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Urban sludge reuse in agriculture: Waste treatment and parasitological risk
Bioresource Technology52 (1995) 37-40
ELSEVIER
0960-8524(94)00149-9
© 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/95/$9.50
U R B A N SLUDGE REUSE IN AGRICULTURE: WASTE TREATMENT A N D PARASITOLOGICAL RISK P. G. G a s p a r d , a * J. Wiart h & J. Schwartzbrod"
"D~partement de Microbiologie, W.H.O. Collaborating Centre, Facult~de Pharmacie, B.P 403, 54001 Nancy Cedex, France hAgence de l'environnement et de Mattrise de l'~nergie, 2 Square Lafayette, B.P 406, 49004Angers, France (Received 25 November 1994; accepted 14 December 1994)
ments, as has been demonstrated in different laboratory studies performed in the field of artificial contamination. However, a viability study of the nematode eggs actually existing in samples is necessary with a view to properly assessing the sanitary state of the sludge recycled in agriculture. The present work was carried out on sludges from various origins to properly evaluate the impact of the different treatments on nematode eggs. An extraction followed by a concentration procedure allowed isolation of eggs, the viability study being then performed on a culture.
Abstract
The development of an extraction-concentration method for the nematode eggs existing in urban treatment-plant sludge made it possible to study the viable nature of these eggs by cultivating them in deionized water at 30°C for 30 days. The criterion selected to show viability was the development of eggs at the larval stage. Sludge samples from various origins (physical, biological and chemical treatments) were analyzed: 93% of the eggs proved to be viable in primary sedimentation sludge. Eggs that can grow have been detected in samples from biological treatments, with, however, a lower frequency for systems calling for lagooning and composting with, respectively, 26 and 25% of viable eggs. Anaerobic digestion gave intermediate results with 66%, whereas a prolonged aeration led to a limited effect, 93% of eggs being considered as viable. A 15 day chemical treatment with lime did not produce complete elimination of viable eggs: the analysis showed 66% of eggs developing at the larval stage. ~. This study thus confirmed that nematode eggs are strongly resistant to most of the classical waste treatments.
METHODS
The viability study consisted of two stages: isolation and concentration of the eggs; culture and counting. Extraction -concentration
The extraction-concentration of eggs from sludge samples was performed in three steps. First, the egg desorption or elution was performed by means of a two-phase treatment consisting of suspending the sample (corresponding to 0-25 g of dry matter) in 20 ml of a detergent solution (SDS at 0-01%). After homogenization, a hydrophobic solvent consisting of 20 ml ethyl acetate was added to the suspension, the mixture being then shaken until an emulsion was obtained. This emulsion was then centrifuged for 6 min at 1200 g. The recovered pellet was suspended in 20 ml water. The second step consisted of two successive filtrations to eliminate the rough debris and to disorganize the pellet. The first filtration was performed on a metallic sieve (mesh opening=500/am), including a washing with 250 ml water to eluate the eggs adsorbed on the particles. The filtrate thus obtained was centrifuged at 600 g for 6 min. The pellet was re-suspended in 20 ml water and submitted to a second filtration on a sieve with a finer mesh opening (100 pm) with the same procedure. The flotation step was performed twice on the pellet with a saturated NaCl solution (specific grav-
Key words: Sewage sludges, treatments, nematodes, egg viability. INTRODUCTION
With urban-sludge recycling being more and more encouraged in agriculture, both a good knowledge of the agronomical value of the product and an assessment of sanitary risks are proving to be necessary. Numerous microorganisms are detected in sludge, and particularly helminth eggs, those currently found (Barbier et al., 1990) being Ascaris, Toxocara and Trichuris eggs for nematodes and Hymenolepis and Tenia eggs for cestoda. These eggs are extremely resistant to the various sludge stabilization treat*Author to whom correspondence should be addressed. 37
R G. Gaspard, J. Wiart, J. Schwartzbrod
38
ity=l.19) added in the ratio of one sediment part to nine solution parts. A 20 s centrifugation at 250 g was effected after homogenization. The supernatant recovered in a 15 ml tube was submitted, after a 5 min settling time, to a second centrifugation. The upper 2.5 ml were then recovered and diluted in deionized water. Two successive washings were then performed in deionized water and the eggs concentrated after a centrifugation at 1200 g for 5 min. Culture and counting
Eggs recovered were cultured in deionized water at 30°C for 30 days. Quantification was obtained by microscopic examination carried out after an ultrasonication (power level, 45 W; repeating cycle 0.9 s during 20 s) and a chemical treatment with Na hypochlorite (2°C). Only Toxocara, Trichuris, Capillaria and Ascaris eggs were counted and the percentage of viable eggs corresponded to the following ratio: number of eggs having reached the larval stage/total number of nematode eggs. Sampling
Urban sludge samples used in agriculture were analyzed and samples containing nematode eggs were
tested to determine the egg viability. Then, the viability study was performed on 26 different samples corresponding to six types of treatment (Table 1). RESULTS All the results are summarized in Table 2. Globally, of the 26 analyzed samples containing nematode eggs, 19 contained viable eggs. The seven samples without live eggs were four compost and three limed sludge. For the 19 samples with live eggs, all types of treatment were represented: fresh sludge, prolonged aeration, anaerobic digestion, lagooning, composting and liming. No egg development inhibition phenomenon was observed in fresh sludge. The test demonstrated there were 93% viable eggs. Biological treatments do not produce a total inactivation of nematode eggs. The samples submitted to such various treatments as prolonged aeration, anaerobic digestion, lagooning or composting still showed very significant percentages of viable eggs~ Prolonged aeration seemed to be totally ineffective with 93% viable eggs recovered, and a lagoon containing 6-year-old sediments still showed 26% viable
Table 1. Sampling for the study of the nematode egg viability
Sludge treatment
Sludge-types
Physical treatment
Primary sedimentation
Biological treatment
Prolonged aeration Anaerobic digestion ( 3 5 ° C ) (15-20 days) Lagooning (6 years) Composting - - fermentation: 4-7 weeks - - maturation: 3 months
Chemical treatment
Liming
Sample numbers
Sludge weight analyzed
Fresh liquid sludge
2
200 g
Liquid- or pasty-sludge Semi-hydrated sludge
9 1
40 g 50 g
Sediment
1
80 g
Compost
6
40 g
Solid sludge
7
20 g
Table 2. Nematode egg viability in urban sludge samples
Treatment
Number of samples analyzed
Number of samples containing viable eggs
Percentage of viable eggs
Physical Primary sedimentation
2
2
93 ___6"
Biological Prolonged aeration Anaerobic digestion Lagooning Composting
9 1 1 6
9 1 1 2
93 ___8 66 + 24 26___14 25 +__32
Chemical Liming
7
4
66 + 32
26
19
--
Total "Confidence limit at 95%.
Sewage treatments and nematode egg death
eggs. Regarding anaerobic digestion, 66% of viable eggs were recovered in the one sample. For the compost, the analysis on a small number of eggs (8) showed a viability of 25% and the chemical treatment with lime after 20 days of storage gave 66% of viable eggs. DISCUSSION
The study of samples directly taken on-site allowed assessment of the impact of the various treatments on the eggs, preceding studies having been, in most cases, only experiments made in laboratories with a surplus of parasitic organisms. The viability analysis of fresh sludge shows no influence of this medium on egg development. The complexity, resistance, as well as selective impermeability of the different shell layers, which make an egg one of the more resistant biological structures (Wharton, 1980), would be the origin of this phenomenon. However, while viability does not seem to be altered, embryonation was never or very rarely observed in any of the sludge samples, whereas eggs developed as soon as they were extracted from the sludge. Clark and Perry (1980) indicated gaseous exchanges at the level of the egg internal layer during embryonation, sludge is low in oxygen and its texture would not make possible those exchanges necessary for egg metabolism, thus preventing any development. Cram (1943) observed for activated sludge that this effluent type was favorable to eggs embryoning. Among the analyzed samples, some eggs were effectively detected with 4-8 cells but the larval stage did not seem to have been reached. This treatment did not alter egg viability. In the case of anaerobic mesophilic digestion, our observations are in accordance with the experimentation of Pike et al. (1983) with Ascaris eggs, where they obtained 50% viable eggs after 20 days at 35°C among the eggs that had not undergone any autolysis. Barbier et al. (1990) show a viability reaching 25% for the same type of treatment. The sediments resulting from a 6 year lagooning showed a survival rate of 26%. Biological factors and time, as well as prolonged anaerobiosis, have a significant impact on egg viability. As far as composting by aeration is concerned, temperatures above 50°C are reached (de Bertoldi et al., 1985), these values represent high temperatures theoretically liable to alter egg viability. Provided this destruction capacity is real it remains variable, for the results obtained. Steer and Windt (1978) thus recommend a maturation of 60 days at 65°C to inactivate all Ascaris eggs, whereas a study of Theis et al. (1978) mentions a degeneration phenomenon with viable eggs found at the end of the composting period. Schwartzbrod et al. (1988) also reported an absence of viability of Ascaris eggs in a 120 days old compost (55-60°C). The large variety of results underlines the difficulty in finding a protocol that would ensure the
39
complete destruction of the infectious nature of eggs. The results of our study also shows that the eggs can be viable after 30 days at 60-70°C. This phenomenon might be explained by the difficulty in reaching a homogeneous compost temperature, as theoretical survival rates depending on temperature described by Feachem et al. (1983) show an inactivation of Ascaris eggs in 10 min at 55°C. Carrington (1985) also recommends a treatment of 2 h at 55°C in the case of pasteurization to get around the protective effect of sludge on eggs. The treatment with lime is not efficient instantaneously and requires a minimum contact time of 2 months with an initial concentration allowing the material to reach a pH of 12"5 (Schuh et al., 1985). With shorter times and lower concentrations of lime, eggs remain viable, as was confirmed by the 66% viable eggs recovered in our samples. The results of this study confirm that Nematode eggs are strongly resistant to the various urbansludge treatments. Moreover, lime and temperature treatments necessitate a strict compliance with homogeneity and specific values for the contact time, temperature and concentration of chemical product to be totally effective. ACKNOWLEDGEMENT
This study was supported by the NANCIE and the Agence de Bassin Rhin-Meuse (France). REFERENCES
Barbier, D., Perrine, D., Duhamel, C., Doublet, R. & Georges, P. (1990). Parasitic hazard with sewage sludge applied to land. Appl. Environ. Mierobiol., 56, 1420-2. Carrington, E. G. (1985). Pasteurisation: effects upon Ascaris eggs. In Inactivation of Micro-organisms in Sewage Sludge by Stabilisation Processes, ed. D. Strauch. Elsevier, London, pp. 121-5. Clarke, A. J. & Perry, R. N. (1980). Egg shell permeability and hatching of Ascaris suum. Parasitology, 80, 447-56. Cram, E. B. (1943). The effect of various treatment processes on the survival of helminth ova and protozoan cysts in sewage. Sewage Works J., 15, 1119-38. De Bertoldi, M., Frassinetti, S., Bianchin, L. & Pera, A. (1985). Sludge hygienisation with different compost systems. In Inactivation of Micro-organisms in Sewage by Stabilisation Processes, ed. D. Strauch. Elsevier, London, pp. 67-76. Feachem, R. G., Bradley, D. J., Garelick, H. & Mara, D. D. (1983). Sanitation and Disease: Health Aspects of Excreta and Wastewater Management. John Wiley & Sons, Chichester. Pike, E. B., Morris, D. L. & Carrington (1983). Inactivation of ova of the parasites Taenia saginata and Ascaris suum during heated anaerobic digestion. Wat. Poll. Control., 82, 501-9. Schuh, R., Phillip, W. & Strauch, D. (1985). Influence of sewage sludge with and without lime treatment on the development of Asearis suum eggs. In Inactivation of Micro-organisms in Sewage Sludge by Stabilisation Processes, ed. D. Strauch. Elsevier, London, pp. 100-12. Schwartzbrod, J., Thevenot, M. T., Collomb, J. & Baradel, J. M. (1986). Parasitologieal study of wastewater sludge.
40
P. G. Gaspard, J. Wiart, J. Schwartzbrod
Environ. Techn. Lett., 7, 155-62. Steer, A. G. & Windt, C. N. (1978). Composting and the fate of Ascaris lumbricoides ova. Water. S.A., 4, 129-33. Theis, J. H., Bolton, V. & Storm, D. R. (1978). Helminth
ova in soil and sludge from twelve U.S. urban areas. J. Wat. Poll. Control. Fed., 50, 2485-93. Wharton, D. A. (1980). Nematode egg shells. Parasitology, 81, 447-63.
ELSEVIER
0960-8524(94)00149-9
© 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/95/$9.50
U R B A N SLUDGE REUSE IN AGRICULTURE: WASTE TREATMENT A N D PARASITOLOGICAL RISK P. G. G a s p a r d , a * J. Wiart h & J. Schwartzbrod"
"D~partement de Microbiologie, W.H.O. Collaborating Centre, Facult~de Pharmacie, B.P 403, 54001 Nancy Cedex, France hAgence de l'environnement et de Mattrise de l'~nergie, 2 Square Lafayette, B.P 406, 49004Angers, France (Received 25 November 1994; accepted 14 December 1994)
ments, as has been demonstrated in different laboratory studies performed in the field of artificial contamination. However, a viability study of the nematode eggs actually existing in samples is necessary with a view to properly assessing the sanitary state of the sludge recycled in agriculture. The present work was carried out on sludges from various origins to properly evaluate the impact of the different treatments on nematode eggs. An extraction followed by a concentration procedure allowed isolation of eggs, the viability study being then performed on a culture.
Abstract
The development of an extraction-concentration method for the nematode eggs existing in urban treatment-plant sludge made it possible to study the viable nature of these eggs by cultivating them in deionized water at 30°C for 30 days. The criterion selected to show viability was the development of eggs at the larval stage. Sludge samples from various origins (physical, biological and chemical treatments) were analyzed: 93% of the eggs proved to be viable in primary sedimentation sludge. Eggs that can grow have been detected in samples from biological treatments, with, however, a lower frequency for systems calling for lagooning and composting with, respectively, 26 and 25% of viable eggs. Anaerobic digestion gave intermediate results with 66%, whereas a prolonged aeration led to a limited effect, 93% of eggs being considered as viable. A 15 day chemical treatment with lime did not produce complete elimination of viable eggs: the analysis showed 66% of eggs developing at the larval stage. ~. This study thus confirmed that nematode eggs are strongly resistant to most of the classical waste treatments.
METHODS
The viability study consisted of two stages: isolation and concentration of the eggs; culture and counting. Extraction -concentration
The extraction-concentration of eggs from sludge samples was performed in three steps. First, the egg desorption or elution was performed by means of a two-phase treatment consisting of suspending the sample (corresponding to 0-25 g of dry matter) in 20 ml of a detergent solution (SDS at 0-01%). After homogenization, a hydrophobic solvent consisting of 20 ml ethyl acetate was added to the suspension, the mixture being then shaken until an emulsion was obtained. This emulsion was then centrifuged for 6 min at 1200 g. The recovered pellet was suspended in 20 ml water. The second step consisted of two successive filtrations to eliminate the rough debris and to disorganize the pellet. The first filtration was performed on a metallic sieve (mesh opening=500/am), including a washing with 250 ml water to eluate the eggs adsorbed on the particles. The filtrate thus obtained was centrifuged at 600 g for 6 min. The pellet was re-suspended in 20 ml water and submitted to a second filtration on a sieve with a finer mesh opening (100 pm) with the same procedure. The flotation step was performed twice on the pellet with a saturated NaCl solution (specific grav-
Key words: Sewage sludges, treatments, nematodes, egg viability. INTRODUCTION
With urban-sludge recycling being more and more encouraged in agriculture, both a good knowledge of the agronomical value of the product and an assessment of sanitary risks are proving to be necessary. Numerous microorganisms are detected in sludge, and particularly helminth eggs, those currently found (Barbier et al., 1990) being Ascaris, Toxocara and Trichuris eggs for nematodes and Hymenolepis and Tenia eggs for cestoda. These eggs are extremely resistant to the various sludge stabilization treat*Author to whom correspondence should be addressed. 37
R G. Gaspard, J. Wiart, J. Schwartzbrod
38
ity=l.19) added in the ratio of one sediment part to nine solution parts. A 20 s centrifugation at 250 g was effected after homogenization. The supernatant recovered in a 15 ml tube was submitted, after a 5 min settling time, to a second centrifugation. The upper 2.5 ml were then recovered and diluted in deionized water. Two successive washings were then performed in deionized water and the eggs concentrated after a centrifugation at 1200 g for 5 min. Culture and counting
Eggs recovered were cultured in deionized water at 30°C for 30 days. Quantification was obtained by microscopic examination carried out after an ultrasonication (power level, 45 W; repeating cycle 0.9 s during 20 s) and a chemical treatment with Na hypochlorite (2°C). Only Toxocara, Trichuris, Capillaria and Ascaris eggs were counted and the percentage of viable eggs corresponded to the following ratio: number of eggs having reached the larval stage/total number of nematode eggs. Sampling
Urban sludge samples used in agriculture were analyzed and samples containing nematode eggs were
tested to determine the egg viability. Then, the viability study was performed on 26 different samples corresponding to six types of treatment (Table 1). RESULTS All the results are summarized in Table 2. Globally, of the 26 analyzed samples containing nematode eggs, 19 contained viable eggs. The seven samples without live eggs were four compost and three limed sludge. For the 19 samples with live eggs, all types of treatment were represented: fresh sludge, prolonged aeration, anaerobic digestion, lagooning, composting and liming. No egg development inhibition phenomenon was observed in fresh sludge. The test demonstrated there were 93% viable eggs. Biological treatments do not produce a total inactivation of nematode eggs. The samples submitted to such various treatments as prolonged aeration, anaerobic digestion, lagooning or composting still showed very significant percentages of viable eggs~ Prolonged aeration seemed to be totally ineffective with 93% viable eggs recovered, and a lagoon containing 6-year-old sediments still showed 26% viable
Table 1. Sampling for the study of the nematode egg viability
Sludge treatment
Sludge-types
Physical treatment
Primary sedimentation
Biological treatment
Prolonged aeration Anaerobic digestion ( 3 5 ° C ) (15-20 days) Lagooning (6 years) Composting - - fermentation: 4-7 weeks - - maturation: 3 months
Chemical treatment
Liming
Sample numbers
Sludge weight analyzed
Fresh liquid sludge
2
200 g
Liquid- or pasty-sludge Semi-hydrated sludge
9 1
40 g 50 g
Sediment
1
80 g
Compost
6
40 g
Solid sludge
7
20 g
Table 2. Nematode egg viability in urban sludge samples
Treatment
Number of samples analyzed
Number of samples containing viable eggs
Percentage of viable eggs
Physical Primary sedimentation
2
2
93 ___6"
Biological Prolonged aeration Anaerobic digestion Lagooning Composting
9 1 1 6
9 1 1 2
93 ___8 66 + 24 26___14 25 +__32
Chemical Liming
7
4
66 + 32
26
19
--
Total "Confidence limit at 95%.
Sewage treatments and nematode egg death
eggs. Regarding anaerobic digestion, 66% of viable eggs were recovered in the one sample. For the compost, the analysis on a small number of eggs (8) showed a viability of 25% and the chemical treatment with lime after 20 days of storage gave 66% of viable eggs. DISCUSSION
The study of samples directly taken on-site allowed assessment of the impact of the various treatments on the eggs, preceding studies having been, in most cases, only experiments made in laboratories with a surplus of parasitic organisms. The viability analysis of fresh sludge shows no influence of this medium on egg development. The complexity, resistance, as well as selective impermeability of the different shell layers, which make an egg one of the more resistant biological structures (Wharton, 1980), would be the origin of this phenomenon. However, while viability does not seem to be altered, embryonation was never or very rarely observed in any of the sludge samples, whereas eggs developed as soon as they were extracted from the sludge. Clark and Perry (1980) indicated gaseous exchanges at the level of the egg internal layer during embryonation, sludge is low in oxygen and its texture would not make possible those exchanges necessary for egg metabolism, thus preventing any development. Cram (1943) observed for activated sludge that this effluent type was favorable to eggs embryoning. Among the analyzed samples, some eggs were effectively detected with 4-8 cells but the larval stage did not seem to have been reached. This treatment did not alter egg viability. In the case of anaerobic mesophilic digestion, our observations are in accordance with the experimentation of Pike et al. (1983) with Ascaris eggs, where they obtained 50% viable eggs after 20 days at 35°C among the eggs that had not undergone any autolysis. Barbier et al. (1990) show a viability reaching 25% for the same type of treatment. The sediments resulting from a 6 year lagooning showed a survival rate of 26%. Biological factors and time, as well as prolonged anaerobiosis, have a significant impact on egg viability. As far as composting by aeration is concerned, temperatures above 50°C are reached (de Bertoldi et al., 1985), these values represent high temperatures theoretically liable to alter egg viability. Provided this destruction capacity is real it remains variable, for the results obtained. Steer and Windt (1978) thus recommend a maturation of 60 days at 65°C to inactivate all Ascaris eggs, whereas a study of Theis et al. (1978) mentions a degeneration phenomenon with viable eggs found at the end of the composting period. Schwartzbrod et al. (1988) also reported an absence of viability of Ascaris eggs in a 120 days old compost (55-60°C). The large variety of results underlines the difficulty in finding a protocol that would ensure the
39
complete destruction of the infectious nature of eggs. The results of our study also shows that the eggs can be viable after 30 days at 60-70°C. This phenomenon might be explained by the difficulty in reaching a homogeneous compost temperature, as theoretical survival rates depending on temperature described by Feachem et al. (1983) show an inactivation of Ascaris eggs in 10 min at 55°C. Carrington (1985) also recommends a treatment of 2 h at 55°C in the case of pasteurization to get around the protective effect of sludge on eggs. The treatment with lime is not efficient instantaneously and requires a minimum contact time of 2 months with an initial concentration allowing the material to reach a pH of 12"5 (Schuh et al., 1985). With shorter times and lower concentrations of lime, eggs remain viable, as was confirmed by the 66% viable eggs recovered in our samples. The results of this study confirm that Nematode eggs are strongly resistant to the various urbansludge treatments. Moreover, lime and temperature treatments necessitate a strict compliance with homogeneity and specific values for the contact time, temperature and concentration of chemical product to be totally effective. ACKNOWLEDGEMENT
This study was supported by the NANCIE and the Agence de Bassin Rhin-Meuse (France). REFERENCES
Barbier, D., Perrine, D., Duhamel, C., Doublet, R. & Georges, P. (1990). Parasitic hazard with sewage sludge applied to land. Appl. Environ. Mierobiol., 56, 1420-2. Carrington, E. G. (1985). Pasteurisation: effects upon Ascaris eggs. In Inactivation of Micro-organisms in Sewage Sludge by Stabilisation Processes, ed. D. Strauch. Elsevier, London, pp. 121-5. Clarke, A. J. & Perry, R. N. (1980). Egg shell permeability and hatching of Ascaris suum. Parasitology, 80, 447-56. Cram, E. B. (1943). The effect of various treatment processes on the survival of helminth ova and protozoan cysts in sewage. Sewage Works J., 15, 1119-38. De Bertoldi, M., Frassinetti, S., Bianchin, L. & Pera, A. (1985). Sludge hygienisation with different compost systems. In Inactivation of Micro-organisms in Sewage by Stabilisation Processes, ed. D. Strauch. Elsevier, London, pp. 67-76. Feachem, R. G., Bradley, D. J., Garelick, H. & Mara, D. D. (1983). Sanitation and Disease: Health Aspects of Excreta and Wastewater Management. John Wiley & Sons, Chichester. Pike, E. B., Morris, D. L. & Carrington (1983). Inactivation of ova of the parasites Taenia saginata and Ascaris suum during heated anaerobic digestion. Wat. Poll. Control., 82, 501-9. Schuh, R., Phillip, W. & Strauch, D. (1985). Influence of sewage sludge with and without lime treatment on the development of Asearis suum eggs. In Inactivation of Micro-organisms in Sewage Sludge by Stabilisation Processes, ed. D. Strauch. Elsevier, London, pp. 100-12. Schwartzbrod, J., Thevenot, M. T., Collomb, J. & Baradel, J. M. (1986). Parasitologieal study of wastewater sludge.
40
P. G. Gaspard, J. Wiart, J. Schwartzbrod
Environ. Techn. Lett., 7, 155-62. Steer, A. G. & Windt, C. N. (1978). Composting and the fate of Ascaris lumbricoides ova. Water. S.A., 4, 129-33. Theis, J. H., Bolton, V. & Storm, D. R. (1978). Helminth
ova in soil and sludge from twelve U.S. urban areas. J. Wat. Poll. Control. Fed., 50, 2485-93. Wharton, D. A. (1980). Nematode egg shells. Parasitology, 81, 447-63.