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Circadian rhythm in Schistosoma haematobium egg excretion
international
Journal
for
Pararitology
CIRCADlAN
1976. Vol. 6. pp. 373-377.
Pergnmon
Press. Printed
in Great
RHYTHM IN SCHZSTOSOA4A EGG EXCRETION
Britain.
HAEMATOBZUAd
J. E. MCMAHON WHO/MRC/Tanzania, (Received
6 February
Box 950, Tanga, Tanzania
1975; in revised form
I7 September
1975)
Abstract-MCMAHON J. E. 1976. Circadian rhythm in Schistosoma haematobium egg excretion. International Journalfor Parasitology 6: 373-377. The usual peak daily egg excretion period in persons harbouring Schistosomn haematobium is the late morning and early afternoon. No evidence was
found to support a hypothesis that the rhythm is mechanical in origin being due to rapid increase of bladder activity in the early morning. A partial reversion of the rhythm occurred when day shift workers changed to night shift. The possible role in the circadian rhythm of host factors-temperature and metabolism and of proteolytic enzyme secretion by miracidia are discussed. INDEX KEY WORDS: Schistosoma hoematobium; egg excretion pattern; diurnal cycle; bladder miracidium; proteolytic enzymes; night activity; circadian rhythm; oviposition; ovamigration; shift; non-viable egg excretion.
INTRODUCTION LITTLE information is available in scientific literature on any possible rhythmic behaviour of adult schisto-
somes or their eggs in the definitive host. This is in contrast to numerous references to circadian and seasonal rhythm of other blood parasites-notably Plasmodium spp. microfilariae and trypanosomes (Worms, 1972). The egg excretion pattern of persons harbouring the digenetic trematode (Schistosoma haematobium) has a diurnal cycle with maximum output in the late morning and early afternoon (Fig. 1). This cycle
are often the main age group responsible for transmission. As hours of playing (and urinating) in water tend to correspond with the peak egg output, such periodicity of egg excretion would favour survival of the species. Conversely very little human water contact occurs during the hours of darkness and early morning when egg output is at its lowest. In order to explain the mechanism of the rhythm, Bell (1969) put forward the hypothesis that it is due to bladder activity of the host rather than to any intrinsic rhythm of the parasite. Bell considered that after waking in the morning there is a rapid increase in bladder activity, with both the frequency of bladder emptying and the rate of urine formation, being increased. The extrusion of a ‘store’ of eggs, which had accumulated in the bladder wall during the previous night, accounted for the egg excretion pattern. The present work was undertaken to investigate this hypothesis
of Bell.
MATERIALS Investigation Time,
h
FIG. 1. Diurnal pattern
of S. haematobium egg excretion of one individual following a normal day’s routine.
has been demonstrated in Egypt (Stimmel & Scott, 1956); East Africa (Jordan, 1959; Bradley, 1963) and Ghana (Onori, 1962). Prior to the investigations in these countries, it had been reported in South Africa that more eggs were found in the afternoon than in early morning specimens (Bennie, 1949). In urinary schistosomiasis endemic areas, children
l-increased
AND METHODS fluid intake
Twelve school pupils (aged 11-15 y) infected with S. haematobium were admitted to a hostel for 76 h. They were randomly divided into 2 groups. All were likely to be harbouring mature and not maturing infections, because all attended an urban school and lived in residential areas where no S. haematobium transmission was occurring and none had been in rural areas for many months. The age of the subjects also made it unlikely that bladder fibrosis or calcification would play an important role in inhibiting egg passage through the tissues. The total urine passed was collected at the specified time, volume and specific gravity were measured and 373
J. E. MCMAHON
374
after thorough mixing a random 10 cma specimen was withdrawn and miracidia counts performed. As the ability of an egg to play an active, rather than a passive, role in its own migration could depend on viability, viable and non-viable eggs were recorded separately. Miracidia were counted using the method described by Davis (1968). Briefly the procedure being as follows: freshly cooled boiled water was added to the centrifuged deposit, thoroughly shaken and hatching performed under artificial light at a constant temperature of 22°C for 30 min (increased time of exposure to light did not result in increased hatching). Miracidia were fixed and stained with alcohol and eosin and following centrifugation and withdrawal of supernatant, all miracidia (hatched and non-hatched) in the final 0.1 ml were counted as a cover slip preparation under the 16 mm objective lens. The work was done by 2 experienced technicians. A built-in random cross-check system was established. The total volume of urine passed by all subjects during the time interval coincident with the egg count was measured. The early morning increased fluid intake for Group 1 was the second and for Group II on the third day (readings of the other 2 days served as controls). On the morning of the increased fluid intake, urine collections were far more frequent than on other days. After passing urine at 5.30 h a total of 2100 cm3 of water was given in divided
I.J.P. VOL.
6. 1976
doses between 5.30 and 8.00 h; 250 cm’ were given at 9.00 and again at lOGO h, after which the fluid intake was not controlled. Egg counts after 5.30 h up to and including 8.00 h were all included in the total 8.00 h count (Fig. 2). The actual counts for more frequent collection periods on the ‘fluid’ day are shown in Fig. 3. Investigation 2-Examination of shift workers Shifts were alternated weekly, the hours of work being 7.00-15.00,15~00-23.00and23.0&7.00 h.Thestudyaimed at following the egg output patterns of 10 adults throughout the three shifts. RESULTS Investigation
1
Results are shown in Figs. 2a and 2b. Infections of Group 1 were lighter than those of Group 2. The actual counts for more frequent collection periods on the ‘fluid’ day are shown in Fig. 3. From these results (Figs. 2 and 3) there is no evidence that increased frequency of contraction and extension of the bladder in the early morning results
in a changed time of the daily peak egg excretion
~ -------
Viable eggs Non-wbleeggs
Group I (a)
FIG. 2 (a & b). Results of the experiment designed to test the effect of greatly increasing the frequency of micturition. Egg counts for similar periods on control and ‘fluid’ days are compared.
I.I.P. VOL.
6. 1976
Circadian rhythm in Schisfosoma haematobium egg excretion
375
12,oooj11.423 ;
f
Group II
Group I Tim, FIG.
h
3. Total egg counts (viable and non-viabIe) at collection periods on the morning of increased fluid intake.
period. Although with Group 1 the peak of nonviable egg excretion occurred earlier folfowing increased fluid intake, this pattern is not repeated with Group 2. Possibly thenumber of non-viableeggs in the sub-mucosa relative to those more deeply situated was greater for Group 1 than 2 and the mechanical activity of the tissues accelerated their passage into the bladder lumen. The total daily output of non-viable eggs was greater for the ‘fluid’ than the control days. This was most marked in 7 subjects with heavy infections in which 18,000 f non-viable eggs were excreted on the ‘fluid’ day and less than 12,000 on both control days. It is feasible that the mechanical activity results in
ova at all levels in the tissues receiving some ‘hurryup’ which is not manifested as any specific pattern.
As the working conditions did not always allow the collecting of all the specimens at the specified times, no attempt has been made to evaluate the results of all 10 individuals. But su~cient specimens were collected to show at least a partial reversion of the diurnal pattern on the afternoon and night shifts and results of subjects whose total urine output was collected for defined periods (Figs. 4 and 5) are very different from the usual diurnal pattern.
302300
Day shifl
Afternoon
shaft
23oc
Night shift
FIG, 4. Mean egg excretion of one subject during the last 2 days of each of 3 shifts.
376
J. E. MCMAHON
Z%W’I
-Subject
on shift
0700 to 1500h
__ _ _ ___ Subject
0” shift
1500
to 2300
-
on shift
2300
to 0700
--
Subject
il h
FIG. 5. Egg excretion pattern from 3 subjects on different
shifts during the same 24 h period. Although the number of persons investigated is too small to draw conclusions, there is suggestive evidence that bodily activity influences the circadian rhythm. DISCUSSION A knowledge of biological rhythms, both on a circadian and seasonal basis, can be of importance in the control of parasitic diseases. This involves a study of the adaptive behavioural patterns of parasite, vector and host and opens a complex field, the exploration of which has hardly begun (McMahon, 1973). Seasonal i~~uenc~ on the production of S. haematobjum and S. mansoni cercariae in Rhodesia were recently described (Shiff, Evans, Yiannakis & Eardley, 1975). The present work is concerned with the daily egg excretion pattern in the definitive host. It has been shown that the diurnal peak of egg excretion in persons undergoing normal daily activities is unaffected by strenuous exercise (Jordan, 1962; Weber, Blair & Clarke, 1967). Important factors in the number of S. ~~e~~~obium eggs excreted from the human host to the exterior, are migration of the schistosomes in the mesenteric vessels, site of oviposition and time taken for subsequent passage through the tissues. It is known that certain drugs result in a shift of schistosomes from the mesenteric vessels to the portal vein and this fact is made use of to treat patients by recovering adult schistosomes by extracorporeal filtration (Kean & Goldsmith, 1969). But it appears that no studies have been undertaken to gain information on any possible circadian pattern of schistosome migration in vertebrate hosts. In the present work only fully emb~onated eggs were found in the urine. Ova of Schistosoma mansoizi
1.3.~.VOL.6. 1976
appear to require 6 days to develop to nlaturity in human tissues and only mature eggs are excreted (Prata, 1957). S. huematobium studies in hamsters show that at least 5 days are needed for ova migration (M. A. Smith, personal communication). It is likely that at least 6 days elapse between oviposition and egg excretion in human bladder tissue. Thus if any intrinsic rhythm of schistosome migratory behaviour, oviposition or ovulation does exist, then the effect of such rhythm on egg excretion is likely to be overshadowed by the time interval for egg passage through the tissues. If neither intrinsic rhythm of the parasite, nor mechanical contraction of the bladder, directly influences the egg excretion pattern, then either other host factors are involved or the egg has some control over its own release. Perhaps secretion of enzymes of the miracidium mediates the active passage of the egg through the tissues. Radioassays for determining the level of proteolytic enzymes in eggs of S. mansoni have recently been reported (Smith, 1974). The lack of immature eggs in both faeces and urine raises another query. Is the egg in some manner able to resist passage through the tissues until it is mature? The lateral spine of S. martsoni would appear to be better situated for such a role than the terminal spine of S. haematobium. Important host factors in egg migration may be temperature (body temperature is from 04-0*6”C lower in the early morning than in the afternoon) or chemical factors related to increased body metabolism. In conclusion these studies suggest that the circadian rhythm of S. haema~obiiim egg excretion is related to body activity of the host. These activities by means of physical or chemical stimuli may trigger the secretion of proteolytic enzymes of miracidia resulting in the extrusion of ova nearest to the bladder lumen. Acknowledgemen&wish to thank Dr. Dion Bell for encouraging me to carry out this study, Dr. Worms and Maire Smith for useful comments and Messrs Mwakanyamale and Maranda for technical assistance.
R~FE~N~ES BELL D. R. 1969. Clinical Trials and diagnositic methods in schisfosomiasis. Annals of the New York Academy ofSciences 160: 593-601. BENNIEI. 1949. Urinary schistosomiasis, the best time to obtain specimens. South African Medical Journal 23: 97-100. BRADLEYD. J. 1963. A quantitative approach to bilharzia. East African Medical Journal 40: 240-249. DAVIS A. 1968. Comparative trials of antimonial drugs in urinary schistosomiasis. BulfeCn of World Health Or~a~~~~~~o~ 38: 197-206. JORDAN P. 19S9. ,4nnrcaI Report of the East African Iptstifute,for Medical Research, Nairobi, p. 25.
I.J.P. VOL.6. 1976
Circadian rhythm in Schisfosoma haemutobium egg excretion
JORDAN P. 1962. A~aal Report of the East A_/%can r~sfitufe~or Medical research, Nairobi, p. 23. KEANB. H. & GOLDSMITEI E. 1. 1969. The recovery of adult schistosomes by extracorporeal filtration Annals of the New York Academy C$ Sciences 160: 713-715. MCMAHONJ. E. 1973. A note on changing concepts of malaria control. Papua New Guinea Medical Journal 16: 78-79. ONORIE. 1962. Observations on variations in Schistosoma haemafob~am egg output and on the relationships between the average egg output of infected persons and the prevalence of infection in a community. Armats of Tropical Medicine and Parasitology 56: 292-296. PRATA A. R. 1957. Biopsia rectal na esquistossomose mansoni. Service National da Educacao Sanifaria, R:o de Janeiro, Brazil, Thesis, p. 159.
377
SHIFF C. J., EVANS A., YIANNAKISC. & EARDLEYM. 1957. Seasonal influenc: on the production of Schisfosomu haematobium and S. mansoni cercariae in Rhodesia. International Journal for Parasitology 5: 119-123. SMITH M. A. 1974. Radioassays for the proteolytic enzyme secreted by living eggs of Schisfosoma mansoni. International Journal for Parasitology 4: 681-683. STIMMELC. M. & SCOTT J. A. 1956. The regularity of egg output of Schistosoma haematobium. Texas Report Biology and Medicine 14: 440-456. WEBER M. C., BLAIR D. M. & CLARKE V deV. 1967. The pattern of schistosome egg distribution in a micturition Row. Central African Journal of ~~edi~i~e 13 : 75-88. WORMSM. J. 1972. Circadian and seasonal rhythm in blood parasites. In Behavioarai Aspects of Parasite Transmission. (Edited by CANNINGE. & WRIGHTC. A.) Academic Press, New York.
Journal
for
Pararitology
CIRCADlAN
1976. Vol. 6. pp. 373-377.
Pergnmon
Press. Printed
in Great
RHYTHM IN SCHZSTOSOA4A EGG EXCRETION
Britain.
HAEMATOBZUAd
J. E. MCMAHON WHO/MRC/Tanzania, (Received
6 February
Box 950, Tanga, Tanzania
1975; in revised form
I7 September
1975)
Abstract-MCMAHON J. E. 1976. Circadian rhythm in Schistosoma haematobium egg excretion. International Journalfor Parasitology 6: 373-377. The usual peak daily egg excretion period in persons harbouring Schistosomn haematobium is the late morning and early afternoon. No evidence was
found to support a hypothesis that the rhythm is mechanical in origin being due to rapid increase of bladder activity in the early morning. A partial reversion of the rhythm occurred when day shift workers changed to night shift. The possible role in the circadian rhythm of host factors-temperature and metabolism and of proteolytic enzyme secretion by miracidia are discussed. INDEX KEY WORDS: Schistosoma hoematobium; egg excretion pattern; diurnal cycle; bladder miracidium; proteolytic enzymes; night activity; circadian rhythm; oviposition; ovamigration; shift; non-viable egg excretion.
INTRODUCTION LITTLE information is available in scientific literature on any possible rhythmic behaviour of adult schisto-
somes or their eggs in the definitive host. This is in contrast to numerous references to circadian and seasonal rhythm of other blood parasites-notably Plasmodium spp. microfilariae and trypanosomes (Worms, 1972). The egg excretion pattern of persons harbouring the digenetic trematode (Schistosoma haematobium) has a diurnal cycle with maximum output in the late morning and early afternoon (Fig. 1). This cycle
are often the main age group responsible for transmission. As hours of playing (and urinating) in water tend to correspond with the peak egg output, such periodicity of egg excretion would favour survival of the species. Conversely very little human water contact occurs during the hours of darkness and early morning when egg output is at its lowest. In order to explain the mechanism of the rhythm, Bell (1969) put forward the hypothesis that it is due to bladder activity of the host rather than to any intrinsic rhythm of the parasite. Bell considered that after waking in the morning there is a rapid increase in bladder activity, with both the frequency of bladder emptying and the rate of urine formation, being increased. The extrusion of a ‘store’ of eggs, which had accumulated in the bladder wall during the previous night, accounted for the egg excretion pattern. The present work was undertaken to investigate this hypothesis
of Bell.
MATERIALS Investigation Time,
h
FIG. 1. Diurnal pattern
of S. haematobium egg excretion of one individual following a normal day’s routine.
has been demonstrated in Egypt (Stimmel & Scott, 1956); East Africa (Jordan, 1959; Bradley, 1963) and Ghana (Onori, 1962). Prior to the investigations in these countries, it had been reported in South Africa that more eggs were found in the afternoon than in early morning specimens (Bennie, 1949). In urinary schistosomiasis endemic areas, children
l-increased
AND METHODS fluid intake
Twelve school pupils (aged 11-15 y) infected with S. haematobium were admitted to a hostel for 76 h. They were randomly divided into 2 groups. All were likely to be harbouring mature and not maturing infections, because all attended an urban school and lived in residential areas where no S. haematobium transmission was occurring and none had been in rural areas for many months. The age of the subjects also made it unlikely that bladder fibrosis or calcification would play an important role in inhibiting egg passage through the tissues. The total urine passed was collected at the specified time, volume and specific gravity were measured and 373
J. E. MCMAHON
374
after thorough mixing a random 10 cma specimen was withdrawn and miracidia counts performed. As the ability of an egg to play an active, rather than a passive, role in its own migration could depend on viability, viable and non-viable eggs were recorded separately. Miracidia were counted using the method described by Davis (1968). Briefly the procedure being as follows: freshly cooled boiled water was added to the centrifuged deposit, thoroughly shaken and hatching performed under artificial light at a constant temperature of 22°C for 30 min (increased time of exposure to light did not result in increased hatching). Miracidia were fixed and stained with alcohol and eosin and following centrifugation and withdrawal of supernatant, all miracidia (hatched and non-hatched) in the final 0.1 ml were counted as a cover slip preparation under the 16 mm objective lens. The work was done by 2 experienced technicians. A built-in random cross-check system was established. The total volume of urine passed by all subjects during the time interval coincident with the egg count was measured. The early morning increased fluid intake for Group 1 was the second and for Group II on the third day (readings of the other 2 days served as controls). On the morning of the increased fluid intake, urine collections were far more frequent than on other days. After passing urine at 5.30 h a total of 2100 cm3 of water was given in divided
I.J.P. VOL.
6. 1976
doses between 5.30 and 8.00 h; 250 cm’ were given at 9.00 and again at lOGO h, after which the fluid intake was not controlled. Egg counts after 5.30 h up to and including 8.00 h were all included in the total 8.00 h count (Fig. 2). The actual counts for more frequent collection periods on the ‘fluid’ day are shown in Fig. 3. Investigation 2-Examination of shift workers Shifts were alternated weekly, the hours of work being 7.00-15.00,15~00-23.00and23.0&7.00 h.Thestudyaimed at following the egg output patterns of 10 adults throughout the three shifts. RESULTS Investigation
1
Results are shown in Figs. 2a and 2b. Infections of Group 1 were lighter than those of Group 2. The actual counts for more frequent collection periods on the ‘fluid’ day are shown in Fig. 3. From these results (Figs. 2 and 3) there is no evidence that increased frequency of contraction and extension of the bladder in the early morning results
in a changed time of the daily peak egg excretion
~ -------
Viable eggs Non-wbleeggs
Group I (a)
FIG. 2 (a & b). Results of the experiment designed to test the effect of greatly increasing the frequency of micturition. Egg counts for similar periods on control and ‘fluid’ days are compared.
I.I.P. VOL.
6. 1976
Circadian rhythm in Schisfosoma haematobium egg excretion
375
12,oooj11.423 ;
f
Group II
Group I Tim, FIG.
h
3. Total egg counts (viable and non-viabIe) at collection periods on the morning of increased fluid intake.
period. Although with Group 1 the peak of nonviable egg excretion occurred earlier folfowing increased fluid intake, this pattern is not repeated with Group 2. Possibly thenumber of non-viableeggs in the sub-mucosa relative to those more deeply situated was greater for Group 1 than 2 and the mechanical activity of the tissues accelerated their passage into the bladder lumen. The total daily output of non-viable eggs was greater for the ‘fluid’ than the control days. This was most marked in 7 subjects with heavy infections in which 18,000 f non-viable eggs were excreted on the ‘fluid’ day and less than 12,000 on both control days. It is feasible that the mechanical activity results in
ova at all levels in the tissues receiving some ‘hurryup’ which is not manifested as any specific pattern.
As the working conditions did not always allow the collecting of all the specimens at the specified times, no attempt has been made to evaluate the results of all 10 individuals. But su~cient specimens were collected to show at least a partial reversion of the diurnal pattern on the afternoon and night shifts and results of subjects whose total urine output was collected for defined periods (Figs. 4 and 5) are very different from the usual diurnal pattern.
302300
Day shifl
Afternoon
shaft
23oc
Night shift
FIG, 4. Mean egg excretion of one subject during the last 2 days of each of 3 shifts.
376
J. E. MCMAHON
Z%W’I
-Subject
on shift
0700 to 1500h
__ _ _ ___ Subject
0” shift
1500
to 2300
-
on shift
2300
to 0700
--
Subject
il h
FIG. 5. Egg excretion pattern from 3 subjects on different
shifts during the same 24 h period. Although the number of persons investigated is too small to draw conclusions, there is suggestive evidence that bodily activity influences the circadian rhythm. DISCUSSION A knowledge of biological rhythms, both on a circadian and seasonal basis, can be of importance in the control of parasitic diseases. This involves a study of the adaptive behavioural patterns of parasite, vector and host and opens a complex field, the exploration of which has hardly begun (McMahon, 1973). Seasonal i~~uenc~ on the production of S. haematobjum and S. mansoni cercariae in Rhodesia were recently described (Shiff, Evans, Yiannakis & Eardley, 1975). The present work is concerned with the daily egg excretion pattern in the definitive host. It has been shown that the diurnal peak of egg excretion in persons undergoing normal daily activities is unaffected by strenuous exercise (Jordan, 1962; Weber, Blair & Clarke, 1967). Important factors in the number of S. ~~e~~~obium eggs excreted from the human host to the exterior, are migration of the schistosomes in the mesenteric vessels, site of oviposition and time taken for subsequent passage through the tissues. It is known that certain drugs result in a shift of schistosomes from the mesenteric vessels to the portal vein and this fact is made use of to treat patients by recovering adult schistosomes by extracorporeal filtration (Kean & Goldsmith, 1969). But it appears that no studies have been undertaken to gain information on any possible circadian pattern of schistosome migration in vertebrate hosts. In the present work only fully emb~onated eggs were found in the urine. Ova of Schistosoma mansoizi
1.3.~.VOL.6. 1976
appear to require 6 days to develop to nlaturity in human tissues and only mature eggs are excreted (Prata, 1957). S. huematobium studies in hamsters show that at least 5 days are needed for ova migration (M. A. Smith, personal communication). It is likely that at least 6 days elapse between oviposition and egg excretion in human bladder tissue. Thus if any intrinsic rhythm of schistosome migratory behaviour, oviposition or ovulation does exist, then the effect of such rhythm on egg excretion is likely to be overshadowed by the time interval for egg passage through the tissues. If neither intrinsic rhythm of the parasite, nor mechanical contraction of the bladder, directly influences the egg excretion pattern, then either other host factors are involved or the egg has some control over its own release. Perhaps secretion of enzymes of the miracidium mediates the active passage of the egg through the tissues. Radioassays for determining the level of proteolytic enzymes in eggs of S. mansoni have recently been reported (Smith, 1974). The lack of immature eggs in both faeces and urine raises another query. Is the egg in some manner able to resist passage through the tissues until it is mature? The lateral spine of S. martsoni would appear to be better situated for such a role than the terminal spine of S. haematobium. Important host factors in egg migration may be temperature (body temperature is from 04-0*6”C lower in the early morning than in the afternoon) or chemical factors related to increased body metabolism. In conclusion these studies suggest that the circadian rhythm of S. haema~obiiim egg excretion is related to body activity of the host. These activities by means of physical or chemical stimuli may trigger the secretion of proteolytic enzymes of miracidia resulting in the extrusion of ova nearest to the bladder lumen. Acknowledgemen&wish to thank Dr. Dion Bell for encouraging me to carry out this study, Dr. Worms and Maire Smith for useful comments and Messrs Mwakanyamale and Maranda for technical assistance.
R~FE~N~ES BELL D. R. 1969. Clinical Trials and diagnositic methods in schisfosomiasis. Annals of the New York Academy ofSciences 160: 593-601. BENNIEI. 1949. Urinary schistosomiasis, the best time to obtain specimens. South African Medical Journal 23: 97-100. BRADLEYD. J. 1963. A quantitative approach to bilharzia. East African Medical Journal 40: 240-249. DAVIS A. 1968. Comparative trials of antimonial drugs in urinary schistosomiasis. BulfeCn of World Health Or~a~~~~~~o~ 38: 197-206. JORDAN P. 19S9. ,4nnrcaI Report of the East African Iptstifute,for Medical Research, Nairobi, p. 25.
I.J.P. VOL.6. 1976
Circadian rhythm in Schisfosoma haemutobium egg excretion
JORDAN P. 1962. A~aal Report of the East A_/%can r~sfitufe~or Medical research, Nairobi, p. 23. KEANB. H. & GOLDSMITEI E. 1. 1969. The recovery of adult schistosomes by extracorporeal filtration Annals of the New York Academy C$ Sciences 160: 713-715. MCMAHONJ. E. 1973. A note on changing concepts of malaria control. Papua New Guinea Medical Journal 16: 78-79. ONORIE. 1962. Observations on variations in Schistosoma haemafob~am egg output and on the relationships between the average egg output of infected persons and the prevalence of infection in a community. Armats of Tropical Medicine and Parasitology 56: 292-296. PRATA A. R. 1957. Biopsia rectal na esquistossomose mansoni. Service National da Educacao Sanifaria, R:o de Janeiro, Brazil, Thesis, p. 159.
377
SHIFF C. J., EVANS A., YIANNAKISC. & EARDLEYM. 1957. Seasonal influenc: on the production of Schisfosomu haematobium and S. mansoni cercariae in Rhodesia. International Journal for Parasitology 5: 119-123. SMITH M. A. 1974. Radioassays for the proteolytic enzyme secreted by living eggs of Schisfosoma mansoni. International Journal for Parasitology 4: 681-683. STIMMELC. M. & SCOTT J. A. 1956. The regularity of egg output of Schistosoma haematobium. Texas Report Biology and Medicine 14: 440-456. WEBER M. C., BLAIR D. M. & CLARKE V deV. 1967. The pattern of schistosome egg distribution in a micturition Row. Central African Journal of ~~edi~i~e 13 : 75-88. WORMSM. J. 1972. Circadian and seasonal rhythm in blood parasites. In Behavioarai Aspects of Parasite Transmission. (Edited by CANNINGE. & WRIGHTC. A.) Academic Press, New York.