Z Parsitenkd (1984)70:147-152

Parasitenkunde
Parasitology Research

Zeitschrift f~ir

,9 Springer-Verlag 1984

Lectin-induced agglutination of trophozoites of different species and strains of Entamoeba

E. Ghadirian* and E. Meerovitch
Institute of Parasitology, Macdonald Campus of McGill University, 21, 111 Lakeshore Road, Ste-Anne-de-Bellevue Que., Canada H9X 1CO

Abstract. In vitro agglutinability of trophozoites of three Entamoeba

histolytica strains, cultivated under axenic conditions in the presence of concanavalin A (Con A), was shown to be related to the degree of their pathogenicity for experimental animals and of the concentration of Con A. Seven strains of E. invadens tested also agglutinated in the presence of Con A, and the degree of agglutination was proportional to the concentration of the lectin. Three strains of E. histolytica-like "Laredo-type" amebae, a strain of E. terrapinae, and a strain of E. moshkovskii agglutinated very slightly, only in the presence of the highest concentration of Con A tested.

Introduction

Of all the species of the genus Entamoeba, only E. histolytica and E. invadens are known to be potentially pathogenic for their hosts. While all the strains of E. invadens are equally pathogenic for carnivorous reptiles (Steck 1963; Baker 1965), strains of E. histolytica differ in their pathogenicity for man. Attenuation of E. histolytica strains after prolonged cultivation in vitro, especially under axenic conditions, may be due to several factors, as yet not fully understood. A good correlation has been found between the pathogenicity of some strains of E. histolytica for man and their ability to produce cecal ulcers in rats (Mizgireva 1966). On the other hand, Bos and Hage (1975) found that all strains of E. histoIytica tested by them, whether from patients or carriers, were pathogenic when inoculated into hamster liver. While the precise reason for the expression of the pathogenic potential by E. histolytica (and by E. invadens) is not known, many factors accompanying the pathogenic (invasive) process have been identified. Among these are the oxidation-reduction potential at the intestinal mucosal surface
* Present addres and offprint requests to." Montreal General Hospital Research Institute 1650

Cedar Avenue, Montreal, Que., Canada H3G 1A4

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Table 1. Strains of Entamoeba histolytica and of E. histolytiea-like strains used in the present study
Species Strains IP-106 200: NIH DKB LA (Laredo) Huff JA PZ IP-1 IP-2 BN SiS SivL 165 ~ References Ghadirian and Meerovitch (1978) Ghadirian and Meerovitch (1979) Deschiens (1937) Dreyer (1961) Beaver et al. (1956) Entner and Most (1965) Meerovitch (1958) Meerovitch (1958) Meerovitch (1958) McConnachie (1958) Gray et al. (1966) Gray et al. (1966) Diamond (1960) Meerovitch (1958) Lachance (1959)

Entamoeba histolytica

Entamoeba histolyticalike "Laredo-type"

Entamoeba invadens

Entamoeba terrapinae Entamoeba moshkovskii

M
FIC

a Originally identified as E. terrapinae by Diamond (1960)

( M e e r o v i t c h et al. 1976), the c o m p o s i t i o n o f the c o n c o m i t a n t bacterial flora in the intestine (Phillips et al. 1958), diet o f the h o s t ( R o s s a n d K n i g h t 1973), a n d the surface m e m b r a n e p r o p e r t i e s o f the a m e b a e themselves. Several studies h a v e s h o w n t h a t t r o p h o z o i t e s o f p a t h o g e n i c strains o f E. histolytica a g g l u t i n a t e d in the p r e s e n c e o f relatively l o w c o n c e n t r a t i o n s o f the lectin, c o n c a n a v a l i n A ( C o n A), while a t t e n u a t e d o r n o n - p a t h o g e n i c strains o f E. histolytica a n d o f o t h e r Entamoeba species did n o t ( M a r t i n e z - P a l o m o et al. 1973; Trissl et al. 1977; B o s a n d v a n de G r i e n d 1977; D a s 1977). T h e p r e s e n t r e p o r t describes a d d i t i o n a l o b s e r v a t i o n s o n the a g g l u t i n a b i l i t y o f t r o p h o z o i t e s o f several strains o f E. histolytica, E. histolytica-like " L a r e d o - t y p e " a m e b a e , E. invadens, E. terrapinae, a n d E. moshkovskii in the p r e s e n c e o f C o n A.

Materials and methods

Trophozoites from 3-day-old axenic cultures of E. histolytica growing in the TPS-1 medium (Diamond 1968) at 37~ C and from 8-day-old axenic cultures of the other species of Entamoeba growing in the TYI-S-33 (Diamond et al. 1978) at 25~ C were used in the experiments. The strains of these amebae that were employed are shown in Table 1. The amebae were washed three times with phosphate-buffered saline, pH 7.2 (PBS) by centrifugation at 200 g for 7 rain. Their number in the final sediment was determined with the aid of a hemocytometer, then they were resuspended in PBS to a final concentration of 5 x 105 cells/ml. The "Laredo-type" strain LA amebae, because of their small size, were resuspended to a concentration of 2 x 106 cells/ml. By the trypan blue exclusion test, the viability of the organisms was estimated at ~ 97%. Solution of the lectin, Con A (Sigma, St. Louis, Missouri) was prepared in PBS. Dilutions of 5, 20, and 40 gg/ml were prepared in this solvent for use in the experiments.

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149

Table 2. Agglutinability of trophozoites of different strains of E. histolytica by concanavalin A Strain Source and year of isolation Agglutination with Con A (gg/ml) ~ 2.5 IP-106 200 :NIH DKB Amebic dysentery, 1965 Amebic dysentery, 1949 Amebic dysentery, 1924 2 1 0 10 3 2 0 20 4 3 1

See Materials and Methods for the scoring system

The agglutination reactions were carried out in Tissue Culture Chamber slides (Lab-Tek Products, Miles Laboratories, Illinois) according to a modification of the method of MartinezPalomo et al. (1973). Volumes of 0.1 ml of suspensions of all species contained 5 x 104 cells, except for the strain LA amebae which were used at a concentration of 2 x 105 cetls/0A ml. The suspensions of amebae were mixed with equal volumes of Con A in PBS. Thus, the concentrations of Con A used in the reactions were 2.5, ~0, and 20 !zg/ml. The chamber slides were agitated for 5 min, then incubated for 30 rain with occasional shaking - E. histolytica at 37~ and the other amebae at 25~ C. At the end of the incubation period, the chamber slides were examined under low power in an inverted microscope. The degree of agglutination was recorded on a scale from 0 to 4, according to the following criteria: 0, no clumps of agglutinated amebae observed; l, few clumps of 4~10 amebae; 2, larger clumps of 10-20 amebae; 3, ~70% of amebae in large clumps of 20-60; 4, almost 90% of amebae in very large clumps of > 60. All the tests were repeated five times, the results representing the averages of five observations. Results and discussion T a b l e 2 shows the results o f exposing the t r o p h o z o i t e s o f the E. histolytica strains to the three c o n c e n t r a t i o n s o f C o n A. It is evident t h a t the agglutination level o f the highly p a t h o g e n i c strain, IP-106, increased progressively in the presence o f increasing c o n c e n t r a t i o n s o f C o n A. T h e 200: N 1 H strain cells a g g l u t i n a t e d s o m e w h a t less, reaching the score o f 3 at C o n A c o n c e n t r a tion o f 20 btg/ml. Strain D K B , originally, but no longer pathogenic, agglutin a t e d only to the score o f i w h e n mixed with the highest c o n c e n t r a t i o n o f C o n A. T a b l e 3 s u m m a r i z e s the results o f tests with the r e m a i n i n g species o f the E. histolytica type. T h e strains o f E. invadens, except for 165 a n d SivL, agglutinated in the presence o f 2.5 l-tg/ml o f C o n A ; the degree o f agglutination was p r o p o r t i o n a l to the c o n c e n t r a t i o n o f the lectin. O f the three strains o f E. histolytica-Iike, " L a r e d o - t y p e " a m e b a e , only two ( H u f f a n d J A ) agglutinated slighlty with 20 g g / m l o f C o n A. Entamoeba terrapinae strain M a n d E. moshkovskii strain F I C reacted very weakly ( < 1 ) only w h e n the highest c o n c e n t r a t i o n , 20 gg/ml, o f C o n A was e m p l o y e d . O u r results c o n f i r m those o f several p r e v i o u s workers. Thus, M a r t i n e z P a l a m o et al. (1973) f o u n d t h a t invasive strains o f E. histolytica were agglutin a t e d strongly b y C o n A, while n o n i n v a s i v e strains agglutinated slightly or n o t at all. T h e y related agglutination to a clustering o f specific receptors

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Table 3. Agglutinability of amebic trophozoites of species other than E. histolytiea by concanavalin A a Species and strain Source and year of isolation Agglutination with Con A (l~g/ml) a 2.5 10 20

E. invadens PZ IP-1 IP-2 165 BM Sis SivL

"Laredo-type" LA Huff JA

Snake, Snake, Turtle, Turtle, Snake, Lizard, Lizard,

1947 1952 1955 1947 1953 1964 1964

1 1 1 0 1 1 0 0 0 0 0 0

3 2 2 1 2 2 1 0 0 0 0 0

4 3 3 2 2 3 2 0 1 1 < 1 <1

Human, 1961 Human, 1956 Human, 1965 Turtle, 1957 Sewage, 1959

E. terrapinae M E. moshkovskii FIC

a See Materials and Methods for the scoring system

for Con A on the cell membrane of the amebae and suggested that virulence is a function of surface properties. The results of Das (1977) were similar to ours, in that trophozoites of invasive strains of E. histolytica and E. invadens were agglutinated in the presence of Con A, and those of noninvasive strains of the former did not. Trissl et al. (1977) determined that pathogenic strains of E. histolytica possess several cell surface properties that differ from those of amebae belonging to species which are either nonpathogenic or not infective for humans. These workers' interpretation of susceptibility of amebae to agglutinate with Con A related the presence on their surfaces of strong and uniform Con A-binding sites and a lack of detectable repulsive charges. Our results differ from those of Trissl et al. with regard to E. invadens strain PZ. Failure of this strain to agglutinate strongly with Con A reported by these workers, and strong agglutination observed by us, may be due to the fact that they used a clone-derived culture and we employed the parent strain. It is conceivable that their clone culture was derived from an ameba with characteristics unlike those of typical E. invadens, i.e., the PZ strain is a mixture of organisms with different biological properties. However, the possibility of variation depending upon conditions prevailing in culture, with or without selection, should also be considered. Our results with respect to the "Laredotype" amebae and E. moshkovskii are in agreement with those of Trissl

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151

et al. ; in addition, E. terrapinae, a commensal of turtles, which is also infective but not pathogenic for snakes, reacts with Con A in the same way as the " L a r e d o - t y p e " amebae and E. moshkovskii.
Acknowledgements. This work was supported by a Natural Sciences and Engineering Research Council grant to E. Meerovitch and a McConnell scholarship to E. Ghadirian. Research at the Institute of Parasitology is supported by the National Science and Engineering Research Council of Canada and the Fonds F C A C pour l'aide et le soutien 5Lla recherche.

References
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Mizgireva MF (1966) Comparative vurulence of Entamoeba histolytica strains isolated from patients and different groups of carriers. Med Parazitol Parazitar Bolez (Moscow) 35 : 201-204 Phillips BP, Wolfe PA, Bartgis IL (1958) Studies on the ameba-bacteria relationship in amebiasis. II Some concepts in the etiology of the disease. Am J Trop Med Hyg 7 : 392-399 Ross GW, Knight R (1973) Dietary factors affecting the pathogenicity of Entamoeba histolytica in rats. Trans R Soc Trop Med Hyg 67:560-567 Steck F (1963) Die Ameobendysenterie der Reptilien. Acta Trop (Berl) 20:115-142 Trissl D, Martinez-Palomo, Arguello C, de la Torre M, de la Hoz R (1977) Surface properties related to Concanavalin A-induced agglutination. A comparative study of several Entamoeba strains. J Exp Med 145 : 65~665 Accepted July 26, 1983