Journal ofUelmlnthology

(1976) 50, 59—63

A demonstration of the presence of anti-snail antibodies in individuals infected with Schistosoma haematobium T. F. H. G. JACKSON and P. P. DE MOOR Institute for Parasitohgy. South African Medical Research Council, P.O. Box 1035, Durban, Natal, South Africa

ABSTRACT An extract of the snail Bulinus (Physopsis) africanus, the intermediate host of Schistosoma haematobium, was used as the antigen in haemagglutination tests in a survey of the prevalence of anti-snail antibodies in various population groups. It was found that sera from known bilharzia-infected individuals and randomly selected individuals from bilhanda endemic areas had significantly higher incidences as well as higher titres of antibodies to this snail antigen than non-infected individuals and individuals from non-endemic areas.

Earlier studies indicated that cercariae of S. haematobium have antigens in common with their snail intermediate host, Bulinus {Physopsis) africanus, incorporated in their surface coat or "glycocalyx" (Jackson, 1976). It seemed possible, therefore, that in the process of invading their definitive hosts, the cercariae might induce an immune response to the snail antigen present in this surface coat. The glycocalyx remains part of the schistosome larva for up to three hours after penetration (Hockley and McLaren, 1973) after which it is lost and the schistosomule has a "naked" tegument until it acquires a coat of definitive host antigen alter several days (Clegg et al., 1971; Clegg and Smithers, 1972). This paper describes the results of haemagglutination tests used to investigate the occurrence of anti-snail antibodies in persons living in areas of varying endemicity of schistosomiasis. MATERIALS AND METHODS Preparation of antigen and cells Sheep red blood cells were collected into an equal volume of Alsever's solution (pH 6*1)* with antibiotics and were stored at 4°C. Cells were then formalinised and tanned as described by Herbert (1967) within three days of collection. In order to minimize cross-reactions, the antigen was partly purified by the following procudure: 0*45 g uninfected and mechanically homogenized fresh snail material was washed three times by suspension in saline, centrifugation and resuspension. The sediment was then homogenized in 3 ml of a solution of 1 % Triton X-100 (BDH) in saline. This suspension was cleared by centrifugation at 32 000 g for 30 minutes and the supernate constituted the purified antigen solution. This solution revealed at least four protein bands when electrophoresed in polyacrilamide gel and stained with amido black. Furthermore, in counter-current electrophoresis against a rabbit anti-snail serum, it gave a band of identity 59

T. F. H. G. JACKSON and P. P. DE MOOR

with Triton-X treated whole cercariae when the two solutions were electrophoresed simultaneously. The snail antigen solution was diluted 1 :5 with phosphate buffered saline (PBS; pH 7-2) and the antigen was then linked to the tanned sheep cells by conventional means (Herbert, 1967). Control cells lacking snail antigen were prepared by diluting 3 ml of 1 % Triton X-100 in saline to 15 ml with PBS, and to this 3 ml of 20% suspension of formalinized tanned cells were added. The two suspensions were incubated overnight at 4°C. They were then washed by centrifugation in PBS and adjusted to a 2% suspension in a 1 :250 dilution of normal rabbit serum in PBS. The suspensions were then ready for use in haemagglutinations The tests were performed in microtiter plates. Serial two-fold dilutions (starting at 1 :2) were made of each serum in two adjacent rows of wells using a 1 :100 dilution of normal rabbit serum in PBS as the diluent, with a total volume of 50/i\ in each well. To one row of wells was added 50 p\ of the antigen-coated cells and to the other row the control cells were added. The results were read after two hours incubation at room temperature in a humidified container and the plates were photographed with a Cordis immunodiffusion camera for permanent records. Selection of test sera (1) Specific antisera Rabbit antisera were raised to uninfected Bulinus (Physopsis) africanns snails, S. haematobium cercariae and to the Triton soluble snail extract described above according to the TABLE 1 Description of the population groups from whom sera were obtained for testing for the presence of anti-snail antibodies.

60

Group

No. of individuals

A

20

African children aged 5-12 years—living in an area of Natal free of schistosomiasis.

B

19

African children aged 5-12 years—selected at random and living in a schistosomiasis endemic area of Natal (disease status unknown).

C

36

African children aged 5-12 years—living in a schistosomiasis endemic area of Natal—all passing S. haematobium ova in their urine and suffering from active schistosomiasis.

D

19

Adult sera—from West Germany; all considered to be free from contact with S. haematobium.

E

20

Adult sera—from blood donors in Holland; all considered to be free from contact with S. haematobium.

F

20

Adult sera—selected at random from Caucasian blood donors in the endemic Natal area (disease status unknown).

G

20

Adult sera—selected at random from adult Coloured blood donors in Cape Town, a non-endemic area of schistosomiasis.

H

20

Adult sera—selected at random from African blood donors in the schistosomiasis endemic area.

I

43

Young adult sera—from known infected African patients (all passing S. haematobium ova in their urine).

Description

Anti-snail antibodies in S. haematobium infections

method of Chase (1967). These were compared by haemagglutination assays to sera from immunologically naive rabbits. (2) Human sera Human sera were assembled from various areas in South Africa and abroad to form comparable groups originating from known infected and healthy individuals. Also included were sera from individuals living in endemic and non-endemic schistosomiasis areas in whom the disease status was unknown. These groups are described in more detail in Table 1. RESULTS Specific antisera The rabbit antisera to Bulinus (Physopsis) africanus, to cercariae and to Triton soluble snail extract all agglutinated the snail-coated red cells to titres in excess of 1 : 512, while the control cells remained negative. Normal rabbit serum did not agglutinate the test or control cells. Selected human sera These results are summarized in Table 2. The groups were compared to each other statistically using the Kolmogorov-Smirnov two-sample test (Siegel, 1956) from which the following conclusions could be drawn. (1) There is a highly significant difference (p> 0-001) between Group A and Group C (i.e. children from an area free of schistosomiasis compared to children known to be infected with the disease). The difference between Groups A and B is only just significant (p> 0-1) as is that between Groups B and C (p> 0*1). TABLE 2 Results of haemagglutination tests performed on 217 selected human sera using sheep red cells coated with snail antigen as the test system. Titres above 1/32 are taken as positive (percentages in parentheses) Group 4 as in Table 1

Total

Titre

Negative Vl28

V256

V512

A

12(60)

7(35)

1(5)

20

B

7(37)

6 (31-5)

6 (31-5)

19

C

2 (5-6)

21 (58-2)

11 (30-6)

D

12 (63)

6(32)

1(5)

19

E

7(35)

11(55)

2(10)

20

F

10 (50)

5(25)

5(25)

20

G

4(20)

11(55)

5(25)

20

H

5(25)

12(60)

2(10)

1(5)

I

3(7)

10(23)

17 (39-5)

9(21)

2(5-6)

36

20 4 (9-5)

43 61

T. F. H. G. JACKSON and P. P. DE MOOR

(2) There are no statistically significant differences between Groups D, E, F and G. (3) There is a highly significant difference between Groups H and I (p> 0-001) (i.e. between African blood donors of unknown disease status from an endemic area and known infected young African adults). (4) There is no significant difference between Groups H and C. The results from sera from non-endemic areas (Groups D to G) were pooled and compared to the other groups as follows. (a) There is no difference between the pooled group (D to G) and Group H. (b) There is no significant difference between the pooled group and Group A. (c) There is a highly significant difference (p> 0-001) between the pooled group and Group I. (d) There is a highly significant difference (p> 0-005) between the pooled group and Group C. DISCUSSION The results indicate that the frequency and titres of anti-snail antibodies are significantly greater in infected individuals than in non-infected individuals. At the population level, too, these antibodies are more prevalent in endemic than in non-endemic areas. The sera originating from blood donors in Holland were all taken from persons that had not visited the tropics, but the proportion of positive results (65 %) was surprisingly high although the titres did not exceed 1 : 128. It is possible, however, that these people may have been exposed to trematode parasites of animals or birds or to other parasites which could give rise to cross-reactions with the snail antigens used in the present study. The sera from people living in the non-endemic region of Cape Town (Group G) gave results similar to the Dutch sera (Group E), but in this instance it might well be that a proportion of these people had visited or even lived in regions of endemic human schistosomiasis at some time during their lives. It would appear that antibodies against snail antigens can occur naturally, but at low titres, even in populations not exposed to infection by human schistosomes. However, more specific immunity to these antigens is induced by schistosome infection and it seems very probable that this immunity is due to the release of snail antigens from the glycocalyx of the cercariae into the tissues of the human hosts shortly after the cercariae have penetrated the skin. However, the possibility of cross-reactions with other parasites or with the adult forms of schistosomes which also have antigen in common with snails (Kemp, et al.t 1975) cannot be entirely excluded. It is perhaps important to note, though, that Kemp et al. pointed out that, in addition to the antigens cross-reacting with adult schistosomes, the cercariae also possessed distinct antigens shared only with their snail hosts. Experiments are currently in progress aimed at identifying the specific snail antigens against which antibodies are produced in humans in naturally occurring infections. ACKNOWLEDGEMENTS We are grateful to Professor G. Piekarski (West Germany), Dr. V. Eijsvoogel (Holland), Professor A. Kipps (Cape Town), Dr. P. Brain (Durban) and Emmaus Mission Hospital, Natal, for assistance with our serum collection. Statistical advice and analyses were provided by Professor H. Linhart, Mr. W. Zucchini and Dr. L. Troskie to whom sincere thanks are due. We are indebted to the South African Medical Research Council for financial support. 62

Anti-snail antibodies in S. haematobium infections

REFERENCES CHASE, M. W. (1967) Production of antiserum. In Methods in immunology and immunochemistry (ed. C. A. Williams and M. W. Chase), 197-306. Academic Press: New York and London. CLEGG, J. A., SMITHERS, S. R. and TERRY, R. J. (1971) Concomitant immunity and "host antigens" associated with schistosomiasis. InternationalJournal for Parasitology, 1, 43-49. CLEGG, J. A. and SMITHERS, S. R. (1972) The effects of immune rhesus monkey serum on schistosomula of S. mansoni during cultivation in vitro. InternationalJournal for Parasitology, 2, 79-98. HERBERT, W. J. (1967) Passive haemagglutination. In Handbook of experimental Immunology (ed. D. M. Weir), 720-744. Blackwell Scientific Publications: Oxford and Edinburgh. HOCKLEY, D. J. and McLAREN, D. J. (1973) S. mansoni: changes in the outer membrane of the tegument during development from cercariae to adult worm. International Journal for Parasitology, 3,13-25. JACKSON, T. F. H. G. (1976) Intermediate host antigens associated with the cercariae of Schistosoma haematobium. Journal of Helminthology, 50,45-47. KEMP, W. M., GREENE, N. D. and DAMIAN, R. T. (1974) Sharing of Cercarienhullen Reaktion antigens between Schistosoma mansoni cercariae and adults and uninfected Biomphalaria pfeifferi. The American Journal of Tropical Medicine and Hygiene, 23, 197-202. SIEGEL, S. (1956) Non-parametric statistics for behavioral sciences. McGraw Hill: New York. Accepted 27th February 1976

63

A demonstration of the presence of anti-snail antibodies in individuals infected with Schistosoma haematobium.

Journal ofUelmlnthology (1976) 50, 59—63 A demonstration of the presence of anti-snail antibodies in individuals infected with Schistosoma haematobi...
345KB Sizes 0 Downloads 0 Views