A serine protease was purified 942-fold from culture supernatant of L. amazonensis promastigotes using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose and continuous elution electrophoresis by Prep Cell, yielding a total recovery of 61%. The molecular mass of the active enzyme estimated by SDS-PAGE under conditions of reduction was 56 kDa and 115 kDa under conditions of non-reduction, suggesting that the protease is a dimeric protein. Additionally, it was found to be a non-glycosylated enzyme, with a pI of 5·0. The optimal pH and temperature of the enzyme were 7·5 and 28 °C respectively, using α-N-ρ-tosyl-L-arginine-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 61% of the enzyme activity was preserved after 1 h of pre-treatment at 42 °C. Haemoglobin, bovine serum albumin (BSA), ovalbumin, fibrinogen, collagen, gelatin and peptide substrates containing arginine in an ester bond and amide substrates containing hydrophobic residues at the P1 site were hydrolysed by this extracellular protease. The insulin β-chain was also hydrolysed by the enzyme and many peptidic bonds were susceptible to the protease action, and 4 of them (L11-V12, E13-A14, L15-Y16 and Y16-L17) were identified. Inhibition studies suggested that the enzyme belongs to the serine protease class inhibited by calcium and manganese and activated by zinc. These findings show that this enzyme of L. amazonensis is a novel serine protease, which differs from all known flagellate proteases characterized.

This review addresses the potential use of neuropeptide receptors for the discovery of anthelmintic agents, and particularly for the identification of non-peptide ligands. It outlines which nematode neuropeptides are known and have been characterized, the published information on drug discovery around these targets, information about existing high- and low-throughput screening systems and finally the likely safety of neuropeptide mimetics.

The entomopathogenic nematode Steinernema carpocapsae is mutualistically associated with the bacterium Xenorhabdus nematophila. Infective Juveniles (IJs) transport X. nematophila cells that provide them with good conditions to reproduce within the insect. In the laboratory, long term stationary-phase culture conditions sometimes lead X. nematophila's variant 1 cells, which were previously isolated from the worms, to spontaneously and irreversibly change into a new phenotypic variant (variant 2). In this paper, we tested the ability of each phenotypic variant to (i) be transmitted by IJs, (ii) to optimize the worm's fitness within the insect, and (iii) to counteract the effect of closely related antagonistic bacteria previously shown as being able to totally prevent S. carpocapsae's reproduction within the insect. We found that IJs did associate with cells of both phenotypes but that the variant 2 cells were preferentially retained by the nematodes when both variants were present in the insect. Both phenotypic variants led to the same fitness of S. carpocapsae in insects not infected by antagonistic bacteria. In insects infected by antagonistic bacteria, both variants were able to provide protection to S. carpocapsae. Nevertheless, this protection depended on the phenotypic variant and the antagonistic bacteria that were co-injected into the insect. Further analysis conduced in vitro showed that this variability could be partly linked to the sensitivity of each antagonistic bacterium to xenorhabdicin, produced by X. nematophila.

Ternidens deminutus (Strongylida) is a parasitic nematode infecting non-human and human primates in parts of Africa, Asia and the Pacific islands. The present study genetically characterized T. deminutus and defined genetic markers in nuclear ribosomal DNA (rDNA) as a basis for developing molecular-diagnostic tools. The sequences of the second internal transcribed spacer (ITS-2) of rDNA were determined for adult specimens of T. deminutus (Nematoda: Strongylida: Oesophagostominae) from the Olive baboon and the Mona monkey.Nucleotide sequence data used in this paper are available in the EMBL, GenBank and DDJB databases under the Accession nos. AJ888729, AJ888730, AF136576, Y10789, Y10790, Y11733, Y11735, Y11736, AJ001594, AJ001599, AJ006149 and AJ006150. The former 2 sequences represent original data reported in this paper. The length and G+C content of the ITS-2 sequences was 216 bp and ~43%, respectively. While there was no sequence variation among individual T. deminutus specimens from the baboon, 6 (2·8%) nucleotide differences were detected in the ITS-2 between the parasite from baboon and that of the Mona monkey, which is similar to the difference (3·2%) between 2 other species of Oesophagostominae (Oesophagostomum bifurcum and O. stephanostomum) from non-human primates, suggesting significant population variation or the existence of cryptic (i.e. hidden) species within T. deminutus. Pairwise comparisons of the ITS-2 sequences of the 2 operational taxonomic units of T. deminutus with previously published ITS-2 sequences for selected members of the subfamilies Oesophagostominae and Chabertiinae indicated that species from primates (including those representing the subgenera Conoweberia and Ihleia) are closely related, in accordance with previous morphological studies. The sequence differences (27–48·3%) in the ITS-2 between the 2 taxonomic units of T. deminutus and hookworms (superfamily Ancylostomatoidea) enabled their identification and delineation by polymerase chain reaction (PCR)-based mutation scanning. The genetic markers in the ITS-2 provide a foundation for improved, PCR-based diagnosis of T. deminutus infections and for investigating the life-cycle, transmission patterns and ecology of this parasite.

The objectives of this study were (1) to examine risk factors for Schistosoma mansoni infection among schoolchildren living in western Côte d'Ivoire, and (2) to carry forward spatial risk prediction and mapping at non-sampled locations. First, demographic and socio-economic data were obtained from 3818 children, aged 6–16 years, from 55 schools. Second, a single stool sample was examined from each child by the Kato-Katz technique to assess infection status of S. mansoni and its intensity. Third, remotely sensed environmental data were derived from satellite imagery and digitized ground maps. With these databases a comprehensive geographical information system was established. Bayesian variogram models were applied for spatial risk modelling and prediction. The infection prevalence of S. mansoni was 38·9%, ranging from 0% to 89·3% among schools. Results showed that age, sex, the richest wealth quintile, elevation and rainfall explained the geographical variation of the school prevalences of S. mansoni infection. The goodness of fit of different spatial models revealed that age, sex and socio-economic status had a stronger influence on infection prevalence than environmental covariates. The generated risk map can be used by decision-makers for the design and implementation of schistosomiasis control in this setting. If successfully validated elsewhere, this approach can guide control programmes quite generally.

We have previously demonstrated that during pregnancy there exists an increased parasiticide activity against Trichinella spiralis newborn larvae (NBL) in infected rats. In this work we analysed the contribution of peritoneal cells from non-infected pregnant rats to the mortality of the NBL in cytotoxicity assays, and evaluated the role of progesterone in this effector mechanism. Our findings suggest that progesterone can induce activation of effector peritoneal cells to destroy the NBL in a rapid and antibody-independent manner. The administration of progesterone to ovariectomized rats also led to a significant decrease in the parasite load of the animals, thus demonstrating that progesterone induces the increase of the parasiticide activity of the leukocytes involved in the mechanisms of NBL death.

The problem of anthelmintic resistance prevents efficient control of parasites of livestock and may soon compromise human parasite control. Research into the mechanisms of resistance and the quest for diagnostic tools to aid control has required research that focuses on field resistance. On the other hand, resistant worms, including those kept in the laboratory, provide useful tools for studying drug action, especially at neuromuscular targets in worms. While the needs and directions of these research aims overlap, this review concentrates on research on drug targets. In this context, resistance is a useful tool for site of action confirmation. For example, correlations between molecular expression studies and resistance assays conducted on whole worms can strengthen claims for sites of anthelmintic action. Model systems such as Caenorhabditis elegans have been very useful in understanding targets but give a limited picture as it is now clear that resistance mechanisms in this worm are different from those in parasites. Accordingly, research on parasites themselves must also be performed. Resistant isolates of the sheep nematode parasite Haemonchus contortus are the most widely used for this purpose as in vivo, in vitro, physiological and molecular studies can be performed with this species. Neuromuscular target sites for the anthelmintics levamisole and ivermectin are the best studied and have benefited most from the use of resistant worm isolates. Resistance to praziquantel and the newer chemical groups should provide new tools to explore targets in the future.

Taeniosis/cysticercosis are important zoonotic infections which continue to be a significant problem in several parts of the world including Latin America, Asia and Africa. Control of these infections has always required the collaboration of both veterinary and medical public health services at a national level and these aspects have prompted the production of this publication jointly by the Food and Agriculture Organisation (FAO), the World Health Organisation (WHO) and the World Organisation for Animal Health (OIE). It has been produced to provide guidance on the design, implementation and standardization of control strategies against these zoonoses. It is a timely update on the previous guidelines produced in 1983 which were of significant importance in providing good overviews of the problems and strategies involved at the time.

Macroparasites are almost always aggregated across their host populations, hence the Negative Binomial Distribution (NBD) with its exponent parameter k is widely used for modelling, quantifying or analysing parasite distributions. However, many studies have pointed out some drawbacks in the use of the NBD, with respect to the sensitivity of k to the mean number of parasites per host or the under-representation of the heavily infected hosts in the estimate of k. In this study, we compare the fit of the NBD with 4 other widely used distributions on observed parasitic gastrointestinal nematode distributions in their sheep host populations (11 datasets). Distributions were fitted to observed data using maximum likelihood estimator and the best fits were selected using the Akaike's Information Criterion (AIC). A simulation study was also conducted in order to assess the possible bias in parameter estimations especially in the case of small sample sizes. We found that the NBD is seldom the best fit for gastrointestinal nematode distributions. The Weibull distribution was clearly more appropriate over a very wide range of degrees of aggregation, mainly because it was more flexible in fitting the heavily infected hosts. Moreover, the Weibull distribution estimates are less sensitive to sample size. Thus, when possible, we suggest to carefully check on observed data if the NBD is appropriate before conducting any further analysis on parasite distributions.

West-central China is an important endemic focus of both alveolar and cystic echinococcosis where several species of intermediate host are commonly infected with Echinococcus granulosus and E. multilocularis. Isolates of E. granulosus were collected from humans and other animals from different geographical areas of Qinghai, Ningxia, Gansu and Sichuan, and genotyped using the mitochondrial DNA marker ATP synthase subunit 6 gene (atp6). The sheep strain (G1 genotype) of E. granulosus was shown to be the only genotype present in sheep, cattle, goats, yaks and humans in the study areas. However, some heterogeneity in the atp6 sequence was evident in a number of the isolates with the most frequent change being a silent substitution (G/A) at position 360 compared with the G1 reference sequence representing isolates collected from the majority of hosts except humans. Two E. multilocularis isolates examined also had sequences that varied from each other and from the reference E. multilocularis atp6 sequence. The genotypic variation we report may reflect phenotypic differences with important consequences in terms of increased host infectivity for hosts by local Echinococcus strains, possibly impacting on the epidemiology and control of echinococcosis. Such adaptations may also result in different sensitivity to drugs or increased virulence for hosts that will impede control efforts and even affect vaccination strategies against Echinococcus.

Infective juveniles (IJs) of the entomopathogenic nematode (EPN) Steinernema feltiae IS-6 can survive exposure to 24% glycerol solution by entering an osmotically desiccated state. Exposure of osmotically desiccated nematodes to extreme temperature assays (40 °C for 10 h and −20 °C for 360 h) resulted in gradual reduction in survival, whereas non-desiccated IJs died within a short exposure to the assay conditions. Through SDS-PAGE, a stress-related protein UNC-87 was found in osmotically desiccated IJs exposed to 40 °C for 3, 6, and 8 h, whose survival rates were 98·9±1·43, 78·5±5·87 and 20·9±4·93%, respectively. The protein was not found in IJs following exposure of osmotically desiccated individuals to 40 °C for 10 h, in which none of the IJs survived. After exposure to −20 °C for 360 h, the survival of osmotically desiccated EPNs with a weak band of UNC-87 was 13·0±3·32%. To identify other responsive proteins that are required for osmotic stress, we used 2-dimensional electrophoresis to analyse the proteins in osmotically desiccated EPNs. The results revealed that 10 novel protein spots and 10 up-regulated protein spots in osmotically desiccated IJs were detected by digital image analysis. Mass spectrometry analysis of 7 significant spots indicated that osmotic stress in desiccated IJs was associated with the induction of actin, Proteasome regulatory particle (ATPase-like), GroEL chaperonin, GroES co-chaperonin and transposase family member. It seems to show actin, UNC-87 and Proteasome regulatory particle may play distinct roles in specific aspects of organization of macromolecular structures under desiccation stress. GroEL and GroES are members of the Hsp60 family of chaperons.

A potential control strategy for nematode infection in sheep is the implementation of a breeding programme to select for genes associated with resistance. The Texel breed is more resistant to gastrointestinal nematode infection than the Suffolk breed, based on faecal egg count, and this difference should enable the identification of some of the genes responsible for resistance. The objective of this study was to determine if variation at the ovine MHC-DRB1 locus was associated with variation in faecal egg count in Suffolk and Texel sheep. Ovar-DRB1 alleles and faecal egg count were determined for Texel (n=105) and Suffolk (n=71) lambs. Eight Ovar-DRB1 alleles, including 1 previously unknown allele, were identified in the Texel breed by sequence-base-typing. Seven Ovar-DRB1 alleles were identified in the Suffolk breed. Two Ovar-DRB1 alleles were common to both breeds, but were among the least frequent in the Suffolk population. In the Suffolk breed 1 Ovar-DRB1 allele was associated with a decrease in faecal egg count and 2 alleles with an increase in faecal egg count. This locus accounted for 14% of the natural variation in faecal egg count in Suffolks. There was no evidence for an association between Ovar-DRB1 alleles and faecal egg count in the Texel breed and the Ovar-DRB1 locus accounted for only 3% of the phenotypic variation in faecal egg count. These results suggest that the Ovar-DRB1 gene plays an important role in resistance to nematode infection in the Suffolk breed. The difference in faecal egg counts between these breeds may be attributable in part to the different allele profile at the Ovar-DRB1 locus.

The effect of Sanguinicola inermis on serum antibody and complement activity in Cyprinus carpio was assessed using an ELISA and haemolytic assays. Possible immune evasion strategies were assessed using immunodetection of host proteins on the surface of the parasite. Carp acclimatized to 20 or 25 °C were infected by exposure to 500 cercariae or injected intraperitoneally with 150 cercariae, and serum monitored over a 63-day period. In cercariae-injected carp, irrespective of time and temperature, a significant increase occurred in complement activity being greatest at 25 °C. In addition, fish exposed to the cercariae of S. inermis and maintained at 20 °C the level of complement activity was significantly higher after 5 weeks compared to controls. At 20 °C intraperitoneal injections of parasites increased serum antibody levels which peaked after 7 days. In contrast, at 25 °C, antibody levels were maintained over 63 days. Exposure of fish to infection did not appear to stimulate antibody production. Immunofluorescence studies revealed ‘host-like’ molecules on the surface of the cercarial body exposed to carp serum and adult flukes obtained directly from the fish or cultured for 24 h in L15 medium. The possible role of ‘host-like’ molecules in immune evasion is discussed and the response at different temperatures is related to infection dynamics.

Immunopathology of Chagas' disease in Balb/c mice infected with 2 Trypanosoma cruzi clones, belonging to the T. cruzi I lineage and presenting different in vitro virulence (P/209 cl1>SO34 cl4) was compared. In the acute phase, evading mechanisms such as parasite-induced lymphocyte polyclonal activation and T cell immunosuppression were higher in mice infected with the clone giving a higher parasitaemia (P/209 cl1). A similar increase of non-specific isotypes was observed in both infections with IgG2a prevalence. Interestingly, CD8+ cell hypercellularity and lymphocyte immunosuppression were observed during the chronic phase (245 days post-infection) in mice infected by the most virulent clone. In the same way, the parasite-specific antibody response was more intense in P/209 cl1-infected mice over the acute phase. During the chronic phase this response remarkably dropped down in SO34 cl4-infected mice exclusively. Finally, P/209 cl1-infected mice presented a more severe inflammation and tissue damage in heart and quadriceps than SO34 cl4-infected mice. This comparative study showed differences between the two clones: a higher virulence in vivo being clearly associated with a greater ability to induce evasion mechanisms and severe tissue damage.

Different parasite taxa exploit different host resources and are often unlikely to interact directly. It is unclear, however, whether the diversity of any given parasite taxon is indirectly influenced by that of other parasite taxa on the same host. Some components of host immune defences may operate simultaneously against all kinds of parasites, whereas investment by the host in specific defences against one type of parasite may come at the expense of defence against other parasites. We investigated the relationships between the species diversity of 4 higher taxa of ectoparasites (fleas, sucking lice, mesostigmatid mites, and ixodid ticks), and between the species richness of ectoparasites and endoparasitic helminths, across different species of rodent hosts. Our analyses used 2 measures of species diversity, species richness and taxonomic distinctness, and controlled for the potentially confounding effects of sampling effort and phylogenetic relationships among host species. We found positive pairwise correlations between the species richness of fleas, mites and ticks; however, there was no association between species richness of any of these 3 groups and that of lice. We also found a strong positive relationship between the taxonomic distinctness of ecto- and endoparasite assemblages across host species. These results suggest the existence of a process of apparent facilitation among unrelated taxa in the organization of parasite communities. We propose explanations based on host immune responses, involving acquired cross-resistance to infection and interspecific variation in immunocompetence among hosts, to account for these patterns.

Fourteen years ago I had the pleasure of reviewing the first edition of Mike Lehane's book. This new edition, long overdue, provides perhaps the most comprehensive, and a very readable, teaching text for medical entomology available today. The title truly reflects the content. Written from a biologist's viewpoint, we are treated to an overview of how blood-feeding insects function rather than the drier, phylogenetic-based treatment that is much more common. The scene is set by two chapters dealing with the importance and evolution of blood-feeding insects. We are then led, chapter-by-chapter through the blood-feeding process from host selection through to the digestion and utilization of a bloodmeal. The penultimate two chapters consider respectively the more intricate interactions of insects with their hosts and the parasites that they transmit. The classification of blood-feeding insects is covered in a final chapter.

During the cyst formation of Trichinella spiralis, the infected muscle cell undergoes basophilic change and apoptosis, which results in nurse cell formation. This study revealed expression kinetics of some apoptosis genes such as p53 and its closely related genes (tumor suppressor genes p53, p53; mouse double minute 2, MDM2; cyclin-dependent kinase inhibitor p21, p21waf). RT-PCR (reverse transcription polymerase chain reaction) results showed that these genes were temporarily expressed in the infected muscles during the cyst formation period, but not in normal muscles (or very low if any), which suggested the involvement of these apoptosis genes in the nurse cell formation. Cysts and neighbouring muscle cells were separately collected and RT-PCR was performed, which suggested that p53 was expressed in the cysts. An immunocytochemical study showed that p53 was expressed in the nucleoplasm of basophilic cell in the cyst and Trichinella larvae, which suggested involvement of these apoptosis genes in the nurse cell formation. The same p53 expression kinetic study was performed on p53 knockout mice. The knockout mice did not express p53 genes, but expressed the other apoptosis genes in the same kinetics with only minor exceptions, suggesting that the expressions of these genes during the cyst formation were more or less p53-independent. There were no differences in the number and morphology of the cysts between the knockout mice and wild type mice. Thus apoptosis seen during the Trichinella cyst formation can be operated in the presence or absence of p53.

Infection of the central nervous system by Taenia solium cysticerci is the cause of human neurocysticercosis, a major neurological infection in the Third World and an emerging infectious disease in the United States. We previously isolated a cysteine proteinase from cysticerci of Taenia crassiceps and demonstrated that it degrades human IgG in vitro. We have now isolated a 48 kDa thiol-dependent proteinase from T. solium. The T. solium enzyme also degrades human IgG, but does not significantly degrade albumin. IgG degradation was inhibited by cysteine proteinase inhibitors, but not significantly by inhibitors of aspartic, serine, or metalloproteinases. The peptide substrate specificity and pH optimum resemble cathepsin L. The Km for the peptide substrate Z-Phe-Arg-AFC was calculated to be 7·0×10−6M, the Kcat was 1·98×105 s−1, and the Kcat/Km 2·84×109M−1 s−1, a value which is within the diffusion control limit for highly catalytic enzymes. We propose that immunoglobulin degradation by the T. solium cysteine proteinase may play a key role in the host-parasite interface and could be employed as a target for chemotherapy.

A major goal of community ecology is to identify and explain non-random patterns of species composition. To date, the search for nested patterns in parasite component communities of the same fish host species has not been attempted, despite the fact that this higher hierarchical level is more relevant to nestedness analyses. The aims of this study are first, to determine the structure of component communities – considering ectoparasites and endoparasites separately – of a marine fish (Sebastes capensis) with an extended geographical distribution along the southeastern Pacific and, second, to explain these patterns by taking into account the extrinsic factors associated with the distribution of this host fish. From April to September 2003 and from April to August 2004, 537 fish were captured from different latitudes along the southeastern Pacific. The component communities of both ectoparasites and endoparasites of this fish host showed significant nested subset patterns. However, the type of nestedness pattern differed between ectoparasites and endoparasites. Ectoparasite component communities of S. capensis show higher species richness between latitude 30°S and 40°S, whereas endoparasite component communities show higher species richness between 40°S and 52°S. A nested pattern in ectoparasite component communities of S. capensis result from the gradual loss of some ectoparasites species southward and northward of the central part of their latitudinal distribution, which can be explained by the interaction of S. capensis with other host fish species from the central Chilean coast. Nestedness in endoparasite component communities of S. capensis is produced by the gains and losses of species toward the south of their latitudinal distribution, caused by changes in their prey-items (intermediate hosts) along their latitudinal distributional range.