RNA interference (RNAi) has become an invaluable tool for the functional analysis of genes in a wide variety of organisms including the free-living nematode Caenorhabditis elegans. Recently, attempts have been made to apply this technology to parasitic helminths of animals and plants with variable success. Gene knockdown has been reported for Schistosoma mansoni by soaking or electroporating different life-stages in dsRNA. Similar approaches have been tested on parasitic nematodes which clearly showed that, under certain conditions, it was possible to interfere with gene expression. However, despite these successes, the current utility of this technology in parasite research is questionable. First, problems have arisen with the specificity of RNAi. Treatment of the parasites with dsRNA resulted, in many cases, in non-specific effects. Second, the current RNAi methods have a limited efficiency and effects are sometimes difficult to reproduce. This was especially the case in strongylid parasites where only a small number of genes were susceptible to RNAi-mediated gene knockdown. The future application of RNAi in parasite functional genomics will greatly depend on how we can overcome these difficulties. Optimization of the dsRNA delivery methods and in vitro culture conditions will be the major challenges.

The treatment of visceral leishmaniasis (VL) in HIV-infected patients is characterized by having a protracted course and frequent relapses, despite the use of adequate anti-leishmanial drugs and effective anti-retroviral therapy. A small subset of patients with significant splenomegaly develops severe cytopaenias and chronic leishmania infection. The use of elective splenectomy is effective for restoring the haematological parameters and reduces the need for blood transfusions but it does not avoid relapsing visceral leishmaniasis.

Cholesterol is a major component of the erythrocyte membrane. In the present study, we investigated the effects of cholesterol reduction in host bovine erythrocytes (RBC) on the growth of Babesia bovis, a major bovine haemoprotozoon. An in vitro growth assay with bovine RBC that had been prepared by pre-treatment with a cholesterol depletion agent (methyl-β-cyclodextrin, MCD) showed that the culture with 5 mm MCD-treated RBC inhibited the growth of B. bovis significantly as compared with that with the control RBC. In further experiments, the treatment with 5 mm MCD was proved to suppress both activities of the parasite, erythrocyte invasion and replication within the infected RBC. In contrast, a slight reduction in the membrane cholesterol by 1 mm MCD treatment promoted both their growth and erythrocyte invasion activity. These results indicate that erythrocyte invasion and replication by B. bovis are affected by the amount of cholesterol in the host erythrocyte membrane.

During a routine health check of a wild-caught North American river otter (Lontra canadensis) small piroplasms were noted within erythrocytes. Analyses of the 18S ribosomal ribonucleic acid (rRNA) gene sequences determined that this was a genetically unique organism most closely related to Babesia microti-like parasites found in other small carnivores. Subsequently 39 wild-trapped North American river otters from North Carolina were tested for the presence of piroplasma deoxyribonucleic acid (DNA) via polymerase chain reaction and piroplasma DNA was detected in 82% (32/39) of these samples. Sequencing of partial 18S rRNA genes from selected cases determined that they were identical to the sentinel case. This report documents the existence of a genetically unique piroplasma in North American river otters and indicates that the prevalence of piroplasma in North Carolina otters is quite high. The pathogenic potential of this organism for otters or other species remains unknown.

Plasmodium malariae, a protozoan parasite that causes malaria in humans, has a global distribution in tropical and subtropical regions and is commonly found in sympatry with other Plasmodium species of humans. Little is known about the genetics or population structure of P. malariae. In the present study, we describe polymorphic genetic markers for P. malariae and present the first molecular epidemiological data for this parasite. Six microsatellite or minisatellite markers were validated using 76 P. malariae samples from a diverse geographical range. The repeat unit length varied from 2 to17 bp, and up to 10 different alleles per locus were detected. Multiple genotypes of P. malariae were detected in 33 of 70 samples from humans with naturally acquired infection. Heterozygosity was calculated to be between 0·236 and 0·811. Allelic diversity was reduced for samples from South America and, at some loci, in samples from Thailand compared with those from Malawi. The number of unique multilocus genotypes defined using the 6 markers was significantly greater in Malawi than in Thailand, even when data from single genotype infections were used. There was a significant reduction in the multiplicity of infection in symptomatic infections compared with asymptomatic ones, suggesting that clinical episodes are usually caused by the expansion of a single genotype.

Multiple invasions (MIs) or infections, i.e. those by more than 1 parasite in the same erythrocyte, could be the result of parasite density or, alternatively, to parasite-related factors or host-related factors. According to the last possibility, to our knowledge, only 3 laboratory studies of malaria have found an increase in the occurrence of MIs when antibodies to parasite antigens were present. Therefore, we tested the possibility that MIs were influenced by the host immune status, using as model the malaria-like parasite Haemoproteus infecting blue tits (Cyanistes caeruleus). Avian hosts infected with Haemoproteus were medicated with primaquine or injected with saline solution and the density of infection and the presence of MIs counted. Medication treatment reduced significantly the density of infection by Haemoproteus in females but not in males. For females, the presence of MIs was positively associated with both the density of infection and the immunoglobulin levels on each capture, but no association was found between the treatment and the presence of MIs. For males, the density of infection but not the immunoglobulin levels was positively associated with the presence of MIs. In addition, medicated males supported more MIs than controls. Our results represent the first line of evidence in the wild for a possible role of the host immune system promoting MIs.

Much evolutionary theory assumes that parasite virulence (i.e. parasite-induced host mortality) is determined by within-host parasite reproduction and by the specific parasite genotypes causing infection. However, many other factors could influence the level of virulence experienced by hosts. We studied the protozoan parasite Ophryocystis elektroscirrha in its host, the monarch butterfly, Danaus plexippus. We exposed monarch larvae to wild-isolated parasites and assessed the effects of within-host replication and parasite genotype on host fitness measures, including pre-adult development time and adult weight and longevity. Per capita replication rates of parasites were high, and infection resulted in high parasite loads. Of all host fitness traits, adult longevity showed the clearest relationship with infection status, and decreased continuously with increasing parasite loads. Parasite genotypes differed in their virulence, and these differences were maintained across ecologically relevant variables, including inoculation dose, host sex and host age at infection. Thus, virulence appears to be a robust genetic parasite trait in this system. Although parasite loads and genotypes had strong effects on virulence, inoculation dose, host sex and age at infection were also important. These results have implications for virulence evolution and emphasize the need for a detailed understanding of specific host-parasite systems for addressing theory.

Two morphologically novel Kudoa species are characterized from brain tissue of fish, Kudoa chaetodoni n. sp. from Chaetodon unimaculatus (Chaetodontidae) and Kudoa lethrini n. sp. from Gymnocranius audleyi and Lethrinus harak (Lethrinidae). Additionally we characterized a 5-spore valve (SV) Kudoa species from the brain of Sillago ciliata (Sillaginidae). Intriguingly, its 18S rDNA sequence was identical to that of the 7 SV Kudoa yasunagai extracted from the brain of a paralichthyid halibut in Japan. These 2 species may either prove to be con-specific, even though morphology and distribution differ, or demonstrate the limit of specific resolution in the small subunit rDNA gene region. Small subunit rDNA sequences from these new species were used in molecular phylogenetic analyses of kudoids to examine congruence of phylogeny with tissue tropism, geographical distribution, and host specificity. There was significant correlation between tissue tropism in the form of well-supported brain and heart-infecting clades. Host specificity and geographical distribution showed some correlations with genotype.

Urinary schistosomiasis is an important source of human morbidity in Msambweni, Kenya, where the intermediate host snail, Bulinus nasutus is found in ponds and water pools. In the past, aquatic habitats in the area have been studied separately; however, recent collections of B. nasutus snails and shells indicated that many of these ponds are in fact connected during and following sufficient rains. Satellite imagery and a geographical information system (GIS) were used to survey the main water courses and potential drainage routes, to locate potential source populations of snails and to determine probable snail dispersal routes. The 2 water bodies implicated as being the most important Schistosoma haematobium transmission foci in the area were found to differ in their degree of connectivity to other B. nasutus source habitats. One pond becomes connected even after normal rains, while the other pond requires prolonged rains or flooding to become connected with source habitats. Consequently, the transmission foci differ in their susceptibility to snail population control measures. Spatially explicit dispersal models that consider the spatial and temporal patterns of connectivity between aquatic habitats will contribute to improved snail surveillance and more focused control for urinary schistosomiasis at a local level.

The phylogenetic relationship of 5 genera, i.e. Diplozoon Nordmann, 1832, ParadiplozoonAchmerov, 1974, InustiatusKhotenovsky, 1978, SindiplozoonKhotenovsky, 1981, and EudiplozoonKhotenovsky, 1985 in the subfamily Diplozoinae Palombi, 1949 (Monogenea, Polyopisthocotylea) was inferred from rDNA ITS-2 region using neighbour-joining (NJ), maximum likelihood (ML) and Bayesian methods. The phylogenetic trees produced by using NJ, ML and Bayesian methods exhibit essentially the same topology. Surprisingly, freshwater species of Paradiplozoon from Europe clustered together with species of Diplozoon, but separated from Chinese Paradiplozoon species. The results of molecular phylogeny and lower level of divergence (4·1–15·7%) in ITS-2 rDNA among Paradiplozoon from Europe and Diplozoon and, on the other hand, high level of divergence (45·3–53·7%) among Paradiplozoon species from Europe and China might indicate the non-monophyletic origin of the genus Paradiplozoon. Also, the generic status of European Paradiplozoon needs to be revised. The species of Paradiplozoon in China is a basal group in Diplozoinae as revealed by NJ and Bayesian methods, and Sindiplozoon appears to be closely related to European Paradiplozoon and Diplozoon with their relationship to Eudiplozoon and Inustiatus being unresolved.

The impact of parasitism on population dynamics is determined in part by the numerical responses of parasites during population fluctuations of their hosts. Vole populations fluctuate in multi-annual cycles allowing such responses to be studied over successive phases of population growth, abundance and decline. We investigate how a helminth community (5 nematode and 7 cestode species) evolved over a full 6-year Water Vole (Arvicola terrestris) population cycle. Brillouin and individual parasite species richness (IPSR) indices were used to measure the numerical response of the parasite community. We report a correlation between levels of parasite intensity and vole population cycle phases. Both indices were consistently higher during pre-decline and decline phases for male and female voles alike. The numerical response of the parasite community suggests that populations may be regulated by parasitism and that studies of this mechanism should allow both for the cyclic or non-cyclic character of the host populations and for the response of the broadest possible set of the local parasite community.

Taxonomic revision by molecular phylogeny is needed to categorize members of the genus Echinococcus (Cestoda: Taeniidae). We have reconstructed the phylogenetic relationships of E. oligarthrus, E. vogeli, E. multilocularis, E. shiquicus, E. equinus, E. ortleppi, E. granulosus sensu stricto and 3 genotypes of E. granulosus sensu lato (G6, G7 and G8) from their complete mitochondrial genomes. Maximum likelihood and partitioned Bayesian analyses using concatenated data sets of nucleotide and amino acid sequences depicted phylogenetic trees with the same topology. The 3 E. granulosus genotypes corresponding to the camel, pig, and cervid strains were monophyletic, and their high level of genetic similarity supported taxonomic species unification of these genotypes into E. canadensis. Sister species relationships were confirmed between E. ortleppi and E. canadensis, and between E. multilocularis and E. shiquicus, regardless of the analytical approach employed. The basal positions of the phylogenetic tree were occupied by the neotropical endemic species, E. oligarthrus and E. vogeli, whose definitive hosts are derived from carnivores that immigrated from North America after the formation of the Panamanian land bridge. Host-parasite co-evolution comparisons suggest that the ancestral homeland of Echinococcus was North America or Asia, depending on whether the ancestral definitive hosts were canids or felids.

Trichinella spiralis is the nematode causative agent of trichinellosis, an intestinal and tissular parasitosis. Even though an early diagnosis during the intestinal phase is essential to limit the infection in humans, to date, there are no available tests to achieve this goal. Based on the immune response generated by the host's intestinal mucosa, the aim of this work was to develop ELISAs to assess the presence of coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes in stool samples of 18 individuals belonging to different outbreaks that have arisen in Argentina. By the methodologies developed in this work it was found that anti-muscle larva excretory-secretory products (ML-ESP) CAbs were detected in 89% of the samples analysed regardless of the time p.i. Anti-ML-ESP IgA, IgG, IgE and IgM were detected in 56%, 56%, 28% and 22% of the individuals respectively. Those samples negative for anti-ML-ESP total immunoglobulins proved positive for anti-adult worm-ESP CAbs. No CAgs were detected in any of the samples. The results obtained in this work indicate that the intestinal immune response in human trichinellosis is featured by all the isotypes of specific immunoglobulins. Furthermore, the detection of antibodies in stool samples, in either the free or complexed form, could be applied to confirm early human trichinellosis.

Entomopathogenic nematode infective juveniles are likely to encounter both uninfected and infected insects and host quality depends on the stage of the infection. We hypothesized that nematode response to infected hosts will change over the course of an infection. Here, we tested this hypothesis by focusing on the influence of host infection status on long-range attraction to host volatile cues. The attraction response of 3 nematode species (Steinernema carpocapsae, S. glaseri and S. riobrave) with different foraging strategies to infected and uninfected insects (Galleria mellonella and Tenebrio molitor) was tested at 24 h intervals from start of infection to emergence of infective juveniles from depleted host. As expected, based on their foraging strategies, S. carpocapsae was not very responsive to hosts, S. glaseri was highly responsive and S. riobrave was intermediate. Generally, the level of attraction did not change with time after infection and was similar between infected and uninfected hosts. An exception was S. glaseri infected T. molitor, which tended to be less attractive to S. glaseri than uninfected hosts. These results suggest that any influence of host infection status on infection behaviour is occurring at subsequent steps in the host-infection process than host attraction, or involves non-volatile cues.

In the present study, we describe the complete mitochondrial (mt) genome of the Atlantic salmon parasite Gyrodactylus salaris, the first for any monogenean species. The circular genome is 14 790 bp in size. All of the 35 genes recognized from other flatworm mitochondrial genomes were identified, and they are transcribed from the same strand. The protein-coding and ribosomal RNA (rRNA) genes share the same gene arrangement as those published previously for neodermatan mt genomes (representing cestodes and digeneans only), and the genome has an overall A+T content of 65%. Three transfer RNA (tRNA) genes overlap with other genes, whereas the secondary structure of 3 tRNA genes lack the DHU arm and 1 tRNA gene lacks the TΨC arm. Eighteen regions of non-coding DNA ranging from 4 to 112 bp in length, totalling 278 bp, were identified as well as 2 large non-coding regions (799 bp and 768 bp) that were almost identical to each other. The completion of the mt genome offers the opportunity of defining new molecular markers for studying evolutionary relationships within and among gyrodactylid species.

The complete nucleotide sequence of the mitochondrial genome was determined for the fish tapeworm Diphyllobothrium latum. This genome is 13 608 bp in length and encodes 12 protein-coding genes (but lacks the atp8), 22 transfer RNA (tRNA) and 2 ribosomal RNA (rRNA) genes, corresponding to the gene complement found thus far in other flatworm mitochondrial (mt) DNAs. The gene arrangement of this pseudophyllidean cestode is the same as the 6 cyclophyllidean cestodes characterized to date, with only minor variation in structure among these other genomes; the relative position of trnS2 and trnL1 is switched in Hymenolepis diminuta. Phylogenetic analyses of the concatenated amino acid sequences for 12 protein-coding genes of all complete cestode mtDNAs confirmed taxonomic and previous phylogenetic assessments, with D. latum being a sister taxon to the cyclophyllideans. High nodal support and phylogenetic congruence between different methods suggest that mt genomes may be of utility in resolving ordinal relationships within the cestodes. All species of Diphyllobothrium infect fish-eating vertebrates, and D. latum commonly infects humans through the ingestion of raw, poorly cooked or pickled fish. The complete mitochondrial genome provides a wealth of genetic markers which could be useful for identifying different life-cycle stages and for investigating their population genetics, ecology and epidemiology.