InrernolionalJournalfnrParosifulog~.

Vol 26, No. 3, pp. 297-302. 1996

Copynght fp 1996 Austrahan Society for Parasitology
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Pathogenic Action of Entamoeba invadens:

Intestinal Epithelium Invasion by
Trophozoites in vitro
J. MORA-GALINDO,* S. RAMIREZ-ROM0 and Ma. M. JAUREGUI-TOPETE

Seccidn de Biologia Celular, Divisidn Biologiu de1Desarrollo, Centro de Investigacidn

Biome’dicade Occidente, I M S S, Apdo. postal l-3838, 44 100 Guadalajara, Jal. A4ksico

(Received II April 1995; accepted 3 October 1995)

Abstract-Mora-GalIndo J., Ramirez-Romo S. & Jauregui-Topete Ma. M. 1996. Pathogenic action of

Entamoeba hadens: intestinal epithelium invasion by trophozoites in vitro. International Journa/ fir
Puvasitology 26: 297-302. Interactions between live Entamoeba invadens trophozoites and guinea-pig caecal
explants were studied. A high percentage of amoebae adhering to the apical surface of epithelial cells was
observed IO-20 min after infection, but no histopathological changes were observed. After 30 min, mild
oedema at the base of the interglandular epithelium and death of some epithelial cells were evident. The
epithelial barrier was invaded by amoebae at desquamating zones and phagocytosis of epithelial cells or
cellular debris was occasionally observed. Invasion of the mucosa and tissue necrosis became more severe with
increased time of incubation. The continuity of epithelial lining was severely compromised after 2 h of
infection and erosive lesions were prominent in the mucosa. These results demonstrate that E. invadens is able
to invade the intestinal epithelium although it reportedly lacks the powerful cytotoxic and cytolytic elements
described for E. /dstolytica.

Key words: Amoehiasis; Entamoeha invadens; Enramoeha hisrol,vtica; epithelium; pathogenesis; light
microscopy.

INTRODUCTION of the mucosal surface, and sometimes deeper

Entamoeba invadens is a pathogenic amoeboid para- leucocyte infiltrates (Geiman & Ratcliffe, 1936; Ishii
site of reptiles (Ratcliffe & Geiman, 1933) which & Noburu, 1971). This suggests a pathogenic beha-
forms trophozoites morphologically similar to those viour similar to that of E. histolytica.
of E. histolytica. The life cycles of these 2 species are McCaul, Poston & Bird (1977) demonstrated the
also considered to be similar (Geiman & Ratcliffe, cytotoxic activity of E. invadens on human hepato-
1936; McConnachie, 1955; Cervantes-Mamoa & cytes maintained in culture, suggesting a phospho-
Martinez-Palomo, 1980). None the less, research on lipase was responsible for this effect. Soluble extracts
the taxonomic relationships of several Entamoeba from E. invadens have a cytotoxic effect on BHK-21
spp. have indicated that E. invadens and E. histolytica cells although intact trophozoites were unable to
may not be closely related (Clark & Diamond, 1991). produce contact lysis (Bos & van den Eijk, 1980). On
Following the oral inoculation of cyst to snakes, it the other hand, homogenates of E. invadens exhibited
has been demonstrated that E. invadens trophozoites considerable haemolytic activity for hamster erythro-
invade the colon, liver and occasionally the distal cytes, but little activity for human erythrocytes
small intenstine and stomach which exhibit necrosis (Lbpez-Revilla & Said-FernLndez, 1980). Protease
activity and the presence of pore-forming materials
*To whom correspondence should be addressed. (amoebopores) have also been reported, and intact
297
 298 J. Mora-Galindo ef al.

trophozites have exhibited some haemolytic activity buffered 1% osmium tetroxide for 1 h and dehydrated
for reptile erythrocytes (Avila, Sgnchez-Garza & through a series of graded ethanol solutions. Small portions
Calderbn, 1985; PCrez-Tamayo er al., 1991; Keller et of mucosa were embedded in Epon 812 resin, and semi-thin
al., 1988). In recent studies, it has been demonstrated longitudinal sections (l-pm-thick) were cut on a Porter-
Blum MT1 or Reichert OmU3 ultramicrotome. The sections
that E. invadens lysates had no effect on both the
were stained with Toluidine Blue and examined by light
transepithelial short circuit current and the trans-
microscopy noting the sites of adhesion, changes in mucosal
mural potential difference in rabbit colonic prepar- morphology and invasion of the epithelial lining by
ations (Navarro-Garcia et al., 1993). trophozoites.
The present investigation examined the effect of
live E. invadens trophozoites on guinea-pig caecal RESULTS
epithelium at the light microscope level, using an in Histological examinations of sections from non-
vitro model previously established for E. histoiytica infected control samples revealed no significant
by Mora-Galindo, Martinez-Palomo & GonzPlez- abnormalities before or after culture when compared
Robles (1982). The pathogenic activity of E. invadens to normal guinea-pig caecal mucosal sections (Mora-
was examined using similar experimental conditions Galindo, Martinez-Palomo & ChBvez, 1978; Snipes,
to those previously used for E. histolytica. 1982). No pathological signs were observed even after
incubation for up to 120 min in BT-S-33 medium. The
MATERIALS AND METHODS epithelial lining was continuous, the absorptive
Amoeba. E. invadens trophozoites of the IP-1 strain were columnar cells showed metachromasia, the goblet
cultured in axenic BI-S-33 medium (Diamond, Harlow & cells were located mainly at the bottom of the crypts
Cunnick, 1978). Trophozoites were harvested at the and a normal heterogeneous cell population was
logarithmic phase of growth by chilling the tubes in an observed in the lamina propria. Extrusion zones of
ice-water bath for 8 min and then centrifuging the cultures interglandular epithelium were recognized by the
for 5 min at 250 g. The cells were resuspended in fresh presence of faintly stained dead cells sloughing from
culture medium at concentrations of 2 x 10’ amoebae/ml. the epithelium.
Amoebae used in our experiments were able to phagocytoze Caecal samples incubated with live E. invadens
and degrade human erythrocytes in vitro (Ramirez-C&do-
trophozoites exhibited slight to moderate pathological
va, Ramirez-Romo & Mora-Galindo, 1990).
Intestinal epithelium. Adult guinea-pigs were anaesthet- changes which increased in severity with longer incu-
ized with pentothal (0.1 ml/l00 g body weight) and the bation times. In caecal mucosa incubated 10 and
caecum was removed through laparotomy. The caecum was 20 min with amoebae, some damaged epithelial cells
cut along taenias in order to obtain sheets of caecal wall. were occasionally observed located at the inter-
These sheets were extensively washed with Hank’s saline glandular epithelium. Amoebae were seen adhering
solution until all the intestinal content was removed. to the surface of both the interglandular epithelium
Cleaned caecal sheets were then gently extended on filter and mouths of crypts, but no tissue damage was
paper with the mucosa facing upwards, and discs of mucosa noted (Fig. 1). The trophozoites were pleomorphic
attached to subjacent layers were cut using a borer. The and contained numerous empty vacuoles; their basal
discs were washed 2 times with BI-S-33 medium and were surfaces followed the apical contour of the epithelial
placed in Using chambers and fasted. They were then
cells. Many amoebae were observed to have agglutin-
maintained in BI-S-33 with the mucosa facing upwards
ated together. Other amoebae were detected partially
(Mora-Galindo et al., 1982).
E.xperimental infections. Medium contained in the muco- within the crypt lumen although the epithelium was
sal side of the chambers was replaced with 0.5 ml of BI-S-33 unaffected. Release of mucin was observed in several
medium containing 1 x IO5 trophozoites/ml. The amoebae goblet cells at the glandular epithelium. The lamina
suspension was gently agitated in order to evenly disperse propria was not affected.
trophozoites on the mucosal surface The infected cultures After 3040 min of incubation, mild oedema
were incubated up to 120 min at 26°C together with non- beneath the interglandular epithelium and some
infected control cultures. Samples of caecal mucosa were damaged epithelial cells were observed. The cells
processed for light microscopic examination immediately exhibited variable morphology and affected cells were
prior to experimental infections and 10,20,30.40, 50,60, 80 intercalated between normal ones. The epithelial
or 120 min after infection.
covering of the glandular epithelium was normal.
Morphoiogicai analysis. At the end of each incubation
Amoebae were observed to evenly coat the caecal
period, mucosal tissues were fixed in situ by adding 0.5 ml
cacodylate-buffered 5% glutaraldehyde for 1 h at room mucosal surface and some trophozoites had invaded
temperature. The fixative was carefully discarded and the the epithelial barrier, crossing the epithelial lining
mucosa rinsed with cacodylate buffer. Mucosal discs were through desquamating zones located at the inter-
detached from the Ussing chambers and carefully rinsed glandular region (Fig. 2). The route of invasion was
with cacodylate buffer. They were post-fixed in cacodylate- strewn with debris left by lysed epithelial cells whose
 Pathogenesis of E. invadens in vitro 299

Fig. 1. Adherence of Entamoeba invadens to caecal epithelium after 20 min incubation in virro.
Amoebae (A) were observed adhering to the interglandular epithelium (I), and to the mouth of
crypts (C). Most goblet cells had discharged their contents (arrow). LP = lamina propria.
Toluidine Blue. Scale bar = 50 urn.
Fig. 2. Adherence and invasion of amoebae after 30 min incubation. Numerous amoebae (A)
came into contact with the epithelial lining but cytotoxic or cytolytic effects were not observed.
Other amobea have invaded the epithelial lining through desquamation zones (arrow) between
undamaged epithelial cells. Toluidine Blue. Scale bar = 50 urn.
Fig. 3. Pathogenic activity of Entamoeba invadens in caecal mucosa. After 50 min incubation
the interglandular epithelium shows groups of dead cells (arrowhead) and subepithelial oedema
(0) although the lining remained intact. Trophozoites adhered randomly to the epithelial
surface. and invasion of mucosa was observed to take place at the interglandular epithelium
(arrow). The glandular epithelium (G) and lamina propria were unaffected. Toluidine Blue.
Scale bar = 100 urn.

apical plasma membranes had become disrupted. The debris was not observed, indeed the majority of
trophozoites displaced the neighbouring epithelial vacuoles within the amoebae were empty. Other
cells gaining access to the basal region (Fig. 2). trophozoites were seen in the lamina propria but the
Phagocytosis of protruding epithelial cells or cellular route of entrance was not distinguished.
 J. Mora-Galindo et al.

Fig. 4. High power view of caecal mucosa after 50 min incubation. One amoeba (1) is adhering to normal interglandular
epithelium, whereas another (2) has crossed the epithelial lining between normal (N) and oedematized epithelium (E). Some
amoebae (*) have even traversed the basement membrane into the lamina propria. Toluidine Blue. Scale bar = 20 nm.
Fig. 5. Ulceration of caecal mucosa 120 min after incubation with E. invadens. The epithelial lining is discontinuous and
numerous dead epithelial cells and debris cover the interglandular area. Other epithelial cells have detached and some are
adhering to amoebae (A). One trophozoite (arrow) is shown in close contact with the basement membrane. The crypt at left
(C) has normal morphology, but the one on the right (D) exhibits considerable basal oedema and destruction of the
epithelial cells. Necrosis and oedema are also observed in the lamina propria (*). Toluidine Blue. Scale bar = 50 pm.

Profuse interstitial oedema was observed at the the lamina propria (Fig. 4) coming into contact with
basal portion of the interglandular epithelial lining in capillaries and cells of the lamina propria but no signs
explants incubated for up to 50 min with live E. of pathological changes of phagocytosis were
invadepzs trophozoites. Many epithelial cells remained observed after 50660 min (Fig. 4).
attached to the basement membrane by thin projec- Severe mucosal erosion was evident in the explants
tions (Fig. 3) while some interglandular epithelial cells after 120 min infection. Tissue destruction involved
were severely damaged or lysed. The glandular epith- large areas of the interglandular epithelium and the
elium did not show any additional changes to those subepithelial upper portion of crypts (Fig. 5).
described in early interaction periods. Subepithelial Amoebae had detached both live and dead epithelial
oedema was also noted in the lamina propria. The cells from the basement membrane, both individually
vacuoles of most amoebae remained empty. and in groups, following their invasion through
Numerous amoebae were observed invading the desquamating zones (Fig. 5). Epithelial cells and
mucosae after 50 min infection and later. Tropho- cellular debris were also occasionally observed to
zoites trespassed the epithelial lining as described have been phagocytozed by amoebae. Many cells in
above causing the epithelial cells to separate from the the glandular lining were pale-coloured and basal
basement membrane, The amoebae then passed into epithelial oedema was observed along some crypts
 Pathogenesis of E. invadens in vitro 301

although basal cells were best preserved (Fig. 5). association with the surface of both the intergland-
Oedema and necrosis were evident throughout the ular and upper glandular epithelium. Progressive
lamina propria. desquamation of epithelial cells was not observed in
mucosa incubated with E. invadens trophozoites.
DISCUSSION With some exceptions, E. invadens trophozoites
Since initial studies on the pathogenesis of E. appeared unable to phagocytoze dead epithelial cells
invadens infections were carried out by Ratcliffe & or portions of them, although erythrocytes were
Geiman (1933), this amoeba species has received little occasionally engulfed. Despite infections by E.
attention as it was perceived to have no public health invadens trophozites, the mucosal lining remained as
importance. In this investigation, we observed that a barrier except for entrance sites of trophozoites at
E. invadens trophozoites were able to damage some desquamating areas, and microulcers. Under similar
guinea-pig caecal epithelial cells located at the experimental conditions, the pathogenic activity of E.
interglandular lining after as little as 30 min incuba- invadens was lower than that of E. histolytica.
tion compared to uninfected controls. However, the The lack of reaction in caecal mucosa treated with
trophozoites did not induce contact cytotoxic effects E. invadens trophozoite lysates (Navarro-Garcia et
on the epithelium in vitro. The mechanisms causing al., 1993) is singular considering our results demon-
epithelial destruction are uncertain. Some amoebic strated the parasite can actively invade the caecal
products liberated into the incubation medium may mucosa. Differences in the effects of whole tropho-
have been cytotoxic especially to the interglandular zoites and lysates on the electrophysiological
epithelium and other toxic products arising from properties of MDCK cells have been previously
tissue necrosis could also be involved. The glandular demonstrated (Martinez-Palomo et al., 1985), the
epithelium was only affected after 100~120 min alterations being faster when live trophozoites were
incubation. Similar findings were reported for E. used. It is evident from this study that E. invadens
histolytica under comparable experimental conditions trophozoites clearly invade the intestinal epithelium
(Mora-Galindo et al., 1982) and after the intracaecal in guinea-pig caecal explants.
inoculation of amoebae into gerbil caecum (Chadee &
Meerovitch, 1985). Acknowledgements-The authors are grateful to Dr Amado
Gontilez-Mendoza for reading the manuscript and Dr
We did not undertake experiments in order to Peter O’Donoghue for constructive comments. J. M.-G. is a
identify the amoebic cytotoxic factors involved with recipient of a Sistema National de Investigadores (Mtxico)
mucosal injury, but morphological findings suggest fellowship. Preliminary results were presented at the 18th
damage to the plasma membrane of epithelial cells. Annual Meeting of the Mexican Society of Microbiology,
This allowed large molecules to leak through as a Acapulco, Gro. MCxico.
consequence of osmotic changes, leading eventually
to cell lysis. Several factors already described in REFERENCES
E. invadens could be involved in cellular damage, like Avila E. E., SLnchez-Garza M. & Caldeion J. 1985. Entn-
phospholipase (McCaul et al., 1977), proteases (Avila moeba histolvtica and Entamoeba invadens: Sulfhydryl-
et af., 1985) and amoebopore (Keller et al., 1988). dependent proteoelytic activity. Journal qf Protozoology
As was demonstrated for E. histolytica (Mora- 32: 163-166.
Galindo et al., 1978), we showed that E. invadens Bos H. J. & Eijk A. A. van. 1980. Serum-inhibited toxicity
of Entamoeba histolytica. Archives de Investigacidn
amoebae were able to invade the caecal epithelial
MPdica (Me’xico) 11 (suppl 1): 135-141.
lining through the interglandular region either by Cervantes-Mamoa A. & Martinez-Palomo A. 1980. Estudio
displacing desquamating epithelial cells or by migrat- de1 ciclo vital de Entumoeba invadens mediante cinemato-
ing through intercellular spaces between normal and grafia espaciada. Archives de Investigacidn MPdica
damaged epithelial cells. Amoeboid movement (Mhxico) 11 (suppl. 1): 3144.
appeared to play an essential role in the invasive Chadee K. & Meerovitch E. 1985. Entamoeba histolytica:
process allowing the amoebae to penetrate the early progressive pathology in the cecum of the gerbil
epithelial barrier and disperse between the cells and (Meriones unguiculatus) American Journal of Tropical
fibres of the lamina propria. Medicine and Hygiene 341 283-29 1.
Despite some similarities in the pathogenic behav- Clark C. G. & Diamond L. S. 1991. The Laredo strain and
other E&amoeba histolytica-like amoebae are Entamoeba
iour of E. invadens and that described for E.
moshkovskii. Molecular and Biochemical Parasitology 46:
histol.vtica (Mora-Galindo et al., 1982), some differ- 11-18.
ences were observed between the species when con- Diamond L. S., Harlow D. R. & Cunnick C. C. 1978. A new
fronted with guinea-pig caecal epithelium in vitro medium for the axenic cultivation of Entamoeba histoly-
under similar experimental conditions. In all periods tica and other Entamoeba. Transactions of the Royal
examined, E. invadens trophozoites were found in Society of Tropical Medicine and Hygiene 12: 43 1432.
302 J. Mora-Galindo et al.

Geiman Q. M. & Ratcliffe H. L. 1936. Morphology and life Mora-Galindo J., Martinez-Palomo A. & Chavez B. 1978.
Cycle of an amoeba producing amoebiasis in reptiles. Interaction entre Entamoeba histolytica y el epitelio cecal
Parasitoiogy 28: 208-228. de1 cobayo. Archives de Investigacidn Midica (Mt!xico) 9
Ishii A. & Noburu Y. 1971 Attempts to infect laboratory (suppl.1): 261-274.
rats and mice and the snake habu Trimersurusflavovir- Mora-Galindo J., Martinez-Palomo A. & Gonzalez-Robles
idis, with Entamoeba invadens. Snake 3: 24-29. A. 1982. Interaction entre Entamoeba histolytica y el
Keller F., Walter C., Lohden U., Hanke W., Bakker- epitelio cecal del cobayo. Estudio cuantitativo. Archives
Grunwald T. & Trissl D. 1988. Pathogenic and non- de Investigacidn Midica (Mkxico) 13 (suppl. 3): 233-243.
pathogenic Entumoeba: pore formation and hemolytic Navarro-Garcia F., Lopez-Revilla R., Tsutsumi V. & Reyes
activity. Journal of Protozoology 35: 359-365. J. L. 1993. Entamoeba histolytica: electrophysiologic and
Lopez-Revilla R. & Said-Fernandez S. 1980. Cytopatho- morphologic effect of trophozoites lysates on rabbit
genicity of Entamoeba histolytica: hemolytic activity of colon. Experimental Parasitology 77: 162-I 69.
trophozoite homogenates. American Journal of Tropical Perez-Tamayo R., Becker I., Montfort I., Ostoa-Saloma P.
Medicine and Hygiene 29: 209-2 12. & Perez-Montfor R. 1991. Role of leukocytes and
Martinez-Palomo A., Gonzalez-Robles A., Chavez B., amoebic proteinases in experimental rats testicular
Orozco E., Fernandez-Castelo S. & Cervantez-Mamoa necrosis produced by Entamoeba histolyrica. Parusitofogy
A. 1985. Structural bases of the cytolytic mechanisms of Research 77: 192-196.
Entamoeba histolytica. Journal of Protozoology 32: Ramirez-Cordova J. J., Ramirez-Romo S. & Mora-Galindo
166175. J. 1990. Quantitative evaluation of intracellular digestion
McCaul T. F., Poston R. N. & Bird R. G. 1977. Entumoeba in Entamoeba invadens. International Journal for Para-
histolytica and Ehtamoeba invadens: chromium release sitology 20: 199-201.
from labeled human liver cells in culture. Experimental Ratcliffe H. L. & Geiman Q. M. 1933. Eleven cases of
Parasitology 43: 342-352. amoebiasis in reptiles. Journal of Parasitology 20: 139.
McConnachie E. W. 1955. Studies on Entamoeba invadens Snipes R. L. 1982. Anatomy of the guinea-pig cecum.
Rhodain, 1934, in vitro, and its relationships to some Anatomy and Embryology 165: 97-l 11.
other species of Entamoeba. Parasitology 45: 452481.