Parasitology Today, vol. 8, no. 8, 1992 273

worm burdens may have been associated with a reduced egg output per worm. Fewer worms may establish after reinfection but produce more eggs per worm. As outlined above, the evidence for this is inconclusive (see point 3). Nevertheless, the reinfection studies in The Gambia also provided evidence that lower levels of reinfection were associated with increases in immune reactivities and such immune responses may regulate (reduce?) parasite populations.

We share the opinion of Gryseels and Polderman that host-related tactors are important epidemiological determinants responsible for regulating schistosome populations in communities. While the results of recent reinfection studies may have highlighted this possibility, the concept is based on the original framework of Bradley ~°. In 1972, he suggested that parasite populations may be regulated by transmission (type I), by the host population by such means as sterile immunity or host mortality (type II) or by premunition or incomplete acquired resistance of individual hosts (type Ill). These types of regulation need not be exclusive. Although one type may predominate, all three may operate to regulate one parasite at one place and time, Type Iit regulation may be particularly

relevant to schistosomiasis, In areas of high transmission, the level of transmission may well be 'excessive', well above the level necessary for parasite persistence in the host population, In such a situation the regulation of parasite numbers by some other factor(s) such a premunition or acquired immunity would be one way of preventing superinfection. Bradley pointed out that, where type III regulation operated, attacks on the life cycle (snail control and chemotherapy for schistosomiasis) would require much effort for any return, offering one possible explanation of the limited effects reported for these approaches. Bradley concluded that, where type Ill regulation was due to partial immunity of the vertebrate host, the best approach to control might be immunization, aiding the natural method of parasite regulation, We do not feel that the profound implications of these conclusions have been fully appreciated and we welcome the opportunity offered by Gryseels and Polderman to bring them forward in discussion.

References I Wilkins, H.A. et al. (1987) Trans. R. Sac. Trap.

Med, Hyg. 81, 29-35 2 Bundy, D,A,P. and Blumenthal, U.J, (1990)in

Parasitism and Host Behaviour (Barnard, C.J, and Behnke, J,M., eds), pp 264-289, Taylor & Francis

3 Cheever, A,W. (I 968) Am. J. Trap. Med, Hyg, 17, 38-64

4 Medley, G. and Anderson, R.M, (I 985) Trans. R. Sac. Trap. Med. Hyg. 79, 532-534

5 Wertheimer, S,P. et al. (1987) Am. J. Trap. Med. Hyg. 37, 79-84

6 Bushara, H.O. et al, (I 980) Am. J. Trap. Med. Hyg. 29, 442~1-5 I

7 Boulanger, D, et al. (1991) Parasite Immunol. 13, 473-490

8 De Jonge, N. et al. (1989) Trans. R. S0c. Trap. Med. Hyg. 83, 368-372

9 Hagan, P. et al. (I 987) Trans. R. Sac. Trap. Med. Hyg. 81,938-946

10 Bradley, D.J, (1972) Trans. R. Sac. Trap. Med. Hyg. 66, 697-708

Paul Hagan Department of Zoology University of Glasgow Glasgow, UK G 12 8QQ

Ursula J. Blumenthal Department of Epidemiology and Population

Sciences London School of Hygiene and Tropical Medicine Keppel Street, London, UK WC I 7HT

H. Andrew Wilkins Medical Research Council Laboratories Fajara, Near Banjul, PO Box 273, The Gambia

Chitinases in Trypanosomatidae: a Cautionary Note

Chitin is a structural polysaccharide comprised of units of N-acetyl-J3-D- glucosamine. It does not occur in either vertebrates or vascular plants but it is a characteristic component of various invertebrate phyla and fungal cell walls. Chitinases (EC 3.2. I. 14) are found in a wide range of organisms, including a large number of non-chitin producers (for review see Ref. I ). They may be of particular significance to parasites of arthropods, which have to penetrate the chitin-rich peritrophic membrane that lines their host's midgut to ensure establishment and/or transmission. Examples of chitinolytic organisms include the bacterium Serratia marcescens 2, the microfilariae of Brugia malayi 3 and the baculovirus Autographa californica NPV (R. Hawtin et al., unpublished). Maudlin and Welburn 4 implicate maternally inherited chitinolytic bacteria in the susceptibility of tsetse flies to infection with trypanosomes. They ascribe this to an indirect effect of the chitinase releasing amino-sugars that inhibit lectin-mediated killing oftrypanosomes in refractory flies, but direct enzyme action, which weakens the peritrophic membrane, may also be involved, Huber et oi. s have shown that ookinetes of Plasmodium gallinaceum produce chitinases that may aid in penetration of the peritrophic membrane of Aedes oegypti. Similarly, Schlein et al, 6 have examined trypanosomatids and suggest that they also produce chitinase to aid

penetration of the peritrophic membrane. In their investigation, a number of Leishmania spp and other trypanosomatids were grown in Dulbecco's modified Eagle's medium with high glucose content, supplemented with 10% foetal calf serum and antibiotics.

We have recently conducted a series of experiments on chitinases in the procyclic stage of Trypanosoma brucei brucei. These were grown in medium SDM79 supplemented with 10% heat-inactivated foetal calf serum 7. Chitinase activity was measured using a fluorometric assay based on 4-methylumbelliferyl oligosaccharides ~, Our results were similar to those of Schlein et al. 6 However, on further investigation it was apparent that the uninoculated medium itself, supplemented with serum, showed chitinolytic activity, When we removed the chitinase activity from the medium by centrifugation with crab shell chitin and used 'cleaned' medium to grow procyclic forms of T. b. brucei, we saw no correlation between increase in chitinase activity and incubation time, as would have been expected if the cells were producing and secreting chitinase as implied by Schlein eta/, 6 No chitinase activity was detected in the harvested trypanosomes, corroborating the results of Schlein et al. 6

While some trypanosomatids do produce chitinases to aid penetration of the peritrophic membrane or to use chitin as a food source, in the light of our results there is no unequivocal evidence for chitinase synthesis by T. lewis~ 6 or T. b. brucei. However, the chitinase activity reported by Schlein et ol. 6 for Leishmania spp is considerably higher than that reported by them for T. lewisi and was also associated

with the Lefshmania cells, suggesting that they synthesize chitinases whereas cells of Trypanosoma spp do not.

Nevertheless, since foetal calf serum has strong chitinase activity, reports of chitinolytic activities in haemoflagellates or other organisms grown in medium supplemented with serum must be interpreted with extreme caution.

Acknowledgement We thank Keith Gull and Trevor Sherwin, University of Manchester, for the supply of cultures of T. b. brucei,

References I Gooday, G.W. ( 1991 ) Biodegradatian I,

177-190 2 Flyg, C, and Bowan, H.G. (I 988) Genet. Res.

52, 51-56 3 Fuhrman, J. et al. (1992) Proc. Natl Acad. Sci,

USA 89, 1548-1552 4 Maudlin, I. and Welburn, S.C. (1988)

Parasitology Today 4, 109- I I I 5 Huber, M. et al. ( 1991 ) Prac. Natl Acad. Sci.

USA 88, 2807-2810 6 Schlein, Y. et al. ( 1991 ) Prac. R. Sac. Land. B:

245, 121-126 7 Brun, R. and Schonenberger, M, (1979) Acta

Trap. 36, 289-292 8 McCreath, K.J. and Gooday, G.W. (1992)

J, Microbial. Methods 14, 229-237

Kevin Arnold Graham Gooday Department of Molecular and Cell Biology Marischal College Aberdeen, UK AB9 I AS

Leslie Chappell (and Kevin Arnold) Department of Zoology Tillydrone Avenue Aberdeen, UK AB9 2TN