Transgenesis is an essential tool for assessing gene function in any organism, and it is especially crucial for parasitic nematodes given the dwindling armamentarium of effective anthelmintics and the consequent need to validate essential molecular targets for new drugs and vaccines. Two of the major routes of gene delivery evaluated to date in parasitic nematodes, bombardment with DNA-coated microparticles and intragonadal microinjection of DNA constructs, draw upon experience with the free-living nematode Caenorhabditis elegans. Bombardment has been used to transiently transfect Ascaris suum, Brugia malayi and Litomosoides sigmodontis with both RNA and DNA. Microinjection has been used to achieve heritable transgenesis in Strongyloides stercoralis, S. ratti and Parastrongyloides trichosuri and for additional transient expression studies in B. malayi. A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developing B. malayi larvae and results in transgene inheritance through host and vector passage. Assembly of microinjected transgenes into multi-copy episomal arrays likely results in their transcriptional silencing in some parasitic nematodes. Methods such as transposon-mediated transgenesis that favour low-copy number chromosomal integration may remedy this impediment to establishing stable transgenic lines. In the future, stable transgenesis in parasitic nematodes could enable loss-of-function approaches by insertional mutagenesis, in situ expression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins.

Large malaria epidemics in the East African highlands during the mid and late 1990s kindled a stream of research on the role that global warming might have on malaria transmission. Most of the inferences using temporal information have been derived from a malaria incidence time series from Kericho. Here, we report a detailed analysis of 5 monthly time series, between 15 and 41 years long, from West Kenya encompassing an altitudinal gradient along Lake Victoria basin. We found decreasing, but heterogeneous, malaria trends since the late 1980s at low altitudes (<1600 m), and the early 2000s at high altitudes (>1600 m). Regime shifts were present in 3 of the series and were synchronous in the 2 time series from high altitudes. At low altitude, regime shifts were associated with a shift from increasing to decreasing malaria transmission, as well as a decrease in variability. At higher altitudes, regime shifts reflected an increase in malaria transmission variability. The heterogeneity in malaria trends probably reflects the multitude of factors that can drive malaria transmission and highlights the need for both spatially and temporally fine-grained data to make sound inferences about the impacts of climate change and control/elimination interventions on malaria transmission.

We conducted the first comprehensive study on the spatiotemporal structure of trematode communities in the large-mouthed valve snail, Valvata macrostoma. A total of 1103 snails were examined monthly between May and October 2007 from Lake Konnevesi, Central Finland, from a shallow (1–2 m deep) and an offshore site (5–6 m deep), located ca. 50–70 m apart. Snails were infected by 10 trematode species. The species composition and prevalence were strikingly different between the sites with high species diversity in the shallow site (all 10 species; total prevalence of sporocysts/rediae 12·1%, metacercariae 55·4%) compared to the deeper site (3 species; prevalence 15·0% and 1·9%, respectively). This difference persisted throughout our study and is probably related to the spatial distribution of bird definitive hosts, whereas the seasonal parasite dynamics are likely to be affected by changes in the age-structure of the snail population. The probability of sporocyst infections increased with snail size, but no such trend was observed in redial or metacercarial infections which decreased with host size. Our results show that generally well-described spatiotemporal differences in trematode infection of molluscs can emerge in very narrow spatial and temporal scales, which emphasizes the importance of these factors in community studies.

Intensity of Ascaris lumbricoides infection was measured in terms of egg counts and worm burden in children 2–10 years of age. The expulsion of A. lumbricoides with a 3-day treatment of mebendazole occurred over 8 days, beginning on the second day of treatment. Ninety-seven percent of the worms were expelled between the second and seventh days. A rapid means of estimating eggs per gram (epg) by the Kato Katz technique correlated well with the method described by Martin & Beaver (1968). In spite of apparent density dependence in egg production, A. lumbricoides egg counts correlated well with worm burdens. It is concluded that, within the context of community surveys, epg is a reasonable means of identifying heavily infected individuals and that epg can be estimated rapidly by a slight modification to the standard Kato Katz technique.

Mice infected with as few as ten eggs of the nematode Trichuris muris are highly resistant to a challenge infection given 5 weeks later. The rate of elimination of challenge larvae is similar in mice immunized by infection with ten or with 300 eggs. Stimulation of immunity to challenge by the low-level infections is dependent upon the persistence of the immunizing worm population and removal of the population at day 14 considerably reduces the level of immunity. Worms developing from a low-level immunizing infection are not eliminated by a ‘self-cure’ response, as is the case with larger infections, and become sexually mature. Adult worms from such an infection are, however, eliminated by the response to a superimposed challenge infection. Repeated infection of mice with small numbers of eggs stimulates an immune response and the worm population is eliminated before patency.

The following list, compiled during my tenure of a grant given by the Nuffield Foundation to the Wildfowl Trust, Slimbridge, Gloucestershire, records species of Protozoa, helminths and Arthropoda found in anatid birds, either by the authors of the papers listed in the References, or by others to whose work these authors refer. It will be seen that there are, after the names of all the parasites, numbers which indicate, by reference to the corresponding numbers in the References, the authors and publications in which the species concerned are recorded. Thus Cotylurus brevis (142, 145*) means that this trematode was recorded from Aix galericulata, the Mandarin Duck, by Dubois (1953) and that Dubois & Rausch (1950a) reported an experimental infection of this duck with it.

Penetration and maturation of Schistosoma mansoni were compared in four strains of mice under controlled conditions, with the C3H as the reference strain. Differences were observed in (1) average numbers of cercariae penetrating, (2) percentage of individual mice penetrated by at least 90% of the available cercariae, (3) worm burdens, (4) maturation of penetrants, and (5) consistency of mice with reference to these characteristics. Criteria 2, 4 and 5 have not been evaluated in this way before.

Best average penetration (47%), greatest consistency, and highest percentage of samples with at least 90% of the cercariae penetrating were in mice of the C3H strain (85%). Of the other strains the Swiss albino strain most resembled strain C3H. Beige and hairless mice were comparatively resistant to penetration since only 60 and 30% respectively of these hosts had at least 90% of the cercariae penetrating. Hairless mice gave the best average worm recovery (23%) and parasite maturation (53%) and beige the poorest (15 and 34%). Mice of the Swiss and beige strains were highly variable in terms of parasite maturation.

The establishment of reproducible differences in infections of hosts at the sub-specific level provides basic information for investigation of factors which influence penetration and maturation and which are thus involved in host specificity and innate resistance in closely related animals.

The authors are grateful to Dr Margaret Deringer, National Cancer Institute, National Institutes of Health, for the hairless mice and to Mildred Walters, Naval Medical Research Institute, for assistance in the production of cercariae.

The germ-cell cycle and method of reproduction in the daughter sporocysts of the bucephalid Cercaria bucephalopsis haimeana (Lacaze-Duthiers, 1854) and the gymnophallid Cercaria dichotoma Lebour, 1911 (non Müller) from the edible cockle, Cardium edule L., have been investigated by the examination of stained sections. A review is given of the principal interpretative theories which attempt to explain the nature of the reproduction and the homology of the germinal sacs of the Digenea. It is suggested that the life-cycle includes a cyclic alternation of homologous generations. Of these, the miracidium/mother-sporocyst and the daughter germinal-sac generations usually reproduce by apomictic parthenogenesis but polyembryony may also occur secondarily in some species.

We are very grateful to Dr Gwendolen Rees and Professor E. W. Knight-Jones for considerable help with the manuscript.

Four monoclonal antibodies, raised against macroschizonts of Theileria parva, were studied to characterize their antigen binding specificity. The indirect fluorescent antibody test showed that the antigen(s) recognized were present in the macroschizont stage of the parasite life-cycle but not in piroplasm, kinete or sporozoite stages. Immunoblot analysis of macroschizont stage antigens suggested that all four antibodies recognized the same antigen. This was a molecule which varied in molecular mass between different parasite stocks, ranging from 68000 to 95000 Da. The antigen was localized by immunoelectron microscopy to the surface of the intracellular macroschizonts.

Interference microscopy has been used to study the rate at which water enters and is lost from larval and adult stages of Ditylenchus dipsaci and D. myceliophagus. It is shown that the cuticle of the dry nematode, irrespective of stage, is completely permeable to water. In contrast, marked intra– and interspecific differences were found in the rate at which stages dry at 0 and 50% relative humidity. All stages of D. myceliophagus dry very rapidly: in this species, death, as a consequence of severe desiccation, appears to be linked to a lack of any marked ability to control water loss. For D. dipsaci, those stages known to survive desiccation will appear to be the slower dryers. In general, the “order of performance” in slowing the rate of drying is the same as the order previously found for survival of desiccation. Both the 4th– and, to a lesser extent, the 3rd–stage larvae of D. dipsaci show evidence of an intrinsic ability to control water loss.

An analysis of the drying curve for 4th–stage larvae of D. dipsaci supports the indications of interference photographs that the rate of drying is slowed by a decrease in permeability of the drying cuticle.

Vacuum desiccation studies further demonstrate the remarkable ability of the 4th–stage larva of D. dipsaci to control its rate of drying. The survival of 4th– and 3rd–stage larvae of D. dipsaci is diminished by very rapid drying, and the survival of other stages of this species and larval and adult stages of D. myceliophagus is precluded. The decrease in survival of the 4th–stage larvae of D. dipsaci is shown to be associated with a more rapid rate of water loss.

The epidemiology of Strongyloides stercoralis was studied in families of clinical (reference) cases and their neighbours at endemic foci in Jamaica. Thirteen foci were studied based on the place of residence of a reference case. For each household of a reference case, the 4 most proximal neighbourhood households (spatial controls) were included in the study. Out of 312 persons contacted 244 were followed up using questionnaires, stool examination and serology. Prevalence of infection based on stool examination was 3·5% and on ELISA 24·2%. Prevalence increased with age but was not related to gender. Reference cases were significantly older than the general study population. The prevalence of infection based on both serology and stool examination was significantly higher in reference than in neighbouring households (the reference cases, themselves, were not included in the analysis). Furthermore, prevalence of infection was highest among persons who shared a bedroom with a reference case and decreased significantly with increasing spatial separation. This is indicative of close contact transmission which has not been previously shown for a geohelminth, but which is common among microparasites.

Two aromatic diamidines, furamidine (DB75) and its phenyl-substituted analogue (DB569), which exhibit trypanocidal activity, were assayed against Trypanosoma cruzi and were found to induce apoptosis-like death characteristics such as nuclear DNA condensation and fragmentation, decreased mitochondrial membrane potential and phosphatidylserine exposure. DB569 displays superior trypanocidal activity compared to furamidine and also had higher ability to induce apoptosis-like death in treated parasites. The present results showing apoptosis-like death in T. cruzi after treatment with both DB75 and DB569 make important contributions to the understanding of the mechanisms of the aromatic diamidines, which represent promising trypanocidal compounds.

Leptospirosis is a zoonotic bacterial disease of global importance. A large spectrum of asymptomatic animal hosts can carry the infection and contribute to the burden of human disease. Environmental sources of human contamination also point to the importance of a hydrotelluric reservoir. Leptospirosis can be caused by as many as 15 different pathogenic or intermediate Leptospira species. However, classification of these bacteria remains complicated through the use of both serological and genetic classification systems that show poor correlation. With the advent of molecular techniques, DNA-based barcoding offers a conceptual framework that can be used for leptospirosis surveillance as well as source tracking. In this review, we summarize some of the current techniques, highlight significant successes and weaknesses and point to the future opportunities and challenges to successfully establish a widely applicable barcoding scheme for Leptospira.

Although there is a growing recognition that the transfer of diseases between humans and non-human primates can be of great significance for conservation biology, there have been only a few studies focusing on parasites. In this study, saddleback (Saguinus fuscicollis) and moustached tamarin (Saguinus mystax) from the rainforest of the Peruvian lowlands were used as models to determine helminth parasite associations between canopy-dwelling primate species and a nearby human settlement. The human population showed high prevalences of infestation with a number of nematodes, including Ascaris lumbricoides (88·9%), Trichuris trichiura (37%) and hookworms (55·6%). However, the ova of these geohelminths were not detectable in tamarin faeces. Thus, no direct parasite transfer from humans to non-human primates could be documented. However, tamarin groups with more frequent contact to humans and their facilities had significantly higher prevalences and egg output of Prosthenorchis elegans, an important primate pathogen, than a forest group. In contrast, a cestode was significantly more common with more egg output in sylvatic than in human-associated groups. Human alteration of the habitat is likely to play a major role in determining the occurrence, prevalence and intensity of helminth infestation of wild non-human primates.

Descriptions of Babesia occultans have previously been restricted to sub-Saharan Africa. Here, we report the finding, for the first time, of this low or non-pathogenic bovine Babesia species in Tunisia, northern Africa. B. occultans DNA was detected by molecular methods in Hyalomma marginatum unfed ticks collected in 3 bioclimatic regions of Tunisia. The near-full-length 18S rRNA gene was sequenced and compared with related sequences retrieved from GenBank. Phylogenetic analysis indicated that other sequences deposited as Babesia sp. could also correspond to B. occultans, suggesting that this species may have a wide distribution in Mediterranean and Asiatic regions, and not only in sub-Saharan Africa as previously described. A B. occultans-specific Reverse Line Blot (RLB) oligonucleotide probe was designed for future epidemiological studies that would help to clarify this possibility.

The dense granule antigen 4 (GRA4) is known as an immundominant antigen of Toxoplasma gondii and, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-γ) production and survived completely against a subsequent challenge infection with a lethal dose of T. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronic T. gondii infections in mice.

Introduction. Evaluation of Leishmania drug susceptibility depends on in vitro SbV susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of SbV-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as SbV-resistant and -sensitive, in order to identify potential resistance markers. Methods. The differential expression of 13 genes involved in SbV metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. Results. Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of SbV-resistant compared to the group of SbV-sensitive parasites (P<0·01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. Discussion. Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro SbV resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro SbV susceptibility assays, but interfering with the gene expression patterns.

The evolutionary significance of chromosome size polymorphism was explored in a representative panel of 26 Trypanosoma cruzi stocks. We tested a progressive model (aCSDI) assuming that the larger the size difference between homologous chromosomes, the more divergent the parasites are. This was contrasted with a non-progressive model (Jaccard's distance), in which any chromosome size difference has the same weight. ACSDI-based dendrograms were very similar to those built-up from multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) data: structuring in 2 major lineages (T. cruzi I and T. cruzi II) and 5 small subdivisions within T. cruzi II was identical, and branching was very similar. Furthermore, a significant correlation (P<0·001) was observed between aCSDI and phenetic distances calculated from MLEE and RAPD data. In contrast, analysis of chromosome size polymorphism with Jaccard's distance generated dendrograms with relatively long branches, causing most branching points to cluster close together, which generates statistically uncertain branching points. Our results thus support a model of progressive chromosome size-variation and show that despite an extensive polymorphism, chromosomal sizes constitute valuable characters for evolutionary analyses. Furthermore, our data are consistent with the clonal evolution model previously proposed for T. cruzi.

The experimental studies of Brucei group trypanosomes presented here demonstrate that the balance of host and parasite factors, especially IFN-γ GPI-sVSG respectively, and the timing of cellular exposure to them, dictate the predominant MP and DC activation profiles present at any given time during infection and within specific tissues. The timing of changes in innate immune cell functions following infection consistently support the conclusion that the key events controlling host resistance occur within a short time following initial exposure to the parasite GPI substituents. Once the changes in MP and DC activities are initiated, there appears little that the host can do to reverse these changes and alter the final outcome of these regulatory events. Instead, despite the availability of multiple innate and adaptive immune mechanisms that can control parasites, there is an inability to control trypanosome numbers sufficiently to prevent the emergence and establishment of virulent trypanosomes that eventually kill the host. Overall it appears that trypanosomes have carefully orchestrated the host innate and adaptive immune response so that parasite survival and transmission, and alterations of host immunity, are to its ultimate benefit.

The aim of this work was to study the structure of the parasite communities of Digeneans of 2 families of Teleost fishes (Sparidae and Labridae) of the Mediterranean sea. We tried to quantify the importance of both the microhabitat requirements of the parasite species and the effect of host biological factors on the parasite communities. We applied, for the first time in parasite community studies, the Canonical Correspondence Analysis (CCA) to analyse (i) the spatial distribution of parasite species within the digestive tract of the hosts; (ii) the host's biological factors (such as diet, host length, gregariousness and abundance) that may influence this spatial distribution of parasite species. Our results showed that potential microhabitats were vacant in the 2 host families studied revealing a lack of niche saturation because either there was little inter- and/or intraspecific competition or there were enough available space and resources within the host. Our results also indicated that the position of the parasite in the digestive tract is much more important than host biological factors for the structure of parasite community. Finally, we highlight the potential use of the CCA method for controlling for phylogenetic constraints in multi-species analyses.