Susceptibility of the snail Biomphalaria alexandrina alexandrina from the UAR and the Sudan to infection with a strain of Schistosoma mansoni from Tanzania*

Bull. Org. mond. San 1970, 43, 809-815 Bull Wid Hith Org. Susceptibility of the Snail Biomphalaria alexandrina alexandrina from the UAR and the Sudan to Infection with a Strain of Schistosoma mansoni from Tanzania* C. C. CRIDLAND 1 A series of laboratory experiments was carried out to determine the susceptibility ofBiomphalaria alexandrina alexandrina, the snail vector ofschistosomiasis in the United Arab Republic and the Sudan, to a strain ofSchistosoma mansonifrom Mwanza, Tanzania. The objective ofthe investigation was to determine whether or not B. alexandrina is refrac- tory orpartially refractory to infection with strains of S. mansoni other than the Egyptian strain. In one series of S exposures, snails from a colony originating in Alexandria, UAR, showed a mean infection rate 4 times that observed when snails from the same colony were exposed to the local strain of S. mansoni. Several investigators have reported Biomphalaria alexandrina alexandrina (Ehrenberg) to be re- fractory or partially refractory to infection with all strains of Schistosoma mansoni Sambon other than the Egyptian strain. Over the past 2 decades work has been done on differences in the suscep- tibility of various intermediate hosts of S. mansoni to different strains of the parasite. Stunkard (1946), Cram, Files & Jones (1947), Files & Cram (1949) and Abdel-Malek (1950) found that Planorbis boissyi Potiez & Michaud (now known as Biom- phalaria alexandrina alexandrina) of Egyptian origin was refractory to the S. mansoni strain of the West Indies. Kikuth & Gonnert (1948) failed to infect B. alexandrina with S. mansoni from Liberia, and de Meillon (1956) reported unsuccessful attempts to infect B. boissyi with South African S. mansoni. However, Cowper (1947) succeeded in infecting B. alexandrina with S. mansoni from Uganda, and Le Roux (WHO, 1957) experimentally infected B. alexandrina with S. mansoni from Kenya and Nigeria. Saoud (1964 1965) experimentally infected B. alexandrina from Egypt with S. mansoni from * This investigation was supported by the Secretariat for Technical Co-.operation with Developing Countries, Ministry of Foreign Affairs, Denmark. 1 Parasitologist, Danish Bilharziasis Laboratory, Char- lottenlund, Denmark. Aden, and he also succeeded in infecting B. alexan- drina with a strain of S. mansoni that originated in Tanzania. All these workers who succeeded in infecting B. alexandrina with strains of S. mansoni other than the Egyptian strain achieved only a low degree of susceptibility. It therefore appeared desirable to undertake observations on the relative susceptibility of B. alexandrina from various parts of the UAR and from one location in the Sudan to a strain of S. mansoni that originated in Mwanza, Tanzania. MATERIALS AND METHODS Five of the B. alexandrina colonies that were studied originated in the UAR (Alexandria, Ismailiya, Suez, Qalyub and Abu Rawaash) and one originated in the Sudan (Jebel Aulia, 56km south ofKhartoum). Colonies of Biomphalaria pfeifferi pfeifferi (Krauss) and B. sudanica tanganyicensis (Smith) that origin- ated in Mwanza, Tanzania, were also studied. From these colonies a total of 1375 juvenile laboratory-bred snails 6-7mm in diameter (8-9 weeks old) were used in the infection experiments. They originated from adults that had not previously been used in any experiments. B. pfeifferi served as a control for the experimental procedure and for the viability of the miracidia. The Mwanza strain 2602 - 809- C. C. CRIDLAND TABLE 1 INFECTION RESULTS FOLLOWING THE EXPOSURE OF B. ALEXANDRINA FROM THE UAR TO A STRAIN OF S. MANSONI FROM TANZANIA No. of snails No. of snails Positive snails Incubation No. of cercariae exposed examined peNo.i shed per snail a No. % ~~~~at262t seprsnia Abu Rawaash colony 25 24 19 79.2 23-71 112 = +++ 25 24 18 75.0 23-82 70 - ++ 25 25 25 100.0 22-29 6 + 25 25 23 92.0 25-58 25 23 20 86.9 23-40 25 23 17 73.9 22-36 25 25 19 76.0 22-70 25 25 10 40.0 24-40 25 25 17 68.0 22-53 25 25 20 80.0 22-74 250 244 188 77.1 - Alexandria colony Suez colony lsmailiya colony Qalyub colony 25 25 25 25 25 25 25 25 25 25 19 18 16 15 12 13 19 19 14 12 0 0 1 0 0 1 0 0 5.3 0 0 6.7 0 0 0 5.3 0 0 0-26 0 0 0-25 0 0 0 0-32 0 0 1 - ++ a See text for explanation. 810 250 I 157 [ 3 [ 1.9 _- SUSCEPTIBILITY OF B. ALEXANDRINA TO "FOREIGN " S, MANSONI of S. mansoni was maintained by the use of B. pfeifferi from Mwanza as intermediate host. The faeces of albino mice that had been exposed in the laboratory to S. mansoni cercariae 3-4 months previously were used as a source of schistosome eggs. The method of snail infection was basically that of Standen (1949) and Cridland (1968). Only freshly hatched and active miracidia were used. Snails were individually exposed once to 6 miracidia in specimen tubes measuring 4 cm in diameter and containing 10 ml of water. One mass exposure was carried out using 25 B. alexandrina (Abu Rawaash) and 25 B. s. tanganyicensis (Mwanza); the snails were placed in the same container and 300 miracidia were added. In addition the same number of each of these species were mass-exposed to the same number of miracidia, but in separate containers. The duration of contact between snails and miracidia was 7 hours. B. pfeifferi from Tanzania were used as controls, each snail being exposed to 5 miracidia. Under standard conditions 10 exposures of 2 colonies and 5 exposures of 3 colonies from 5 different localities were performed, as were 10 exposures of a colony from one location in the Sudan. At the end of the experiments a simultaneous exposure was made with batches of 25 snails from each of the 5 Egyptian colonies. The water in the specimen tubes was maintained at the same temperature (26-28°CQ as the water from which the snails were taken prior to exposure. This temperature was maintained throughout the period of 90 days during which the snails were under observation. The positive snails from each batch were separated and placed in an aquarium for 14 days, after which they were examined individually for cercariae. The number of cercariae shed daily by each snail was graded as follows: under 30= +, 30-300 = ++, over300= +++. The mortality during the pre-patent isolation and during the period of observation was recorded. Snails that died after the 20th day of exposure were dissected and examined for sporocysts and cercariae. Only the snails shedding cercariae were counted as positive, sporocyst-development not being counted as infection. RESULTS The results, summarized in Tables 1 and 2, revealed that the Abu Rawaash colony was highly susceptible, with a mean infection rate of 77.1 %. TABLE 2 INFECTION RESULTS FOLLOWING THE EXPOSURE OF B. ALEXANDRINA FROM THE SUDAN (KHARTOUM COLONY) TO A STRAIN OF S. MANSONI FROM TANZANIA No. of snails No. of snails Positive snails Incubation No. of cercariae exposed examined period (day shed per snaila 'C 25 24 14 58.3 25-90 25 25 8 32.0 23-43 25 25 6 24.0 23-53 25= +++ 25 24 13 54.2 22-81 25 20 6 30.0 24-69 31 = ++ 25 21 11 52.4 23-55 25 19 9 47.4 25-31 36 = + 25 18 6 33.3 26-33 25 22 12 54.5 25-77 25 25 7 28.0 25-45 250 223 92 41.3 - a See text for explanation. 81 C. C. CRIDLAND The number of cercariae shed was also high, 59.6% of the snails being graded as +++ and only 3.2 % as +. Of the 4 remaining colonies those from Suez and Alexandria showed a low to moderate degree of susceptibility, with mean infection rates of 21.6% and 34.6%, respectively. The numbers of cercariae shed in these 2 colonies were markedly different, and the mortality rate was high to mode- rate (25.6%) in the Suez colony and comparatively low (14.4%) in the Alexandria colony. Snails from Qalyub and Ismailiya showed low susceptibility, and none of the snails in these colonies shed suffi- cient cercariae to be graded +++. The morta- lity was high-37.2 % and 39.2%, respectively. Only I snail in each of 3 batches from Qalyub shed cercariae, and the highest infection rate obtained was 6.7%. Retarded infection was com- mon in the refractory snails, and 9% of them were found to harbour daughter sporocysts. In the 4 negative experiments on the Ismailiya colony there was a retarded infection rate of 5 %. The Khartoum colony showed a moderate in- fectivity rate, and the mortality was low, only 10.8% of the snails dying during the experiments. No retarded infection was found. The results of the simultaneous experiment (Table 3) followed the pattern of those listed in Tables 1 and 2, although the infection rate was be- low average. The simultaneous mass exposure of B. alexandrina and B. s. tanganyicensis gave infection rates of 25.0% and 55 %, respectively, and the separate mass exposure of these species the rates of 48.0% and 60.0%, respectively. The mortality rate of snails during and after the development period of the parasite was lower among snail populations with higher infection rates. DISCUSSION From the results given in Tables 1 and 3 it is clear that under experimental conditions the Abu Rawaash snails were highly susceptible to infection with a strain of S. mansoni from Tanzania. Snails of this colony had the shortest pre-patent period (22 days); of the snails that shed, 61 % were observed during the first week and 9% during the second. In the batch showing a 100% infection rate, all the snails produced cercariae within 8 days. Con- sidering the heavy emergence of cercariae, the mortality was very low, only 6 snails out of 250 (2.4%) dying during the experiments. The infection persisted over a long period, during which 112 snails (59.6 %.) shed 300-1000 cercariae daily. Only 6 snails (3.2%) shed less than 30 cercariae per day. It is unusual to obtain such a high infection rate when exposing a local vector snail to a " foreign " strain of S. mansoni, especially with B. alexandrina from the UAR. If it had occurred in B. pfeifferi, which is a versatile vector serving as intermediate host for several, if not all, strains of S. mansoni in Africa and the Western hemisphere, this high infection rate would have been understandable. The mean infection rates for both the Qalyub and the Ismailiya colonies were low, the Qalyub colony showing the lowest infectivity of all groups studied: only 3 snails out of 157 examined became infected, giving a mean infection rate of 1.9% TABLE 3 RESULTS OF SIMULTANEOUS EXPOSURE OF BATCHES FROM 5 DIFFERENT COLONIES OF B. ALEXANDRINA FROM THE UAR TO A STRAIN OF S. MANSONI FROM TANZANIA Colony No. of snails No. of snails __Positive snails | period(dnayso) exposedexamined No. at 26-28'C Abu Rawaash 25 25 20 80.0 23-27 Alexandria 25 22 5 22.7 28-41 Suez 25 16 3 18.8 28-44 lsmailiya 25 16 1 6.3 0-42 Qalyub 25 16 0 0.0 0 Controls (B. pfeffleri from Tanzania) 25 23 22 95.7 22-24 812 SUSCEPTIBILITY OF B. ALEXANDRINA TO " FOREIGN " S. MANSONI (or 5.8% if the negative experiments are discounted). In contrast to the highly susceptible snails from Abu Rawaash, the Qalyub snails proved refractory in 7 of 10 experiments. Previous infection experi- ments with snails from Qalyub had shown them to be highly susceptible to infection with S. mansoni from the UAR (Cridland, 1968). This is of interest since the rate of infection of Abu Rawaash snails with the Tanzanian S. mansoni (77.1 %) approached their rate of infection with S. mansoni from the UAR (85 O). Only 1 batch out of 5 in the Ismailiya colony shed cercariae, and the infection rate obtained was 17.6%-an unusual result, since the miracidia used in the 4 negative exposures gave positive results with other colonies of snails exposed on the same day, and since the control snail B. pfeifferi was highly susceptible to the same miracidia. The infection rate of 17.6% was nearly as great as that obtained when snails from this colony were exposed to S. mansoni from the UAR (18.2 Y.). The number of cercariae shed by the Qalyub and Is- mailiya colonies was small; no positive snails shed more than 300 per day, and most shed less than 30. The results obtained with the Alexandria snails were unexpected. They showed a moderate degree of susceptibility, with a mean infection rate of 34.6%. In 1 experiment the infection rate was as high as 57.1 %, and no refractory tendency occurred in this colony. Cridland (1968) exposed 300 snails from this colony to S. mansoni from the UAR, and in 12 different exposures achieved the low mean infection rate of 8.8 %, the highest rate being 12.5 %. These results clearly demonstrate that under ex- perimental conditions the snails from Alexandria were more susceptible to the Tanzanian strain of S. mansoni than to S. mansoni from the UAR. This finding does not agree with those of Files (1951), who states that " The compatibility of host and parasite from a given endemic area is to be expected since the life cycle is maintained in that area ". However, Chin-Tsong Lo (1970), after exposing species of the genus Bulinus to a strain of Schistosoma haemato- bium (Bilharz) from the UAR, noted that snail- parasite specificity is not a necessary development 'in a given area, and that snails from the same area as the parasites do not always show greater compa- tibility with them than do snails from different areas. It is well known that Bulinus bulinus truncatus (Audouin) exhibits this phenomenon. The author has on several occasions, working with S. haema- tobium from the UAR, obtained an infection rate of 56.0% in B. truncatus from Iraq, but only the low rates of 8% and 8.3%, in B. truncatus from Giza and Faium, UAR, respectively. The Suez snails showed the lowest cercarial output. The emergence of cercariae was slow and only 60% of the infected snails were shedding 2 weeks after the emergence of cercariae began. Although the Khartoum colony (Table 2) had a short pre-patent period (24 days), it had a longer incubation period than the other snails tested, and in 1 batch the last cercaria emerged 90 days after exposure. The author has not, unfortunately, been able to test B. alexandrina from Khartoum with the local strain of S. mansoni, but on 3 occasions he has experimentally infected this species with S. mansoni from the UAR, obtaining a mean infection rate of 37.6%, slightly lower than the mean rate of 41.3% achieved with the Tanzanian S. mansoni. In the mass exposure of B. alexandrina from Abu Rawaash and of B. s. tanganyicensis, the infection rate in the former was approximately half the rate in the latter, showing that the miracidia were more at- tracted to their local snail host than to the "foreign" B. alexandrina. No mortality occurred in the latter species, whereas B. s. tanganyicensis showed a fairly high mortality, again leading to the conclusion that the local snails had a greater attraction for the miracidia. The results of the separate mass exposures showed a similar pattern, although under these conditions the infection rate in B. alexandrina was approxi- mately twice the rate found when it was exposed together with B. s. tanganyicensis. The latter showed only a slight difference in infection rate when ex- posed separately from and together with B. alex- andrina. The mortality rate was low in both species. Saoud (1965) experimentally infected B. s. tanganyicensis from Mwanza to the local strain of S. mansoni and obtained an infection rate of 61.59%. This compares well with the results obtained in the present investigation. The results of the present study showed striking differences in the susceptibility of the different snail colonies to the Tanzanian strain of S. mansoni. It can be concluded that under experimental condi- tions B. alexandrina from the UAR was highly susceptible in only 1 colony and was less susceptible, or partially refractory, in other colonies. Attempts were also made to infect B. alexandrina from Abu Rawaash and from 5 other Egyptian colonies with strains of S. mansoni from Liberia, 4 813 C. C. CRIDLAND the Democratic Republic of the Congo, St Lucia (West Indies), and Madagascar, using 3 batches of 25 snails for each of the 4 strains of S. mansoni, but with negative results. The author (Cridland, 1968) succeeded in infecting B. alexandrina from the UAR with a Liberian strain of S. mansoni, obtaining the low infection rate of 0.7%, and from Abu Rawaash with a strain from western Uganda, obtaining the low infection rate of 1.3 %; the numbers of snails were 150 and 75, respectively. Negative results were not obtained when the snail colony from Khartoum was exposed to S. mansoni from the Democratic Republic of the Congo. In a series of 4 exposures to batches of 25 snails a mean infection rate of 7.3% was achieved. Snails from Khartoum also proved susceptible to the Egyptian S. mansoni with an infection rate of 40%, but proved refractory to S. mansoni from the West Indies. Successful experimental infection in the laboratory does not necessarily correspond to epidemiological facts, and likewise negative results do not prove that a similar occurrence would take place in nature, hence the important need for parallel field investi- gations. Wright (1967) has stated that the " rela- tionship between larval schistosomes and their snail hosts is extremely delicate and, in many cases, is strain-specific so that development of the next larval stages will not occur even in an unusual strain of the normal host species ". The relationship between snails and parasites from different endemic areas calls for additional explanation. The author has no adequate explanation to offer and is in complete agreement with Wright. ACKNOWLEDGEMENTS My grateful thanks are due to Dr G. MandahlmBarth, Danish Bilharziasis Laboratory, Charlottenlund, Denmark, who criticized the manuscript. RtSUMI8 RECEPTIVITE DU MOLLUSQUE BIOMPHALARIA ALEXANDRINA ALEXANDRINA ORIGINAIRE DE LA REPUBLIQUE ARABE UNIE ET DU SOUDAN A L'INFECTION PAR UNE SOUCHE TANZANIENNE DE SCHISTOSOMA MANSONI On a etudi6 au laboratoire la receptivit6 de six colonies de Biomphalaria alexandrina originaires de la Republique arabe unie (5 souches) et du Soudan (1 souche) a l'6gard d'une souche de Schistosoma mansoni en provenance de Mwanza (Tanzanie). Cinquante experiences ont ete effectu6es, chacune utilisant 25 mollusques exposes indi- viduellement a 6 miracidiums. La souche d'Abu Rawaash s'est revelee la plus recep- tive, avec un taux moyen d'infection de 77,0% et une 6mission cercarienne sup6rieure i 300 unit6s par jour chez 59,6% des sp6cimens. La mortalit6 a ete de 2,4% seule- ment. Chez les mollusques originaires de Qalyub et d'Ismallia, les taux moyens d'infection n'ont atteint que 1,9 et 3,9% respectivement. Les 6missions de cercaires ont 6t6 peu intenses et la mortalite s'est 6tablie a 37,2 et 39,2% respectivement. Les souches de Suez et d'Alexan- drie ont fait preuve d'une r6ceptivit6 interm6diaire, avec des taux moyens d'infection de 21,5 et 34,6%. Les mol- lusques d'Alexandrie ont emis sept fois plus de cercaires que leurs cong6neres de Suez. La mortalit6 a ete de 25,6% pour la souche de Suez et de 14,4% pour la souche d'Alexandrie. Le taux d'infection de 34,6% observ6 chez B. alexan- drina originaire d'Alexandrie apres contact avec la souche tanzanienne de S. mansoni est inattendu, ce meme mol- lusque, expos6 a la souche locale du schistosome, n'ayant contracte l'infection que dans la proportion de 8,8% en moyenne. B. alexandrina de Khartoum (Soudan) a present6 un taux moyen d'infection de 41,3% et une mortalit6 de 10,8%. L'exposition simultan6e des cinq souches egyptiennes de B. alexandrina a la souche tanzanienne de S. mansoni a donne des resultats concordant, dans l'ensemble, avec ceux des experiences isolees; les taux d'infection 6taient cependant legerement inferieurs. Exposes ensemble a l'infection par 300 miracidiums de S. mansoni de Tanza- nie, 25 B. alexandrina et 25 B. tanganyiensis ont presente des taux d'infection de 25 et 55% respectivement. Expo- sees isolement, les deux especes ont ete infect6es dans la- proportion de 48 et 60 . Ces donn6es font ressortir les fortes variations de la receptivite de diverses souches de B. alexandrina a l'infec- tion par la souche tanzanienne de S. mansoni. Dans les conditions exp6rimentales decrites, une seule souche s'est montree tres r6ceptive, les autres etant moins sen- sibles ou partiellement refractaires a l'infection. 814 SUSCEPTIBILITY OF B. ALEXANDRINA TO "FOREIGN S. MANSONI 815 REFERENCES Abdel-Malek, E. (1950) Amer. J. trop. Med., 6, 887 Chin-Tsong Lo (1970) Malacological Review, 2, 135 Cowper, S. G. (1947) Ann. trop. Med. Parasit., 41, 173 Cram, E. B., Files, V. S. & Jones, M. F. (1947) Nat. Inst. Hlth Bull., 189, 81 Cridland, C. C. (1968) Bull. Wld Hlth Org., 39, 955 Files, V. S. (1951) Parasitology, 41, 264 Files, V. S. & Cram, E. B. (1949) J. Parasit. 35, 555 Kikuth, W. & G6nnert, R. (1948) Ann. trop. Med. Parasit., 42, 256 Meillon, B. de (1956) Med. Klin., 51, 670 Saoud, M. F. A. (1964) Trans. roy. Soc. trop. Med. Hyg., 58, 288 Saoud, M. F. A. (1965) J. Helminth, 4, 403 Standen, 0. D. (1949) Ann. trop. Med. Parasit., 43, 268 Stunkard, H. W. (1946) J. Parasit., 32, 539 WHO (1957) Wld Hlth Org. tech. Rep. Ser., No. 139 Wright, C. A. (1967) The schistosome life-cycle. In: Mostofi, F. K. (ed.), Bilharziasis. Berlin, Springer, pp. 3-7