJly in autoclaved

TYI-S-33 (biosate, iron, and ~erum) medium (Diamond et al., 1978), sllpplemented

with 100 units/mL of penicillin and 100 #Lg/mL of dihydrostreptomycin slilphate salt.

Animais

Six to 10 week old male Mongolian gerbils (Meriones unguiculatus) , from

Tumblebrook Farms (West Brookfield, U.S.A.), were used throughout this study. •

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30

They were maintained under standard laboratory conditions and provided with

laboratory chow and water ad libitum. One week after arrivaI, each gerbil was trea.ted

by oral gavage with 15 mg of metronidazole (Rhône-Poulenc, Montreal, Canada) for

three consecutive days and then allowed to rest for 10 days. This ensured that the

gerbils were free of intestinal infections.

Ail inoculations were done orally, to unanaesthetized gerbils. With live G.

lamblia inoculations" ~~h gerbil was infected with 1 x Hf trophozoites.

Preparation of the G. lamblia and E. histolytica Trophozoite Extracts

Trophozoites (from a 72-h culture) were dislodged from the walls of culture

vessels by immersion in an ice bath for 15-30 min. They were then sedimented by

centrifugation at 800 g for 10 min, at 4° C, and washed 5 times in sterile phosphate

buffered saline (PBS) (pH 7.2). Trophozoites were then lysed in sterile PBS by

sonication, using a Sonic Dismembrator (Fisher Scientific, Montreal, Canada) at

maximum output, for 10 min, in an ice bath. The sonicated material was cleared of

insoluble matter by eentrifuging at 23,300 g for 20 min, at 4° C, and the supemat.ant

eollected as the erude soluble extract.

Quantification of Trophozoites in the Gerbil Smalt Intestine

The method of Belosevic and Faubert (1983), with modifications, was ll)ed 'to

determine trophozoite numbers in the smalt intestine. Briefly, gerbils were killed and

the small intestine removed and divided into three equal sections. The segments were

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31

slit longitudinally and each placed in 6 mL cold PBS. They were left in a shaking iee

bath for 1 h to dislodge the trophozoites from the intestinal mucosa. The intestinal

sections were then removed, the liquid centrifuged at 800 g for 10 min and the pellet

resuspended in cold PBS. The total number of trophozoites recovered from each

segment was then determined using a haemocytometer.

Measurement of Intestinal Disaccharidases

- Preparation of Intestinal Homogcnate -

Homogenates were prepared as previously described (Belosevic et al., (989),

with modifications. Briefly, the gerbils were killed and the smalt intestine removed and

divided into three sections. The segments were placed in ice cold distilled water and

were each flushed with 50 mL of distilled water. The segments wcre then slit

longitudinally and the mucosa scraped off with a glass microscope slide. The mucosa

from all three sections were combined, weighed (wet weight), and placed in four

volumes of ice cold distilled water. The mucosal scrapings were then homogenized

using a Con Torque power unit at maximum speed (Eberbach Corp., Ann ArboT,

U.S.A.). Homogenates were stored, without prior centrifugation, at - 7(J' C until used

in the assay.

- Assay for Intestinal Disaccharidases -

Disaccharidase activity of homogenated mucosa was measured using the glucose

oxidase peroxidase assay (Dahlqvist, 1968), as modified by Belosevic el al. (1989),

which is based on a colour reaction with the glucose liberated by a disaccharidase in

, j

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32

60 min. The assay consisted of adding to e.ach weIl of a 96-well Nunc microwell plate

(Gibco BRL, Burlington, Canada) 0.01 mL of an appropriately diluted mucosal sample

(hornogenate) and 0.01 mL substrate-buffer solution of a disaccharidase to be measured,

in quadruplicate. The plate was then incubated at 37° C in humidified atmosphere for

60 min. After incubation, 0.3 mL tris-glucose oxidase reagent was added to each weIl

and the plate incubated for an additional 60 min. The plate was then read at 415 nm

using a Bio-Tek microplate reader (Mandel Scientific, Guelph, Canada). For each

assay, 8 wells of reagent blank and a glucose standard series (2, 6, and 10 p.g glucose)

in quadruplicate were also done. Disaccharidase activity is expressed as units/mg

protein in the mucosal sample (U/mg), where units represent p.moles of disaccharide

hydrolyzed/min .

- Reagents -

AIl chemicals were obtained commercially. 0-(+ )-Glucose, 11-lactose, maltose,

0-( + )-trehalose, maleic acid, glucose oxidase, o-dianisidine, peroxidase, and triton X-

100 were purchased from Sigma. Sucrose was obtained from Fisher Scientific and tris

(ultra pure) was purchased from ICN Biomedicals Canada Ltd. (St. Laurent, Canada).

Collection of G. lamblia Excretory /Secretory Products

The me.dium from a 72-h trophozoite culture was dr..canted and, in order to

remove any residual TYI-S-33 medium, the culture flask was quickly washed with

warm maintenance medium. This medium consisted of RPMI 1640 with L-glutamine

(Gibco BRL), supplemented with 11.4 mM L-cysteine Hel (Fisher Scientific), 10% fetaI

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33

bovine serum (Flow Laboratories, Toronto, Canada), 20 mM N-2-

hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (Sigma), 100 units/mL of

penicillin (Sigma) and 100 JLg/mL of dihydrostreptomycin sulphate sait (Sigma) (Guy

et al., 1991). The culture vessel, with the attached trophozoites, was then tilled with

the maintenance medium and placed at 37° C for 6 h, in order to allow for the

maximum production of trophozoite products. Next, trophozoites were sedimented by

centrifugation at 800 g for 10 min. The supernatant was collected, a sample was

examined microscopically, and it was centrifuged again iftrophozoites were seen. This

was repeated until no trophozoites were found in the supernatant. The final supernatant

was then lyophilized and resuspended in PBS, for use as the in vitro-rele-ased products

of the parasite.

Measurement of Protein Concentration

Protein concentrations were determined ,B described by Lowry el al. (1950,

using 0.2 mL of sample and a final reaction volume of 1.3 mL. A standard curvc was

prepared using freshly dissolved bovine serum albumin (BSA).

Statisticaj Analysis

Statistical significance was determined using the Mann-Whitney V-test. The

probability level of P < 0.05 was considered significant.

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34

RESULTS

Primary Infection with Live G. lamblia Trophozoites

In order to establish the basis of disaccharidase deficiencies in giardiasis and

provide a reference for co m pari son , the effects of a primary G. lamblia infection on the

activities of lactase, maltase, sucrase, and trehalase in gerbils were examined. For ail

4 disaccharidases, there was a 43 % to 78 % reduction in activity, starting on day 10 p. L

(Fig. 1). This time corresponds to the beginning of the acute phase of the infection,

as the highest trophozoite burden in the small intestine was found from day 9 to day 12

p. i. (data not shown). The reduction in enzyme activity was maintained well past the

elimination phase of the infection (around day 30 p.L), as it was detected even on day

50 p. i. This pattern of decreased activity was found with ail disaccharidases measured,

with the exception of maltase. Although the activity of maltase was decreased from day

10 p.L. by day 50 p.i. it had returned to normal levels. The reduction in sucrase

activity varied the most over the course of the infection (43% to 78% reduction), white

that of trehalase remained the most consistent (61 % to 76% reduction).

For ail the experiments in our study, there was a great deal of variation between

individual gerbils in a particular test group and also over time. The activity of

disaccharidases is known to be modified by a number of factors such as age, nutritional

status, hormones and pancreatic secretions (Goda and Koldovsky, 1988). In addition,

it is important to note that although the 4 enzymes examined are intestin" •

disaccharidases, they are not all affected in the same way as they are located in different

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LACTASE MALTAS~ 35

- 0.066 2.' lUI

S 0.060 2.2

'- 0.0'6 2.0 :;:, -~ O.O~O 1.8 ~ '.-4 1.11 :> 0.035

• .-4 ~ 1.4 c:,) 0.030 < 1.2 • CI) 0.025 !Il 1.0 Ils

OC 0.020 • • '.-4 • 0.8 • ... 0.016 Ils 0.11 ..c:::

c:,) c:,)

0.010 0.4 Ils

0.005 !Il 0.2 '.-4

~ 0.000 0.0 .. 10 20 40 50 • 10 20 .0 60

SUCRASE TREHALASE 0.26 0.018 -~ 0.24 S

"- 0.22 0.014

:;:, - 0.20 0.012 ~ ~ 0.18 '.-4 :> 0.16 • 0.010 '.-4 ~ c:,) O." < 0.008 CI) 0.12 !Il Ils 0.10 0.008 • • OC .... 0.06 ... Ils

..c::: 0.06 0.004

t.l t.l O.Of Ils 0.002 !Il 0.02 .... ~ 0.00 0.000 .. 10 20 40 50 • 10 20 40 50

Days Post-Infection Days Post-Infection

Fig. 1. Disaccharidase activities over time in gerbils with a primary infection of live G. lamblia trophozoites. Each gerbil was infected with 1 x 1 rJ trophowites. Con trois were uninfected, age-matched gerbils. Results are expressed as mean ± S.E , n =4 for uninfected controis <.) and n=5 for infected groups (181). * Differences between control and infected groups were significant, P < 0.05.

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36

areas of the mucosal brush border (Dahlqvist and Semenza, 1985).

Challenge with Live G. lamblia Trophozoites

Since gerbils are immune to challenge infections, we investigated the effects on

the activities of lactase, maltase, sucrase, and trehalase over lime in gerbiJs challenged

with G. lamblia. We were particularly interested in determining how carly after a

challenge infection enzyme activities could be affected. Therefore, disaccharidase

activities were measured as carly as 1 h (0.042 days) post-challenge (p.c.), and

compared to age-matched controls which received a primary infection but were not

challenged. Sucrase activity remained comparable to controllevels (Fig. 2). However,

the activities of lactase and trehalase were reduced on day 4 p.c. A decrease in the

activity of maltase was observed on both days 2 and 4 p.c. The reduction in lactase,

maltase, and trehalase activities in the challenged animais ranged between 33% and

59%. These decreases in enzyme activity occurred in the absence of trophozoites, as

the challenge infection does not establish in the gerbil gut (no trophozoites detected).

Challenge with the Soluble Extract of G. lamblia Trophozoites

Gerbils were challenged with 0.1 mg of the soluble extract of G. lamblia

trophozoites and the activities of the 4 enzymes under study were measured on days l,

2, 4, and 10 p.c. The activity of sucrase did not differ from controllevels on any of

these days. Howcver, the activities of lactase, maltase, and tre:.alase were reduced by

as much as 60% on day 4 p.c. (Fig. 3). In addition, the activity of maltase was also

LACTASE MALTASE 37

• - 0.032 tI!

1.8

S 0.028 '-P - 0.02. ~ .. J .... > 0.020 .... 1.0

[ • 1><

1.2

+J t.)

< O.OUS C!) • III «1 0.012 't:I .... ~ «1 0.008

.cl t.)

•

L l [ IX ><:

IX )(

>< IX ><: x Xi ><: Xi x x x x

>< X x IX X X

>< IX IX >< ><

0.8

0.8

0.4

t.) 0.00. «1

III .... ~

0.000

I)c I)c IX 1>< 1>< IX I)c lx rx 1>< I)c pc

I)c lx t>< 1><

0.2

0.0 CIl .. co co .. co .. co Ct .. .. Ct .. .. ... .. CI CI .. CI CI N on "! .. CI

... on Ct .. .. .. .. .. .. N III "! .. CI CI .. .; .; .; ... oi .. .; .; .; ... N .. .; .; .. N

SUCRASE TREHALASE

- 0.18 0.012 bD e 0.18 '- 0.010 ::J • - 0.14

~ ~ 0.12 .. 0.008 > ..

~ ~ 0.10 t.)

< 0.008

~ X

1 )(

C!) 0.08 Xi

><: III X «1

L ><

'tI 0.06 IX IX IX .. 0.004 IX l

• ~ x l x «1 II

..c:I 0.0. lx 1>< IX t.) ~ lx 1>< (.) 0.002 lx 1>< i><

lx lx IX >< CIl 0.02 1>< )< III 1><

I>c lx IX 1>< .,.. IX lx i>C

~ I)c IX IX 1>< 0.00 0.000 i>C

N .. co .. .. co Ct N Ct Ct .. Ct Ct ~ .. ... la CI .. .. co CI ... on .. CI Q CI .. ft N ... ' "! .. co CI .. N ... CI .. CI "! .; .; .; ... oi .. CI .; .; CI N .. .; . N .. "

Days Post-Challenge Days Post-Challenge

Fig. 2. Disaccharidase activities over time in gerbils challenged with live G. lamblia trophozoites. Gerbils were challenged with 1 x H1 trophozoites 50 days after a primary G. lamblia infection with the same dose. Controls were age-matched gerbils which received a primary infection but were not challenged. Results are expressed as mean ± S.E., n=4 for unchallenged controls (.) and n=5 for challcnged groups (~).

• * Differences between control and challenged groups were significant, P < 0.05 .

•

•

•

0.022 -~ Si 0.020

~ 0.018 -!' 0.0111

'> o.oa .... ~

Q 0.01~ <

-ea.I

0.010

0.008

0.0011

0.00.

0.002

0.000

0.1.

e '" 0.12 ~ -~ 0.10 ~ .... ~ .... t: 0.08

-< 0.08

0.02

0.00

38 LACTASE MALTASE

1.11

1..

1.2

1.0

0.11

0.11

o .•

0.2

0.0 CONTROL 0.1 0.075 0.05 CONTROL 0.1 0.075 0.05

SUCRASE TREHALASE 0.010

0.008

0.0011

0.00.

0.002

0.000 CONTROL 0.1 0.075 0.05 CONTROL 0.1 0.0'15 0.05

Antigen Dose (mg) Antigen Dose (mg)

Fig. 3. Disaccharidase activities in gerbils challenged orally with varying amounts of the soluble extract (antigen) of G. lamblia trophozoites 50 days after a primary infection with 1 x 1(1 live trophozoites. Controi gerbils were age-matched and received a primary infection. but were not challenged. Results are for day 4 post-challenge and are expressed as mean + SE. n =4 for unchallenged controls <.) and n =5 for antigenchallenge.d groups (lX!). .je Differences between control and challenged groups were significant. P < 0.05.

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39

reduced on day 2 p.c. (data not shown). These patterns, observed with the 0.1 mg

antigen dose, are similar to those found with the live challenge.

In order to determine whether the effect of the soluble extract on disaccharidase

activities could be obtained with lower doses, immune gerbils were challenged with

0.075 mg or 0.05 mg total of the extract. Enzyme activities were then measured on

day 4 p.c., as this was the time when the disaccharidases were most affected by the 0.1

mg extract challenge. When the antigen dose was 0.075 mg, only the lactase and

maltase activities were significantly lower th an the unchallenged controls (Fig. 3).

However, the mean reductions in these enzyme activities were not as great as in the

group of animais challenged with 0.1 mg of the soluble extract of the trophozoites.

With an antigen dose of 0.05 mg, there were no significant differences found between

challenged and control groups for any of the disaccharidases under study. However,

sucrase activity remained unaffected by the antigen, regardless of the dosage.

Challenge with the Soluble Extract of E. histolytica Trophozoites

To determine whether the effects of the G. lamblia soluble extract on the

disaccharidases are specifie to this parasite, gerbils immune to Giardia were challenged

with 0.1 mg of the soluble extraet of E. histolytica trophozoites. Disaccharidase

activities were measured on days 2 and 4 p.c. and for ail 4 enzymes the levels observed

in E. histolytica-challenged groups were comparable to control values (Table 1) .

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Table 1. Disaccharidase activities in gerbils previously exposed to G. ~amblia and challenged with a soluble extract of E. histolytica trophozoites. Disaccharidase activities are the average values (n =4, for controls; n =5, for test groups) in U/mg ± SE. Gerbils were challenged orally with 0.1 mg of the soluble extract of E. histolytica trophowites 50 days following a primary infection with 1 x 106 G. lamblia trophowites. Controls were age-matched gerbils which received a primary G. lamblia infection, but were unchallenged.

Enzyme Days Post- U nchallengr.d Challenged with E.

Challenge Control histolytica*

2 0.013 ± 0.001 0.015 ± 0.002

Lactase --------------------------------------------------------------4 0.018 + 0.001 0.013 ± 0.003

2 1.32 + 0.13 0.96 ± 0.16

Maltase 4 1.29 + 0.10 1.02 ± 0.08

2 0.12 ± 0.01 0.12 ± 0.02

Sucrase 4 0.10 ± 0.01 0.098 ± 0.011

2 0.0048 ± 0.0008 0.0059 ± 0.0005

Trehalase 4 0.0080 + 0.0008 0.0059 ± 0.00 15

III Differences between control and test groups were not significant, P < 0.05

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41

Challenge with G. lamblia Excretory ISecretory Products

Since the mainly somatie antigens of the soluble extract were able to affect

disaccharidase activities, we wanted to study whether metaboIic excretory/secretory

products released by live G. lamblia trophozoites could also affect the enzyme

activities. Therefore, previously infected gerbils were challenged .')rally with O. l mg

of the in vitro-released products of the parasite. Challenging immune gerbils with the

excretory/secretory products did not affect the activities of maltase and trehalase (Table

2). However, on day 4 p.c. the activity of lactase was decreased by 27%, while there

was a 33% reduction in sucrase activity.

In addition, uninfected gerbils were inoculated with O. 1 mg of the

excretorylsecretory products, to determine whether these products could affect

disaccharidase activities in naive animais. Enzyme activities measured on day 10 p.L

(the time when activities were first reduced in the primary infe.ction) were comparable

to controllevels (data not shown).

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42

Table 2. Disaccharidase activities in gerbils previously exposed to G. lamblia on day 4 post-challenge with in vitro-released excretory/secretory products. Disaccharidase activities are the average values (n=5) in U/mg ± SE. Fifty days following a primary infection with 1 x 1 ()6 G. lamblia trophozoites, gerbils were challenged with 0.1 mg of the supernatant of a fully grown G. lamblia culture maintained in modified RPMI for 6 h. Controls were age-matched gerbils, previously infected with G. lamblia, which were challenged with 0.1 mg of unconditioned modified RPMI.

- = Enzyme RPMI Control Culture Supernatant

Lactase 0.015 ± 0.001 0.011 + 0.001*

Maltase 0.77 ± 0.14 0.64 ± 0.10

Sucrase 0.11 ± 0.01 0.074 + 0.009*

8Trehalase 0.0048 ± 0.0004 0.0046 + 0.0008

* Differences between control and test groups were significant, P < 0.05

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43

DISCUSSION

Since diarrhea is a major consequence of G. lamblia infections, it is important

to examine the malabsorption underlying much of it. Unabsorbed sugars lead to

diarrhea by causing net fluid accumulation in the intestinal lumen (Launiala, 1968). In

this respect, disaccharidase deficiencies play a key role in understanding the pathogenic

mechanisms which lead to intestinal dysfunction in giardiasis. In fact, symptotns of

sugar malabsorption in other intestinal diseases/disorders are identical to several found

in Giardia infections, including abdominal distension, cramps, and flatulence, as weil

as diarrhea (Ravich and Bayless, 1983). Disaccharidase enzymes are located on or

within the microvilli of the small intestinal brush border and are involved in the final

stage of carbohydrate digestion. In this study, we focused particularJy on differences

in disaccharidase activity following various challenge inoculations in gerbils immune

to G. lamblia.

The activities of lactase, maltase, sucrase and trehalase decreased during a

primary G. lamblia infection in gerbils, beginning on day 10 p.L and remaining low

for the duration of the study (with the exception of maltase). These results differ from

the findings of Belosevic et al. (1989), who reported a decrease in trehalase activity

beginning only on day 20 p.i. Furthermore, they reported that ail disaccharidases had

recovered by day 40 p.i., except for lactase. The longer period of decreased enzyme

activity in our study may be due to the larger infective dose (l x 1()6 trophozoites) given

to each gerbil, compared to that used by Belosevic and co-workers (2 x lOS

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44

trophozoites). It is important to note that the presence of the parasite in the small

intestine was not found to be necessary in order to maintain the disaccharidase

deficiencies. The parasite was eliminated from the gut by day 30 p.i., but the decreases

in enzyme activity persisted beyond this time. These findings are supported by work

done on malabsorption in Giardia-infected humans. Clinical studies have shown that

there are patients in which the parasite has been eradicated and yet the malabsorption

persists (Brasitus, 1983). Il is possible that these impairments of enzyme activity are

due to a combination of direct damage of enterocytes by the attachment of G. lamblia

trophozoites, followed by an effective host mucosal immune response. However, the

precise mechanism has yet to be elucidatcd.

During a challenge infection with live G. lamblia trophozoites, we also observed

decreases in the activities of lactase, maltase and trehalase. These deficiencies were of

short duration, compared to the primary infection. Although enzyme levels were

measured as early as 1 h p.c. and up to 20 days p.c., thcy were depressed only on days

2 or 4 p.c. Belosevic and colleagues (1989), also using G. lamblia-challenged gerbils,

reported lactase, sucrase and trehalase deficiencies beginning on day 1 p.c. and

remaining low for several weeks. However, their results were compared to uninfected

gerbils as controls. We chose to use gerbils which had received a primary infection,

but no challenge, as our control group. In this case, any decreases in enzyme activity

due to the primary infection were not taken into account in our analysis of the

secondary exposure to the parasite. For the disaccharidases which were affected, the

deficiencies occurred even though G. lamblia trophozoites did not establish in the gerbil

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45

gut upon challenge. Perhaps this led to the impairments not being persistent. Several

studies have examined the link between Iltucosal damage by the parasite and intestinal

dysfunction, to explain disaccharidase deficiencies. Buret and colleagues (1992), llsing

the gerbil model for G. lamblia, concluded that a loss of brllsh border surface area led

to impaired digestion and absorption in giardiasis. Other studies, examining the

enzyme deticiencies in Giardia infections, found that villous atrophy resulted in the

decreases in disaccharidase activity (Buret et al., 1990; Khanna (II al., 1990).

However, Jennings and co-workers (1976) found humans suffering from giardiasis who

had morphologically normal mucosa and low lactase levels, and it has been establishcd

that villous shortening is only variably present in both human and experimental

giardiasis (Adam, 1991). Although the process leading to the decline in disaccharidase

activities is unknown, our results indicate that the effects are not immediate.

The challenge of immune gerbils with 0.1 mg of the soluble extract of G.

lamblia trophozoites resulted in reductions in disaccharidase acti vit y which were

comparable in terms of the levels and patterns to those induced by the live parasite in

a challenge infection. These gerbils were challenged with considerably less Giardia

protein compared to the amounts used by Belosevic et al. (1989). In this study, we

wanted to see how closely we could correlate the amount of the extract given with the

d{lsage used in the live challenge and still obtain impairments of disaccharidase activity.

\\ e decide.d lo tirst challenge the animais with 0.1 mg of the cxtract. Under the

method used for the preparation of the soluble extract, this amount was equivalent to

4 times the number of trophozoites lIsed for the live challenge. The effr,ct on the

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46

enzymes was lost when challenging with 0.05 mg of the extract. However, our results

also show that it is possible to link the amount of the extract dose with the effects on

the disaccharidase activities. Decreasing the antigen dose used to challenge the gerbils

led to smaller enzyme deficiencies. E. histolytica, like G. lamblia, is an intestinal

prOlozoan parasite which produces energy by fermentation. They are two of the few

eukaryotes with an anaerobic metabolism (Adam, 1991). Despite these similarities,

disaccharidase activities were not affected when gerbils previously exposed to G.

lamblia were challenged with a soluble extraet of E. histolytica trophozoites. A study

using an in vitro rabbit model of acute intestinal amebiasis found that exposing colon

segments to E. histolytica trophozoite Iysates resulted in distinct mucosal damage

(Navarro-Garcfa et al., 1993). Therefore, the inability of the E. histolytica extract to

induce reductions in enzyme aetivity in our study quite strongly suggests that the

decreases observed with a Giardia challenge are specifie to this parasite.

The fact that we did obtain reduetions ln disaecharidase activity in Giardia

extract-challenged gerbils indieates that the live parasite is not needed and suggests that

there may be an immunologieal basis for the decreases. Specifie G. lamblia surface

antigens, which are not found on E. histolytica trophozoites, may be targets of the anti

Giardia immune response. Other researchers, using a murine model, have found that

T eells, upon reaeting with an antigen, can release lymphokines whieh damage

rmcrocytes (Mowat and Ferguson, 1981). Such damage could result in a decline in

disaccharidase activities. However, although T cells are known to play a role in the

immune response to Giardia, this process has yet to be examined in giardiasis .

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47

Macrophage interaction with Giardia antigens could also affect enzyme levels, since

mediators su ch as superoxide radicals and lysosomal proteases, released by

macrophages, have been implicated in mucosal damage in G. lamblia-infected mice

(Goyal et al., 1993).

The results obtained with the in vitro-released excretory/secrctory prodllcts of

the parasite were not consistent with the live parasite challenge and the Giardia extract

challenged findings. Sucrase activity was decreased, whercas it was not affccted by a

ch?Jlenge with either the live parasite or the soluble trophozoite extract. Similarly,

maltase and trehalase were not impaired by a challenge with the metabolic

excretory/secretory products, yet they had been when the parasite was llsed. These

results suggest that Giardia metabolic products do not likely play a major role in the

disaccharidase deficiencies observed in giardiasis. Perhaps these t~xcretory/secretory

products contain proteolytic activity which affects the enzymes in a differential manncr.

The excretory/secretory products have been reported to include antigenic, slirfacc

derived mateT/al, with a major polydisperse substance (Nash el al., 1983). Howcvcr,

the presence of a proteinase has not been detcrmined. Flirthermore, althuligh intestinal

damage related to a Giardia exotoxin is an attractive hypothesis, there is no evidcncc

of such a product (Chavez et al., 1986). Further characterization of the effects of the

excretory/secretory products on enzyme activity, as weil as the components of these

products, is necessary.

The data presented here demonstrate that the disaccharidase deficiencies in

gerbils previously exposed to G. lamblia are a parasite-specific cffect and can occur in

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48

response to antigen challenge in a dose-dependent manner. Although there are

indications that the deriine in disaccharidase levels in the challenge infection is the

result of the host's mucosal immune response to a Giardia antigen(s), the precise

mechanism by which Giardia produces diarrhea and malabsorption have yet to be

characlerized. Specifically, therc is a need to find the mediators involved in this

process .

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49

REFERENCES

Adam R.D. 1991. The biology of Giardia spp. Microbiol. Rev. 55: 706-732.

Belosevic M. and Faubert G.M. 1983. Giardia muris: correlation between oral dosage, cOllfse of infection and trophozoite distribution in the mOllse small intestine. Exp. Parmitol. 56: 93-100.

Belosevic M., Faubert G.M. and Mac1ean J.O. 1989. Disaccharidase activity in the small intestine of gerbils (Meriones un~uiculalus) during primary and chalienge infections with Giardia lamblia. GUI 30: 1213-1219.

Belosevic M., Faubert G.M., Mac1ean J.O., Law C. and Croll N.A. 1983. Giardia lamblia infections in Mongolian gerbils: an animal mode\. J. Infi'ct. Dis. 147: 222-226.

Brasitus T.A. 1983. Parasites and malabsorption. Clin. Gastromterol. 12: 495-510.

Buret A., Gall D.G., Nation P.N. and OIson M.E. 1990. Intestinal prolozoa and epithelial ceU kinetics, structure and function. Parasitol. Today 6: 375-380 .

Buret A., Hardin J.A., Oison M.E. and Gall D.G. 1992. Pathophysiology of small intestinal mal absorption in gerbils infected with Giardia lamblia. Gastroenterology 103: 506-513.

Chavez B., Knaippe F., Gonzalez-Mariscal L. and Martinez-Palomo A. 1986. Giardia lamblia: electrophysiology and ultrastructure of cytopathology in culture.d epithelial cells. Exp. Parasitol. 61: 379-389.

Craun a.F. and Jakubowski W. 1987. Status of watel'borne giardiasis outbreaks and monitoring methods. In: Procel~dings o/the International Symposium on Wa/(>r Related Hea/th Issues. Tate C.L. Jr. (ed.). American Watei Resources Association: Bethesda, pp. 167-174.

Dahlqvist A. 1968. Assay of intestinal disaccharidases. Anal. Biochem. 22: 99-107.

Dahlqvist A. and Semenza G. 1985. Disaccharidases of srnall-intestinal mucosa. J. Pediatr. Gastroenterol. Nulr. 4: 857-865.

Daniels C.W. and Belosevic M. 1992. Oisaccharidase activity in the small intestine of susceptible and resistant mice after primary and challenge infections with Giardia muris. Am. J. Trop. Med. Hyg. 46: 382-390 .

Diamond L.S., Harlow D.R. and Cunnick C.C. 1978. A new medium for the axenic

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cultivation of Entamoeba hiltolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyp,. 72: 431-432.

Ferguson A., Gillon J. and Ai Thamery D. 1980. Intestinal abnormalities in murine giardiasis. Trans. R. Soc. Trop. Med. Hyg. 74: 445-448.

Gillin F.D. and Oiamond L.S. 1979. Axenically cultivated Giardia lamblia: growth, attachment and the role of L-cysteine. In: Waterborne Transmission of Giardiasis. Jakubowski W. and HoffJ.C. (OOs.). U.S. Environmental Protection Agency: Cincinnati, pp. 270-272.

Goda T. and Koldovsky O. 1988. Dietary reglilation of small intestinal disaccharidases. Wld. Rev. NUlr. Diel. 57: 275-329.

Goyal R., Mahajan R.C., Ganguly N.K., Sehgal R., Gorowara S. and Singh K. 1993. Macrophage-mediated enterocyte damage in BALBlc mice infected with different strains of Giardia lamblia. Scand. J. Gastroenterol. 28: 845-848.

Guy R.A., Bertrand S. anrl Faubert G.M. 1991. Modification of RPMI 1640 for use in in vitro immur,ological studies of host-parasite interactions in giardiasis. .1. Clin. Microbici. 29: 627-629.

Hartong W.A., Gourley W.K. and Arvanitakis C. 1979. Giardiasis: clinical spectrum and fllnctional-structural abnormalities of the small intestinal mucosa. Gastroenterology 77: 61-69.

Jennings W., Rowland R., Hecker R., Gibson G.E., Fitch R.J. and Reid D.P. 1976. The significance of lower jejunal disaccharidase levels. Aust. N.Z. J. Med. 6: 556-560.

Khanna R., Joshi K., KumKum, Malik A.K. and Vinayak V.K. 1990. An ultrastructural analysis of changes in surface architecture of intestinal mucosa following Giardia lamblia infection in mice. Gastroenterol. Jpn. 25: 649-658.

Launiala K. 1968. The effect of unabsorbed suc rose and mannitol on small intestinal flow rate and mean transit time. Scand. J. Gastroenterol. 3: 665-671.

Lewis P.D. Jr., Belosevic M., Faubert G.M., Curthoys L. and Mac1ean J.O. 1987. Cortisonc-induced recrudescence of Giardia lamblia infections in gerbils. Am. J. Trop. Med. Hyg. 36: 33-40.

Lowry O.H., Rosebrough NJ., Farr A.L. and Randall R.J. 1951. Protein measurements with folin phenol reagent J. Biol. Chem. 193: 265-275.

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Mowat A.MeL. and Ferguson A. 1981. Hypersensitivity reaetions in the small intestine. 6. Pathogenesis of the graft-versus-host reaetion in the small intestinal mueosa of the mouse. Transplalllation 32: 238-243.

Nash T.E., Gillin F.O. and Smith P.D. 198j. Excretory-secretory products of Giardia lamblia . ./.lmmunol. 131: 2004-2010.

Navarro-Garcia F., L6pez-Revilla R. and Tstusumi V. 1993. Dose- and time-dependent functional and structural damage to the colon mucosa by EntamOl'ba histolytica trophozoite lysates. P{lfasitol. Res. 79: 517-522.

Ravich W.J. and Bayless T.M. 1983. Carbohydrate absorption and malabsorption. Clin. Gasfroenterol. 12: 335-356.

Smith P.D., Gillin F.D., Spira W.M. and Nash T.E. 1982. Chronic giardiasis: studies on drug sensitivity, toxin production and host immune responsc. Gastroenterology 83: 797-803.

Wilson H.S.P., Stauffer S.J. and Walker T.S. 1982. Waterborne giardiasis outbrcakAlberta. Cano Dis. Wkly. Rpt. 8: 97-98.

Wright S.G. and Tomkins A.M. 1977. Quantification of the lymphocyte infiltrate in jejunal epthelium in giardiasis. Clin. Exp. Immunol. 29: 408-412.

Yardley J.H., Takano J. and Hendrix T.R. 1964. Epithelial and other mucosallesions of the jejunum in giardiasis. Jejunal biopsy studies. Bull. Johns Hopkins Ho.\'p. 115: 389-406.

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CONNECTING STATEMENT

ln Manuscript l, the effects on disaccharidase activity in gerbils during G.

lamblia infections was described. Il was found that challenging previously-infected

animais with a erude soluble extract of G. lamblia trophozoites resulted in decreases

in enzyme activity similar to those obtained with a live challenge infection. Therefore,

in the study presented in Manuscript 2, an attempt was made to purify the specifie

fraction of the trophozoite extract responsible for these disaccharidase deficiencies .

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MANUSCRIPT 2

IDENTIFICATION OF A FR.ACTION OF

GIARDIA LAMBLIA TROPHOZOITE EXTRA CT

ASSOCIATED WITH DISACCHARIDASE

DEFICIENCIES IN MONGOLIAN GERBILS

Shawn R. Mohammed and G. M. Faubert


McGiII University

Macdonald Campus

21 ,111 Lakeshore Road

Ste. Anne de Bellevue, Quebec

Canada H9X 3V9

(submitted for publication)

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ABSTRACT

The effects on disaccharidase activities of challenging gerbils previously exposed

to Giardia lamblia with fractions of the crllde trophozoite extract were examined. Gel

filtration by Sephacryl S-200 HR chromatography of the soluble extract rcsulted in 3

major protein fractions (FI, F2, F3). Only a challenge with fraction FI (0.1 mg total

dose) was found to induce disaccharidase deficiencies. Boiling FI prior to challenge

did not change this effect on the enzyme activities. However, the decTeases were not

obtained when the total FI dose was reduced to 0.05 mg. Column chromatography of

fraction FI under dissociating and reducing conditions resulted in 2 further fractions:

Fla and Flb. Challenging immune gerbils with Flb led to impairments of

disaccharidase activity similar to those obtained with FI. A challenge with Fla had no

effect on disaccharidase ac:tivities. Protein analysis of the crude extrac~, as weIl as the

fractions of the extract, revealed severa! high and low moleclliar weight bands, with

proteins of the cTude extract ranging from 14 to 200 kDa. These findir.gs indicate that

a constituent(s) of fraction Flb is the portion of the parasite which induces

disaccharidase deficiencies in immune gerbils. This fraction consists of proteins

ranging in molecular weight from 32 to 200 kDa. In addition, the G. lamblia fraction

involved in the decreases in enzyme activity is heat-stable .

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INTRODUCTION

Giardia lamblia is a gastrointestinal parasite of humans and other mammalian

species, with a worldwide distribution. It is a common cause of diarrhea in day-care

centres, institutionalized persons, homosexuals and travellers to developing nations

(Key stone el al., 1978; Brodsky el al., 1974; Schmerin ('1 al., 1978). This tlagellated,

binucleate parasite adheres to the brush border of the host's enterocytes by means of

a ventral sucking disk. In the gut lumen, the trophozoites differentiate into the

intermediate encysting trophozoite form and then to cysts (Campbell and Faubert,

1994). One nuclear division OCCUTS in the cyst or during its formation, so that

tetranucleate cysts are excreted with the feces. Many giardial infections are weil

tolerdted and asymptomatic in human patients. However, wh en iIIness is evident, the

spectrum of symptoms is wide and ranges from acute, self-limiting gastroenteritis to

protracted and debilitating malabsorption (Shandera, 1990).

In giardiasis, it has been established that there can be considerable

malfunctioning of the epithelium of the small intestine. For example, fat and vitamin

B12 malabsorption has been described in human giardiasis (Hoskins et al., 1967; Wright

et al., 1977). Vitamin A deficiency has been found in children infected with G.

lamblia (Mahalanabis el al., 1979). Also, temporary disaccharidase deficiencies have

becn well documented in this disease. Buret et al. (1990) reported decreases in maltase

and sucrase activities in mice, following a primary infection with G. murÎ.'l. ft was

suggested that these disaccharidase deficiencies were due to a diffuse shortening of

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56

brush border mierovilli. Studies on G. lamblia infections in mice found decreases in

several brush border enzymes throughout the eour~e of infection (Nain et al., 1991).

The degree of decline in these enzyme aetivities correlated weIl with the number of

trophozoites in the jejunum, with the most severe reductions in enzyme activity

oceurring during the peak phase of infection. Using the gerbil as an animal model,

Belosevic and eol!eagues (1989) observed decreases in disaceharidase activity during

both primary and challenge infections with G. lamblia. The iovolvement of the host's

immune response in the deficiencies was suggested by the ability of a erude extraet of

the trophozoites to ioduee these impairments of enzyme activity in immune gerbils

(Belosevic et al., 1989). Recent work in our laboratory has confirmed these previous

findings (Manuseript 1). We also showed that decreasing the extract dose used to

challenge the gerbils led to smaller reduetions in enzyme aetivity. In addition, it was

determined that the effects of the extraet in a challenge inoculation are specifie to the

parasite, since challenging animals previously exposed to G. lamblia with a erude

extract of Entamoeba histolytica did not result in impairments of enzyme activity

(Manuscript 1). However, the component(s) of the G. lamblia crude extract involved

in the disaccharidase deficicncies, as weIl as the mechanism(s) leading to sueh

reductions in enzyme activity, are unknown. G. lamblia is consldered to be

antigenically complex, yet little is known about the structures and properties of these

antigens (Chaudhuri et al., 1988).

Cevallos and Farthing (1992) reported a strain-dependent reduction in

disaeeharidase aetivities in G. lamblia-infected rats. The antigenic differences between

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these strains may have led to the variable effects on the enzyme activities. The present

study was undertaken in order to identify the specifie fraction of the G. lamblia crude

antigen extract responsible for inducing the disaccharidase deficiencies observed in

immune gerbils. ln addition, we undertook an initial characterization of this fraction .

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MATERIALS AND METROnS

Parasites

G. lamblia trophozoites, WB strain (American Type Culture Collection no.

30957), originally isolated from a symptomatic patient (Smith et al., 1982) were used

throughout this study. Trophozoites were cultured axenically in filter-sterilized TYI-S-

33 (trypticase, yeast extract, iron, and serum) medium adapted for G.lamblia (Gillin

and Diamond, 1979), wW, 10% adult bovine serum (Sigma Chemical Co., St. Louis,

U.S.A.), and supplemented with 100 units/mL of penicillin (Sigma) and 100 Ilg/mL of

dihydrostreptomycin sulphate salt (Sigma), in the absence of bovine bile. Twice

weekly, the trophozoites were passaged.

Animais

Six to 10 week old male Mongolian gerbils (Meriones unguiculatus) , originating

from Tumblebrook Parms (West Brookfield, U.S.A.), were used in this study. They

were maintained under standard laboratory conditions and provided with laboratory

chow and water ad libitum. One week after arrivai, gerbils were each tre.ated with 15

mg of metronidazole (Rhône-Poulenc, Montreal, Canada), by oral gavage, for 3

consecutive days and th en allowed to rest for 10 days. This treatment was done to

ensure that the gerbils were free of intestinal infections.

AU inoculations were done orally, to unanaesthetized gerbils. In the primary

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infection, each gerbil was inoculated with 1 x 1()6 live G. lamblia trophozoites.

Preparation of the Crude Extract of G. lamblia Trophozoites

Trophozoites (from a 72-h culture) were dislodged from the wans of culture

vessels by immersion in an ice bath for 15-30 min. They were sedimented by

centrifugation at 800 g for 10 min, at 4° C, and washed 5 times in sterile phosphate

buffered saline (PBS) (pH 7.2). Trophozoites, suspended in sterile PBS, were th en

lysed by discontïnuous sonication in an ice bath, using a Sonic Dismembrator (Fisher

Scientific, Montreal, Canada) at maximum output, for 10 min. The sonicated material

was cleared of insoluble matter by centrifuging at 23,300 g for 20 min, at 4()C, and the

supernatant collected as the crude soluble extract.

Column Chromatography

Samples were fractionated by gel filtration chromatography. For this purpose,

approximately 80 mL of preswollen Sephacryl S-200 HR gel (Pharmacia LKB

Biotechnology, Uppsala, Sweden), wet bead diameter 25-75 #-tm, were packed into a 1.8

x 50 cm glass column (Bio-Rad, Mississauga, Canada). The void volume of the

column was determined by applying Blue Dextran 2000 and the column calibrated with

known molecular weight (MW) marker of gel filtration proteins (Pharmacia). For each

run, at 4° C, 1-2 mL of sample (with 10% glycerol) was applied to the gel bed and a

flow rate of 5 mL/h maintained using a peristaltic pump (Pharmacia). Fractions of 2

mL each were collected and an elution profile obtained by measuring the optical density

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60

(0.0.) at 280 nm and plotting these values against the fraction numbers. According

to the 0.0. values, e.}(·h peak and trailing eluates were pooled separately. These

pooled fractions were th en dialyzed, using Spectra/Por 6 Membrane (MW Cut-Off:

1(00) (Spectrum Medical Industries Inc., Los Angeles, U.S.A.), against distilled water

for 24 h, at 4° C. Following dialysis, fractions were concentrated by lyophilization and

resuspended in PHS. The fractions were stored at - 70° C until used.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

(SDS-PAGE)

Samples were electrophoresed in a Mini-PROTEAN II vertical slab cell (Bio

Rad) using the discontinuous system of Laemmli (1970). Protein samples were

separated through a 4% stacking gel and 10% separating gel. Bio-Rad molecular

weight standards were run simultaneously. The gel was stained with 0.25% Coomassie

Brilliant Blue R-250 dye (Bio-Rad).

Measurement of Intestinal Disaccharidases

- Preparation of Intestinal Homogenate -

Homogenates were prepared as previously described (Belosevic et al., 1989),

with modifications. Briefly, the gerbils were killed and the small intestine removed and

divided into three sections. The segments were placed in ice cold distilled water and

each was flushed with 50 mL of distilled water to clear it of intestinal debris. The

segments were then slit longitudinally and the mucosa scraped off with a glass

l • 61 1

microscope slide. The mucosa from all three sections were combined, weighed (wct

weight), and placed in four volumes of ice cold distilled water. The mucosal scrapings

were then homogen\zed using a Con Torque power unit at maximum speed (Eberbach

Corp., Ann Arbor, U.S.A.). Homogenates were stored, wlthout prior centrifugation.

at - 7(1 C until used in the assay.

- Assay for Intestinal Disaccharidases -

Disaccharidase activity of homogenated mllcosa was measured using the glucose

oxidase peroxidase assay of Dahlqvist (1968), as modified by BelosevÎC el al. (1989),

which is based on a colollr reaction with the glucose liberated by a particular

disaccharidase in 60 min. The assay consisted of adding to each weil of a 96-well

• Nunc microwell plate (Gibco BRL, Burlington, Canada) 0.01 mL of appropriately

diluted mucosal sample ~homogenate) and 0.01 mL sub~trate-buffer solution of a

disaccharidase to be measured, in quadruplicate. The plate was th en incubatcd at 37"

C in humidified atmosphere for 60 min. After incubation, 0.3 mL tris-glucose oxidase

reagent was added to each well and the plate incubated for an additional 60 min. The

plate was then read at 415 nm using a Bio-Tek microplate reader (Mandel Scicntitic,

Guelph, Canada). For each assay, 8 wells of reagent blank and a glucose standard

series (2, 6, and 10 Ilg glucose) in quadruplicate were also donc. Disaccharidase

activity is expressed as units/mg protein in the mucosal sample (U/mg), where units

represent f,tmoles of disaccharide hydrolyzed/min.

- Reagents -

• AlI chemicals were obtained commercially. 0-( + )-G1ucose, fi-lactose, maltose,

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62

D-( + )-trehalose, maleic acid, glucose oxidase, o-dianisidine, peroxidase, and triton x-

100 were purchased from Sigma. Sucrose was obtained from Fisher Scientific and tris

(ultra pure) was purchased from ICN Biomedicals Canada Ltd. (St. Laurent, Canada).

Measurement of Protein Concentration

PrC'tein concentrations were determined as described by Lowry et al. (1951),

using 0.2 mL of sample and a final reaction volume of 1.3 mL. For each protein

as say , a standard curve was prepared using freshly dissolved bovine serum albumin

(BSA).

Statistical Analysis

Statistical significance was determined using the Mann-Whitney U-test.

Significancc was assigned at the probability level of P < 0.05 .

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63

RESULTS

Fractionation of the Soluble Extract of G. lamblia Trophozoites

The crude soluble extract of G. lamblia trophozoites was subjected to column

chromatography using Sephacryl S-200 HR gel filtration in order to fractionate its

components. Three light-absorbing peaks were observed, one of which appeared in the

void volume and another at the end of total column volume (Fig. 1). Eluted materials

were cotlected to give fractions FI (> 150,000 MW), F2 (- 67,000 MW), and F3

« 14,000 MW), as indicated in Fig. 1. These were pooled from fractions 16-22, 23-

29, and 34-43, respectively.

Challenge with the Fractions of the G. lamblia Soluble Extract

To determine whether any one of the fractions of the soluble extract (described

above) could affect disaccharidase activity in gerbils previously exposed to G. lamblia,

each animal was challenged with 0.1 mg of FI, F2, or F3. The animais wcrc

challenged on day 50 post-infection, which is past the elimination of the parasite from

the gerbil small intestine (Manuscript 1). The activities of the enzymes lactase,

maltase, sucrase, and trehalase were then measured on days 2 and 4 post-challenge

(p.c.), as these were the days on which disaccharidase activity was affecte.d during

challenge inoculations with the whole crude extract (Manuscript 1). As shown in Fig.

2, the activity of sucrase was not affected by any of the 3 fractions of the extract, as

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64

~ c::I ~

~ fil ~

rQ -< ~ = 1 1 1 1 0.8 Ft 0.6 t-------t

~ 0.4 ,...., a 0.2

~ 0.09

0 0.08 ~ N ........ 0.07 ~

o+J 0.06 .,.. rn ~ 0.05 4> ~ ..... 0.04-as (J 0.03 .,..

o+J ~ 0.02 0

0.01

0.00 5 la 15 20 25 30 35 40 45 50

Fraction Number

Fig. 1. Chromatograph of the soluble extract of sonicated G. lamblia trophozoites. Approximately 1.8 mg of the soluble extract were run through a Sephacryl S-200 HR column at a flow rate of 5 mLlh, using a buffer of 0.15 M PBS, 0.02% sodium azide (pH 7.2). Fractions were collected and pooled to give FI, F2, and F3, as indicated. The column was calibrated with Blue Dextran 2000 (BD; > 2 x 106 MW), Aldolase (Ald; 158,000 MW), Bovine Serum Albumin (BSA; 67,000 MW), and Ribonuclease A (RnA; 13,700 MW) .

•

•

•

- 0.012 taG S 0.010

....... P 0.018 -~ 0.018 '004

.!: O.OU ~

~ 0.012

" 0.010 ID Ils

'1:1 .... ... Ils .cl g g Ils ID .... =

0.008

0.008

0.00'

0.002

o.ooo~-

_ 0.18 '-II

~ 0.18 ;:, - 0.1'

~ .... 0.12 ~ ....

+1 Co) 0.10 < : 0.08 at ~ 0.08 ... as .cl 0.0.

Co) Co) Ils 0.02 ln .... ~ 0.001---

LACTASE

2

SUCRASE

2

Days Post-Challenge

65

MALTASE

1.8

1.' 1.2

1.0

0.8

0.8

o .•

0.2

O.OL_-2

TREHALASE 0.010

0.0011

0.008

0.007

0.008

0.000

0.00'

0.003

0.002

0.001

0.0001---2

Days Post-Challen,e

Fig. 2. Disaccharidase activities in gerbils challenged with different fractions of the soluble extract of G. lamblia trophozoites. Each gerbil was cha.llenged with 0.1 mg of Ft (IZ]), F2 (IXI), or F3 (ISI) 50 days after a primary infection with 1 x 106 live trophozoites. Control gerbils <.) were age-matched and received the primary infection, but were not challenged. Results are expressed as mean ± SE, n =4 for unchallenged controls and n =5 for fraction-challenged groups. * Differences between control and challenged groups were significant, P < 0.05.

•

•

•

66

levels on both days 2 and 4 p.c. remained comparable to controls. However, the

activities of lactase, maltase, and trehalase were reduced by as much as 47% on day

4 p.c. in Fl-challenged gerbils. In addition, an FI challenge also led to a deerease in

maltase activity on day 2 p.c. On the other hand, challenging immune gerbils with

either F2 or F3 had no effeet on any of the disaccharidase activities.

In order to determine whether the effeet of fraction FIon enzyme activities is

dose-dependent, immune gerbils were challenged with 0.075 mg or 0.05 mg total of FI.

Disaccharidase activity was th en measured on day 4 p.c., the time when the most

extensive effects on the enzymes were observed with the 0.1 mg FI challenge. Sucrase

activity remained unaffeeted by lhis fraction of the soluble extract, regardles~ of the

dosage (Fig. 3). However, lactase and maltase activities were significantly reduced

with an FI ,Jose of 0.075 mg, compared to unchallenged controls. These decreases in

disaccharidase activity were similar to the reduced levels observed in the gerbils

challenged with 0.1 mg of FI. There were no reductions in the activities of any of the

4 enzymes when an FI dose of 0.05 mg was given to the animais.

Gerbils which had a prior Infection with G. lamblia were also challenged with

0.1 mg of FI which had previously been boiled at 100° C for 5 min. Disaccharidase

activity was then measured on day 4 p.c. and compared to age-matched control animais

which had reeeived a primary infection, but were not challenged. Deereases in lactase,

maltase, and trehalase activities were deteeted, while sucrase activity did not

significantly differ from control levels (data not shown). These findings were similar

to those obtained with FI which was not boiled.

•

•

•

_ 0.011 III El 0.010

....... P 0.0111 -~ O.OUI +' .... .. 0.010& .... ..., ~ 0.012

CU 0.010 la

~ 0.0011 .... :; 0.0011

~ 0.000& C)

= 0.002 .... .::1 0.000

_ 0.10& lai S ~ 0.12 '-'"

b 0.10 .... ~ .... t 0.011 ..:

CU la 0.01 .. ~ .... J.4 0.00& .. ~ C) CJ 0.02 .. la ....

.::1 0.00

LACTASE

CONTROL 0.1 0.075 0.06

SUCRASE

CONTROL 0.1 0.076 0.011

Fraction Fl DOlic (ml)

67 MALTASE

1.11

1.0&

1.2

1.0

0.8

0.1

0.0&

0.2

0.0 CONTROL 0.1 0.0711 0.011

TREHALASE 0.010

0.001

0.008

0.007

0.001

0.0011

0.000&

O.OOS

0.002

0.001

0.000 CONTROL 0.1 0.076 0.01

Fraction Fl Doae (ml)

Fig. 3. Disaccharidase activities in gerbils challengcd with varying amounts of fraction FI of the soluble extract of G. lamblia trophozoites. Gerbils were challenged 50 days following a primary infection with 1 x 106 live trophozoites. Control gerbils were agematched and received a primary infection, but were !lot challenged. Results are for day 4 post-challenge and are exprcssed as mean ± S.E , n =4 for unchallenged controls (.> and n=5 for Fl-challenged groups (~). * Differences between control and challenged groups were significant, P < 0.05.

•

•

•

68

Fractionation of FI

In order to further purify the fraction of the G. lamblia extract responsible for

inducing the disaccharidase deficiencies, fraction FI was subjected to gel filtration

chromatography using a Sephacryl S-200 HR column. A buffer consisting of the

dissociating agent guanidine Hel (Sigma) and the reducing agent DL-dithiothreitol

(Sigma) was used to achieve further separation of FI, by disrupting non-covalent

interactions and breaking disulfide bonds in this fraction. Two peaks were observed,

one of which eluted with the void volume and the other also appeared in the high

molecular weight (> 67,000 MW) region of the separation (Fig. 4). Fractions 15-19

and 20-25 were collected separately to give pooled fractions Fla and Flb, respectively.

Challenge with Fractions Fla and Flb

To determine whether disaccharidase activity could be affected by one of the

fractions of FI (obtained as described above), immune gerbils were each challenged

with 0.1 mg total of either Fla or Flb. Enzyme activities were then measured on day

4 p.c. The activities of ail 4 enzymes remain<~ comparable to controllevels in Fla

challenged animais (Fig. 5). However, challenging with fraction Flb resulted in

significant decreases in the activities of lactase, maltase, and trehalase. These

reductions ranged from 39% to 71 %. Sucrase activity was also unaffected by an Flb

challenge.

•

•

•

69 ~ - ~ Il

Q fil ~ Â IZI IZI Q = 1 1 1 1

0.12 Fla t---t

0.11 -... a 0.10

= 0.09 0 cc 0.08 ru '-'

~ 0.07

+J ...... 0.06 rn ~

0.05 Q)

~ Flb

~ 0.04-

= t.> ...... 0.03 ~ ~ 0.02

0 0.01

0.00 5 10 15 20 25 30 35 40 45 50

Fraction Number

Fig. 4. Chromatograph of fraction FI of the soluble extract of sonicated G. lamblia trophozoites. Approximately 2 mg of Ft were applied on a Sephacryl S-200 HR column (1.8 x 50 cm) at a f10w rate of S mL/ho The eluent consisted of 2 M guanidine-HCl, 2.5 mM dithiothreitol and 0.02% sodium azide, in PBS (pH 7.2). Fractions were collected and pooled to give Fla and Flb, as indicated. The column was calibrated with Blue Dextran 2000 (BD; > 2 x 106 MW), Bovine Serum Albumin (BSA; 67,000 MW), Ovalbumin (aval; 43,000 MW), and Ribonuclease A (RnA; 13,700 MW).

•

•

•

_ 0.010 ... S 0.011

"'" ~ _ 0.011

!' 0.01" .... ~

::: 0.012 g

~ 0.010 CU : 0.001 ." .... ~ .. ~ CI CI .. ., .... ~

0.001

0.001

0.000

_ 0.11 III S 0.10

'"', ~ 0.01 -!' 0.01 .... ~ 0.07 .... .., ~ 0.01

CU 0.05 ., .. ." .... ~ .. ~ CI CI .. ., .... ~

0.0"

o.os

0.01

0.01

0.00

CONTROL

CONTROL

70 LACTASE MALTASE

l."

l.2

l.0

• 0.8

0.1

0.4

0.8

0.0 'la 'lb CONTROL ria 'lb

SUCRASE TREHALASE 0.001

0.001

0.007

0.008

0.001 •

Il.003

1~.002

0.001

0.000 rla 'lb CONTROL rla 'lb

Fraction Fraction

Fig. 5. Disaccharidase activities in gerbils challenged with fractions Fla and Flb. Gerbils were each challengro with 0.1 mg of Fla or Flb 50 days following a primary infection with 1 x 1()61ive trophozoites. Control gerbils were age-matched and received a primary infection, but were not challenged. Results are for day 4 post-challenge and are expressed as mean ± SE, n=4 for unchallenged controls <.) and n=5 for FlalFlb-challenged groups (181). * Differences between control and challenged groups were significant, P < 0.05.

•

•

•

71

SDS-PAGE

In order to analyze the protein constituents of the crude extract of sonicated G.

lamblia trophozoites and of the partially purified extract fractions, samples were

compared following SDS-PAGE. Several high and low molecular weight bands were

seen (Fig. 6). The erude extraet showed approximately 20 discernable protein bands,

with molecular weights ranging from 14 to 130 kDa. However, the banding pattern of

fraction FI was in the 19 to 200 kDa range and F2 was in the smaller regiol1 of 30 to

55 kDa. Fraction F3 showed only one protein band, of 20 kDa. For both Fla and

Flb, a number of bands were detected, mai ni y in the molecular weight region of 32 to

200 kDa. However, there were 2 faint yet discrete bands seen in fraction Fla which

were absent from Flb. These had molecular masses of 25 kDa and 27 kDa (arrows).

In addition, major bands of 30 kDa and 33 kDa were secn in ail samples, with the

exception of fraction F3.

•

•

•

kDa 200-

97.4-

21.5-

1 2 3

72

4 5 6

Fig. 6. SDS-PAGE of the crude extract of G. lamblia trophozoites and partially purified fractions of the extract. The gel was stained with Coomassie brilliant blue R-250. Molccular weight standards are shown on the left. Lane 1: erude extract; 2: FI; 3: F2; 4: F3; 5: Fla; 6: Flb .

•

•

•

73

DISCUSSION

G. lamblia is the most corn mon intestinal protozoan parasite of humans and the

acute to chronic diarrhea which it can cause is often associated with intestinal

malabsorption (Farthing, 1992). Even when Giardia is not sllspected, cases of

malabsorption may be due to latent giardiasis, as the organism can be found in the

intestine withoutappearing in feces (Kamath and Murugasu, 1974). The disaccharidase

deficiencies which contribute to the malabsorption of nutricnts in Giardia infections are

therefore an important area of investigation.

In this study, we examined the contribution of the parasite to host malabsorption

of disaccharides in giardiasis. Our chromatographie profile of Giardia protcins is

supported by work done by Chaudhuri and colleagues (1988), who obtained a similar

3·peak separation of the G. lamblia soluble extract, using Sephacryl S-300 gel fi Itration.

Moreover, these researchers found that maximum antigenic activity, as determined by

counter immunoelectrophoresis, was associated with their high molecular weight

fraction, which eluted in the void volume and corresponds to our fraction FI.

Following separation of the G. lamblia soluble extract through Sephacryl S-200 HR

chromatography, we wer~ able to clearly implicate fraction FI in the disaccharidasc

deficiencies in immune gerbils. The patterns of lactase, maltase and trehalase

reductions on days 2 and 4 p.c. in the FI-challenge mimic those obtained in previous

studies with live- and extract-challenged animaIs (Manuscript 1). The fact that we were

able to decrease the amount of F l used to challenge gerbils and lose the effect on ail

•

•

•

74

the enzymes with a 0.05 mg dose, as was found in an identical study using the cru de

ex tract (Manuscript 1), served to confirm the importance of fraction FI in the

disaccharidase deticiencies. In addition, the constituent of FI involved in the

impairments of disaccharidase activity is heat-stable since the effects of Fion the

enzyme activities were not altered by boiling FI prior to the challenge.

We were able to partially purify FI into 2 fractions, Fla and Flb, again usillg

a Sephacryl S-200 HR column. The use of an eluent containing the dissociating agent

guanidine HCI and the reducing agent dithiothreitol allowed us to disrupt non-covalent

interactions and break disulfide bonds in FI, and therefore further separate giardial

proteins. The ability of a challenge with Flb, but not Fla, to induce the sa me

disaccharidase deficiencies in immune gerhils as when challenging with FIor the

complete trophozoite extract (Manuscript 1), indicates that we have isolated a fraction

of the parasite with a constituent(s) that is intimately linked to disaccharide

malabsorption.

The complexity of the soluble extract of G. lamblia was demonstrated by the

SDS-PAGE protein analysis. The present study revealed that approximately 20 protein

bands, ranging from 14 to 130 kDa, cou Id be detected. These findings are supported

by those of other researchers, who have reported between 20 to 28 protein bands with

molecular weights ranging from a low of 10 kDa up to 140 kDa (Moore et al., 1982;

Smith et al .• 1982; Chaudhuri et al .• 1988). We also observed sorne very faint bands

in the rcgion between 130 and 200 kDa. Such poorly discernable high molecular

weight bands have been reported by Smith and colleagues (1982), as weIl. Using an

•

•

•

75

extract of the Pl strain of G. lamblia trophozoites, Kumkum and co-workers (1988)

were able to clearly detect bands with molecular weights up to 205 kDa. Such

differences in the bands observed through SDS-PAGE analysis are likely the result of

inter-strain antigenic variation. It should be noted that both inter- and intra-strain

antigenic variation could contribute to the variability of symptoms seen in giardiasis.

Perhaps infections with Giardia strains which possess the relevant protel\1(s) present in

Flb lead to disaccharidase deficiencies in the hosto Conversely, an infœtion wilh il

strain which lacks the key portion of fraction Flb would not affect disaccharide

absorption. The protein profiles also revealed that the separation of the trophozoite

extract and fraction FI, using gel filtration, was not ideal. There were fractions

obtained which shared proteins of similar molecular weight. This probably rcsuItcd due

to contamination from preceding fractions during the chromatographie separation.

However, given the inability of fraction F2 proteins to affect enzyme activity, il is

likely that any proteines) in Flb responsible for the disaccharidase deficieneies is in the

55 to 200 kDa region.

The role of immune mechanisms III producing malabsorption and intestinal

symptoms is unclear. Daniels and Belosevic (1992) found that challenging micc with

an extract of G. muris led to more depressed disaccharidase activity in susceptiblc

animais as compared to resistant mice. These authOls suggested that this finding could

be due to an immune responsc to a particular parasite antigen in the susceptible micc

which differs from the response to anothcr antigen by the reslstant mice. Thereforc,

selective immune recognition of the relevant parasite antigen, present in fraction FI b,

•

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76

could ultimately result in enzyme deficiencies. The specific type of immune response

to Flb which could cause a decline in disaccharidase activities rema:ns unknown.

Based on other studies on intestinal damage, the disaccharidase deficiencies in giardiasis

may be linked to a mucosal mast cell response to an :-, h antigen. Perdue and Gall

(1987) implicated mast cell mediators in the abnorma:ities of intestinal function

observed in anaphylactic rats. In these rats, there is significant disruption of the

intestinal mucosa which is linked tl' mllcosal mast ccli degranulation (Patrick et al.,

1988). In addition, Curtis et al. (1990) demonstrated decreased disaccharidase activity,

as weil as decreased brush border microvillus surface area and vilIus height, in rats

following chronic antigen challenge. Mucosal mast cell involvement was sllggested by

mast cell proliferation and degranulation. lt is certain, however, that since the Flb

challenge in the present study was done orally this would imply that in order to

stimulate any type of host mucosal immune response, fraction Ftb remained

immunogenically intact upon arrivai in the smalt intestine.

It is important to note that there are possible non-immune mechanisms which

could contribute to disaccharidase deficiencies. For example, Giardia trophozoites are

known to possess proteinase activity (Hare el al., 1989). A particular proteinase could

be present in fraction Flb which affects the enzyme levels as observed. Giardia

trophozoites also exhibit lectin activity. Plant lectins have been shown to be capable

of directly damaging rat enterocytes, producing brush border abnormalities similar to

those seen in giardiasis (Lorenzsonn and Olsen, 1982). It is therefore possible that

Giardia lectins are present in fraction Flb and contribute to host microvillus damage,

•

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77

with subsequent disaccharidase impairment. In addition, it has been found that ~he

variant surface proteins (VSPs) which are present on Giardia trophozoites contain

metal-binding domains, sorne of which bind zinc (Thompson et al., 1993). Fraction

Flb may contain a VSP(s) which competes with the host for zinc in the intestine,

causing a decrease in enzyme activity. It is apparent, though, that whatever the

constituents of FI b which are involved in the disaccharidase deficiencies, they are able

to evade degradation by the enzymes of the gut, through an unknown mechanism.

The present study has implicated fraction Flb of the G. lamblta trophozoite

extract in the disaccharidase deficiencies observed in immune gerbils. It seems

probable that without the relevant component of Flb, Giardia strains cannot cause

disaccharidase deficiencies in infected hosts. However, further investigation of the

mechanism(s) of interaction between Flb and the mucosal epithelial ceUs, inc1uding the

specifie portion of fraction Flb involved in the intestinal dysfunction, is necessary .

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78

REFERENCES

Belosevic M., Faubert G.M. and Maclean J.O. 1989. Oisaccharidase activity in the smalt intestine of gerbils (Meriones unf.:lIiculatus) during primary and challenge infections with Giardia lamblia. Gur 30: 1213-1219.

Brodsky R.E., Spencer H.C. and Schultz M.G. 1974. Giardiasis in American travelers to Soviet Union. J. Infect. Dis. 130: 319-323.

Buret A., Gall D.G. and OIson M.E. 1990. Effects of ruurine giardiasis on growth, intestinal morphology, and disaccharidase activity. J. Parasitol. 76: 403-409.

Campbell J.O. and Faubert G.M. 1994. Comparative studies on Giarclia lamblia encystation in vitro and in vivo. J. ParasilOl. 80: 36-44.

Cevallos A.M. and Farthing M.J .G. 1992. Small intestinal functional damage in experimental giardiasis is strain-dependent. Gastroenterology 102: A602.

Chaudhuri P.P., Pal S., Pal S.C. and Das P. 1988. Studies on Giardia lamblia trophozoite antigens using sephacryl S-300 column chromatography, polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay. In: Advances in Giardia Research. Wallis P.M. and Hammond B.R. (cds.). University of Calgary Press: Calgary, pp. 191-194.

Curtis G.H., Patrick M.K., Catto-Smith A.G. and Gall D.G. 1990. Intestinal anaphylaxis in the rat: effect of chronic antigcn exposure. Gastroent(lrolo~y 98: 1558-1566.

Dahlqvist A. 1968. Assay of intestinal disaccharidases. Anal. Biochem. 22: 99-107.

Daniels C. W. and Belosevic M. 1992. Oisaccharidase activity in the small intestine of susct::ptible and resistant mice after primary and challenge infections with Giardia muris. Am. J. Trop. Med. Hyg. 46: 382-390.

Farthing M.J.G. 1992. New perspectives in giardiasis. J. Med. Microbiol. 37: 1-2.

Gillin F.D. and Diam(lnd L.S. 1979. Axenically cultivated Giardia lamblia: growth, attachment and the role of L-cysteine. In: Waterborne Transmission of Giardiasis. Jakubowski W. and HoffJ.C. (eds.). U.S. Environmental Protection Agency: Cincinnati, pp. 270-272.

Hare O.F" Jarroll E.L. and Lindmark D.G. 1989. Giardia lamblia: characterization of proteinase activity in trophozoites. Exp. Parasitol. 68: 168-175.

J

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79

Hoskins L.C., Winawer S.1., Broitman S.A., Gottlieb L.S. and Zarncheck N. 1967. Clinical giardiasis and intestinal malabsorption. Gastroenterology 53: 265-279.

Kamath K.R. and MJrugasu R. 1974. A comparative study of four methods for detecting Giardia lamhlia in children with diarrheal disease and malabsorption. Guslroenterology 66: 16-21.

Keystone J .S., Krajdell S. and Warren M.R. 1978. Person-to-person transmission of Giardia lamblia in day-care nurseries. Cano Med. Assoc. J. 119: 241-248.

Kumkum, Khanna R., Khuller M., Mehta S. and Vinayak V.K. 1988. Plasma membrane associated antigens of trophozoites ofaxenic Giardia lamblia. Trans. R. Soc. Trop. Med. Hyg. 82: 439-444.

Laemmli U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680-685.

Lorenzsonn V. and Olsen W.A. 1982. ln vivo responses of rat intestinal epithelium to intraluminal dietary lectins. Gustroenterology 82: 838-848.

Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J. 1951. Protein measurements with folin phenol reagent. J. Biol. Chem. 193: 265-275.

Mahalanabis D., Simpson T.W., Chakraborty M.L., Ganguli C., Bhattacharjee D.K. and Mukherjee K.L. 1979. Malabsorption of water miscible vitamin A in children with giardiasis and ascariasis. Am. J. Clin. NUlr. 32: 313-318.

Moore G.W., Sogandares-Bernal F., Dennis M.V., Root D.M., Beckwith D. and van Voorhis D. 1982. Characterization of Giardia lamblia trophozoite antigens using polyacrylamide gel electrophoresis, high-performance liquid chromatography, and enzyme-Iabeled immunosorbent assay. Vel. Parasitol. 10: 229-237.

Nain C.K., Dutt P. and Vinayak V.K. 1991. Alterations in enzymatic activities of the intestinal mucosa during the course of Giardia lamblia infection in mice. Ann. Trop. Med. Parasitol. 85: 515-522.

Patrick M.K., Dunn I.J., Buret A., Miller H.R.P., Huntley J.F., Gibson S. and Gall D.G. 1988. Mast cell protease release and mucosal ultrastructure during intestinal anaphylaxis in the rat. Gastroenterology 94: 1-9.

Perdue M.H. and Gall D.G. 1987. Mucosal mast cells and the intestinal epithelium. Adv. Exp. Med. Biol. 216A: 645-652 .

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S,chmerin M.J., Jones T.C. and Klein H. 1978. Giardiasis: association with homosexuality. Ann. lm. Med. 88: 801-803.

Shandera W.X. 1990. From Leningrad to the day-care center - The ubiquitous Giardia lamblia. West. J. Med. 153: 154-159.

Smith p.n., Gil1.in F.O., Kaushal N.A. and Nash T.E. 1982. Antigenic analysls of Giardia lamblia from Afghanistan, Puerto Rico, Ecuador, and Oregon. II/fi'et. Immun. 36: 714-719.

Smith p.n., Gillin F.O., Spila W.M. and Nash T.E. 1982. Chronic giardiasis: studies on drug sensitivity, toxin production and host immune rt~sponse.

Gastroenterology 83: 797-803.

Thompson R.C.A., Reynoldson J.A. and Lymbery AJ. 1993. Giardia - from molecules to disease and beyond. Parasitol. Today 9: 313-315.

Wright S.G., Tpmkins A.M. and Ridley O.S. 1977. Giardiasis: clinical and therapeutic aspects. Gut 18: 343-350.

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81

GENERAL DISCUSSION

Infections with G. lamblia are now considered an important cause of

gastroenteritis (Shandera, 1990) and dis:a.ccharidase deficiencies have been consistently

identified as one of various abnormalitr\~s of small intestinal pathology in giardiasis

(Jennings et al., 1976). Since unabsorbea sugars lead to diarrhea by causing net fluid

accumulation in the intestinal lumen (Launl\\la, 1968), disaccharidase deficiencies play

a key role in understanding the pathogeol'Ic mechanisms which lead to intestinal

dysfunction in giardiasis. ft has been suggest\,j that the changes in the small intestine

of Giardia-infected individuals, including the 1 mpairments of disaccharidase activity,

are due to both a direct effect of the parasite and to antigen-specific host immune

responses, such as immune complexes and delayed-type hypersensitivity (Gillon et al. ,

1982). However, other host factors, such as diet, gut motility and nutritional status

could also contribute to the intestinal pathology (Ferguson et al., 1980). At present,

there is Iittle knowledge of the relative contributions of parasite and host-related factors

to the disaccharidase rleficiencies in giardia;'ls. Therefore, this project involved a closer

examination of the process by which G. lamblia causes these reductions in enzyme

activity, using the gerbil model of the human disease.

In Manuscript l, a more extensive characterization of the effects on

disaccharidase activity in G. lamblia-infected gerbils was reported on. For all 4

disaccharidases examined, the activities were reduced during a primary G. lamblia

infection. These enzyme deficiencies were first observed during the acute phase of the

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82

infection, but persisted weB past the elimination of the parasite from the smalt intestine.

These findings indicate that the disaccharidase deficiencies are not dependent on the

persistent presence of the parasite in the gut. In facto studies on giardiasis in humans

have found that there are patients with continued malabsorption following eradication

of the parasite (Brasitus, 1983). It is possible that the enzyme deficiencies continue due

to the initial enterocyte destruction by trophozoite attachment and to indirect intestinal

damage by an effective host mucosal immune response. Mucosal disruption would

necessarily affect the disaccharidases present there. In a challenge infection with live

G. lamblia trophozoites, the activities of the disaccharidases (with the exception of

sucrase) were also decreased. However, the reductions in enzyme activity in this case

were of short duration, occurring only on days 2 and 4 p.c., perhaps bccause G.

lamblia trophozoites do not establish in the gerbil gut upon challenge. In addition,

similar impairments of disaccharidase activity were obtained when gerbils were

challenged with 0.1 mg of the soluble extract of G. lamblia trophozoites. The effects

of the extract challenge were dose-dependent and indicate that the live parasite is not

needed to obtain the enzyme deficiencies. These findings suggest that there may lle an

immunological basis for the decreases, involving a host response to G. lamblia surface

antigens. Since challenging gerbils previously expost'.d te G. lamblia with an extract

of E. histolytica had no affect on disaccharidase activities, it seems that the decreases

observed with a Giardia challenge are specifie to this parasite. Also, it seems that G.

lamblia metabolic products do not play a major role in the disaccharidase deficiencies

observed in giardiasis as the trophozoite excretory/secretory products affected these

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83

enzymes in a manner which was inconsistent with the live parasite challenge.

Manuscript 2 focused on studies involving the partial puriftcation and

characterization of a specific fraction of the G. lamblia crude extract responsible for

inducing the disaccharidase deficiencies in immune gerbi:s. Gel filtration using a

Sephacryl S-200 HR column allowed for the se!Jaration of the crude extract of G.

lamblia trophozoites into 3 fractions. Only a challenge with fraction FI affected

disaccharidase activities in immune gerbils, with decreased levels observed on days 2

and 4 p.c. The patterns of the reductions in enzyme activity with the FI-challenge

were similar to those obtained with live- and extract- challenged animais and indicated

the importance of fraction FI in the disaccharidase deficiencies. The component of FI

involved in the impairments of enzyme activity was determined to be heat-stable .

Sephacryl 5-200 HR gel chromatography of fraction FI, using an eluent co.mlÏnirz the

dissociating agent guanidine-HCI and the reducing agent dithiothreitol, allowed for the

further separation of G. lamblia proteins. Of the 2 fractions obtained following this

separation, only a challenge with fraction Flb resultui in disaccharidase deficiencies,

similar to those observed in F 1- or crude extract- challenged gerbils. These results

indicate that with Flb we have partially purified the fraction of the parasite responsible

for inducing disaccharidase deficiencies in immune animals. nrotein analysis of the

erude extract and the fractions of the extract reve.aled many high and low molecular

weight banùs, with any protein(s) in Flb responsible for the decreases in enzyme

activity in the 55 to 200 kDa region.

The role of immune mechanisms in producing disaccharidase deficiencies in G .

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84

lamblia infections is not known. Garside and colle,aglles (1991) found that interferon-

a/{3 (IFN-a/{3) contributes to intestinallesions in l1lice with graft-verslls-host re,lction

(GVHR). It was suggested that IFN-a/j3 callsed mucosal damage through the induction

of natural killer (NK) ceU activity. Intestinal damage by NK cells could thcrcfore lcaù

to reductions in disaccharidase activity in giardiasis. Another possible immune

mechanism which could result in disaccharidase deficiencies in Giardia infections is the

disruption of the intestinal l1lucosa by mast cell mediators. Mast cell degranulation may

occur in response to a Giardia antigen and lead to intestinal abnormalities, as has bcen

found in rats following chronic challenge with egg albumin (Curtis l'I al., 1990). Otllcr

researcher~, using rnice with GVHR, have found that T cells, after reacting wlth an

antigen, can release lymphokines which damage enterocytes (Mowat and Fcrguson,

1981). Such damage could again result in a decline in disaccharidase activities.

Moreover, there are many other possible immune mechanisms whlch could produce

impairments of enzyme activity, however there has yet to be a link found bctwccn a

particular ho st l1lucosal immune response and the disaccharidase deti il~ncics in

giardiasis.

There are several possible- avenues for future research in this arca. For

example, more sophisticated purification techniques can be used in studies to isolate the

specifie component of G. lamblia which is responsible for the decreascs in enzyme

activity. Also, disaccharidase levels can be measured following the lIlhibition of factors

such as free radical::;, mast ceIl degranulation/n1ediators, IFN-al{3, or Iysosomal

proteases. This would aid in determining which immune mediators are involved in the

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85

disaccharidase deficiencies. Studying the cellular infiltration in the small intestine

during challenge inoculations would also assist in specifying the type of immune

mechanism associated with the reductions in disaccharidase activity. Another area

which deserves further investigation is the effects of G. lamblia excretory/secretory

products on the small intestine.

Although many aspects of the process leading to disaccharidase deficiencies in

giardiasis have yet to be examined, it is hoped that the findings presented here can be

used in developing therapeutic strategies for malabsorption and, ultimately, for G.

lamblia infections .

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86

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