Evaluation of an overlay technique for the recovery of Schistosoma mansoni cercariae

AN OVERLAY TECHNIQUE FOR RECOVERING SCHISTOSOME CERCARIAE 125 Evaluation of an overlay technique for the recovery of Schistosoma mansoni cercariae DONALD G. SANDT 1 Abstract The consistent recovery of schistosome cercariae from turbid water may be an important observation in the study of schistosomiasis transmission. The phototactic behaviour of cercariae was used as the basis of a technique in which cercariae were induced to migrate into a layer of clear water overlying the turbid sample; the clear layer was then filtered and the cercariae counted. The recovery rate, however, does not appear to justify the use of this technique, at least in the field. The consistent recovery of schistosome cercariae under all conditions of water turbidity may be an important observation in the study of the transmis- sion of schistosomiasis. Pesigan et al. (1958) noted higher infection rates with Schistosoma japonicum in sentinel mice exposed in an infected stream after heavy rains, and Rowan (1965) suggested that tur- bidity may serve " as cover for cercariae against predation by fish". Several authors have investigated the recovery of cercariae in relation to turbidity. Rowan (1957) con- sidered 16 ppm of suspended material the maximum level for direct filtration with a Schleicher & Schull S & S 404 paper filter; at higher levels of turbidity, flocculation with aluminium sulfate and sodium bicarbonate could be used but some cercariae were lost. Sandt (1972a) using a SX40A-25 glass-silicone filter, extended this limit and found that particle size rather than total turbidity was the determining factor in the maximum volume of water that could be fil- tered by one filter and in the rate ofcercarial recovery. Barrett & Ellison (1965,) Butler et al. (1967), and Olivier (1966) attempted to eliminate the fine parti- cles that cause blockage of the filter by means of con- tinuous centrifugation. However, published data indicate that recoveries became erratic with increas- ing turbidity of the water and that cercariae were unaccountably lost. 1 Formerly Special Staff Member, The Rockefeller Foun- dation, on assignment to the Research and Control Depart- ment, St. Lucia (West Indies). Klock (1961) reported a high recovery accuracy (98 %), regardless of the level of turbidity, achieved with a phototactic method, but the apparatus proved to be cumbersome for use in the field and it was never tested extensively. In this communication, a modifica- tion of Klock's method is described, and data from laboratory tests are presented. Material and methods In order to verify Klock's finding that, after at least 15 min in the dark, cercariae exhibit a strong vertical migration when exposed to intense light, preliminary tests were carried out in a black plastic tube measuring 7.6 cm in height by 3.8 cm in diameter, fitted with a light-proof top with an air-vent to allow the removal of samples in the dark. From an undisturbed lighted pool containing S. mansoni cercariae, 50-ml samples were transferred to the tube and then withdrawn as two 25-ml aliquots after various lengths of time and under varying conditions: light, darkness, darkness followed by light, and light followed by agitation (filling and emptying the pipette). The two aliquots represented the bottom and top portions of the sample; the total distance from the bottom of the tube to the meniscus at 50 ml when the tube con- tained 50-ml of water was 5.7 cm. The numbers of cercariae in the two aliquots were determined by filtration and staining. Results Results of the preliminary tests (Fig. 1) indicated the response of cercariae to light and agitation. A minute after transfer to the apparatus under condi- tions of either light or darkness, 87% of the cercariae were found in the upper 25 ml; they then began to return to a near-equal distribution (Fig. 1; control). Assuming an even distribution at 0 time, 37% of the cercariae migrated from the bottom to the top of the tube in the first minute after initial stimulation. Agitation after 5 and 20 min in the light resulted in 75% recoveries 1 min later, and exposure to light after 5 and 20 min in the dark resulted in 87% re- coveries 1 min later. A combination of 5 min in the 2880B BRIEF COMMUNICATIONS Fig. 1. Results of preliminary tests on the response of S. mansoni cercariae to varying conditions of light, darkness, and agitation for various lengths of time. dark followed by both exposure to light and agitation (not shown in Fig. 1) also yielded 87% recoveries. Several overlay procedures were then tested in an attempt to recover cercariae from a shallow 2-layered water column that had a high-turbidity sample of water on the bottom and a clear layer of water on top. The apparatus illustrated in Fig. 2 was designed OVFTLams OVgAY UUP Fig. 2. The overlay chamber in cross-section. as a result of these tests. Cercariae in a 70-litre pool were mixed by means of air bubbles for 1 h prior to sampling in order to simulate field conditions; 7 litres of this clear sample water or of turbid (200 Jackson turbidity units) sample water were then pumped into the overlay chamber. The overlay chamber was a tray measuring 57.2 x 46.4 x 10.8 cm, constructed of polyethylene and polymethyl metha- crylate. The depth of water in the chamber was 3.2 cm. Simultaneously, clear water was pumped through the overlay input into a sealed compartment inside the chamber and up into 48 tubes so that it spread out over the sample water to a depth of 2.5 cm. This layer of water was then drawn off by gravity 1 min later and was filtered through a SX40A-25 filter. The entire operation tookapproxi- mately 4 min. Recovery of cercariae by the overlay technique was fairly consistent, regardless of turbidity, but was lower than that required for operations in the field. The recovery rate was 19.2 0.7% cercariae with clear sample water and 26.6 + 6.4% cercariae with turbid (200 Jackson turbidity units) sample water. In addition, a large number of detached tails (20.4 % calculated as a percentage of the total recovery in- cluding whole cercariae and tails) indicated that the cercariae were separating prior to filtration, since direct filtration of the sample water alone showed a breakage rate of only 5.9 %. Reduction of turbidity was, however, rapid and efficient-namely, 75% at 1 min of overlay time and 80-90% at slower rates. Since only the very fine particles from the turbid water sample mixed with the overlay water, it was very easy to examine the filters. The results of these tests were later confirmed by a different estimation method for cercariae (Sandt, 1972b). Discussion The low rate of recovery, in conjunction with the high rate of breakage, of cercariae does not seem to justify use of the overlay technique in preference to direct filtration and flocculation. Since the technique utilizes a behavioural response that depends on the physiological condition of the cercariae, it may, however, prove to be useful for some purpose not related to recovery of cercariae in the field. ACKNOWLEDGEMENTS This work was carried out with the support, and under the auspices, of the Research and Control Department, St Lucia (West Indies), and the Rockefefler Foundation. 90 LIGHT a)90 LIH AGITATION 80 Lo C.,' 60 40. E 0 50 1 23 5 6 10 LIGHT o90 DA ,,-80 0) 40) 70 60 50 Time (min ) WHO 20600 126 AN OVERLAY TECHNIQUE FOR RECOVERING SCHISTOSOME CERCARIAE 127 The author thanks Dr Peter Jordan, Director, and Dr Robert F. Sturrock, Research and Control Depart- ment, St Lucia, for their advice and helpful criticism, and Mr Anthony Aquille for technical assistance. REFERENCES Barrett, P. D. & Ellison, P. R. (1965) Cent. Afr. J. Med., 11, 338-340 Butler, J. M. et al. (1967) Publ. Hlth Rep. (Wash.), 82, 250-252 Klock, J. W. (1961) Bull. Wld Hith Org., 25, 738-740 Olivier, L. (1966) Amer. J. trop. Med. Hyg., 15, 882-885 Pesigan, T. P. et al. (1958) Bull. Wld Hith Org., 18, 481-578 Rowan, W. B. (1957) J. Parasit., 43, 696-697 Rowan, W. B. (1965) Bull. Wld Hlth Org., 33, 63-71 Sandt, D. G. (1972a) Bull. Wld Hlth Org., (in press) Sandt, D. G. (1972b) Bull. Wid Hith Org., (in press) The seroepidemiology of rubella in Kuala Lumpur, West Malaysia S. K. LAM 1 Abstract Rubella antibody rates in the female population of Kuala Lumpur were lower than those reported from temperate countries, though similar to rates found in other tropical countries excepting Sin- gapore. Among the major ethnic groups, the im- munity status of the Chinese was higher than that of the Malay and Indian groups. Since the isolation of rubella virus by two groups of workers (Weller & Neva, 1962; Park- man et al., 1962), many seroepidemiological studies have been carried out in different countries. In most areas surveyed, at least 80 % of the women in the population were already immune by the time they reached child-bearing age, i.e., 15 years and over (Rawls et al., 1967; Dowdle et al., 1970). Exceptions to this were found in Trinidad, Jamaica, a rural area of Japan (Rawls et al., 1967), Panama and Peru (Dowdle et al., 1970), and Hawaii (Sever et al., 1965), in all of which the antibody rates were lower than 80%. Rawls et al. (1967) noted that the antibody rate was also high in Singapore, but this finding was based on a small population sample. In a survey in Bangkok and Dhonburi, Thailand, Thongcharoen et al. (1970) found an overall antibody rate of 52.7 %. Apart from these last two countries, 1 Department of Bacteriology, Faculty of Medicine, University of Malaya. Kuala Lumpur. Malaysia. no other serological investigation on rubella has been carried out in the tropical countries of south- east Asia. Rubella has never been reported in epidemic proportions in Malaysia although congenital ru- bella is occasionally seen. From November 1969 to July 1970 there was a minor outbreak of the disease in Singapore with several cases of con- genital rubella (Tan et al., 1970). This outbreak was confined to Singapore and did not spread to Malaysia although the two countries are linked by a short causeway. The object of the survey described in this paper was to determine the immune status of women in Kuala Lumpur, the capital of Malaysia, and to compare the findings with those obtained in other countries. As Malaysia is a cosmopolitan country, the findings were also analysed according to the three major ethnic groups-Malays, Chi- nese, and Indians, which comprise approximately 50 %, 37 %, and 11 %, respectively, of the total population of 8.7 million in West Malaysia. MATERIALS AND METHO;DS Over a period of 2 years 697 serum samples were collected from female patients attending the University Hospital in Kuala Lumpur. The ages of the patients ranged from 1 to 50 years; 83.1 % of them fell in the child-bearing age group (15-44 2880c 11