Parasite enzymes involved in proteolysis and amino acid metabolism have attracted considerable attention over the last decade. Nevertheless, current knowledge is extensive for just a few parasites and several enzymes. Most enzymes remain largely unexplored. This review concentrates upon a selection of the better studied enzymes and the potentially valuable approaches now being adopted in their study. We present a personal view on the most suitable strategies for exploiting this area of parasite biochemistry with novel antiparasite drugs. The content of the review reflects our own work and interests, but we have aimed to include a sufficiently broad range of topics so that this overview serves as a useful introduction for those new to the subject.

There have been several reviews that provide good coverage of the appropriate literature (Barrett, 1991; McKerrow et al. 1993; North & Lockwood, 1995; Sakanari et al. 1995; Robertson et al. 1996; Vial, 1996; Coombs & Mottram, 1997; Walker & Barrett, 1997), therefore we detail here just some of the publications and refer readers to the reviews quoted for further information. This treatise mainly highlights progress made in studies with parasitic protozoa. Parasitic worms present more difficult problems for drug designers and there has been only limited progress to date in this area of biochemistry; we include here mention of just some of the more exciting advances so far.

When Small-Ear-Miniature pigs subcutaneously injected once with frozen oncospheres of Taiwan Taenia, Korea Taenia, T. saginata or T. solium emulsified with Freund's complete adjuvant (1·6 × 104/0·4 ml) were challenged orally with 1·6 × 104 viable eggs of Taiwan Taenia 41 days later, they all showed strong resistance compared with pigs vaccinated with T. solium. Most pigs (5/8) of the former 3 groups harboured no cysticerci. The number of cysticerci was 5·5±9·1 (mean±S.D.), whereas pigs of the T. solium group and control group harboured 160±30·1 and 661±637·2 cysticerci, respectively. All cysticerci recovered from vaccinated pigs and most cysticerci in control pigs were degenerated or calcified at 36–55 days after oral egg challenge. These results strongly suggest that oncospheres of Taiwan Taenia and Korea Taenia are very similar to T. saginata in their immunogenicity in pigs.

The effects of a range of FMRFamide-related peptides (FaRPs) on isometric contractility were tested using isolated muscle strips from the liver fluke, F. hepatica. The neuropeptides tested were the molluscan FaRPs, FMRFamide and FLRFamide, the turbellarian FaRPs, RYIRFamide and GYIRFamide, the cestode peptides, NPF and GNFFRFamide, and the nematode FaRPs, AF-1 (KNEFIRFamide), AF-2 (KHEYLRFamide), AF-8 (KSAYMRFamide), and PF-4 (KPNFIRFamide). Dose-response experiments were undertaken at a concentration range of 5 nM–5 μM for all of the neuropeptides tested. FMRFamide and AF-8 caused statistically significant increases in the amplitude and frequency of contractions at concentrations of 0·5 μM and 5 μM. FLRFamide and AF-2 also caused significant increases in contraction frequency at concentrations of 0·5 μM and 5 μM, although a significant increase in amplitude of contraction was observed only at a concentration of 5 μM. GYIRFamide increased both amplitude and frequency significantly at concentrations of 50 nM, 0·5 μM and 5 μM. RYIRFamide significantly increased frequency of contractions at concentrations of 0·5 μM and 5 μM, but failed to have a significant effect on contraction amplitude. AF-1 at a concentration of 5 μM increased contraction amplitude, but failed to have an effect on frequency at any of the concentrations used. PF-4 caused a statistically significant increase in both the amplitude and frequency of contractions at a concentration of 5 μM. NPF and GNFFRFamide had no effect on the in vitro motility of F. hepatica over the range of concentrations tested. The results are discussed in the light of possible structure–activity relationships in the FaRPs tested.

The distribution of Varroa jacobsoni on clustered, overwintering workers of Apis mellifera was investigated. The majority of mites were found between the 3rd and 4th ventro-lateral tergites of the abdomen with a significant preference for the left side of the host. This position would enable the mites to place their mouthparts in close proximity to the central portion of the bees' ventriculus. This may allow the mites access to nutrients at higher concentrations than would occur elsewhere in the haemolymph. At the start of winter, most infested bees carried only a single mite but towards the end increasing numbers of bees carried 2 or more mites. There was also an increase in the mite : bee ratio and more mobile mites (i.e. those moving about on the bees) were recorded. These results suggest that the mites suffer a lower overwinter mortality than the bees and are capable of transferring to a new host either before the original host dies or before it falls to the floor of the hive. This will have consequences for the transfer of diseases by Varroa within the hive.

The mechanism of linkage of phosphorylcholine (PC) to excretory–secretory products (ES) of adult Brugia pahangi has been investigated. Biosynthetic radio-isotope labelling of ES with [3H]choline followed by SDS–PAGE/fluorography revealed a smear of molecular weight approximately 40–100 kDa which loses its radiolabel following exposure to N-glycosidase F, but not mild alkali. PC is thus attached to this smear of molecules via N-type glycans, a mechanism of linkage previously observed with respect to PC–ES of Acanthocheilonema viteae. Western blotting analysis of non-radiolabelled ES demonstrated the existence of additional PC–ES which were insensitive to N-glycosidase F, but not to alkali. This second group of molecules is therefore likely to contain PC linked to O-glycans. Filarial nematodes may thus utilize 2 classes of glycan for attachment of PC. Examination of B. pahangi and A. viteae whole worm extracts by Western blotting indicated that their PC content could not be cleaved by N-glycosidase F and hence the use of N-type glycans may be restricted to a subset of ES products. The implications of these findings with respect to developing inhibitors of PC attachment for use as anti-filarial drugs are discussed.

The host–parasite relationship in taeniosis due to Taenia solium is practically unknown. Monoclonal antibodies were prepared against whole extracts of adult T. solium parasites and evaluated with tapeworms recovered from experimentally infected hamsters and with cysticerci from naturally infected pigs. With one antibody, mAb 4B3, it was possible to identify, purify and partially characterize a T. solium myosin. Some findings indicate that it corresponds to conventional myosin or myosin type II such as: purification with KCl, high molecular weight, size, structure (dimeric protein with globular and long tail portions), reaction with commercial anti-myosin antibodies, distribution in muscle fibres of parasites and cross-reactivity with antibodies against paramyosin from T. solium cysticerci. The reaction of the mAb was only with taeniids and not with other parasites. Also myosin was detected in faeces of infected animals and in supernatants of parasite cultures. Its presence in biological fluids may be useful for diagnosis of infected hosts.

A large field study has been performed in the Cochabamba region of Bolivia with the aim of comparing the polymerase chain reaction (PCR) with other diagnostic methods for Chagas' disease. The amplification of Trypanosoma cruzi-specific kinetoplast DNA sequences in blood samples was compared with classical serological methods, specific IgM detection and direct parasite visualization for 268 school children in a single village where Chagas' disease transmission is active. Of 113 children positive by classical serology or buffy coat examination, 106 were detected by PCR (sensitivity: 93·8%). We did not observe any significant difference of PCR sensitivity between initial (IgM and/or buffy coat positive) and indeterminate stage (only IgG positive) patients. Among the remaining 155 children unconfirmed as chagasic (who were either only IgM positive, IgG-, IgM-, and buffy coat-negative) only 1 case was PCR positive. This case may be due to DNA contamination, or to a very recent infection not detected otherwise, or to specific immune depression. These results show that PCR is a very sensitive parasitological test for Chagas' disease in active transmission regions. The future follow-up of the possibly infected patients who were only IgM-positive should clarify the interest of PCR and IgM tests in the detection of starting infections.

This paper describes the localization of paramyosin immunoreactivity in Schistosoma japonicum and represents the first comparative immunolocalization study among schistosome adult, cercariae and lung schistosomula by electron microscopy. A polyclonal antibody was utilized to immunolabel paramyosin or paramyosin-like proteins. Paramyosin was localized within the muscle layer of all 3 developmental stages. Furthermore, paramyosin was localized within granules of the post-acetabular glands of cercariae, and within the tegument matrix and surface of lung schistosomules. Adults and cercariae did not display any detectable paramyosin on the surface or within the tegument. The possible functions of paramyosin within S. japonicum and the relevance of these findings in relation to the reported protective properties of paramyosin as an anti-schistosome vaccine target molecule are discussed.

A metabolic pathway known as the mannitol cycle has been identified in Eimerian parasites. The pathway is a shunt off of the glycolytic pathway at fructose-6-phosphate (F6P). Two enzymes convert F6P to mannitol and two other enzymes are responsible for converting mannitol back to F6P when it is utilized. Although the pathway is present in various stages of the parasite the most apparent role of this pathway is in the sexual portion of the life cycle, particularly in the formation of oocysts. Extremely high concentrations of mannitol, approaching 0.3 M, are present in unsporulated oocysts. Mannitol functions as the endogenous energy source for oocysts to sporulate in the environment outside of the host. An inhibitory protein which inactivates the first enzyme of the mannitol cycle has been isolated from an oocyst derived inhibited enzyme complex and is believed to prevent the futile cycling of F6P during the maturation of oocysts. Evidence of the vital role of mannitol in the development and maturation of Eimeria tenella oocysts has been facilitated through the use of the drug Nitrophenide™, a known anticoccidial which has now been found to be an inhibitor of one of the enzymes responsible for the biosynthesis of mannitol in the parasite. This compound prevents the formation of oocysts and at lower doses reduces mannitol levels in shed oocysts. In addition, oocysts with reduced mannitol levels fail to complete the sporulation process lending further evidence for this polyol's role in the parasite.

An indirect immunocytochemical technique combined with confocal scanning laser microscopy has been used to demonstrate immunoreactivities to the nonapeptide, RPPGFSPFR (bradykinin, BK) and the endogenous flatworm regulatory peptide, GYIRFamide in the nervous system of the monogenean, Diclidophora merlangi. In addition, a simultaneous double-labelling technique was employed to examine possible co-localization of GYIRFamide- and neuropeptide F (NPF) immunoreactivities, using antisera to the C-terminal nonapeptide-amide of NPF (Moniezia expansa, FAIIGRPRF.NH2). BK immunostaining was restricted to a small population of nerve cells and associated fibres within the ventral nerve cords and to 2 pairs of nerve cells innervating the cirrus and the pharynx, respectively. No immunopositive nerve cells and fibres were identified within the brain or in association with the female reproductive apparatus. In contrast, GYIRFamide staining was abundant throughout the central and peripheral nervous systems, and appeared similar to the staining pattern revealed using an FMRFamide antiserum. GYIRFamide immunoreactivity was localized to nerve cells and fibres within the paired cerebral ganglia and the longitudinal ventral, dorsal and lateral nerve cords and their numerous interconnecting transverse commissures. The plexuses of the buccal suckers, pharynx and clamps of the haptor were strongly immunopositive for GYIRFamide, as were nerve cells innervating the ootype, the oviduct and the vitelline reservoir of the reproductive apparatus. Double-labelling experiments indicated an apparent co-localization of GYIRFamide and NPF immunoreactivities.

When crude extracts of Spirometra mansoni plerocercoid (sparganum) were analysed by SDS–polyacrylamide gel electrophoresis (PAGE)/immunoblot using patients' sera, IgE antibodies reacted specifically with 21, 27 and 53 kDa proteins. The 21 and 27 kDa proteins have been previously characterized as cysteine proteases. In this study, the 53 kDa protein was confirmed, by immunoprecipitation, to induce a specific IgE response. The protein was purified by affinity chromatography using an IgG1 (κ2) type mAb. The protein was partially sensitive to peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F (endo F) digestion. It exhibited an endoproteinase activity in a thiol-dependent manner preferentially degrading benzoyloxycarboxyl-phenylalanyl-arginyl-4-methoxy-β-naphthylamide (Z-phe-arg-MNA) of a panel of substrates tested. This endoprotease activity was maximal at pH 6·5 and in 0·1 M sodium phosphate. The proteolytic activity was inhibited by 10−5ML-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) and 1 mM iodoacetamide (IAA), and potentiated by dithiothreitol (DTT, 5 mM).

The impact of diets varying in type and level of carbohydrates resistant to endogenous enzymes and lignin on the establishment and location of Ascaris suum and Oesophagostomum dentatum was investigated experimentally. Fifty worm-free pigs, from a specific pathogen-free farm were used. The animals were assigned randomly to 5 diets and infected with 600 infective A. suum eggs and 6000 infective larvae of O. dentatum per pig. The diets consisted of a traditional ground barley plus protein feed (diet A), commercial full-constituent pelleted feed (diet B), barley flour plus protein (diet C), barley flour, inulin (Raftiline® ST, ORAFTI, Tienen, Belgium), sugar beet fibre plus protein (diet D), and barley flour, wheat bran, and protein (diet E). The faecal egg excretion was followed and the pigs were slaughtered at 8 weeks p.i. and samples taken from the small and large intestine. Intestinal contents were analysed for worm burdens, worm location and female worm fecundity along with the concentration of insoluble (chromic oxide) and soluble (polyethylene glycol-4000) markers, lignin, non-starch polysaccharides (NSP) and organic acids. In all diet groups A. suum worm burdens were low and comparable, whereas the O. dentatum worm burdens were significantly higher in pigs fed the diets with high levels of NSP and lignin (diets A and E) than in pigs fed diets B, C, and D. The present study suggests that a diet rich in lignin and insoluble NSP's provides favourable conditions for the establishment of O. dentatum in the large intestine of pigs while it is unlikely that the concentration of short-chain fatty acids and pH plays any major role.

We studied flea assemblages on rodents in different habitats of the Ramon erosion cirque in the Negev Desert to examine whether host–habitat relations influence flea spatial distribution. Eleven flea species parasitizing 12 rodent species were recorded. There was significant positive relationship between flea species richness and body mass of the host species; no relationships were found between relative richness of flea assemblage and either the number of habitats occupied by the host species or the size of host geographical range. The differences in pattern of flea parasitism among habitat types within host species were determined by both environmental features of a habitat and the specific pattern of habitat use by rodents. There was replacement of Xenopsylla conformis by Xenopsylla ramesis on Meriones crassus and Gerbillus dasyurus among different habitats. The results of ordination of the flea collections from each individual host demonstrated that the flea assemblages were segregated mainly along 4 axes, which explained 86% of total variance. Each of the ordination axes corresponded with a change in flea species composition. The directions of these changes were (1) among-hosts within a habitat and (2) among-habitats within a host.

Natural anti-proteases (α1-protease inhibitor (α1-PI; α1-antitrypsin) and α2-macroglobulin (α2-M)) were found in the blood of rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis. The α2-M inhibited Cryptobia salmositica proteases and was significantly higher in brook charr than in rainbow trout. Under in vitro conditions it took longer for the same number of parasites to neutralize the α2-M in charr than in trout blood. The haemolysis which occurred when C. salmositica was incubated in the blood of rainbow trout was due to neutralization of α2-M. This in vitro study also showed that it was the metalloprotease of C. salmositica that lysed red blood cells and the plasma of the two species of fishes initially prevented haemolysis by inhibiting the proteolytic activity. We suggest that the natural plasma α2-M plays an important role in defence against cryptobiosis in fishes.

Inhibitors of sterol and phospholipid biosynthesis in kinetoplastid parasites such as Trypanosoma cruzi, the causative agent of Chagas' disease, and different species of Leishmania have potent and selective activity as chemotherapeutic agents in vitro and in vivo. Recent work with the sterol C14α-demethylase inhibitor D0870, a bis triazole derivative, showed that this compound is capable of inducing radical parasitological cure in murine models of both acute and chronic Chagas' disease. Other inhibitors of this type, such as SCH 56592, have also shown curative, rather than suppressive, activity against T. cruzi in these models. Leishmania species have different susceptibilities to sterol biosynthesis inhibitors, both in vitro and in vivo. Leishmania braziliensis promastigotes, naturally resistant to C14α-demethylase inhibitors such as ketoconazole and D0870, were susceptible to these drugs when used in combination with the squalene epoxidase inhibitor terbinafine. Inhibitors of Δ24(25) sterol methyl transferase have been shown to act as potent antiproliferative agents against Trypanosoma cruzi, both in vitro and in vivo. New inhibitors of this type which show enhanced activity and novel mechanisms of action have been synthesized. Recent work has also demonstrated that this type of enzyme inhibitors can block sterol biosynthesis and cell proliferation in Pneumocystis carinii, a fungal pathogen which had previously been found resistant to other sterol biosynthesis inhibitors. Ajoene, an antiplatelet compound derived from garlic, was shown to have potent antiproliferative activity against epimastigotes and amastigotes of Trypanosoma cruzi in vitro; this activity was associated with a significant alteration of the phospholipid composition of the cells with no significant effects on the sterol content. In addition, alkyllsophospholipids such as ilmofosine, miltefosine and edelfosine have been shown to block the proliferation of T. cruzi and Leishmania and alter both the phospholipid and sterol composition. These results indicate the potential of lipid biosynthesis inhibitors as useful therapeutic agents in the treatment of leishmaniasis and Chagas' disease.

The blood and broncho-alveolar leucocyte (BAL) responses, to a primary Necator americanus infection, were studied in male BALB/c and NIH mice. Following percutaneous infection, a significant blood leucocytosis occurred in both the BALB/c and NIH strains. The peak response occurred, in both strains, on day 10 p.i. and this was reflected in the eosinophil response which peaked at the same time-point. The change in blood eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. In BALB/c mice, however, infection elicited a significantly more intense total leucocyte, lymphocyte and eosinophil response than in NIH mice. In contrast, the BAL response was more intense in the NIH strain. Peak BAL responses were observed between days 12 and 16 p.i., in both strains, and the change in broncho-alveolar eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. The relationship between the observed leucocyte responses and resistance to the migrating larvae of a primary infection is discussed.

Discocotyle sagittata displays a clearly defined nocturnal egg-hatching rhythm. When eggs of D. sagittata were incubated in alternating 12 h periods of light and darkness at 13 °C, the majority of larvae hatched within the first 2 h of darkness. Larvae were rarely recovered after 4 h of darkness, and none hatched in the light. Reversal of the light/dark cycle resulted in immediate reversal of the hatching rhythm, so that larvae emerged at the beginning of the new dark period. This suggests that hatching is a direct response to exogenous cues and not endogenously driven. In continuous darkness hatching was arrhythmic; hatching occurred in continuous light although total numbers of larvae emerging were suppressed. Field experiments confirmed that eggs exposed to the natural day/night cycle throughout development also demonstrated the precise hatching rhythm seen under controlled conditions. Mechanical disturbance played no part as a hatching stimulus. However, larvae hatched in the presence of both host mucus and gill tissue. D. sagittata does not show an egg-laying rhythm. The hatching responses have adaptive advantage related to the host's activity pattern. Trout are generally visual predators, inactive in darkness, providing a stationary target for emerging larvae responding either to darkness or to host hatching factors.

To study immunological responses in filarial infections, interleukin-2 receptor α (IL-2Rα) expression in the peripheral blood lymphocytes (PBL) of Lewis rats infected with Brugia pahangi was observed by flow cytometry. During infection, no increase in IL-2Rα was observed in either microfilaraemic or amicrofilaraemic rats. The PBL of rats were stimulated with phytohaemagglutinin (PHA) and the frequency of IL-2Rα-positivity was examined. After microfilariae appeared, the increase in the rate of IL-2Rα-positivity in microfilaraemic rats was less than in amicrofilaraemic rats. The anergy of IL-2Rα expression in microfilaraemic rats was restored when their lymphocytes were cultured with normal rat serum. IL-2Rα expression in normal uninfected rats decreased when cultured with serum from microfilaraemic rats. These results suggest that microfilariae-related factors in rat serum suppress PHA-induced IL-2Rα expression in PBL and thus inhibit cell proliferation and host immunity.

Leishmania and other trypanosomatid protozoa require reduced pteridines (pterins and folates) for growth, suggesting that inhibition of these pathways could be targeted for effective chemotherapy. This goal has not yet been realized, indicating that pteridine metabolism may be unusual in this lower eukaryote. We have investigated this possibility using both wild type and laboratory-selected antifolate-resistant strains, and with defined genetic knockouts of several pteridine metabolic genes. In Leishmania, resistance to the antifolate methotrexate is mediated through several mechanisms singly or in combination, including alterations in transport leading to reduced drug influx, overproduction (R-region amplification) or point mutation of dihydrofolate reductase-thymidylate synthase (DHFR-TS), and amplification of a novel pteridine reductase (PTR1, encoded by the H-region). All of the proteins involved are potential targets for antifolate chemotherapy. Notably, parasites in which the gene encoding dihydrofolate reductase (DHFR) has been deleted (dhfr-ts− knockouts) do not survive in animal models, validating this enzyme as a target for effective chemotherapy. However, the properties of pteridine reductase 1 (PTR1) suggest a reason why antifolate chemotherapy has so far not been successful in trypanosomatids. PTR1, by its ability to provide reduced pterins and folates, has the potential to act as a by-pass and/or modulator of DHFR inhibition under physiological conditions. Moreover, PTR1 is less sensitive to many antifolates targeted primarily against DHFR. These findings suggest that successful antifolate chemotherapy in Leishmania will have to target simultaneously both DHFR and PTR1.

The activation of peritoneal macrophage effector functions after therapy with free PZQ and PZQ incorporated in liposomes (lip.PZQ) was studied in the Mesocestoides corti–mouse model system. Each drug formulation was administered to an infected group of mice in 6 daily doses from day 14 p.i. Phagocytic activity of macrophages increased significantly after the administration of both drug formulations, more after lip.PZQ with an earlier peak observed for PZQ (day 3) than for lip.PZQ (day 6). Empty liposomes had no significant effect. The average counts of ingested particles in phagocytosing cells were significantly higher only after lip.PZQ administration. The pattern of changes in phagocytic activity correlated with the reduction of parasite numbers in the peritoneal cavity, with the highest observed on day 6 after therapy with lip.PZQ. Phagocytosis of lip.PZQ in vivo stimulated significantly the respiratory burst in peritoneal macrophages, with the highest concentration of superoxide anions recorded on day 1 after the last dose, whereas therapy with PZQ itself did not increase this process significantly. The capacity for the respiratory burst declined in all groups with progressing infection. It is proposed that the phagocytic activity of peritoneal macrophages after therapy was stimulated indirectly as a consequence of activation of the specific immune response. The larvicidal effect of lip.PZQ on the tetrathyridia in the peritoneal cavity was synergistic with the phagocytic activity and might be the result of double action of drug and superoxide anions generated during the respiratory burst stimulated by this drug formulation.