January 2007 Vol 7 correct A.indd ABSTRACT ObjectiveS: (a) To determine the natural infection rate of Bulinus truncatus and Biomphalaria pfeifferi snails with trematodes’ cercariae. (b) To determine the emergence and rhythmicity of cercariae. (c) To elucidate the high-risk time for man and other animals to acquire infection. Methods: Snails were collected from Dawar El Mahadi Agricultural Scheme, Khartoum State, identified in the laboratory, kept at room temperature and fed on lettuce. The snails were screened weekly for six weeks for natural infection and infected snails were kept in the dark. The swimming patterns and resting position of the freshly emerged cercariae were studied using a stereomicroscope. The rhythmicity of the different types of cercariae was studied by screening three sets of 5 naturally infected snails under fluorescent light from 07.00 to 9.00 and similar sets from 9.00 to 07.00. Results: Out of ,257 screened Buli- nus truncatus, 87 (4.9%) shed four types of cercariae. The highest prevalence of natural infection (9.5%) was by schistosome cercariae followed by amphistome (2.5%), xiphidiocercariae (2.4%) and lastly by avian cercariae (0.5%). However, out of 200 screened B. pfeifferi, 22 (%) shed only xiphidiocercariae. The rhythmicity studies showed that the emergence of schistosome cercariae increased steadily from 07.00 to reach its peak at .00-3.00. The emergence rhythms of avian cercariae are similar to those of the schistosome, but with an early peak at 09.00-.00. The xiphidiocercariae and amphistome cercariae started with high rate of emergence at 07.00. and decreased gradually to very low levels or complete disappearance, respectively, around sunset. Conclusion: Information on cercarial rhythmicity and chronobiological characteristics are thought to be useful in avoiding water contact during high-risk time of infection and may be helpful in the identification of closely related species and strains of cercariae. Key words: Bulinus, Biomphalaria, prevalence, cercarial rhythms, schistosomes, amphistome, avian, xiphidiocercariae. Laboratory Studies on the Prevalence and Cercarial Rhythms of Trematodes from Bulinus truncatus and Biomphalaria Pfeifferi Snails from Khartoum State, Sudan Abdel Aziz M Ahmed1, Nidal A Ibrahim1, *Mohamed A Idris2 في املثقبة الديدان ذوانب (تواتر) نسبة ونظم لدراسة جتارب مختبرية ترنكيتص البولينص قواقع حتديد خروج . (ب) املثقبة الديدان بذوانب فايفري ــا وبيومفيالري ترنكيتص البوالينص قواقع في الطبيعية االصابة ــبة نس (أ)حتديد ــص: الهدف: امللخ املهدي دوار من القواقع ــات عين الطريقة: جمعت . املثقبة ــدان الدي لالصابة بذوانب خطورة ــر االكث الوقت حتديد املثقبة. (ج) ــدان الدي ــب ذوان ــم ونظ ، ــابيع اس ــتة ولفترة س ــبوعيا اس القواقع قمنا بفحص . باخلس البلهارزيا ومتت تغذيتها مبختبر القواقع ــظ وحف وتصنيف ــرز ف ومت ، ــوم ــة اخلرط بوالي بفحص ثالث اتلفة الذوانب نظم بدراسة قمنا م. َسِّ ُا باهر اتلفة الذوانب وجلوس سباحة دراسة أجرينا . في الظالم املصابة القواقع وحفظنا 7 مساء الى من الساعة فحصنا مجموعات متشابهه و كذلك ، 7 مساء الى 7 صباحا من الساعة ــية الفلورنس االضاءة حتت ــية خماس مجموعات البلهارزيا ذوانب وسجلت ، 1257 مجموع من الذوانب من أنواع بأربعة مصابة بوالينص ترنكيتص (14.9%) 187 ألنتائج: وجدنا . 7 صباحا الساعة 200 بايومفيالريا مجموع ومن .(5%) الطيور ذوانب وأخيرا (2.4%) ــركاريا ، والذيفيدوس (2.5%) ــتوم االمفيس ذوانب (%9.5)، تلتها ــبة نس أعلى 7 صباحا الساعة من اخلروج بدأت البلهارزيا ذوانب أن بينت نظم الذوانب ــة دراس . ــركاريا الذيفيدوس بذوانب فقط مصابة (11%) 22 ان وجد فايفري وصلت لكن ، البلهارزيا ذوانب لنظمية متشابهة كانت الطيور ذوانب نظمية أن كما 11صباحا الى الواحدة بعد الظهر. الساعة بني ما ذروتها لتصل تدريجيا تناقصت ثم 7 صباحا الساعة خروجها معدالت اعلي بدات ــتوم ــيركاريا واالمفيس ذوانب الذيفيدوس أما . 9-11 صباحا ــاعة الس بني ذروتها التعرض لعدم فائدة كبيرة ذو املثقبة الديدان سلوك ذوانب حول املعلومات اخلالصة: توفر . غروب الشمس مع بالتوالي توقفت متاما قليلة أو العداد في تصنيف تساعد قد والنظم اخلروج دراسة أن كما . الذوانب أثناء ذروة وجود االبتعاد عن املياه امللوثة طريق عن ذلك و البلهارزيا ، خاصة ، بها لالصابة . الذوانب لهذه املتشابهة واالنواع االجناس 1Schistosomiasis Research Laboratory, Faculty of Science, University of Khartoum, Sudan, 2Department of Microbiology & Immunology, College of Medicine & Health Sciences, Sultan Qaboos University, P.O. Box 35, Al Khod 123, Muscat, Sultanate of Oman *To whom correspondence should be addressed. Email: midris@squ.edu.om SULTAN QABOOS UNIVERSITY MEDICAL JOURNAL DECEMBER 2006 VOL 6, NO. 2 SULTAN QABOOS UNIVERSITY© O R I G I N A L S T U D Y A B D E L A Z I Z M A H M E D , N I D A L A I B R A H I M A N D M O H A M E D A I D R I S 66 SCHISTOSOMES ARE HELMINTHES OF THE class Trematoda that alternate generations, with a sexual phase in definitive mammalian hosts and an asexual phase in intermediate snail hosts. In humans, these blood flukes reside in the mesenter- ic and vesical venules. They have a life span of many years and daily produce large numbers of eggs, which must traverse the gut and bladder tissues on their way to the lumens of excretory organs. To understand the transmission dynamics of the disease, it is necessary to highlight essential segments in the process of ac- quiring an infection. Thus, knowledge of cercarial in- fection rates, not only in individual habitats and the area as a whole, but also in relation to seasonal popu- lation changes, is crucial.1,2 Other segments like the susceptibility of the snail to infection and the effects of such infection, the pattern and number of cercarial production and the factors influencing cercariae are of paramount importance in aspects related to the trans- mission of schistosomiasis. As relatively few studies were conducted on the production and dynamics of trematodes cercariae in Africa, the main objectives of this study were to de- termine the prevalence, highlight the cercarial emer- gence of trematodes from naturally infected snails and to elucidate the high risk time for man and other ani- mals to acquire infection. M E T H O D S SN A I L C O L L E C TI O N Four field surveys were conducted for systematic snail sampling. A standardized scoop was used for sampling some minor canals in Dawar El Mahadi agricultural scheme, Khartoum State. In each field survey, the col- lected snails were pooled in plastic bowls and lined by grass cover from the canals. The collected snails were transported to the Schistosomiasis Research Labora- tory, University of Khartoum, where different snail species were isolated, cleaned and a series of labora- tory experiments were conducted. PR E VA L E N C E O F N AT UR A L I N FE C TI O N The collected snails were screened weekly, for six weeks, for the presence of trematodes natural infec- tion. Patches of (20-30) cleaned snails were put in small glass containers containing about 20 ml of warm water (30-38°C) and placed under artificial light from 10:00 to 13:00 to enable cercarial emergence. The glass containers showing cercariae were identified and the snails were screened individually for cercarial shed- ding. Snails (Bulinus and Biomphalaria) observed to shed any trematode cercariae were placed in separate plastic bowls containing 5 liters of dechlorinated water and kept in the dark at room temperature (30-35°C). O B SE RVATI O N O F L I V I N G UN STA I N E D C E R C A R I A E Immediately after cercarial shedding the snails that liberated the same type of cercaria were placed in the same plastic bowls. The different freshly emerged cer- carial specimens were placed, separately, in a water volume sufficient to allow their normal behavioural pattern. The swimming patterns and resting position of the cercariae were observed using a stereomicro- scope of 8-40 times of magnification. To observe the cercarial developmental pattern, a piece of cellophane was introduced to enable cercarial encystation on ex- ternal substrate (if any). The identification of cercariae to the major type level was carried out by transfer- ring living and unstained cercariae onto a glass slide. A cover slip was added and excess water was drawn off by applying a strip of filter paper to one side of the cover slip. STA I N I N G A N D I D E N TI FI C ATI O N O F C E R C A R I A E The different types of cercariae were collected from naturally infected snails by emergence then prepared in permanent slides using Ehrlich’s Haematoxylin and were identified following Frandsen and Christensen.3 It was observed that the morphological features in the stained cercariae are more easily observable than in unstained ones. The diagnostic characteristics used in the identification of the cercariae included: general ap- pearance, tegument, body suckers, alimentary system, excretory system, gland cells, genital primordium and cercarial tail. Four types of cercariae (human schisto- some, xiphidiocercaria, amphistome and avian) were identified. C E R C A R I A L E ME R GE N C E A N D RY TH MI C I T Y Of each cercarial type, three sets of five naturally in- fected snails were placed in small beakers each with 15 ml of warm water (34-40°C) and placed under fluores- cent light from 07:00 to 19:00. The water with cercar- iae was changed in concurrent sets every two hours. Three samples, 50 micro litres each, of the cercarial suspension in the beaker, were taken by a Finn pipette into small Petri dishes. Drops of iodine were added to kill and stain the cercariae, which were then counted L A B O R AT O R Y S T U D I E S O N T H E P R E VA L E N C E A N D C E R C A R I A L R H Y T H M S O F T R E M AT O D E S F R O M B U L I N U S TR U N C AT U S A N D B I O M P H A - L A R I A P F E I F F E R I S N A I L S F R O M K H A R T O U M S TAT E , S U D A N 67 under a dissecting microscope. Averages of the cer- carial numbers in the 3 samples were calculated. Then the total numbers of cercariae in the suspension were calculated as follow The mean number of cercariae in 50 microlitres X 20 X volume of suspension in millilitres. Other similar sets, replications and calculations were carried for cer- carial emergence from 19:00 to 07:00. STATI STI C A L A N A LY SI S Based on the distribution of the observation, paired t-test and Wilcoxon matched pairs, signed ranks tests were used to determine the level of significance in the differences of related samples of paired observations. In addition, all normally distributed related samples of more than two observations were treated with Fried- man twoways ANOVA. R E S U L T S PR E VA L E N C E O F N AT UR A L I N FE C TI O N Out of 1,257 screened B. truncatus snails, 187 (14.9%) shed four different types of cercariae. The highest prevalence of natural infection was shown by schisto- some cercariae (9.5%) followed by amphistome (2.5%), xiphidiocercariae (2.4%) and lastly by avian cercariae (0.5%). The observed differences in the snails’ infec- tion rates were found to be statistically significant (p < 0.05). Out of 200 B. pfeifferi screened snails, 22 (11%) liberated only xiphidiocercariae. C E R C A R I A L PR O D U C TI O N Table 1 shows the means and the ranges of cercarial production from naturally infected B. truncatus snails. The number of xiphidiocercariae liberated daily was more than 62% of the total cercariae. At the very com- mencement point, xiphidiocercariae were more than twice the total number of other emerging cercariae, as shown in Table 2. Amphistome cercariae were the least observed in both aforesaid parameters. Statisti- cal analysis revealed high significant differences (P < 0.001) in the rate of different cercarial emergence. Fur- ther analysis, via Scheffe test, reassured that such dif- ferences were mainly attributed to xiphidiocercariae. I N N ATE R H Y TH M S Tables 2 summarises the patterns of innate diurnal rhythms of the four types of cercariae, liberated from naturally infected B. truncatus. The individual types of cercariae showed a remarkable constant daily perio- dicity, though day-to-day irregularity in numbers of cercariae shed was observed. In the naturally infected snails, cercariae emergence began at sunrise, around 07:00. In schistosome cercariae, the emergence in- creased to reach a peak at 11:00-13:00 and gradually decreased to disappear around sunset at 19:00. The avian and xiphidiocercariae were like the schistosome in the general pattern, but with an early peak at 09:00- 11:00. However, avian cercariae showed substantial nocturnal emergence. On the other hand, the amphis- tome cercariae started from the highest rate of emer- gence at sunrise to sharp decrease at early afternoon and to complete disappearance around sunset. D I S C U S S I O N The full understanding of the transmission pressure of a trematode infection in a given area must include knowledge of cercarial production and dynamics. The present study emphasized that the local strain of B. truncatus is susceptible to the local S. haematobium strain. Also, to the best of our knowledge a single study was conducted more than 20 years ago in North Gezira, Sudan and gave 3.3% patent infection of B. truncatus by S. haematobium4 which is much lower than the 9.5% reported in this study. The reported nat- ural infection (0.2%) of B. pfeifferi snails by S. mansoni in the Sudan was by far less,5,6 but comparable to the prevalence of natural infection (7.9%),7 in B. arabica, a strain of B. pfeifferi,8 from the Sultanate of Oman. Cercarial emergence is the final product of many biotic and abiotic factors.9 Productivity also depends on the host-parasite compatibility.10 If a strain of snail is very receptive, the parasitic process will be longer and the cercarial productivity greater.11,12 The maxi- mum mortality rate was observed among snails with high natural infection, immediately after the first trial for cercarial shedding. The intensity of the snails’ nat- ural infection was expressed in their multiple infec- tions as well as the high frequent shedding of cercari- ae. Such a mortality rate was expected, since infected snails are comparatively more sensitive to environ- Table 1: Means and ranges of cercarial emer- gence from naturally infected B. truncatus (snail/day) Type of cercariae Means and ranges Schistosome cercariae 135 (53 - 252) Xiphidiocercariae 827 (198 - 2430) Amphistome cercariae 82 (14 - 106) Avian cercariae 285 (274 - 291) A B D E L A Z I Z M A H M E D , N I D A L A I B R A H I M A N D M O H A M E D A I D R I S 68 mental changes than non-infected snails. It seems that the temperature and the water quality are the factors behind the increased death rates among the naturally infected snails.12 The longevity of infected snails in the field is determined by different factors such as: age of the snail, super-infection and effect of schistosome miracidia and other parasites. However, the lifespan of the naturally infected snails could not be elucidated in the field, since the date on which the snails became infected was not known.9 Studies on trematode emergence in freshwater systems suggest that the larval emergence from snails varies according to the circadian cycle and is optimized to enhance the probability of successful transmission.13-17 The present study proved that the cercarial shedding pattern of S. haematobium, although it peaked early (11:00-13:00), does not differ from those reported by other workers. Cercariae of S. mansoni in the Gunaid scheme peaked 13:00-15:00,3,9 while the same species in the Gezira scheme peaked 12:00-14:00.1 It seems that the peak of the S. haematobium cercarial rhythm coincides with the peak of human water-contact activ- ities during the day. The cercariae aves are like schisto- somes in this manner, but with an earlier peak, 09:00- 11:00, moving to a complete disappearance around dusk time. The cercarial emergence rhythm is closely corre- lated with periods of activity of the most permissive host. Human schistosomes have a diurnal emergence rhythm. S. rodhaini, which infects wild rodents, has a nocturnal emergence. The ultradian emergence rhythm of S. margrebowiei, which peaks at dawn and dusk, is thus perfectly adapted to antelopes com- ing to water pools early in the morning and late in the evening. This variability of emergence rhythms, documented at the interspecific level, was recently observed at intraspecific level by the discovery of chronobiological polymorphism in populations of S. mansoni from a single endemic area.18 In Sudan, the schistosome cercarial emergence peaks vary over an interval of twelve hours from one scheme to another, between 6:00 to 18:00, depending on the Schistosoma population considered.1 Findings on cercarial emergence rhythmicity might reveal micro-evolutionary shifts taking place in schistosome populations. Obviously, data on cer- carial chronobiological characteristics may well prove of value for the identification of closely related species and strains.8 Thus, thorough systematic observations are crucial to elucidate these characteristics. Further- more, such studies might explain the role of different factors affecting the cercarial rhythmicity and produc- tion dynamics, especially in the field. A C K N OW L E D GE ME N TS We would like to thank with great appreciation Pro- fessor E. B. Taha, Minster of Science and Technology, for a generous financial support for the Schistomiasis Research Laboratory. We are also grateful to the labo- ratory staff for their cooperation. R E F E R E N C E S 1. Ahmed AA. Micro epidemiological factors influenc- ing transmission pressure of schistosomiasis in Gunaid Scheme, Sudan. J Natl Sci 2002; 2:22-32. 2. Ahmed AA. Schistosomiasis in sugar cane schemes, Su- dan. J Natl Sci 2003; 3:9-17. 3. Frandsen F, Christensen NO. An introductory guide to the identification of cercariae from African freshwater snails with special reference to cercariae of trematode species of medical and veterinary importance. Acta Tropica 1984; 41:181-202. 4. Babiker SM, Blankespoor HD, Wassila M, Fenwick A, Daffalla AA. Transmission of Schistosoma haemato- Table 2: Emergence rhythms of cercariae from naturally infected B. truncatus Time of day(Hours) Number and types of cercariae schistosome cercariae Xiphidio-cercariae Amphistome cercariae Avian cercariae 07:00 - 09:00 140 ± 24 1430 ± 51 264 ± 33 293 ± 29 09:00 - 11:00 160 ± 9 1849 ± 108 118 ± 26 570 ± 32 11:00 - 01:00 220 ± 43 421 ± 84 21 ± 3 201 ± 26 13:00 - 15:00 140 ± 11 236 ± 47 6 ± 2 163 ± 9 15:00 - 17:00 12 ± 3 115 ± 23 2 ± 0.3 110 ± 6 17:00 - 19:00 2 ± 0.5 86 ± 17 0.0 90 ± 8 19:00 - 07:00 60 ± 3 2.0 ± 0.3 230 ±24 2480 ± 92 L A B O R AT O R Y S T U D I E S O N T H E P R E VA L E N C E A N D C E R C A R I A L R H Y T H M S O F T R E M AT O D E S F R O M B U L I N U S TR U N C AT U S A N D B I O M P H A - L A R I A P F E I F F E R I S N A I L S F R O M K H A R T O U M S TAT E , S U D A N 69 bium in North Gezira, Sudan. J Trop Med Hyg 1985; 88 :65-73. 5. Ahmed AA, Babiker A, El Tash LA, Shiff CJ. Develop- ment of a modified baited trap for detection of schis- tosome cercariae in Sudanese irrigation canal systems. Acta Tropica 2002; 82:363-368. 6. Hilali AH. Transmission of Schistosoma mansoni in the Managil area, Sudan. Ph.D. thesis, Department of Zool- ogy, Faculty of Science, University of Khartoum, 1992 7. Mone H, Mouahid G, Shaban MA, et al. Ecological and molecular studies on emerging schistosomiasis man- soni in Dhofar Governorate, Sultanate of Oman. Trop Med Int Health 2003; 8:269-276. 8. DeJong RJ, Morgan JA T, Wilson WD, et al. Phylogeog- raphy of Biomphalaria glabrata and B. pfeifferi, impor- tant intermediate hosts of Schistosoma mansoni in the New and Old World tropics. J Molec Eco 2003; 12:3041- 3056. 9. Ahmed AA. Epidemiological observations of intesti- nal Schistosomiasis in the Gunaid Sugar Cane Scheme, Central state, Sudan. Ph.D thesis, 1998, Department of Zoology, Faculty of Science, University of Khartoum. 10. Niemann GM, Lewis F A. Schistosoma mansoni: Influ- ence of Biomphalaria glabrata size on susceptibility to infection and resultant cercarial production. Exp Para- sitol 1990; 70:286-292. 11. Barbosa FS. Survival and cercariae production of Bil- harzian Biomphalaria glabrata and B. straminea in- fected with Schistosoma mansoni. J Parasitol 1975; 61: 151-152. 12. Mouahid A, Coombes C. Genetic variability of Schisto- soma bovis cercarial production according to miracidial dose. J Helminth 1987; 61:89-94. 13. Pages J R, Theron A. Analysis and comparison of cercar- ial emergence rhythms of Schistosoma haematobium, Schistosoma intercalatum, Schistosoma bovis, and their hybrid progeny. Int J Parasitol 1990; 20:193-198. 14. Combes C, Fournier A, Mone, H, Theron A. Behaviors in trematode cercariae that enhance parasite transmis- sion: patterns and processes. Parasitol 1994; 109:S3- S13. 15. Pechenik JA, Fried B. Effect of temperature on survival and infectivity of Echinostoma trivolvis cercariae: a test of the energy limitation hypothesis. Parasitol 1995; 111: 373-378. 16. N’Goran E, Bremond P, Sellin E. Intraspecific diversity of Schistosoma haematobium in West Africa: chrono- biology of cercarial emergence. Acta Tropica 1997; 66: 35-44. 17. Fingerut TJ, Zimmer AC, Zimmer KR. Patterns and processes of larval mergence in an Estuarine Parasite System. Biol Bull 2003; 205:110-120. 18. Shiff JC. Population genetics of Schistosoma haemato- bium. Measurement of parasite diversity using RAPD- PCR. Exp Parasitol 2000; 96:47-51.