Plasmodium falciparum parasites use multiple ligand-receptor interactions to invade human erythrocytes. Variant expression levels of members of the PfRh and PfEBA ligand families are associated with the use of different erythrocyte receptors, defining invasion pathways. Here we analyse a major polymorphism, a large sequence deletion in the PfRh2b ligand, and erythrocyte invasion profiles in uncultured Senegalese isolates. Parasites vary considerably in their use of sialic acid-containing and protease-sensitive erythrocyte receptors for invasion. The erythrocyte selectivity index was not related to invasion pathway usage, while parasite multiplication rate was associated with enhanced use of a trypsin-resistant invasion pathway. PfRh2b protein was expressed in all parasite isolates, although the PfRh2b deletion was present in a subset (~68%). Parasites with the PfRh2b deletion were found to preferentially utilize protease-resistant pathways for erythrocyte invasion. Sialic acid-independent invasion is reduced in parasites with the PfRh2b deletion, but only in isolates derived from blood group O patients. Our results suggest a significant role for PfRh2b sequence polymorphism in discriminating between alternative erythrocyte receptors for invasion and as a possible determinant of virulence.

Plasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.

Thioredoxin reductase (TrxR), a NADPH-dependent disulfide oxidoreductase, is vital in numerous cellular processes including defence against reactive oxygen species, cell proliferation and signal transduction. TrxRs occur in 2 forms, a high Mr enzyme characterized by those of mammals, the malaria parasite Plasmodium falciparum and some worms, and a low Mr form is present in bacteria, fungi, plants and some protozoan parasites. Our hypothesis is that the differences between the forms can be exploited in the development of selective inhibitors. In this study, cyclodextrin- and sulfonic acid-derived organotelluriums known to inhibit mammalian TrxR were investigated for their relative efficacy against P. falciparum TrxR (PfTrxR), a high Mr enzyme, and Trichomonas vaginalis TrxR (TvTrxR), a low Mr form of TrxR. The results suggest that selective inhibition of low Mr TrxRs is a feasible goal.

Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5′-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4·7±1·0 μm but a fraction of about 40–50% was insensitive to CrATP. Remarkably, at low substrate concentration (0·2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0·33±0·10 μm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2′ disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites.

Cysteine peptidases of protozoa have been implicated in a variety of biological events, and the expression of these enzymes is modulated in response to distinct stimuli, including environmental changes and differentiation. In the present work, we have examined the expression of cysteine peptidases from Herpetomonas samuelpessoai grown at distinct temperatures and during dimethylsulfoxide (DMSO)-elicited differentiation. We demonstrated that a 45 kDa cysteine peptidase had its activity reduced during the parasite growth at 37°C in comparison to 26°C, and when cultured up to 72 h in the presence of DMSO. The modulation in the 45 kDa cysteine peptidase expression is connected to the differentiation process, since both temperature and DMSO are able to trigger the promastigote to paramastigote transformation in H. samuelpessoai. The possible immunological similarity of H. samuelpessoai proteins with well-known cysteine peptidases produced by trypanosomatid pathogens, including cruzipain (Trypanosoma cruzi) and cysteine peptidase b (cpb) from Leishmania mexicana, was also investigated, as well as with calpain molecules. The protein cellular lysate of H. samuelpessoai reacted with antibodies raised against cpb of L. mexicana and calpain of Drosophila melanogaster; however, no reaction was observed against cruzipain. The 35 kDa cpb-like protein had its expression diminished in DMSO-treated parasites, while the 80 kDa calpain-like molecule was enhanced and an additional 30 kDa calpain-related polypeptide was exclusively observed in these cells. Fluorescence microscopy and flow cytometry analyses corroborated these data. The results described above add H. samuelpessoai to the list of parasites whose differentiation seems to be correlated with cysteine peptidase expression.

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.

Aiming to further characterize the haemocyte subsets in Biomphalaria snails, we have performed a detailed flow cytometric analysis of whole haemolymph cellular components using a multiparametric dual colour labelling procedure. Ethidium bromide/acridine orange fluorescence features were used to first select viable haemocytes followed by flow cytometric morphometric analysis based on the laser scatter properties (forward scatter-FSC and side scatter-SSC). Our findings demonstrated that B. glabrata (BG-BH, highly susceptible to S. mansoni) and 2 strains of B. tenagophila (BT-CF, moderately susceptible and BT-Taim, resistant to S. mansoni) have 3 major circulating haemocyte subsets, referred to as small, medium and large haemocytes. The frequency of small haemocytes was higher in BG-BH, while medium haemocytes were the most abundant cell-type in both B. tenagophila strains. Schistosoma mansoni infection resulted in early reduction of large and medium circulating haemocytes followed by an increase of small haemocytes. Although parasite infection induced haemocyte alterations in all Biomphalaria strains, the response was particularly intense in BT-Taim, the parasite-resistant snail. Interestingly, the trematode infection induces changes in haemocytes with less granular rather than in those with more granular profile. The results indicated that, in B. tenagophila of Taim strain, circulating haemocytes, especially the medium and high subset with less granular profile, are very reactive cells upon S. mansoni infection, suggesting that this cell subset would participate in the early parasite destruction observed in this snail strain.

We report the discovery of a biological rhythm in the reproductive behaviour of the tick Bothriocroton hydrosauri that was absent in Amblyomma limbatum, a species that occurs on the same species of reptile host. Female B. hydrosauri mated in autumn or winter delayed oviposition until the following spring, while there was no diapause in conspecific females mated in spring or early summer. Initiation of ovipositional diapause in ticks is usually related to photoperiodic stimuli, but this was not the case for B. hydrosauri. The sinusoidal pattern in pre-oviposition times of B. hydrosauri females mated in different months in the laboratory suggests an internal seasonal time-keeping mechanism. We hypothesize that hormones imbibed by females during their bloodmeal may provide environmental cues associated with the induction of diapause. Irrespective of the mechanism underlying the rhythm, diapause by B. hydrosauri females mated during autumn or winter is of adaptive advantage because it synchronizes oviposition with favourable environmental conditions for egg hatching and increases the chance of larvae finding a host. The lack of a similar biological rhythm in A. limbatum may be a reflection of the different environmental conditions this species experiences throughout most of its range as compared with B. hydrosauri.

Different lineages experience different rates of phenotypic diversification, resulting in greater or lower variance in the expression of phenotypic traits among the species within a lineage. Here, morphological diversification is investigated in 14 different trematode families, based on a dataset comprising morphometric data on body size and 4 anatomical structures (oral sucker, ventral sucker, pharynx, cirrus sac) from 386 species. Three hypotheses are tested and subsequently rejected based on the empirical evidence. First, the degree of morphological variation in all traits within a trematode family, measured as the coefficient of variation among species, appears independent of the average body size of species belonging to that family. Second, patterns of morphological diversification appear similar whether endothermic or ectothermic vertebrates are used as definitive hosts. Third, phylogenetically older trematode lineages did not display greater morphological variation than younger, more derived ones, ruling out evolutionary time as an explanation. The results are consistent with developmental constraints acting on morphological diversification, since for some pairs of traits, variation in one trait is not independent of variation in another trait. More importantly, across most families, variation in body size was significantly more pronounced than variation in the relative sizes of the other morphological features. Trematode body size therefore varies widely while the general body architecture of the family is maintained. The fact that the evolution of the body plan is more conservative than that of body size suggests that the range of morphologies that can evolve in trematodes is constrained.

Nippostrongylus brasiliensis larvae are particularly susceptible to immunological attack during the pre-lung stage of primary and secondary infections in mice. Whilst most of the common laboratory strains of mice are permissive hosts for the parasite, in this study we report for the first time, the strong resistance of naïve FVB/N mice to N. brasiliensis. Damage to larvae is evident within the first 24 h of infection and this may be critical to later larval development and reproductive success. Inflammatory responses in the skin, and larval escape from this tissue were comparable in susceptible CBA/Ca and resistant FVB/N mice, with most larvae exiting within 4 h of a primary infection. Lung larval burdens were also similar between strains, but larvae recovered from FVB/N mice were smaller and less motile. In FVB/N mice, larval colonization of the gut was impaired and worms produced very few eggs. However FVB/N mice did not show enhanced resistance to Heligmosomoides bakeri (also known as Heligmosomoides polygyrus), a nematode largely restricted to the gut. Damage done in the pre-lung or lung stages of infection with N. brasiliensis is likely to contribute to ongoing developmental and functional abnormalities, which are profoundly evident in the gut phase of infection.

Angiostrongylus vasorum is a nematode parasite of sylvan and domestic species of the family Canidae. It has a broad but patchy distribution worldwide, and there is evidence for geographical spread and increasing incidence of infection in recent years. While historically Angiostrongylus-like nematodes identified in dogs and foxes have been described as A. vasorum in Europe and Angiocaulus raillieti in South America, more recent taxonomic revision has amalgamated these into a single species, A. vasorum. Here we report, for the first time, the molecular characterization of isolates of A. vasorum from Germany, Portugal, Denmark and the United Kingdom on the basis of the mitochondrial COI gene and the second ribosomal internal transcribed spacer. When compared with isolates from Brazil, sequence analysis revealed 2 distinct genotypes. Estimated rates of evolution based on COI sequences for both nematode and host are consistent with the hypothesis that the presence of A. vasorum in South America is a result of an ancient evolutionary event. Angiostrongylus vasorum in South America potentially represents a separate species to that observed in Europe.

Host-mediated responses and parasite density-dependent processes can have a major influence on the growth and fecundity of parasitic nematodes. However, host characteristics and parasite intensity consistently change during the course of an infection and these could affect worm length and number of eggs in a non-constant way. We used a free-living population of rabbits naturally infected with Trichostronglyus retortateformis and examined how adult nematode length and the number of eggs in utero were affected by host characteristics (i.e. age, sex, breeding status) and parasite intensity, in a seasonal environment, between 2004 and 2007. Nematode length and number of eggs in utero decreased exponentially with host age; in contrast, parasite intensity increased, peaked in juveniles and decreased in older hosts. These patterns were consistent between rabbit cohorts. A negative relationship was observed between parasite intensity and nematode length, as well as number of eggs. Nematode length was strongly affected by nematode sex and host age, while the number of eggs was mainly influenced by nematode length. The direct influence of host-mediated effects appeared quantitatively more important than parasite density dependence in controlling length and egg production in naturally infected wild rabbits. However, their relative contribution changed during the course of the infection such that, while host immunity still influenced worm numbers, the direct effect of density-dependent interactions contributed the most at high parasite intensities.