Studies on heterologous immunity in schistosomiasis*

Bull. Org. mond. Sante 1969, 41, 225-232 Bull. Wld Hith Org. Studies on Heterologous Immunity in Schistosomiasis * 3. Further Observations on Heterologous Immunity in Mice M. A. AMIN 1 & G. S. NELSON 2 Previous studies on heterologous immunity in mice and rhesus monkeys have shown that immunization with Schistosoma bovis, S. mattheei or S. rodhaini cercariae can limit the severity of infection resulting from subsequent challenge with S. mansoni. The present report describes further evidence of the protection afforded by heterologous immunization. It has been demonstrated that mice immunized with S. mattheei can withstand S. mansoni infections that are lethal to normal mice and that the degree of immunity developed is influenced by the level of maturation and the duration of the initial infection, the size and number ofimmunizing doses, the route ofexposure and the sex of the parasite. It has been suggested that the severity of human schistosomiasis may be decreased as a result of pre- vious exposure to animal schistosomes. This type of heterologous immune response in man has been named " zooprophylaxis " by Nelson, Teesdale & Highton (1962). Amin, Nelson & Saoud (1968) and Nelson et al. (1968) have shown that both mice and rhesus monkeys can develop a marked degree of immunity against challenge with Schistosoma man- soni when they have been immunized by infection with the bovine schistosomes, S. mattheei and S. bo- vis, and the rodent schistosome, S. rodhaini. The immunity was shown by the reduction in both the expected worm burdens and the egg loads. The pre- sent study was designed to see if an initial infection with S. mattheei could protect mice against an other- wise lethal infection of S. mansoni and to identify some of the factors influencing the development of heterologous immunity in mice. MATERIALS AND METHODS A South African strain of S. mattheei maintained in Bulinus (Physopsis) africanus and Mastomys natal- ensis was used as the immunizing agent, and a Puerto * From the London School of Hygiene and Tropical Medicine, London, England. This investigation was gener- ously supported by the World Health Organization and the Tropical Medicine Research Board of the United Kingdom through the Ministry of Overseas Development. 1 Present Address: Stack Medical Research Laboratory, P.O. Box 287, Khartoum, Sudan. 2 Professor of Helminthology, London School of Hygiene and Tropical Medicine. Rican strain of S. mansoni maintained in Biompha- laria glabrata and white mice was used for challenge infections. The experimental animals were all 6-week- old, male T.O. strain albino mice weighing 20 g-25 g each. The techniques used for infecting mice and for tissue egg counts were those of Saoud (1965) and Nelson & Saoud (1966). Adult schistosomes were recovered by the perfusion technique of Smithers & Terry (1965). The adult worms were stained in acetic- acid-alum-carmine for species identification in mixed infections. The statistical analysis of mouse survival was done by the X2 test, the numbers of mice alive and dead in the immunized, challenged groups and in the challenge controls being compared 12 weeks after challenge. Differences between 2 means were tested for statistical significance by Student's t-test. The analysis was made after logarithm transformation (and not logit transformation as incorrectly stated by Nelson et al., 1968) so that the assumption of normality could be better justified; a P value of 0.05 or less was considered to be significant. For each experiment the details of the timing, immunization challenge and results are shown in Tables 1-5. RESULTS Effect of immunization with S. mattheei on the sur- vival of mice subsequently exposed to an otherwise lethal infection of S. mansoni Preliminary experiments indicated that 300 S. man- soni cercariae would kill non-immunized mice in the acute phase of disease (i.e., shortly after the prepatent 2363 225 4 M. A. AMIN & G. S. NELSON period). For the main experiment, 68 mice were divided into 3 groups; 18 mice served as challenge controls and 20 as uninfected controls. The 30 re- maining mice were immunized with 50 S. mattheei cercariae each; altogether, 7 mice died during the immunizing period, death occurring shortly after the ninth week. The remaining immunized mice were divided into 2 groups; 17 were challenged with 300 S. mansoni cercariae each along with the challenge control mice and the other 6 served as immunized but not challenged controls. None of the uninfected controls or the immunized, non-challenged controls died during the observation period of 15 weeks from challenge. All the challenge controls were dead 12 weeks after challenge, by which time only 5 of the immunized, challenged mice were dead. In both groups, deaths occurred 8 weeks after the challenge infection and the 12 sur- viving mice of the immunized group were autopsied 3 weeks later. Statistical analysis of the results showed that there was a highly significant difference between the survival rates of the immunized, challenged mice and the challenge controls (P<0.001). The reduc- tions in the worm burdens and the tissue egg loads of the immunized mice were further evidence of this protection. As indicated in Table 1, the mean recoveries of S. mansoni adult worms from the challenge controls and the immunized, challenged mice were 94.0 and 17.3, respectively. The difference between these 2 means was highly significant with a P value of less than 0.001. No statistical differences were observed between the mean worm recoveries and tissue egg counts of S. mattheei from the immunized, challenged mice and the immunized, non-challenged controls. A 0 (a, C E 0 0(. 00 0 0 .0 a) CL c 0 0._0D S c 0 0 ._ M 0 x -a ._ r- 0s Effect of the duration of the immunizing period on heterologous immunity The design and the results of these experiments are shown in Table 2. The mice were immunized with 50 cercariae of S. mattheei and challenged with 150 cercariae of S. mansoni. No statistically valid evidence of immunity was obtained when the chal- lenge was made 0, 1 and 3 weeks after the initial infections, but at 6, 9, 15, and 30 weeks a highly significant immunity was evident from the reduc- tions in both the S. mansoni worm burdens and the egg loads of the immunized mice compared with the controls. The effect of immunization was slight after an interval of 6 weeks but very marked at 9, 15 and 30 weeks, showing that the age of the initial infection at the time of challenge is an important 226 O-. 0 0S S M1 N CO Ine LO co N CO CO CO CO I CO t m rX Oa, co ,CO coc 01 CO X co co I4 o (C 0 0D COO § § I Lu -j co -j z or nco D z M Lu 2 Lu C- M UJ I _- aDC- a:u~ 0 F- UJ Lu z zo 0 CM CL) 0 U)Lu 0 0j Lu 0l 0 _ it cc, 0 00 E C L UJ .0 O e 0 Xu -E) Cll '00Lu_ 0.000 0 F aLos# 0 0 0 0 0 @ 0E 0 _M___ E0 )Z Om. en 0 C- Co lES .0 °=C-o .= ci 0 0 SU C0 '30 MU 00 z E n) n l) I o o COw Co STUDIES ON HETEROLOGOUS IMMUNITY IN SCHISTOSOMIASIS. 3 0- 0 0.t M 0o: O o0OD 4- u0cOD 0C co 0 0 cm lec 0c M 0 0- 0 0 - I C 00m Col0 ) P 0 0 w W _ C4tO C C c' 0 0) 0 04 o Nno _| e L co m CDNlco tos |N 00Qco _ 04 0 4 cm _ _ _ IV I Qo) le cm co| C%_ r- C 0 1 | Z l C w co 0o 04en 0 L o| 0|___E_|___of | _NN _ ._ ____ __ ____N__ ____ |____ ______ 0-|-e f ___I __ | _ __ _I __ __ -N __ - N_ __ j ______0oZt E0 {n I 4 Q o I I01n 0DD | |~ e : II I o I o ^ o I > : e | o I r 0 00~~C~T- ou)0 w 04 r- co40co0 z° 20 -o___ _to_ |__-It od T- Ir o o r o - 0 t In 0 Co w Loc W: 04-co 0 0 _ -N -0_)000 0 Co 0 N 04 CD0 co 0 0 co c*CL ° u iii o || f n DcoQ 0 In I n | l _l- X4 co co c 0 0 c z 04 C o 04 C '- C 04 C LOQ c cm I 4 c c Q aCDo 0 0 In 0 '- 0 04 0) '- 0 CDl InI~ ad ld r la a lcoI~ ad lo ol C404 N co v co I- le0c 0 :Pu0 00 00E o0) c ~ ~ c r-dr- 0Cd0 L ) c 0 w- t t 0~~~~~~~~~~~~~~~~~~~~~~0 0 0i 0 l Inm CO r 0 0) Co m 0l - oco mlt cm co a co mlc -~~~c m c 4 r-. ~ ~ ~ ~ ~ - 0 0 0~~~C "t 0 0 0) Co Co 00CM - 0o In 0 0 0 '04 C 0 co 0 0) C 00)0t 00 00 0 04 ) 4n0) In 0 0)0C4 o~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~C ~~~~~ 0 ~~~ ~ ~ le c m o0r-c C-0C co R0 r 0 0 t 0 0 0 COU 4 0. LL 0 0 U' o co o0 0) In 0lc CD ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~~C __v 00j 0 z *' U.)- 0) Lu 0 z - z Cl) 0 CD 0 -j 0 LU I z 0 a 0 c-A CQz L - L-0 z 0 I I- 0 i- 0 LU LI- LU 227 2 0 co 0 c ._ 0 (ax 2 z0 o 0 a) 0 0 0 1- 0 C2- 0 -a 4._ cmE esi 0 1 0 0 40W W M La M. A. AMIN & G. S. NELSON O co ° LO I %_ _- 06 to XI ow I., - Co LD 04 IO O N I o MO O 0 0 -LO tn C- VI C C-3 8 a le Me " X 0 0))0 I C4 o> 0 4 CO 1W M _- S 0 U r- C4 U,) .rl U co r0 cLi co Z Co o oCCoC4i I. co co co IO, C':'DCh I- lw v C I s co 0) co _- VI cO 0 0o O 0 0 - Eow 0 Z. 00 .,3a)QC'S-'= X .- @ - 4- a ca C M= w w W L A.o,0 v v <0 > -.~ .'3-_ a) a) a X @ X O z E ' a)q) _ factor in the development of acquired heterologous immunity. There was also a reciprocal effect on the S. mat- theei. The S. mattheei worm burdens in the immu- nized, challenged mice and the immune controls were significantly different only at 9 weeks after challenge (P<0.001) but S. mattheei egg densities were reduced in the immunized, challenged mice in the groups challenged 6 weeks, 9 weeks and 30 weeks after primary infection (P<0.01; P<0.001; P<0.01). Effect of the size of the immunizing infection on heterologous immunity In the following experiments, inoculations of 10 and 20 S. mattheei cercariae were used to immunize the mice. Table 3 shows the experimental design and results; it can be seen that the mean recoveries of S. mansoni were less in the immunized mice than in the challenge controls, for both levels of immu- nizing infection. Student's t-test showed that the difference between the means was not significant at the 5% level with an immunizing infection of 10 cer- cariae but that it was significant when the infecting dose was 20 cercariae (P<0.01), and the number of S. mansoni eggs per immunized mouse was sig- " nificantly reduced (by 58.9%) below that of the control (P<0.01), suggesting that a minimum of 3 pairs of S. mattheei is required to stimulate a significant immune response in the mouse. Recoveries of S. mattheei adult worms from the ° immunized, challenged mice and the immune con- trols were not significantly different in either experi- ment but, with immunizing doses of 20 cercariae, the mean S. mattheei egg load was reduced in the immunized, challenged mice to 65.9% of that in the non-challenged controls. A a) a) S M 0 0 U) a) a) 0 (a) 00l. x -a -C a) Effect of the method of infection on heterologous immunity This experiment compared the immunizing effect of single and triple infections by both the percu- taneous and intraperitoneal routes. The 5 immuni- zation schedules used, and the results, are shown in Table 4. After being immunized, the mice were divided into 2 lots; half the mice, together with a group of challenge controls, were each challenged with 150 cercariae of S. mansoni and the remaining mice served as immunized, non-challenged controls. Statistical analysis of the results showed that the difference between the S. mansoni worm burdens of the immunized mice and the challenge controls were significant, with P values of less than 0.001 in the 228 *4) 0 4 0. CI) Eo 01 C Ue t _ 0 U) UJ- _) ___e_ 0.-0 U)o C) 0 z I- z 2 20 CD 0 -J0 aJ I- z 0Co Oi mLcl I- N 2 LU I 2 LU LA U. 0 E 0 0 C co 0 a) 0 Ie .0U L. I- O'E CI,0)0o 0o M, C) I o I ° 2 2 'WI VI' STUDIES ON HETEROLOGOUS IMMUMTY IN SCHISTOSOMIASIS. 3 -0 ~-.-0 LI0C 4u0C1 00.2C ~01", 01 1m O. 1-z I-- 1O to0 10 r- 1l_ (E % aI- `|_1 IIIX 0 co|_|00 coCEC CL r- 0r-. co) c o1 _1 8 I|81 "~~~~~~~o- coSo IuW 1 eo28II _ 8 01 ° C41 | IN _ N 0 0.J 0 01 1 co co01 -a 0.N Io>°T100 cor- 10 CD ILl 1-0J-_I _1V- lo ZzzCo X'- °e I -° I |eco I o ° 0> E LI I '0 =o COj 10J~~~~~~a 10 CD (s)laa^)~ ~Ce 10u 0 0. -0 000eifi ° -I I O 0_ o5,.. E $ a_ _co LI co') 04 C-4 C 0 F '00 ~~~~~C')"0i110C 1 - ~~~~~~CO14 0)1 00 C'EI ~ 4 co 4 VI'_- LI 4 c0qC0I o0 'I Cc "I 0 LI 04 04no CM 01 0 l - t C0 11 ' a -0 lbL-_ _ _ _ _ 0O-C 0 OUCI=C I (810oM) IBAJo1u I 01C1 1 104 0- *-0a .-0WE0 0'- .0 I t O r- 0 CX .E 000 0 0I C') S C 0(D 01 CL to (D 0 ._ CL) 0 C (L at Q CO XC Cj C 0 ._ 0. group given a single percutaneous inoculation and less than 0.01 in the groups given a single intraperi- toneal inoculation or multiple percutaneous inocula- tions. The difference between the groups immunized with single infecting doses by the 2 different routes and then challenged is also statistically significant. Analysis of S. mansoni tissue egg counts showed similar results, suggesting that, under the conditions of the experiment, percutaneous exposure to a single infection induces a higher level of resistance than one conferred by intraperitoneal inoculation, and that a single percutaneous exposure was more effec- tive than a triple exposure with the same total number of parasites. Although approximately equal numbers of S. mat- theei worms were recovered from the various groups, the egg densities were much lower in mice infected intraperitoneally or given multiple inoculations by the percutaneous route than in those given 1 percu- taneous dose. The low egg counts in mice given repeated infecting doses are probably due to the fact that some of the females, particularly those of the last infection, had not reached sexual maturity at the time of challenge. The low egg counts in mice given a single intraperitoneal inoculation suggests that the development of the worms administered by this route was much slower than in those inoculated by the natural percutaneous route. Immunizing effect of single-sex infections By infecting snails with a single miracidium it is possible to obtain batches of cercariae of one sex only which can then be used to produce single-sex infections in mice. Mice were given an immunizing infection of either 50 male or 50 female cercariae of S. mattheei. Half the mice in each group were subsequently challenged with 150 cercariae of S. man- soni at the same time as the challenge controls. The experimental design and results of this experi- ment are shown in Table 5. Fewer S. mansoni worms and eggs were recovered from all the immunized mice than from the challenge controls but statistical analysis of the results showed that the difference between the means of the worm burdens and the egg loads of the mice immunized with female S. mansoni cercariae and the challenge controls was not significant. On the other hand, the differences between the mice immunized with male cercariae and the controls were highly significant (P<0.001). An interesting incidental observation was that mice immunized with female S. mattheei were found to have some S. mattheei eggs in their tissues. 229 -o z Fz 0 0 -J0 z 0 LU D LUII z 0 p LU z I z 0 LU F z 0 z -J 0 0 z J U 0 LU 0. F LU zj M. A. AMIN & G. S. NELSON az I N° 04 co CD 04 04 ' I' eq uz o 0 0 C- I Co 1 (8U, o 0o: o I co, 0mC. c0 CD pC In04 a; oI e al CO COl COC00 0 O, I co c co aw CO Os 04 04 CR C D CD cI co co adn cmCO 04 04 cor r- r'. M Ln I n In a) C7 0- C co 0- - ) OE- - 0 -,--0 0 00aa Ufl LO c#i 0 E '~ E ILA. DISCUSSION 0C _ c a) a0) c (U 0 0Fz .) 0) C)0 a} 0 2 0 a) Q a) 0 a2) 0 0a) C M The present experiments indicate that heterologous immunity to S. mansoni can be demonstrated by the reduction in the worm burdens and the egg loads and by the ability of immunized mice to survive infections that are lethal to normal mice. The degree of immunity was influenced by the duration of the initial infection, the size and the number of the immunizing infecting doses, the route of exposure and the sex of the parasites. An interval of at least 6 weeks between initial infection and challenge was necessary for significant immunity to develop and the effect of immunization was increased when the immunizing period was extended to 9, 15 or 30 weeks. Similar time intervals have been shown to be neces- sary in homologous immunity to S. douthitti (Kagan, 1952) and S. mansoni (Sadun & Lin, 1959; Hunter et al., 1962) indicating that the initial infections must reach maturity before immunity to challenge infections can be demonstrated. This is in line with the recent studies by Smithers & Terry (1967), who have shown that the adult worms are mainly respon- sible for stimulating acquired homologous immunity to S. mansoni in rhesus monkeys. The duration of schistosome immunity may vary in different host-parasite relationships since, in the present study, immunity was still at a highly sig- nificant level when the challenge was made after 30 weeks, whereas, for example, in hamsters the immunity was lost after the twelfth week while in white rats it was not lost until between 32 and 63 weeks (Ritchie et al., 1963). According to Kagan (1952), Thompson (1954), Wang et al. (1958) and Hsu & Hsii (1963), resistance to schistosome infections is increased by repeated immunization. On the other hand, Hunter et al. (1962) found that a number of cercarial exposures is no more effective in producing immunity to chal- lenge infection than is a single exposure to the same total number of cercariae. This was also shown in the present experiments; a single exposure of 50 cer- cariae of S. mattheei conferred a much higher level of immunity in mice against challenge with S. man- soni than did the same number given in 3 exposures, and with 20 cercariae the effect of immunization produced by the multiple-exposure technique was no greater than that produced by a single immuniz- ing exposure. Both the size of the immunizing dose and the time intervals are of significance in deter- mining the degree of immunity since, when the time interval between the last immunizing dose and the 230 00 -o co E6 4)0 0 o cm E 0 0 0) C e) u_U 'c z' u U C 0( E wU a) 0 0 0 0 _) LU 0 0 L Cl(D ms 0 400 C =' 0. CQ0 e a 0 60o 0 - a) 0SOM eJI Q z I- z V) 0 0 -j0 X LUJ It M z 0 U') z 0 ULUILa. z -j Xx U z I z 0 - z0 - N - La. 'Z z IL) -J I.- a) 0 a)0o 2, 0Jt)E.- STUDIES ON HETEROLOGOUS IMMUNITY IN SCHISTOSOMIASIS. 3 challenge was prolonged, a better immunizing effect was achieved. A similar result was obtained by Hsu, Hsu & Osborne (1965) in studies on rhesus monkeys infected with S. japonicum. The low level of immunity observed in mice immunized intraperitoneally was probably due to the slow and inadequate development of the initial infection. The extension of the earlier parts of the life-cycle at the expense of the later parts would tend to cause less immunity as the adult phase is the most immunogenic part of the life-cycle (Smithers & Terry, 1967). The importance of the sex of the immunizing parasites was demonstrated in our experiments since immunization with male cercariae conferred a marked degree of heterologous immunity (although less than that stimulated by a mixture of male and female cer- cariae) whereas female worms were less effective in protecting mice. There was only a marginal difference in the number of adult worms recovered but the num- ber of eggs produced was only 10% of the number in the controls. These observations corroborate those obtained in mice and rhesus monkeys by Vogel & Minning (1953) and Lin, Ritchie & Hunter (1954) for S. japonicum and Smithers (1962) for S. mansoni. Although Kagan (1952) failed to protect mice with males of S. douthitti, he observed stunting of the female worms of the challenge infection. On the other hand, Oliver & Schneidermann (1953) were unable to demonstrate any resistance to S. mansoni after a single exposure of mice to male cercariae and Szumlewicz (1964) failed to protect mice with irradiated male S. mansoni cercariae although she had some success with irradiated female cercariae. Our experiments with inoculations of cercariae of a single sex indicate that immunity can be produced by male worms alone and that eggs are not necessary. This supports the conclusions of Smithers & Terry (1967), who have shown that adult worms transferred directly into the hepatic portal system of normal rhesus monkeys stimulated a marked degree of immunity, in contrast to the absence of immunity in monkeys inoculated with several thousand living eggs. This is especially important as the eggs are responsible for most of the pathological changes associated with schistosomiasis. It might prove pos- sible to develop a live vaccine against human schisto- somiasis incorporating male bovine cercariae if it could be demonstrated that this type of vaccine would be non-pathogenic. Another possibility would be to exploit this phenomenon in bovine schistoso- miasis (Nelson et al., 1968) by vaccinating domestic animals with male cercariae of a human species of schistosome. R,1SUM1 ETUDE DE L'IMMUNITE, HETEROLOGUE DANS LA SCHISTOSOMIASE: 3. NOUVELLES OBSERVATIONS RELATIVES A L'IMMUNITE HETEROLOGUE CHEZ LA SOURIS Au cours de precedentes recherches portant sur l'immu- nit6 hterologue chez la souris et le singe rhesus, on a montre que l'exposition prealable des animaux d'expe- rience a des cercaires de Schistosoma bovis, S. mattheei ou S. rodhaini attenuait la gravite d'une infection ulterieure par S. mansoni. Les experiences actuelles ont eu pour but de rechercher si une infection initiale par S. mattheei est capable de proteger la souris contre une infection letale par S. mansoni et de mettre en lumiere les facteurs qui influent sur l'acquisition de l'immunite heterologue. Une exposition unique a 50 cercaires de S. mattheei confere a la souris un degre eleve d'immunite contre une infection d'epreuve par S. mansoni. On note une reduction significative de la charge parasitaire et du nombre d'ceufs presents dans les tissus et les souris traitees survivent a des infections qui tuent les animaux temoins non immunises. L'intervalle separant l'exposi- tion immunisante de l'exposition d'epreuve joue un r6le important dans l'acquisition de l'immunitd. Aucune protection n'est observee lorsque l'infection d'epreuve est pratiqu6e apres un intervalle de 0, 1 ou 3 semaines; une nette immunit6 se manifeste apres 6 semaines, et l'immunite est quasi absolue apres 9, 15 ou 30 semaines. L'importance de la dose immunisante apparait si l'on considere que l'inoculation d'une dose de 10 cercaires de S. mattheei ne provoque qu'une faible reponse immu- nitaire, alors qu'apres administration d'une dose de 20 a 50 cercaires, la souris bendficie d'une immunite solide ou meme pratiquement absolue. La voie utilisee pour l'administration de la dose immunisante doit egalement retenir l'attention. La voie percutanee permet d'obtenir une immunite plus forte que la voie intraperitoneale. Par ailleurs, en cas d'utilisation de la voie percutanee, les resultats sont meilleurs si l'on recourt a une exposition unique plut6t qu'a une serie d'expositions successives, la dose totale de cercaires restant la meme. Enfin, l'exposition de la souris a des cercaires males de S. mat- theei assure a l'animal une meilleure protection que l'immunisation par des cercaires femelles. Cette derniere observation montre clairement qu'un degre significatif d'immunite het6rologue peut etre obtenu en l'absence d'oeufs, ce qui permet d'entrevoir la possibilite de pre- parer un vaccin destine it la prophylaxie de la schisto- somiase humaine ou animale. 231 232 M. A. AMIN & G. S. NELSON REFERENCES Amin, M. A., Nelson, G. S. & Saoud, M. F. A. (1968) Bull. Wld Hlth Org., 38, 19-27 Hsu, H. F., Hsu, S. Y. Li & Osborne, J. W. (1965) Nature (Lond.), 206, 1338-1340 Hsii, S. Y. Li & Hsu, H. F. (1963) Z. Tropenmed. Parasit., 14, 506-512 Hunter, G. W., Grandall, R. B., Zickafoose, D. E. & Purvis, Q. B. (1962) Amer. J. trop. Med. Hyg., 11, 17-24 Kagan, I. G. (1952) J. infect. Dis., 91, 147-158 Lin, S. S., Ritchie, L. S. & Hunter, G. W. (1954) J. Para- sit., 40, 42 Nelson, G. S., Amin, M. A., Saoud, M. F. A. & Tees- dale, C. (1968) Bull. Wld Hlth Org., 38, 9-17 Nelson, G. S. & Saoud, M. F. A. (1966) Trans. roy. Soc. trop. Med. Hyg., 60, 429-430 Nelson, G. 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