Experimental amoebic infection in guinea-pigs immunized with low grade amoebic infection

Experimental amoebic infection in guinea-pigs immunized with low grade amoebic infection

347 TRANSACTIONS OF THE ROYAL Experimental SOCIETY OF TROPICAL amoebic MEDICINE AND HYGIENE, VOL. 74, No. 3, 1980 infection in guinea-pigs ...

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74, No.

3, 1980

infection in guinea-pigs grade amoebic infection




P. JAIN, S. SAWHNEYAND V. K. VINAYAK* Division of Experimental Parasitology, Department of Experimental Medicine, Post-Graduate Institute of Medical Education and Research, [email protected], India


Guinea-pigs were immunized with a low grade amoebic infection, either intracaecally or intramesenterically. The immunized animals were challenged with a virulent strain of Entamoeba histolytica. Some of the animals, immunized via the intracaecal route were challenged repeatedly. 10 to 30% of immunized animals developed caecal lesions compared with 92 -8 % of non-immunized animals. The percentage of protection in animals challenged repeatedly was greater than in those challenged once. Neither immunized nor nonimmunized guinea-pigs developed hepatic abscesses. It appears that prior low grade infection could lead to resistance to subsequent amoebic challenge.

Boeck & Drbohlav medium and the other was the axenic strain (NIH:200) maintained in Diamond’s TPS-I medium (DIAMOND, 1968). Immunization procedure (i) Immunization tith

monoaxenic amoebae: The animals were inoculated intracaecally with 1,000 to 2,000 monoaxenic amoebae per animal. Seven days later these animals were treated withMetronidazole, given orally via catheter, at the rate of 40 mg/kg/day for seven days. (ii) Immunization with axenic amoebae: Guineapi& -were injected with 1,000 to 2,000 live axenic amoebae per animal via the mesenteric vein. Sensitization


Amoebic patients show variable levels of circulating antibodies which appear to have no protective role (KRUPP, 1974). Repeated infection inducing an acquired immunity is not known and few reports on this aspect are available. FAUST(1932) reported that some dogs inoculated with Entamoeba histolytica developed acute amoebic dysentery while in others there were no symptoms. The organisms disappeared almost immediately from the intestine in 11.5 y0 of infected animals. THOMPSON & LILLIGRAN (1948) indicated the role of immunity in amoebiasis, in uncured infected dogs on subsequent challenges. They observed protection in all animals but one. In 1952 SWARTZWELDER & AVANT assessedthe degree of resistance to reinfection with E. histofytica by treating the previous infection. They observed protection in about 80 % of animals, to both homologous and heterologous strains of E. histolytica. Recently MEEROVITCH et al. (1978) observed protective immunity in hamsters immunized intradermally with live axenic amoebae. The present study was undertaken to determine the host response to amoebic infection in guineapigs, previously immunized by low grade infection with monoaxenic E. histolytica isolate as well as axenic E. histozytica (NIH : 2000).


This was checked by an intradermal test as described by KIRPATRICK et al. (1972). The criteria of MADDISON et aZ. (1968) were applied when considering a positive dermal reaction, i.e. an area of induration and/or erythema of 1.0 cm diameter. Blood from each animal was collected for study of the level of humoral antibody titres before challenge. Amoebic infection

All the animals, immunized with live amoebae either intracaecally or intramesenterically, were challenged intracaecally with 80,000 to 100,000 amoebae of a virulent strain (H-25) of E. histozytica. The amoebae were inoculated as described by WOOLFE(1963), with a few modifications (VINAYAK et al., 1977). Groups of animals

The animals were divided into five groups. No immunization, but the amoebic infection by a virulent strain (H-25) of E. histotvtica was established intracaecally. ” Group ZZ: The animals, immunized with monoaxenic amoebae, were challenged with one dose of a virulent strain (H-251 . , of

Group I:

E. histolytica.


and Methods


Guinea-pigs three to four weeks old, weighing 150 to 200 g and free of natural amoebic infection were used. The absence of natural amoebic infection was confirmed by repeated stool examination. E. histolytica strains Two strains of amoebae were used for immunization: one was grown monoaxenically in a modified

Group III:

The animals immunized with monoaxenic amoebae, were challenged twice, intracaecally, with an interval of one week.

* Reprints from: Dr. V. K. Vinayak, Assistant Professor, Division of Experimental Parasitology, Department of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India.




of caecal




No. of animals infected


No. of animals with caecal lesions

by by by by


12 ( 85.7%)

in immunized




1:: IV V

2.0 1.33 l-66 1.78

lOO.O”/, 50.0%

33.3% lOi.O% 66.6%


and cellular



Humoral immune response Mean IHA titre* After Before CIEP challenge challenge reactors 82 5: 54 57 60

3 (25.0%)


and unimmunized



: 0


Histopathological grading of caecal lesions Grade I Grade II Grade III

Average Neal score



monoaxenic amoebae with one challenge monoaxenic amoebae with two challenges monoaxenic amoebae with three challenges axenic amoebae with one challenge.

of caecal lesions


and unimmunized

No. of animals with liver abscess

20 (100.0%)

99 14


1:: IV V

in immunized

14 (100.0%)

I-Control group II-Immunization III-Immunization IV-Immunization V-Immunization




1:: IV V



and hepatic

No. of animals inoculated




300 84 634 84



16.7% 0 00

in immunized

Granulomatous lesion

0 3Z.3%

and unimmunized


Cell-mediated immune response (Percentage migration inhibition)


Animals with caecallesions-35.0

75.0% 88.8%

Animals without lesions-10.0

f 15 *O% + 5 .O%



Control animals-5.0

f 3 *O%

*Reciprocal of highest serum dilution

Group IV: Group V:

At autopsy

The animals, immunized with monoaxenic amoebae, were challenged thrice at one-week intervals. These animals, immunized via the mesenteric vein (by live axenic amoebae), were challenged with one dose of a virulent strain of E. histolytica (H-25).

Seven days after the last challenge, the animals of all groups were killed with ether. Just before killing, blood samples via cardiac puncture were collected for determination of humoral antibody

and cell-mediated response. The lesions in the caecum and liver were sought. Caecal lesions were classified as suggested by NEAL (1951) and histologically graded as suggested by VINAYAK et al. (1977). Any area of lesion in the caecum or liver was fixed in 10% form01 saline and tissues were processed and stained with haematoxylin and eosin and periodic acid Schiff. Zmmunological studies (i) Humoral immune

response: Blood samples, collected before and after challenge, were studied for the humoral antibody response by the indirect

P. JAIN et al. haemagglutination test (IHAT) (PRAKASHet al., 1970) and countercurrentimmunoelectrophoresis (CIEP) test (KRuPP, 1974) employing axenic amoebic antigen. (ii) Cell-mediated immune (CMI) response: CM1 response was studied at the time of killing by the macrophage migration inihbition test (ORLTZORITZ et al., 1975). Specific axenic amoebic antigen and non-specific phytohaemagglutinin (PHA) mitogen were employed as antigens. Results The number of animals studied in different groups and the frequency of caecal lesions and liver involvement is shown in Table I. In Group I, i.e. the control group where the animals were challenged without prior immunization, 13 of 14 animals developed caecal lesions. All the immunized animals in Groups II, III, IV and V, had a positive delayed skin reaction before challenge. Six of 20 animals of Group II had caecal lesions but amoebae could be demonstrated in the caecal contents of all the inoculated animals. Only one of nine animals in Grout III and one of seven animals in Group IV showed caecal lesions. Amoebae were seen in the caecal contents of seven of nine animals of Grouu IV and all of nine animals of Group III. In Group V, in which animals were immunized with axenic amoebae via the mesenteric vein, three of 12 animals developed caecal lesions. E. histolytica trophozoites were present in the caecal contents of 12 of 14 animals. None of the animals in any of groups had liver abscesses. Average Neal scores obtained were 3.92, 2.0, 1.33, 1.66 and 1.78 in Groups I, II, III, IV and V respectively. Neal scores so obtained were statistically significant as compared to control animals (I?< .Ol). Histopathologically, in general, the caecal ulcers were either of Grade I (i.e. amoebae failed to penetrate the muscularis mucosae) or of Grade II (i.e. amoebae crossing muscularis mucosae and appearing in the submucosa). One of the six animals with ulcers in Group II had Grade III ulcer fi.e. amoebae reaching to-the muscular coat or invading it (Table II)). Only one animal of Group V had a granulomatous lesion. The detailed histopathology of these grades has been described elsewhere (JAIN et al., 1979). Humoral immune response was checked by IHA and CIEP tests. The IHA titres, before challenge, were 1: 0, 1: 56, 1: 54, 1: 57 and 1: 60 in groups I, II, III, IV and V respectively; after challenge, the titres rose to 1: 82, 1: 84, 1:300, 1: 634 and 1:84 in Groups I, II, II, IV and V respectively. On the whole, the CIEP test became positive whenever the IHA titres were 1:64 to 1: 1024. The agreement between the two tests w& 80.0 to 100.0% (Table III). The CMI response, as judged by macrophage migration inhibition is shown in Table III. The CM1 was slightly depressed in animals with caecal lesions (35.0 & 15.0 % inhibition) and was normal in animals with no lesions (10 -0 k 5.0 y0 inhibition).


Discussion The induction of immunity to amoebic infection by live trophozoites has been reported by FAUST (1932), THOMPSON & LILLIGRAN (1948), SWARTZWELDER & AVANT (1952) and MEEROVITCH et al. (1978). The results of the present study also indicate that protective immunity can be established when a small dose of amoebae is given intracaecally or intramesenterically, that a dose of live trophozoites is a better immunogen than are killed trophozoites (SWARTZWELDER & MULLER, 1950; KRUPP, 1974; JAIN et al., 1979) and that, because of better immunogenicity, protection afforded by live amoebae occurred in more animals (KRUPP, 1974; JAI-N et al., 1979). This has been further substantiated by the less severe caecal lesions in animals immunized with live amoebae. Recently MEEROVITCHet al. (1978) have reported 90.0% protection in animals immunized with live axenic amoebae. This report compares favourably with our results even though a different route of immunization was employed, MEEROVITCHet al. (1978) using the intradermal route while we used the intracaecal or intramesenteric route. MEEROVITCH et al. (1978) injected 20 million axenic amoebaein 0.1 ml of TPS-I medium intradermally. Localized lesions were seen in these vaccinated animals after four days. 20 days after injection the lesions started receding and the animals were challenged intrahepatically. In the present study, the animals, immunized via the intracaecal route were also challenged repeatedly. The protection in the animals with one challenge dose was 70 *0 % while 88 *9 y0 and 85 ~4y0 of animals challenged, twice and three times respectively, were completely refractory to amoebic infection. Thus the protection in animals with repeated challenge was greater than that in animals challenged once. It is thus evident from this study that immunization by low grade amoebic infection could make the host immune to subsequent challenge. Acknowledgement The authors gratefully acknowledge the help of Mr. Harjindar Singh Kamboj in typing the manuscript. References Diamond, L. S. (1968). Technique of axenic cultivation of Entamoeba histolytica (Schaudinn, 1903) and Entamoeba histolytica like amebae. Journal of Parasitology, 54, 1047-1056. Faust. E. C. (1932). Exoerimental amebiasis in do&. Americhn $urnal

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Krupp, I. M. (1974). Comparison of countercurrentimmunoelectrophoresis with other serological tests in the diagnosis ofamebiasis. American 3ournal of Tropical Medicine and Hygiene, 23, 27-30.





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Oritz-Oritz, L., Zamacona, C., Sepulveda, B. & Ycapin, N. R. (1975). Cell-mediated immunity in patients with amebic abscess of the liver. f”ni;;ca~and




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