Studies on heterologous immunity in schistosomiasis

Bull. Org. mond. Sante 1972, 47, 591-600 Bull. Wld Hlth Org. Studies on heterologous immunity in schistosomiasis 6. Observations on cross-immunity to Ornithobilharzia turkestanicum, Schistosoma bovis, S. mansoni, and S. haematobium in mice, sheep, and cattle in Iran J. MASSOUD1 & G. S. NELSON 2 Experiments were carried out in mice, cattle, and sheep to investigate the possibility that heterologous immune reactions may occur between the schistosomes prevalent in man and domestic animals in Iran. Immunization with Ornithobilharzia turkestanicum from cattle produced a considerable degree of immunity in mice against challenge with Schisto- soma bovis, S. haematobium, and S. mansoni. The results of immunizing cattle with 0. turkestanicum, S. bovis, and S. haematobium were even more striking; there was a reduction of30-40 % in the number ofadult worms and a proportionally greater reduction in the tissue egg counts. Sheep developed a less marked immunity. Supplementary experi- ments on homologous immunity showed that mice developed a considerable degree of immunity against S. bovis. The results of the heterologous immunity experiments with S. haematobium and S. bovis are ofparticular interest as both parasites often occur in the same area andare often transmittedby the same snail host, man andcattle being exposed to the cercariae of both species simultaneously. The reciprocal immunity produced by these infections may be mutually beneficial in limiting the severity of schistosomiasis in man and domestic animals in the endemic areas. Previous papers in this series have demonstrated a significant heterologous immune response between human and animal schistosomes in mice, monkeys, cattle, and sheep (Nelson et al., 1968; Amin et al., 1968; Amin & Nelson, 1969; Hussein et al., 1970; Preston et al., 1972). The purpose of the study report- ed here was to extend these observations by investigat- ing the heterologous immune reactions between ani- mal and human schistosomes in Iran, with a view to assessing their possible importance in nature. In the preliminary experiments in mice Ornithobilharzia turkestanicum was used as an immunizing agent. In subsequent experiments observations were made on cross-immunity to 0. turkestanicum, Schistosoma bovis, and S. haematobiumn in calves and sheep and the levels of immunity produced by both homologous and heterologous systems were compared. 1 Present address: P.O. Box 1310, School of Public Health, Teheran, Iran. 2Present address: Professor of Medical Helminthology, London School of Hygiene and Tropical Medicine, London, England. EXPERIMENTAL MATERIAL Four species of schistosome were used: the local strains of 0. turkestanicum, S. bovis, and S. haema- tobium, and a Puerto Rican strain of S. mansoni for comparison with previous observations by Nelson et al. (1968) and Amin et al. (1968). The calves and sheep were obtained from areas in Iran where there is no schistosomiasis. The mice were 6-week- old T.O. strain albinos. HETEROLOGOUS IMMUNITY STUDIES IN MICE Each experiment was separately controlled and followed the same design as that used in previous studies (Table 1). Groups of immunized and chal- lenged, challenge control, and immunized but not challenged animals were used. Following immuniza- tion with one or more exposures of various doses of 0. turkestanicum cercariae the animals were chal- lenged 9 weeks later with 300 cercariae of S. bovis 2938 591- 5 592 J. MASSOUID & G. S. NELSON 4D C6ID I~~~~~~~ 00 0 0 0 U~~~o a~e T- U-~) CoN N Nq Lo 20 0 = CD 0 0 0 0 0 00 ~ 0 0 j 00 0 0 00bc 6~ 0 0 0 N C tC ~ 0 00 0 00 0 0~~~~~~~~~~ C~~~~~~~~~~~~~~~~~v 0~~~~~~~~v 00 . C CD CD o' mr' -D C6C C~~~~~~C lb ~~.20 ~ ~ C' I, I1 0 0 C 7U 0 CaO '. N - 0 ~ 0 C0 r-C14 C) CD00 v- OQ 30 O 0 0~~~~ 0 0~~~~C) DC *~~~~~ 0 .r ~ ~ C) C = C U) ~~~< Ci~~~~ m (Aj LO v- 0 = o coCl % CD E ) ' D ' 0 0 4) 0'- '- ~~ 0'- 0' r- = E ECL 0 0 = -~ 0 ~~~0 0 0W0CD U0o Z 0 0- C 'a ) C: U) E co r' o ( Eo o. o 0 0~~~~~xx . o *O.C ~~~~- 0.00o --ui STUDIES ON HETEROLOGOUS IMMUNITY IN SCHISTOSOMIASIS. 6 or of S. haematobium, or 150 cercariae of S. mansoni. The cercariae at each exposure were all taken from the same suspension obtained from a large number of infected snails. Experiment 1: mice immunized with 0. turkesta- nicum (100 cercariae) and challenged with S. bovis (300 cercariae) The recoveries of schistosomes showed that the mean worm burden of S. bovis in the immunized group was slightly lower than in the challenge con- trol group. The S. bovis egg counts in the liver and gut, and total egg counts per mouse, were markedly lower (50%) in the immunized group than in the challenge control group. The differences between the means in the liver, gut, and total egg counts were all statistically significant (P<0.05, P<0.01, and P<0.02, respectively). Experiment 2: mice immunized with 0. turkestani- cum (3 x 50 cercariae) and challenged with S. bovis (300 cercariae) Immunization was carried out by 3 repeated inocu- lations of 50 0. turkestanicum cercariae at 4-week intervals. The challenge exposure with S. bovis cercariae was carried out 4 weeks after the last exposure to 0. turkestanicum cercariae. As indicated in Table 1 the mean recovery of S. bovis from the challenge infection was again slightly lower in the immunized and challenged group than in the challenge control group. Analysis showed that the liver, gut, and total egg counts per mouse in the immunized and challenged group were 55 % lower than in the challenge control group. Student's t-test showed that the differences between the means of the eggs in liver and gut and the total eggs of these two groups were significant (P <0.05, P <0.02, and P <0.02 respectively). The total num- ber of eggs per female was markedly lower in the immunized group. Experiment 3: mice immunized with 0. turkestani- cum (200 cercariae) and challenged with S. bovis (300 cercariae) There was a reduction of 33 % in worm burdens of S. bovis in the immunized group. The liver and total egg counts per mouse showed significant reduc- tions (P<0.05 and P<0.05), but the gut egg count did not. The number of eggs per female was also higher in the non-immunized group. Experiment 4: mice immunized with 0. turkestani- cum (3 x 50 cercariae) and challenged with S. hae- matobium (300 cercariae) Although the reduction in the number of adult S. haematobium (9 %) was not significant, there was a significant reduction (42%) in the liver egg counts (P <0.01) and in total eggs per mouse (P <0.05). Experiment 5: mice immunized with 0. turkestani- ckm (50 cercariae) and challenged with S. mansoni (150 cercariae) There was a reduction of 27% in the number of S. mansoni adults but the tissue egg counts were reduced by only 7%. An interesting observation was the presence of 0. turkestanicum eggs in the immunized animals although none were found in the control mice infected with 0. turkestanicum. Experiment 6: mice immunized with 0. turkestanii cum (200 cercariae) and challenged with S. manson- (150 cercariae) With the increased cercarial dose the mean total tissue egg count showed a 38% reduction in the immunized group. This reduction and the reductions in the liver and gut egg counts were all statistically significant (P<0.02, P<0.05, and P<0.05 respec- tively). 0. turkestanicum eggs were seen in both the immunized and control animals. Comment on experiments in mice The results show a considerable degree of hetero- logous immunity with all the systems tested except when a low dose of 0. turkestanicum was used against challenge with S. mansoni. The experiments with 0. turkestanicum showed that although it reached maturity in mice it usually produced only a few eggs when given alone; in combined infections with S. bovis or S. haematobium, however, a con- siderable number of 0. turkestanicum eggs were found in the tissues. A similar phenomenon of heterospecific stimulation, in which S. mattheei re- sponded to the presence of S. mansoni, was reported by Taylor et al. (1969). A single exposure to 0. turkestanicum cercariae was as effective as multiple exposure in producing immu- nity. This is in contrast to our previous observations on S. mattheei and S. mansoni (Amin & Nelson, 1969). Moreover, there was no apparent increase in effectiveness when the immunizing dose was in- 593 J. MASSOUTD & G. S. NELSON Table 2. Effect of heterologous immunity on recoveries 0 of adult. turkestanicum, S. bovis, and S. haematobium in calves and sheep Experi- . . Mean adult worm recoveries ReductionEmeri- Cercarial dose: immunizing in adulteno. versus challenge species G roup Aa Group B Group C c worms O.t. S.b. O.t. Sb. O.t. S.b. S.b. 7. Calves 8 000 0. turkestanicum 3 251 - 2 186 1 966 - 3 156 38 versus 5 000 S. bovis S.b. O.t. S.b. O.t. S.b. O.t. O.t. 8. Calves 5000 S. bovis versus 3 530 - 3 210 2 283 - 3 251 30 8 000 0. turkestanicum S.h. S.b. S.h. S.b. S.h. S.b. S.b. 9. Calves 3 x 7 000 S. haematobium - - 334 1 819 - 3 156 42 versus 5 000 S. bovis S.h. O.t. S.h. O.t. S.h. O.t. O.t. 10. Calves 3x7 000 S. haematobium - - 422 2230 - 3251 31 versus 8 000 0. turkestanicum O.t. S.b. O.t. S.b. O.t. S.b. S.b. 11. Sheep 5 000 0. turkestanicum 1 774 - 1 574 1 974 - 2 151 8 versus 5 000 S. bovis Sb. O.t. S.b. O.t. S.b. O.t. O.t. 12. Sheep 5000 S. bovis versus 2151 - 1 992 1 000 - 1 774 44 5 000 0. turkestanicum a Immune control group (immunized but not challenged). b Immunized and challenged group. c Challenge control group (challenged only). creased from 100 to 200 cercariae. It is possible that with this system there is a minimum effective immunizing level below 100 cercariae similar to the threshold recorded for S. mansoni by Smithers & Terry (1965) in rhesus monkeys. HETEROLOGOUS IMMUNITY STUDIES IN CALVES AND SHEEP The calves and sheep were divided into groups of 2 or 3 animals. The challenge control group in one experiment served as the control for the other experiments. The immunizing and challenging doses of cercariae consisted of a single exposure of 8 000 cercariae of 0. turkestanicum or 5 000 S. bovis in calves and 5 000 cercariae of 0. turkestanicum or S. bovis in sheep. The calves immunized with S. haematobium were given 3 inoculations of 7 000 cercariae in each inoculation at 4-week intervals. Autopsy was performed 9 weeks after the chal- lenge exposure. The mean worm recoveries (Table 2) and egg counts per gram of tissue (Table 3) were the criteria used to evaluate the degree of resistance. Experiment 7: calves immunized with 0. turkestani- cum (8 000 cercariae) and challenged with S. bovis (5 000 cercariae) The calves were divided into 3 groups. Group A consisted of 3 calves that were exposed to 8 000 cer- cariae of 0. turkestanicum but not challenged; they were killed 9 weeks after exposure. Group B con- sisted of 3 calves each exposed to 8 000 cercariae of 0. turkestanicum and challenged 9 weeks later with 5 000 cercariae of S. bovis; they were killed after a further 9 weeks. Group C, which served as controls for the calves in group B, consisted of 2 calves each exposed to only 5 000 cercariae of S. bovis. The number of adult S. bovis recovered from the immunized calves was 38% lower than that recovered 594 STUDIES ON HETEROLOGOUS IMMUNITY IN SCHISTOSOMIASIS. 6 Table 3. Effect of heterologous immunity on tissue egg densities of 0. turkestanicum, S. bovis, and S. haematobium in calves and sheep Experiment Cercarial dose: immunizing emTissues Mean egg counts per g of tissue ReductionEprmno. versuia dse immunizing examined in eggsno. versus challenge species for eggs Group Aa Group B b Group C c (%) O.t. S.b. O.t. S.b. O.t. Sb. S.b. 7. Calves 8000 0. turkestanicum liver 404 - 112 872 - 3076 71.7 versus 5 000 S. bovis small intestine 17 567 - 10 300 735 - 2 138 65.6 large intestine 0 - 0 1123 - 4 721 76.2 S.b. O.t. S.b. O.t. S.b. O.t. O.t. 8. Calves 5000 S. bovis versus liver 3076 - 2 133 156 - 404 61.4 8 000 0. turkestanicum small intestine 2 138 - 1 266 4 001 - 17 567 77.2 large intestine 4 721 - 943 0 - 0 - S.h. Sb. Sh. S. S.h. Sb. Sb. 9. Calves 21 000 S. haematobium liver - - 0 2 150 - 3076 30.1 versus 5 000 S. bovis small intestine - - 0 1 175 - 2 138 45.0 large intestine - - 0 430 - 4721 90.9 S.h. O.t. S.h. O.t. S.h. O.t. O.t. 10. Calves 21 000 S. haematobium versus liver - - 8 333 - 404 17.6 8 000 0. turkestanicum small intestine - - 16 3 019 - 17 567 82.8 large intestine - - 20 0 - 0 - O.t. Sb. O.t. Sb. O.t. Sb. Sb. 11. Sheep 5 000 0. turkestanicum versus liver 1 095 - 723 7 815 - 9 757 19.9 5 000 S. bovis small intestine 8 880 - 6 950 3 919 - 4 635 15.4 large intestine 0 - 0 3 684 - 5 005 26.4 Sb. O.t. Sb. O.t. Sb. O.t. O.t. 12. Sheep 5000 S. bovis versus liver 9 757 - 24 073 138 - 1 095 87.4 5 000 0. turkestanicum small intestine 4 635 - 14 752 6 912 - 8 880 22.2 large intestine 5 005 - 8 215 0 - 0 - a Immune control group (immunized but not challenged). b Immunized and challenged group. c Challenge control group (challenged only). from the challenge control group. There were reductions in tissue egg counts of 72%, 66%, and 76% in the liver, small intestine, and larger intestine, respectively. Experiment 8: calves immunized with S. bovis (S 000 cercariae) and challenged with 0. turkesta- nicum (8 000 cercariae) The design of this study was similar to that of the previous experiment, except that there were 3 animals in groups B and C while group A had 2 animals. The number of immunizing S. bovis cercariae was 5 000, challenged by 8 000 cercariae of 0. turkestanicum. The mean number of 0. turkestanicum adults in group B was 30% lower than that in group C. The reductions in tissue egg counts per gram of liver and small intestine were 61 % and 77 %, respectively. Experiment 9: calves immunized with S. haemato- bium (3 x 7 000 cercariae) and challenged with S. bovis (5 000 cercariae) Calves in group B were immunized with 3 doses of 7 000 S. haematobium cercariae at 4-week inter- vals, making a total of 21 000 cercariae per calf. The challenge was the same as in experiment 7. The mean recovery of S. bovis worms in group B was 42% below that in group C. Distinguishing S. haematobium from S. bovis was relatively easy, since the S. haematobium worms were almost all very small and immature with undeveloped internal organs, but there may have been some errors owing to the stunting effect of the immunity on S. bovis. The S. bovis egg counts in the liver, small intestine, and large intestine were all reduced in the immunized animals (by 30%, 45 %, and 91 %, respectively.) 595 J. MASSOUE) & G. S. NELSON Experiment 10: calves immunized with S. haema- tobium (3 x 7000 cercariae) and challenged with 0. turkestanicum (8 000 cercariae) The mean recovery of adult 0. turkestanicum was 31 % lower in group B than in group C. The corresponding reduction in tissue egg counts was 17% in the liver and 82% in the small intestine. Experiment 11: sheep immunized with 0. turkesta- nicum (5 000 cercariae) and challenged with S. bovis (4 000 cercariae) The sheep proved to be highly susceptible to both parasites. The reduction in the S. bovis worm burden in group B was 8 %. Tissue egg counts were reduced by 20% in the liver, 15 % in the small intes- tine, and 26% in the large intestine. Experiment 12: sheep immunized with S. bovis (5 000 cercariae) and challenged with 0. turkesta- nicum (5 000 cercariae) In the reverse experiment the immune effect was more impressive, with a reduction of 440% in adult 0. turkestanicum and a reduction in tissue egg counts of 870% in the liver and 2200 in the small intestine. However, these experiments should be repeated be- cause of the extreme variation between the 2 immu- nized animals; in one the total number of adult 0. turkestanicum recovered was 432, with no eggs in the liver and a very low egg count of 350 eggs per gram of small intestine, whereas the other sheep in group B had 1 569 worms and 13 475 eggs per gram of small intestine. Comment on experiments in calves and sheep Detailed accounts of the pathology of 0. turkesta- nicum and S. bovis in naturally infected and experi- mentally infected cattle, sheep, and goats will be pub- lished elsewhere (Massoud, unpublished data). A striking difference between these 2 parasites is the marked localization of 0. turkestanicum adults in the upper mesenteric veins; most of the eggs are found in the duodenum, with very few in the liver and none in the large intestine. On the other hand, S. bovis and S. mattheei worms are distributed widely through- out the large and small bowel and the liver is often seriously affected. 0. turkestanicum is therefore much less pathogenic than these other bovine schisto- somes. It is of considerable interest that the calves exposed to this relatively benign parasite developed a marked resistance to challenge with S. bovis at cercarial densities that would normally have seriously affected them. The results with S. haematobium were even more striking as this parasite is generally regarded as being noninfective to livestock. Exposure of the calves to large doses of S. haematobium showed that a considerable number of worms develop to the adult stage. In the 4 animals exposed to 21 000 cer- cariae the worm recovery rates were 640 (3 %), 204 (0.9%), 418 (1.9%), and 250 (1.2%). Only in the first animal were eggs recovered, the counts being 16 per g in the liver, 33 per g in the small intestine, and 40 per g in the large intestine. The eggs were all deformed and blackened and con- tained no miracidia. These animals were infected with both S. haematobium and 0. turkestanicum, so this may be another example of heterospecific stimulation. The report by McKenzie (1970) of finding natural infections of S. haematobium and S. mansoni in sheep in Southern Rhodesia may have a similar explanation although in Southern Rhodesia there is a possibility of confusion with small eggs of S. mattheei, which would be difficult to distinguish from S. haematobium. The most effective heterologous immunity was developed by the cattle given 3 exposures to S. haema- tobium and challenged with 0. turkestanicum. There was also good cross protection against S. bovis, which again showed that a nonpathogenic schisto- some can give considerable protection against infec- tion with a pathogenic form. Hsu et al. (1966) had some success with the reverse experiment in pro- tecting rhesus monkeys against S. haematobium by repeated immunization with an Iranian strain of S. bovis. The results of the experiments in sheep were less dramatic but again some protection was demon- strated. SUPPLEMENTARY HOMOLOGOUS IMMUNITY EXPERIMENTS IN MICE AND CALVES The main purpose of this series of studies has been to observe the interaction of different species of animal and human schistosomes. As there was abundant material available in Iran, however, the opportunity was taken to study the effect of homo- logous immunity to S. bovis and to 0. turkestanicum (see Table 4). Experiment 13: mice immunized with S. bovis (100 cercariae) and challenged with S. bovis (300 cercariae) The experiment followed the same pattern as experiments 1-6. A marked immunity developed, 596 STUDIES ON HETEROLOGIOUS MMUNITY IN SCHISTOSOMIASIS. 6 Table 4. Homologous immunity to S. bovis in mice and to 0. turkestanicum and S. bovis in calves Mean adult worm Reduc- Mean egg counts perg Red Experiment Cercarial dose: immunization recoveries a ie tion in no. versus challenge Group Group Group worms for eggs Group Group Group eggs Aa B b C c (% A B C (% 13. Mice 100 S. bovis versus 7 12 34 65 whole mouse 7000 29000 57000 49.0 300 S. bovis 14. Calves 1 000 S. bovis versus - 3 242 2 893 0 liver - 1 168 1 438 18.8 4000 S. bovis small intestine - 1 832 3453 46.9 large intestine - 508 2 116 76.0 15. Calves I 000 0. turkestanicum versus - 1 400 2 434 42 liver - 113 110 0 4 000 0. turkestanicum small intestine - 3 260 12 000 72.8 large intestine - - - - a Immune control group (immunized but not challenged). b Immunized and challenged group. c Challenge control group (challenged only). with a 66% reduction in the adult S. bovis burden (P <0.001) and a reduction of 49% in total eggs per mouse. The P values for the differences in the egg counts in the liver and gut and in total egg counts per mouse were P <0.2, P <0.02, and P <0.05, respectively. Experiment 14: calves immunized with S. bovis (1 000 cercariae) and challenged with S. bovis (4 000 cercariae) Calves were divided into immunized and non- immunized groups. There was no significant differ- ence in the number of adult worms recovered from the immunized and control animals; in fact there were more in the immunized group-3 200 against 2 900. The tissue egg counts were reduced in the immunized animals by 19% in the liver, 47% in the small intestine, and 76% in the large intestine. Experiment 15: calves immunized with 0. turke- stanicum (1 000 cercariae) and challenged with 0. turkestanicum (4 000 cercariae) The mean number of 0. turkestanicum adults recovered in the immunized group was 1 400 com- pared with 2 434 in the control group, a reduction of 42%. The immunity had no effect on the egg densities in the liver but there was a reduction of 73% in the egg counts from the small intestine. Comment on experiments 13-15 The experiment with S. bovis in mice showed that this system could be used for studying homologous immunity. This is in contrast to the system of S. mansoni in mice, which has given disappointing results in homologous immunity studies (Smithers, 1962). The demonstration of a well-developed homolo- gous immunity in cattle against both S. bovis and 0. turkestanicum is of interest not only in relation to its possible significance in the field but also because of the potential value of the model for studying immu- nity in a normal host-parasite system. All previous studies on homologous immunity in schistosomiasis have been of artificial systems, usually S. mansoni in mice, rhesus monkeys, or rats, and there has been a tendency to assume that the findings are of direct relevance to immunity in schistosomiasis in man or domestic animals. DISCUSSION The results of these experiments with 0. turkesta- nicum, S. bovis, S. mansoni, and S. haematobium confirm our earlier observations that there is con- siderable cross resistance between different species of schistosome (Amin et al., 1968; Nelson et al., 1968; Amin & Nelson, 1969; Hussein et al., 1970; Preston et al., 1972). The accumulated evidence suggests that previous exposure to animal schisto- somes may be of significance in protecting man from the more serious effects of S. mansoni, S. haemato- bium, or S. japonicum infections. This phenomenon, which is of considerable importance in many infec- tious diseases, has been named " zooprophylaxis " and is defined as " the prevention or amelioration of disease in man as a result of previous exposure to natural infections of animal origin" (Nelson et al., 1962). The experiments described here and those 597 598 J. MAssoUD & G. S. NELSON reported by Hussein et al. (1970) and Preston et al. (1972) on S. mansoni versus S. mattheei in calves and sheep clearly demonstrate that the reverse type of heterologous immunity may be of considerable importance in protecting cattle and sheep from the more serious effects of animal schistosomiasis. The heterologous responses occur not only between human and animal parasites, e.g., S. haematobium and S. bovis, but also between different species of animal schistosome, e.g., 0. turkestanicum and S. bovis. There is increasing evidence that schistosomiasis can be a serious economic problem in the livestock industry. S. mattheei is widespread in southern Africa, causing severe outbreaks in cattle and sheep (Le Roux, 1929; Condy, 1960; Strydom, 1963; Hurter & Potgieter, 1967; Lawrence, 1968; McCully & Kruger, 1969; Reinecke, 1970; Lawrence & Condy, 1970). In Central and North Africa S. mat- theei is replaced by S. bovis; serious consequences of this disease have been reported from cattle in East Africa (Dinnik & Dinnik, 1965) and the Sudan (Malek, 1969; Hussein, 1971).1 It is also a problem in Corsica (Brumpt, 1930), Sardinia (Coluzzi et al., 1965), Israel (Lengy, 1962), Iraq (McHattie & Chad- wick, 1932), and Iran (Arfaa et al., 1965; Massoud, unpublished data). Other species of schistosome that are of importance in the livestock industry include S. japonicum, S. spindale, S. nasalis, S. incognitum, Ornithobilharzia bomfordi, and 0. turkestanicum (see, for example, Lapage, 1962). The problem of schistosomiasis in livestock is probably greatly underestimated as most studies have been carried out on animals slaughtered in abattoirs. In many areas only the relatively fit animals-those that have developed some degree of immunity-are accepted for slaughter. It may there- fore be that many young animals die of the infection and that older animals seriously affected by the dis- ease fail to reach the abattoirs. This is certainly true of animals living in the epizootic conditions 1 See also Hussein, M. F. (1969) The pathology of spon- taneous and experimental bovine schistosomiasis. A study of the spontaneous disease and of experimental infestations of calves and mice with Schistosoma bovis and Schistosoma mattheei, Ph. D. Thesis, University of London. reported from South Africa and the Sudan. There is an obvious need to reassess the economic signifi- cance of schistosomiasis in livestock and to evaluate the role of immunity in limiting the severity of the disease. Our experiments on the interaction of schistosomes suggest that the simultaneous transmission of S. bovis and S. haematobium in the same area may be to the mutual benefit of man and his livestock in reducing the effects of schistosomiasis. In Iran S. haemato- bium and S. bovis are transmitted by the same inter- mediate snail host and the campaign to limit the human disease by the application of molluscicides has resulted in a simultaneous decrease in the infection rates in cattle and man (Massoud & Nelson, 1972). It may therefore be too late to assess the significance of heterologous immunity between S. haematobium and S. bovis in this par- ticular area. However, there are many other areas in the Middle East and Africa where field studies could be carried out on the interaction of different species of schistosome. Further studies are also necessary to develop optimum immunization pro- cedures in cattle and sheep with a view to field trials to test the efficacy of heterologous immunity in areas where schistosomiasis is an important vet- erinary problem. In previous studies (Hussein et al., 1970; Preston et al., 1972) we found that S. mansoni was a useful immunizing agent against S. mattheei in cattle and sheep, but a few of the animals produced patent S. mansoni infections that might potentiate its transmission to man in areas where Biomphalaria snails are prevalent. The present studies confirm the previous observations of Kuntz & Malakatis (1955) with goats, McHattie et al. (1933) and Saeed 2 with sheep, and Saoud (1966) with pigs that S. haemato- bium is unlikely to produce viable eggs in livestock. Sufficient adult worms develop in cattle to stimulate immunity without causing pathological effects and without producing viable eggs. This parasite could therefore be used as an innocuous imnmunizing agent with very little risk of transmission to man. ' Saeed, A. A. (1970) Observations on the pathogenesis of schistosomiasis in mice, calves and sheep, Ph. D. Thesis, University of London. ACKNOWLEDGEMENTS We are indebted to Dr Martin Taylor for his help with the analysis of the data and preparation of the manuscript. This study would have been impossible without the assistance of many members of the technical staff of the Bilharzia Pilot Project, Dezful, Iran, and the London School of Hygiene and Tropical Medicine, STUDIES ON HETEROLOGOUS IMMUNITY IN SCHISTOSOMIASIS. 6 599 London, England. The investigations were supported by the Government of Iran through the Institute of Public Health Research, Teheran, by the World Health Organi- 2ation, and by the Tropical Medicine Research Board of the United Kingdom through the Ministry of Overseas Development. RESUME tTUDE DE L'IMMUNITE HETtROLOGUE DANS LA SCHISTOSOMIASE: 6. OBSERVATIONS SUR L'IMMUNITE CROISEE ENVERS ORNITHOBILHARZIA TURKESTANICUM, SCHISTOSOMA BOVIS, S. MANSONI ET S. HAEMATOBIUM CHEZ DES SOURIS, DES MOUTONS ET DES BOVINS EN IRAN De precedentes etudes ont montre qu'un degre notable d'immunite protectrice h6terologue apparait chez des souris, des singes et des bovins infectes par diverses especes de schistosomes d'origine humaine ou animale. Des recherches similaires ont e faites chez des souris, des bovins et des moutons exposes A l'infection par des schistosomes frequernment rencontres en Iran. L'infection de souris par Ornithobilharzia turkestanicum leur confere une protection elevee contre une infection d'epreuve par Schistosoma bovis, S. haematobium et S. mansoni. Chez les animaux exposes au prealable A 100-200 cercaires d'O. turkestanicum, puis A 300 cercaires de S. bovis, 300 cercaires de S. haematobium ou 150 cer- caires de S. mansoni, on note une reduction de la charge parasitaire de 23-33% pour S. bovis, 9% pour S. haemato- bium et 20-27% pour S. mansoni, tandis que le nombre d'ceufs pr6sents dans les tissus est reduit de 50-56%, 42 %, et 7-38% respectivement. Chez les bovins, des r6sultats plus concluants encore ont ete obtenus. Apres immunisation par exposition A 21 000 cercaires de S. haematobium, suivie d'une in- fection d'epreuve par 5 000 cercaires de S. bovis, le nombre d'adultes de ce dernier schistosome est reduit de 42%, le nombre d'aeufs presents dans le foie de 30% et celui des ceufs deceles dans le gros intestin de 91 %. Une immunite croisee du meme ordre se manifeste en cas d'immunisation suivie d'infection d'epreuve par 0. turkestanicum et S. bovis d'une part, S. bovis et 0. turkestanicum d'autre part, enfin S. haematobium et 0. turkestanicum. L'immunisation de moutons par 0. turkestanicum leur confere une protection de moindre niveau contre l'infection par S. bovis, mais de bons resultats sont observes lorsqu'on inverse l'ordre d'exposi- tion aux schistosomes. D'autre part, lorsque des souris exposees A 100 cer- caires de S. bovis sont soumises ensuite a une infection d'epreuve par 300 cercaires du m6me parasite, la charge parasitaire en adultes est reduite de 65% et le nombre d'aeufs presents dans les tissus de 49 %. Chez des bovins exposes successivement A 1 000 cercaires puis A 4 000 cer- caires de S. bovis, le nombre de vers adultes n'est pas diminue mais la quantite d'ceufs est reduite de 19% dans le foie et de 76% dans l'intestin. 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