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Protective effects of crude and chromatographic fractions of axenic Entamoeba histolytica in guinea-pigs
483 TRANSACTIONS OFTHEROYALSOCIETYOFTROPICALMEDICINEAND IIYGIENE,VOL.~~,NO.~,~~SO
Protective
effects
Division of Experimental
of crude Entamoeba
and chromatographic fractions histolytica in guinea-pigs
of axenic
V. K. VINAYAK, S. SAWHNEY, I?. JAIN AND R. N. CHAKRAVARTI Parasitology, Department of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India
immunogenic compared to other fractions (SAWHNEY 1979). It was thought worthwhile to study the protective abilities of crude and chromatographic fractions of axenic amoebic antigen (NIH: 200) in an experimental amoebic infection in guineapigs.
Summary Axenic amoebic antigen was chromatographed on Sephadex G-200. Two peaks divided into three fractions were obtained which were concentrated by lyophilization. Guinea-pigs were given a series of subcutaneous injections of crude Entamoeba kistolytica antigen and its chromatographic fractions mixed with Freund’s complete adjuvant. After three immunizing doses, the animals were challenged with intracaecal injection of a virulent isolate of amoebae (Strain No. H-25) and- were killed seven days later. The antibody response was studied by indirect haemagglutination (IHA) and countercurrent immunoelectrophoresis (CIEP) before intracaecal inoculation and at the time of killing. Fraction I, which had the highest molecular weigh?, stimulated high levels of antibodies in guinea-uigs and showed 9‘i ?;I protection. Only one animal of I2 inoculated had gross lesions and these were very mild. The caecal contents of three of the 11 anima!s without gross lesions were positive for amoebae but no tissue invasion was observed. Partial protection was observed with other antigens--305;, with whole amoebae, 18:; with Fraction II and 30 9: with Fraction III. In most there were multiple ulcers of the deep penetrating type and amoebae either penetrating and invading the muscular layer or the lytic process extending up to the muscular layer. In one animal, immunized with whole amoebic antigen, multiple hepatic lesions were also observed and trophozoites of E. kistolytica were demonstrated histologically.
The gel filtration of the amoebic antigen was carried out as described elsewhere (SAWHNEY et al., 1979). 5 ml of axenic antigen containing 30 mg of protein was applied to the top of a 45 r: 2.5 cm column of $ebhadex G-200: The elution was carried out by employing 0.05 M tris buffer, pH 7 * 5. The typical chromatographic fraction so obtained is shown in the figure. The fractions were concentrated by lyophilization and the lyophilized material was reconstituted in 5 ml of normal saline.
Introduction Scanty information is available on immunity to Entamoeba kistolytica infection. It is apparent to clinicians that patients do have repeated infections, in spite of high levels of anti-amoebic antibodies. Alternatively, it is rare for a patient who has been successfully treated for amoebic liver abscess to have a second liver abscess, indicating that acquired resistance to amoebic infection is possible. It is very difficult to prove the development of acquired resistance in an individual with a history of amoebiasis, but studies in an experimental mohel could be helpful. The antibodv titres could be high because of ihe presence, in t6e amoebic extract;of certain antigenic determinants which elicit high antibody levels but, at the same time, there could be certain antigenic determinants which are functional although eliciting a low antibody response. We have previously observed that the chromatographic Fraction I of an axenic amoebic extract is highly
and challenge of experimental animals Three to four-week-old guinea-pigs (250 to 300 n) were divided into five groups. Group 1 were con& animals without imm&zaiion but challenged with 100,000 virulent amoebae (Strain No. H-25) intracaecally (VINAYAK et al., 1977). The strain had been isolated from a patient with amoebic dysentery. The animals in Groups 2,3,4 and 5 were immunized resnectivelv. with crude antigen and Fraction I, II ani III. l&al volumes of antigen and complete Freund’s adjuvant were emulsified for 5 to 8 min. Animals were given weekly subcutaneous injections of 0.6 ml of this emulsion for three weeks. Seven days after the last injection, blood samples were taken for measurement of antibody response by indirect haemagglutination (PRAKASH et al., 1969) and counter-current immunoelectrophoresis tests (KRUPP, 1974a). One week after the last injection each of these animals were injected intracaecally with 100,000 virulent amoebae. Seven days later,
et al.,
Material Antigen
and Methods
preparation
The antigen used was a sonicated extract of axenically grown E. kistot’ytica (NIH:200). Amoebae from a 72-hour culture in TPS-I medium were washed thrice in sterile normal saline, concentrated by centrifugation at 850 g for 5 to 7 min and sonicated for 5 min at 20 Kc in an MSE ultrasonic disintegrator. The sonicated material was centrifuged at 12,000 L g for half an hour at 4°C. Protein concentration was determined by LOWRY'S technique (1951). The supernatant was then lyophilized and then fractionated on Sephadex G-200. Separation and concentration on Sepkadex G-200 column
Immunization
of antigenic
components
484
PROTECTIVE EFFECTS OF AXENIC E. histolytica
Table I-Antibody response in guinea-pigs Entamoeba histolytica
immunized
FRACTIONS IN GUINEA-PIGS
with crude and chromatographic
BEFORE CHALLENGE Immunization antigen Group
IHA titre No. of (no. of animals guinea- in parentheses) Pii9
1 No immunization
14
Neg. (14)
2 Whole antigen
14
1:64 (3) 1:256 (2) 1:1024 (3) 1:4096 (2)
Mean IHA titre 0
1:2542
1: > 16000 (1) n.d. (3) 12
1:256 (2) 1:1024 (5) 1:4165 1:4096 (2) 1: > 16000 (2) n.d. (2)
4 Fraction II
12
1:64 (3) Neg. (9)
1:16
1164 (3) Neg. (7)
1:19
10
of axenic
AFTER CHALLENGE
CIEP (no. of animals in parentheses)
IHA titre (no. of animals in parentheses)
Neg. (14)
Pos. (3) Pos. (2) F2
is;
Mean IHA titre 1:41
1:1024 (8) 1:4096 (3) 1: > 16000 (3) 1:4819
CIEP (no. of animals in parentheses)
2.~(‘8”,’ Pos. (8) Pos. (3) Pos. (3)
pas: (1) n.d. (3)
3 Fraction I
5 Fraction III
fractions
1:1024 (1) 1:4096 (6) 1:8800 1: > 16000 (5)
Pos. (1) Neg. (11)
Pos. (2) Neg. (8)
1:59
Pos. (1)
Pos. (5) Neg. (6) 1:64 (6) 1:256 (2) Neg. (2)
1:90
Pos. = positive; Neg. = negative; n.d. = not done
the animals were killed. Just before killing, they were bled via cardiac puncture and, at killing, lesions in the caecum and liver were sought. Caecal lesions were classified as suggested by NEAL (1951) and were further graded histologically as suggested by VINAYAK et al. (1977). Any area of lesion in the caecum and or liver was fixed in 10% form01 saline. Tissues were processed and stained with haematoxylin and eosin and FAS. Results Elution Profile The elution profile of E. histolytz’ca (NIH:200 strain) indicates that there are two peaks divided into three fractions when 30 mg of protein is put on a column of Sephadex G-200. The recovery was only 74%. The replication of results was excellent with each column run. Fraction I contained 14% of the total protein and Fractions II and III had 17% and 41 y0 protein respectively. Immunization and results of challenge of guinea-pigs The blood samples, collected one week after the last immunizing dose, were tested by the indirect haemagglutination and counter-current immunoelectrophoresis test (KRUPP, 1974a) to determine the level of antobodies. None of the animals had any antibody titre before immunization. After immunization, but before challenge, the animals immunized with whole amoebic antigen had IHA titres ranging from 1:64 to 1:16000, with an arithmetic mean IHA titre of 1:2542. Samples from
three animals could not be tested. All the animals with IHA titres, had a positive CIEP test. For animals immunized with Fraction I had IHA titres ranging from 1:256 to 1:16000 and those immunized with Fraction II and Fraction III had very low titres, i.e. 1:64. Nine animals immunized with Fraction II and seven animals immunized with Fraction III failed to show any haemagglutinating antibodies. Here again, the results of the CIEP test compared well with those of the IHA test (Table I). The antibody response in serum samples collected just before killing showed a twofold increase in the mean IHA titres in animals immunized with whole amoebic antigen and Fraction I; mean IHA titres ranged from I:64 to I:256 in animals immunized with Fractions II and III and the titre was only 1:64 in the control group of animals which were not immunized. After challenge, there were five, seven and two animals in Groups 1, 4 and 5 respectively which showed no haemagglutinating activities (Table I). At post-mortem examination of all these animals, seven days after intracaecal inoculation with amoebae, all organs were checked for gross lesions and the caecal contents for the presence of E. histolytica trophozoites. In the control group (no immunization), 13 (92.130/,) of the guinea-pigs showed gross caecal lesions and large numbers of amoebae could be demonstrated in the caecal contents. One animal, although showing no gross lesions, had a caecal content positive for amoebae.
Table II-Gross caecal lesions of axenic Entamoeba histolytica Immunizing
antigen
in animals
No. of animals
v. K. VINAYAK
et al.
immunized
with
485 crude
and chromatographic
No. gross lesions, caecal contents
Gross lesions in caecum Positive
fractions
Negative
Positive E.h. +
Negative E.h. -
No immunization
14
13
1
:.
0
Fraction antigen I Whole Fraction II Fraction III
12 14 12 10
1: 10
11 4 2 3
3 2 2
8” 0 1
Table
III-Protective
Immunization material Groups 1 No immunization 2 Whole antigen 3 Fraction I 4 Fraction II 5 Fraction III
Table
Group
IV-Protective
Immunization
7
effects of crude Animals with caecal lesions/ animals inoculated
and chromatographic
Grading of lesions Average caecal score
Grade I
Grade II
5.31 5.21 1.17 5.69 5.20
6 (46.2%) 0 (0.0%) l(lOO*O%) 3 (30.0%) 4 (57.1%)
7(53.8x) 2(20.0X) 0 (0.0%) 5(50.0X) 3(42*9%)
13/14 (92.9 %) 10/14(71.4%) l/12 (8.3 %) 10/12(83*3 %) 7/10(70*0 %)
effects of crude antigen,
antigen
Whole antigen Fraction I Cont. whole antigen
of E. histolytica antigen
fractions
Fraction
I and concentrated
Protein concentration 1 .2 mg/animal (containing 0.17 mg of Fraction I) 0.5 mg/animal 3 .6 mg/animal (containing 0.5 mg of Fraction I)
Lesions were 3 to 5 cm in diameter and covered with a sheath of slough which could be easily peeled off, revealing deep ulcers with a necrotic and congested floor. The animals usually had multiple lesions (Table II). Ten (71.41%) of the 14 animals immunized with whole amoebic antigen had gross caecal lesions. There were no lesions in 30 “/o of animals, indicating that there was partial protection. The caecal contents of all these 10 animals were also positive for amoebae. The size of the lesions ranged from 1 *O to 5 .O cm. There were usually one or two caecal lesions in each animal but in three there were ulcers throughout the caecum. The caecal contents of the infected animals were semi-fluid to watery. In two of the four animals without .gross lesions, amoebae were present in the caecal contents. Amoebae were not detected histologically in the caecal mucosa of these four animals. 12 animals were immunized with Fraction I.
Grade III ;Q;*;m; * o.
whole amoebic
Animals with caecal lesions/ animals inoculated
antigen
Percentage protection
10/14
29%
l/l2
92 o/ /0 91 ‘X
l/11
Only one had a gross lesion which was very small, approximately 0.5 cm in diameter. Although three of the 11 animals without gross lesions had caecal contents positive for amoebae, these were few in number and no tissue invasion was observed. Animals immunized with Fractions II and III showed partial protection-18% and 30%, respectively. The lesions presented as shallow weers, irregular in shape and from 1.0 to 5 .O cm in diameter. The ulcers were covered by a mutinous sheath; this sheath could be easily peeled off revealing an uneven surface consisting of areas of congestion intermingled with areas of superficial necrosis. There were multiple lesions in all the animals with gross lesions (Table II). The protective effect, the average Neal scores and grade of lesions are shown in Table III. 92.30% of non-immunized animals had caecal lesions. The amoebae had broken the muscularis mucosae and the ulcers had extended in to the submucosa where
486
PROTECTIVEEFFECTSOF AXENIC E. histolytica FRACTIONSIN GUINEA-PIGS
many amoebae could be seen. Only 30 %, protection was achieved in the animals immunized with whole amoebic antigen. Ulcers were of the deep penetrating type and very extensive and there was deep necrosis of the mucosa. At a few of the necrotic places brown pigment could also be seen. Amoebae were found to be infiltrating into the submucosa and forming flask-shaped ulcers with necrotic debris. In 80% of the animals with ulcers amoebae had either penetrated and invaded the muscular layer or the lytic process initiated by the amoebae had extended up to the muscular layer. Only one animal immunized with Fraction I had lesions which were very slight. There was superficial necrosis of the mucosa and amoebae were confined to the mucosa only. Interglandular stroma and granular tissue were found to be infiltrated with cells, mostly mononuclear histocytes and lymphocytes with some polymorphs. Amoebae were confined to the muscularis mucosae with invasion to the submucosa which showed some degree of oedema (Table III). Since Fraction I could urotect 92”;, of the animals it was thought worthwhile to concentrate the whole amoebic antigen so that it contains 0.5 mg of Fraction I. Of the 11 animals immunized with concentrated whole amoebic antigen only one (9.00,6) had caecal lesions which were also very slight (Table IV). One of the animals immunized with whole amoebic antigen also had multiple hepatic lesions. Histologically, the hepatic lesions consisted of a central zone of cytolysed granular material surrounded by degenerated liver cells, a few leucocytes, connective tissue cells, erythrocytes and, occasionally, trophozoites of E. histolytica. Parenchymatous cells were compressed at the periphery of the lesions, and amoebae could be seen either at the periphery of the abscess cavity or slightly away from it. The presence of amoebae was confirmed by staining the tissue with PAS stain and counterstaining with haematoxylin. I
Discussion The findings clearly show that the extract of E. histolytica contains a mixture of different antigenic molecules. Experiments utilizing these antigenic determinants obtained after column chromatography indicated that Fraction I had the potential functional capability. This fraction has a molecular weight of 650,000. A similar type of fraction has also been reported by ALIKHAN & MEEROVITCH (1968) and KRUPP (1974b). These authors also concluded that Fraction I is composed of proteins of molecular weights in the range of 650,000. Immunization of animals with this high molecular weight fraction could protect 92.0”; of them; in only one of 12 animals did mild, superficial lesions develop. At the same time, in the control group, lesions occurred in 92 * 13 96 of animals, indicating the virulence sta us of the isolate of E. histolytica employed. Partial protection -17 $L, and 30 %-was obtained with Fractions II and III respectively. In a study by KRUPP (1974b) it was pointed out that the crude extract of amoebae protected 70 “/ of animals although we only achieved protection in 3Oq/, of animals immunized with the
crude antigen. It is difficult to explain the variations between the results of our study and those of KRUPP (1974b), although the isolate of amoebae used was the same in both. The difference in results could possibly be explained on the basis of protein content of the immunizing doses. We used the crude preparation containing 1.2 mg protein/ animal but KRUPP (1974b) used 5.6 mg protein/ animal. The amount of protein, and therefore protective antigen, seems to be very critical; when we concentrated the protein in the crude extract to contain 3.6 mg per animal, 91 y0 of the animals showed protection. 3.6 mg protein in crude extract contains 0.5 mg of protein equivalent to that in Fraction I. We have already demonstrated that Fraction I contains 14% of the total protein. Thus, the similarity in the results in animals immunized with Fraction I and those immunized with concentrated crude amoebic extract could be due to the amount of protein content of Fraction I in both the preparations. There were animals in the various groups which had no gross caecal lesions although the caecal contents were positive for amoebae. It seems that amoebae in such a situation failed to invade the mucosa so as to produce gross caecal lesions. Thus they were in a commensal state. Such an observation is similar to that in amoebic patients who are asymptomatic, although passing cysts of E. histolytica in the stool. In one of the animals we observed multiple abscesses in the liver. There was a central zone of necrosis in which amoebic trophozoites could be demonstrated in a haematoxylin preparation and were confirmed by PAS staining. Antibody response in amoebic patients has been very well documented and persistence of antibodies after cure has been measured and reported (KRUPP, 1970, KRUPP & POWELL, 1970, 1971). It is a consensus that the antibodies do not contribute to the protection of the host as reinfections commonly occur, even when the antibody titres are relatively
0.7 1
14
COLUMN DIMENSION 45x2.5MM SAMPLE SIZE -30 mg PROTEIN/5ml 0.05 M TRIS HCI BUFFER. - PH-7.5 FLOW RATE 3-4 ml/10 MINUTE
30
44
70
ELUTION VOLUME Chromatographic pattern of axsnic Enumorba histolytka snti@mn on sephadex O-200 gel. Fig. 1. Chromatographic pattern of axenic Emamoeba antigen on Sephadex G-200 gel.
hirtolyrica
V. K. VINAYAK et al. high. In the present study, it has been clearly shown that the animals immunized with Fraction I had a high antibody titre and, at the same time, showed 92% protection; animals immunized with crude amoebic extract though also showed high antibody levels but protection was observed in only 30 %. The antibody level in this group of animals seems to be due to the host immune response to a non-protective component of the antigen. It thus seems that circulating antibodies to the Fraction I component of the amoebic antigen might have some protective role. Further work on this is under progress. Cellular immunity may also be playing some part in the control of the infection. ORTIZ-ORTIZ et al. (1975), SAVANAT et al. (1973) and HARRIS & BRAY (1976) have shown that cellular immunity is altered in patients with amoebic liver abscess. JAIN et al. (1980) recently observed that the cellular immunity was not markedly altered in experimentally infected guinea-pigs with only caecai lesions. HARRIS & BRAY (1976) reuorted almost identical findings in clinical cases of intestinal amoebiasis. De&led studies of this aspect in both clinical cases and experimentally infected animals are being carried out in this laboratory. .
I
^
References
Alikhan, Z. & Meerovitch, E. (1968). A comparative study of the antigen of some of the “histolytica A qualitative and type” strains of Entamoeba: quantitative evaluation of antigens by indirect haemagglutination, gel precipitation and immunoelectrophoresis. American Journal of Tropical Medicine
and Hygiene,
17, 528-539.
Harris, W. G. & Bray, R. S. (1976). Cellular sensitivity in amoebiasis-preliminary results of lymphocyte transformation response to specific antigen and to nitrogen in carrier and disease states. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70, 340-343. Jain, P., Sawhney, S. & Vinayak, V. K. (1980). Experimental amoebic infection in guinea pigs immunized with low grade amoebic infection. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, (in press). Krupp, I. M. (1970). Antibody response in intestinal and extra-intestinal amoebiasis. American Journal of Tropical Medicine and Hygiene, 19, 57-62. Krupp, I. M. & Powell, S. J. (1971). Comparative
487
study of the antibody response in amoebiasis. Persistence after successful treatment. American Journal of Tropical Medicine and Hygiene, 29, 421-424. Krupp, I. M. (1974a). Comparison of counterimmunoelectrophoresis with other serological tests in the diagnosis of amoebiasis. American Journal of Tropical Medicine and Hygiene, 23, 27-30. Krupp, I. M. (1974b). Protective immunity to amoebic infection demonstrated in guinea pigs. American Journal of Tropical Medicine and Hygiene,
23, 355-360.
Lowery, 0. H., Rosebrough, N. J., Parr, A. L. & Randall, R. J. (1951). Protein measurement with folin phenol reagent. Journal of Biological Chemistry,
193, 265-275.
Neal, R. A. (1951). Some observations variation of virulence and response to therapy of strains of Entamoeba histolytica Transactions of the Royal Society of Medicine
and Hygiene,
on the chemoin rats. Tropical
44, 439-442.
Ortiz-Ortiz, L., Zamacone, G., Sepulveda, B. & Kapin, N. R. (1975). Cell mediated immunity in patients with amoebic abscess of the liver. Clinical
Immunology
and
Immunopathology,
4,
127-134. Prakash, O., Tandon, B. N., Bhalla, I., Ray, A. K. & Vinayak, V. K. (1969). Indirect haemagglutination and immobilization tests and their evaluation in intestinal and extraintestinal amoebiasis. AmericanJournal of Tropical Medicine and Hygiene,
18, 670-675.
Savanat, T., Viviyamond, P. & Nimitmongkoe, N. (1973). Blast transformation of lymphocytes in amoebiasis. AmericanJournal of Tropical Medicine and Hygiene, 22, 705-710. Sawhney, S., Chakravarti, R. N., Jain, I-‘. & Vinayak, V. K. (1979). Immunogenicity of axenic Entaantigen and its fractions. moeba histolytica Transactions of the Royal Society of Tropical Medicine
and Hygiene,
74, 26-29.
Vinayak, V. K., Naik, S. R., Sawhney, S., Sehmi, N. & Chhuttani, P. N. (1977). Pathogenicity of Entamoeba histolytica: virulence of strains of amoebae from symptomatic and asymptomatic of Medical cases of amoebiasis. Indian Journal Research, 66, 935-941. Accepted for publication
2nd September,
1979.
Protective
effects
Division of Experimental
of crude Entamoeba
and chromatographic fractions histolytica in guinea-pigs
of axenic
V. K. VINAYAK, S. SAWHNEY, I?. JAIN AND R. N. CHAKRAVARTI Parasitology, Department of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India
immunogenic compared to other fractions (SAWHNEY 1979). It was thought worthwhile to study the protective abilities of crude and chromatographic fractions of axenic amoebic antigen (NIH: 200) in an experimental amoebic infection in guineapigs.
Summary Axenic amoebic antigen was chromatographed on Sephadex G-200. Two peaks divided into three fractions were obtained which were concentrated by lyophilization. Guinea-pigs were given a series of subcutaneous injections of crude Entamoeba kistolytica antigen and its chromatographic fractions mixed with Freund’s complete adjuvant. After three immunizing doses, the animals were challenged with intracaecal injection of a virulent isolate of amoebae (Strain No. H-25) and- were killed seven days later. The antibody response was studied by indirect haemagglutination (IHA) and countercurrent immunoelectrophoresis (CIEP) before intracaecal inoculation and at the time of killing. Fraction I, which had the highest molecular weigh?, stimulated high levels of antibodies in guinea-uigs and showed 9‘i ?;I protection. Only one animal of I2 inoculated had gross lesions and these were very mild. The caecal contents of three of the 11 anima!s without gross lesions were positive for amoebae but no tissue invasion was observed. Partial protection was observed with other antigens--305;, with whole amoebae, 18:; with Fraction II and 30 9: with Fraction III. In most there were multiple ulcers of the deep penetrating type and amoebae either penetrating and invading the muscular layer or the lytic process extending up to the muscular layer. In one animal, immunized with whole amoebic antigen, multiple hepatic lesions were also observed and trophozoites of E. kistolytica were demonstrated histologically.
The gel filtration of the amoebic antigen was carried out as described elsewhere (SAWHNEY et al., 1979). 5 ml of axenic antigen containing 30 mg of protein was applied to the top of a 45 r: 2.5 cm column of $ebhadex G-200: The elution was carried out by employing 0.05 M tris buffer, pH 7 * 5. The typical chromatographic fraction so obtained is shown in the figure. The fractions were concentrated by lyophilization and the lyophilized material was reconstituted in 5 ml of normal saline.
Introduction Scanty information is available on immunity to Entamoeba kistolytica infection. It is apparent to clinicians that patients do have repeated infections, in spite of high levels of anti-amoebic antibodies. Alternatively, it is rare for a patient who has been successfully treated for amoebic liver abscess to have a second liver abscess, indicating that acquired resistance to amoebic infection is possible. It is very difficult to prove the development of acquired resistance in an individual with a history of amoebiasis, but studies in an experimental mohel could be helpful. The antibodv titres could be high because of ihe presence, in t6e amoebic extract;of certain antigenic determinants which elicit high antibody levels but, at the same time, there could be certain antigenic determinants which are functional although eliciting a low antibody response. We have previously observed that the chromatographic Fraction I of an axenic amoebic extract is highly
and challenge of experimental animals Three to four-week-old guinea-pigs (250 to 300 n) were divided into five groups. Group 1 were con& animals without imm&zaiion but challenged with 100,000 virulent amoebae (Strain No. H-25) intracaecally (VINAYAK et al., 1977). The strain had been isolated from a patient with amoebic dysentery. The animals in Groups 2,3,4 and 5 were immunized resnectivelv. with crude antigen and Fraction I, II ani III. l&al volumes of antigen and complete Freund’s adjuvant were emulsified for 5 to 8 min. Animals were given weekly subcutaneous injections of 0.6 ml of this emulsion for three weeks. Seven days after the last injection, blood samples were taken for measurement of antibody response by indirect haemagglutination (PRAKASH et al., 1969) and counter-current immunoelectrophoresis tests (KRUPP, 1974a). One week after the last injection each of these animals were injected intracaecally with 100,000 virulent amoebae. Seven days later,
et al.,
Material Antigen
and Methods
preparation
The antigen used was a sonicated extract of axenically grown E. kistot’ytica (NIH:200). Amoebae from a 72-hour culture in TPS-I medium were washed thrice in sterile normal saline, concentrated by centrifugation at 850 g for 5 to 7 min and sonicated for 5 min at 20 Kc in an MSE ultrasonic disintegrator. The sonicated material was centrifuged at 12,000 L g for half an hour at 4°C. Protein concentration was determined by LOWRY'S technique (1951). The supernatant was then lyophilized and then fractionated on Sephadex G-200. Separation and concentration on Sepkadex G-200 column
Immunization
of antigenic
components
484
PROTECTIVE EFFECTS OF AXENIC E. histolytica
Table I-Antibody response in guinea-pigs Entamoeba histolytica
immunized
FRACTIONS IN GUINEA-PIGS
with crude and chromatographic
BEFORE CHALLENGE Immunization antigen Group
IHA titre No. of (no. of animals guinea- in parentheses) Pii9
1 No immunization
14
Neg. (14)
2 Whole antigen
14
1:64 (3) 1:256 (2) 1:1024 (3) 1:4096 (2)
Mean IHA titre 0
1:2542
1: > 16000 (1) n.d. (3) 12
1:256 (2) 1:1024 (5) 1:4165 1:4096 (2) 1: > 16000 (2) n.d. (2)
4 Fraction II
12
1:64 (3) Neg. (9)
1:16
1164 (3) Neg. (7)
1:19
10
of axenic
AFTER CHALLENGE
CIEP (no. of animals in parentheses)
IHA titre (no. of animals in parentheses)
Neg. (14)
Pos. (3) Pos. (2) F2
is;
Mean IHA titre 1:41
1:1024 (8) 1:4096 (3) 1: > 16000 (3) 1:4819
CIEP (no. of animals in parentheses)
2.~(‘8”,’ Pos. (8) Pos. (3) Pos. (3)
pas: (1) n.d. (3)
3 Fraction I
5 Fraction III
fractions
1:1024 (1) 1:4096 (6) 1:8800 1: > 16000 (5)
Pos. (1) Neg. (11)
Pos. (2) Neg. (8)
1:59
Pos. (1)
Pos. (5) Neg. (6) 1:64 (6) 1:256 (2) Neg. (2)
1:90
Pos. = positive; Neg. = negative; n.d. = not done
the animals were killed. Just before killing, they were bled via cardiac puncture and, at killing, lesions in the caecum and liver were sought. Caecal lesions were classified as suggested by NEAL (1951) and were further graded histologically as suggested by VINAYAK et al. (1977). Any area of lesion in the caecum and or liver was fixed in 10% form01 saline. Tissues were processed and stained with haematoxylin and eosin and FAS. Results Elution Profile The elution profile of E. histolytz’ca (NIH:200 strain) indicates that there are two peaks divided into three fractions when 30 mg of protein is put on a column of Sephadex G-200. The recovery was only 74%. The replication of results was excellent with each column run. Fraction I contained 14% of the total protein and Fractions II and III had 17% and 41 y0 protein respectively. Immunization and results of challenge of guinea-pigs The blood samples, collected one week after the last immunizing dose, were tested by the indirect haemagglutination and counter-current immunoelectrophoresis test (KRUPP, 1974a) to determine the level of antobodies. None of the animals had any antibody titre before immunization. After immunization, but before challenge, the animals immunized with whole amoebic antigen had IHA titres ranging from 1:64 to 1:16000, with an arithmetic mean IHA titre of 1:2542. Samples from
three animals could not be tested. All the animals with IHA titres, had a positive CIEP test. For animals immunized with Fraction I had IHA titres ranging from 1:256 to 1:16000 and those immunized with Fraction II and Fraction III had very low titres, i.e. 1:64. Nine animals immunized with Fraction II and seven animals immunized with Fraction III failed to show any haemagglutinating antibodies. Here again, the results of the CIEP test compared well with those of the IHA test (Table I). The antibody response in serum samples collected just before killing showed a twofold increase in the mean IHA titres in animals immunized with whole amoebic antigen and Fraction I; mean IHA titres ranged from I:64 to I:256 in animals immunized with Fractions II and III and the titre was only 1:64 in the control group of animals which were not immunized. After challenge, there were five, seven and two animals in Groups 1, 4 and 5 respectively which showed no haemagglutinating activities (Table I). At post-mortem examination of all these animals, seven days after intracaecal inoculation with amoebae, all organs were checked for gross lesions and the caecal contents for the presence of E. histolytica trophozoites. In the control group (no immunization), 13 (92.130/,) of the guinea-pigs showed gross caecal lesions and large numbers of amoebae could be demonstrated in the caecal contents. One animal, although showing no gross lesions, had a caecal content positive for amoebae.
Table II-Gross caecal lesions of axenic Entamoeba histolytica Immunizing
antigen
in animals
No. of animals
v. K. VINAYAK
et al.
immunized
with
485 crude
and chromatographic
No. gross lesions, caecal contents
Gross lesions in caecum Positive
fractions
Negative
Positive E.h. +
Negative E.h. -
No immunization
14
13
1
:.
0
Fraction antigen I Whole Fraction II Fraction III
12 14 12 10
1: 10
11 4 2 3
3 2 2
8” 0 1
Table
III-Protective
Immunization material Groups 1 No immunization 2 Whole antigen 3 Fraction I 4 Fraction II 5 Fraction III
Table
Group
IV-Protective
Immunization
7
effects of crude Animals with caecal lesions/ animals inoculated
and chromatographic
Grading of lesions Average caecal score
Grade I
Grade II
5.31 5.21 1.17 5.69 5.20
6 (46.2%) 0 (0.0%) l(lOO*O%) 3 (30.0%) 4 (57.1%)
7(53.8x) 2(20.0X) 0 (0.0%) 5(50.0X) 3(42*9%)
13/14 (92.9 %) 10/14(71.4%) l/12 (8.3 %) 10/12(83*3 %) 7/10(70*0 %)
effects of crude antigen,
antigen
Whole antigen Fraction I Cont. whole antigen
of E. histolytica antigen
fractions
Fraction
I and concentrated
Protein concentration 1 .2 mg/animal (containing 0.17 mg of Fraction I) 0.5 mg/animal 3 .6 mg/animal (containing 0.5 mg of Fraction I)
Lesions were 3 to 5 cm in diameter and covered with a sheath of slough which could be easily peeled off, revealing deep ulcers with a necrotic and congested floor. The animals usually had multiple lesions (Table II). Ten (71.41%) of the 14 animals immunized with whole amoebic antigen had gross caecal lesions. There were no lesions in 30 “/o of animals, indicating that there was partial protection. The caecal contents of all these 10 animals were also positive for amoebae. The size of the lesions ranged from 1 *O to 5 .O cm. There were usually one or two caecal lesions in each animal but in three there were ulcers throughout the caecum. The caecal contents of the infected animals were semi-fluid to watery. In two of the four animals without .gross lesions, amoebae were present in the caecal contents. Amoebae were not detected histologically in the caecal mucosa of these four animals. 12 animals were immunized with Fraction I.
Grade III ;Q;*;m; * o.
whole amoebic
Animals with caecal lesions/ animals inoculated
antigen
Percentage protection
10/14
29%
l/l2
92 o/ /0 91 ‘X
l/11
Only one had a gross lesion which was very small, approximately 0.5 cm in diameter. Although three of the 11 animals without gross lesions had caecal contents positive for amoebae, these were few in number and no tissue invasion was observed. Animals immunized with Fractions II and III showed partial protection-18% and 30%, respectively. The lesions presented as shallow weers, irregular in shape and from 1.0 to 5 .O cm in diameter. The ulcers were covered by a mutinous sheath; this sheath could be easily peeled off revealing an uneven surface consisting of areas of congestion intermingled with areas of superficial necrosis. There were multiple lesions in all the animals with gross lesions (Table II). The protective effect, the average Neal scores and grade of lesions are shown in Table III. 92.30% of non-immunized animals had caecal lesions. The amoebae had broken the muscularis mucosae and the ulcers had extended in to the submucosa where
486
PROTECTIVEEFFECTSOF AXENIC E. histolytica FRACTIONSIN GUINEA-PIGS
many amoebae could be seen. Only 30 %, protection was achieved in the animals immunized with whole amoebic antigen. Ulcers were of the deep penetrating type and very extensive and there was deep necrosis of the mucosa. At a few of the necrotic places brown pigment could also be seen. Amoebae were found to be infiltrating into the submucosa and forming flask-shaped ulcers with necrotic debris. In 80% of the animals with ulcers amoebae had either penetrated and invaded the muscular layer or the lytic process initiated by the amoebae had extended up to the muscular layer. Only one animal immunized with Fraction I had lesions which were very slight. There was superficial necrosis of the mucosa and amoebae were confined to the mucosa only. Interglandular stroma and granular tissue were found to be infiltrated with cells, mostly mononuclear histocytes and lymphocytes with some polymorphs. Amoebae were confined to the muscularis mucosae with invasion to the submucosa which showed some degree of oedema (Table III). Since Fraction I could urotect 92”;, of the animals it was thought worthwhile to concentrate the whole amoebic antigen so that it contains 0.5 mg of Fraction I. Of the 11 animals immunized with concentrated whole amoebic antigen only one (9.00,6) had caecal lesions which were also very slight (Table IV). One of the animals immunized with whole amoebic antigen also had multiple hepatic lesions. Histologically, the hepatic lesions consisted of a central zone of cytolysed granular material surrounded by degenerated liver cells, a few leucocytes, connective tissue cells, erythrocytes and, occasionally, trophozoites of E. histolytica. Parenchymatous cells were compressed at the periphery of the lesions, and amoebae could be seen either at the periphery of the abscess cavity or slightly away from it. The presence of amoebae was confirmed by staining the tissue with PAS stain and counterstaining with haematoxylin. I
Discussion The findings clearly show that the extract of E. histolytica contains a mixture of different antigenic molecules. Experiments utilizing these antigenic determinants obtained after column chromatography indicated that Fraction I had the potential functional capability. This fraction has a molecular weight of 650,000. A similar type of fraction has also been reported by ALIKHAN & MEEROVITCH (1968) and KRUPP (1974b). These authors also concluded that Fraction I is composed of proteins of molecular weights in the range of 650,000. Immunization of animals with this high molecular weight fraction could protect 92.0”; of them; in only one of 12 animals did mild, superficial lesions develop. At the same time, in the control group, lesions occurred in 92 * 13 96 of animals, indicating the virulence sta us of the isolate of E. histolytica employed. Partial protection -17 $L, and 30 %-was obtained with Fractions II and III respectively. In a study by KRUPP (1974b) it was pointed out that the crude extract of amoebae protected 70 “/ of animals although we only achieved protection in 3Oq/, of animals immunized with the
crude antigen. It is difficult to explain the variations between the results of our study and those of KRUPP (1974b), although the isolate of amoebae used was the same in both. The difference in results could possibly be explained on the basis of protein content of the immunizing doses. We used the crude preparation containing 1.2 mg protein/ animal but KRUPP (1974b) used 5.6 mg protein/ animal. The amount of protein, and therefore protective antigen, seems to be very critical; when we concentrated the protein in the crude extract to contain 3.6 mg per animal, 91 y0 of the animals showed protection. 3.6 mg protein in crude extract contains 0.5 mg of protein equivalent to that in Fraction I. We have already demonstrated that Fraction I contains 14% of the total protein. Thus, the similarity in the results in animals immunized with Fraction I and those immunized with concentrated crude amoebic extract could be due to the amount of protein content of Fraction I in both the preparations. There were animals in the various groups which had no gross caecal lesions although the caecal contents were positive for amoebae. It seems that amoebae in such a situation failed to invade the mucosa so as to produce gross caecal lesions. Thus they were in a commensal state. Such an observation is similar to that in amoebic patients who are asymptomatic, although passing cysts of E. histolytica in the stool. In one of the animals we observed multiple abscesses in the liver. There was a central zone of necrosis in which amoebic trophozoites could be demonstrated in a haematoxylin preparation and were confirmed by PAS staining. Antibody response in amoebic patients has been very well documented and persistence of antibodies after cure has been measured and reported (KRUPP, 1970, KRUPP & POWELL, 1970, 1971). It is a consensus that the antibodies do not contribute to the protection of the host as reinfections commonly occur, even when the antibody titres are relatively
0.7 1
14
COLUMN DIMENSION 45x2.5MM SAMPLE SIZE -30 mg PROTEIN/5ml 0.05 M TRIS HCI BUFFER. - PH-7.5 FLOW RATE 3-4 ml/10 MINUTE
30
44
70
ELUTION VOLUME Chromatographic pattern of axsnic Enumorba histolytka snti@mn on sephadex O-200 gel. Fig. 1. Chromatographic pattern of axenic Emamoeba antigen on Sephadex G-200 gel.
hirtolyrica
V. K. VINAYAK et al. high. In the present study, it has been clearly shown that the animals immunized with Fraction I had a high antibody titre and, at the same time, showed 92% protection; animals immunized with crude amoebic extract though also showed high antibody levels but protection was observed in only 30 %. The antibody level in this group of animals seems to be due to the host immune response to a non-protective component of the antigen. It thus seems that circulating antibodies to the Fraction I component of the amoebic antigen might have some protective role. Further work on this is under progress. Cellular immunity may also be playing some part in the control of the infection. ORTIZ-ORTIZ et al. (1975), SAVANAT et al. (1973) and HARRIS & BRAY (1976) have shown that cellular immunity is altered in patients with amoebic liver abscess. JAIN et al. (1980) recently observed that the cellular immunity was not markedly altered in experimentally infected guinea-pigs with only caecai lesions. HARRIS & BRAY (1976) reuorted almost identical findings in clinical cases of intestinal amoebiasis. De&led studies of this aspect in both clinical cases and experimentally infected animals are being carried out in this laboratory. .
I
^
References
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