In Colombia, Plasmodium resistance to antimalarials such as chloroquine and antifolates is a serious problem. As a result, the national Colombian health authorities are monitoring the efficacy of alternative drugs and schemes. The study of genetic polymorphisms related with drug resistance is required in the region. In vitro responses to chloroquine, quinine, mefloquine, amodiaquine, desethylamodiaquine, artesunate and dihydroartesunate were carried out by HRP ELISA. SNP analysis in Pfcrt and Pfmdr1 genes was performed by PCR-RFLP in 77 samples from the North West region of Colombia. In vitro resistance to chloroquine was high (74%), followed by mefloquine (30%) and desethylamodiaquine (30%). A positive correlation between the IC50 of paired drugs was also detected. The allele Pfmdr1 N86 (wild) was present in 100% of the samples and 1246Y (mutant) in 92%. However, their presence did not correlate with in vitro drug resistance. Presence of the mutations K76T and N75E in Pfcrt was confirmed in all samples. Analysis of 4 codons (72, 74, 75 and 76) in pfcrt confirmed the presence of the haplotypes CMET in 91% and SMET in 9% of the samples.

For the evaluation of the epidemiology of Theileria equi and Babesia caballi in a herd of 510 horses in SW Mongolia, several mathematical models of the transmission dynamics were constructed. Because the field data contain information on the presence of the parasite (determined by PCR) and the presence of antibodies (determined by IFAT), the models cater for maternal protection with antibodies, susceptible animals, infected animals and animals which have eliminated the parasite and also allow for age-dependent infection in susceptible animals. Maximum likelihood estimation procedures were used to estimate the model parameters and a Monte Carlo approach was applied to select the best fitting model. Overall, the results are in line with previous experimental work, and add evidence that the epidemiology of T. equi differs from that of Babesia spp. The presented modelling approach provides a useful tool for the investigation of some vector-borne diseases and the applied model selection procedure avoids asymptotical assumptions that may not be adequate for the analysis of epidemiological field data.

A panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html ‘Microsatellite Locus Extractor’, and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1·6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.

Schistosomes are complex parasitic helminths with discrete life-cycle stages, adapted for survival in their mammalian and snail hosts and the external aquatic environment. Recently, we described the fabrication and use of a microarray to investigate gender-specific transcription in Schistosoma japonicum. To address transcriptional differences, 8 gender-associated gene transcripts identified previously by the microarray analysis were selected for further study. First, differential transcription patterns were investigated in 4 developmental stages using real-time PCR. Subsequently, we undertook functional analysis of a subset of 4 transcripts encoding metabolic enzymes, so as to correlate gender-associated transcript levels with enzyme activity in protein extracts from adult worms. The 8 characterized molecules serve as a basis for further investigation of differential gene expression during the schistosome life-cycle and for studying the sexual dimorphism of adult worms. Continual refinement and annotation of the microarray used in the current study should support future work on these aspects.

The lectin-inhibitory sugars D-glucosamine (GlcN) and N-acetyl D-glucosamine (GlcNAc) are known to enhance susceptibility of the tsetse fly vector to infection with Trypanosoma brucei. GlcNAc also stimulates trypanosome growth in vitro in the absence of any factor derived from the fly. Here, we show that GlcNAc cannot be used as a direct energy source, nor is it internalized by trypanosomes. It does, however, inhibit glucose uptake by binding to the hexose transporter. Deprivation of D-glucose leads to a switch from a metabolism based predominantly on substrate level phosphorylation of D-glucose to a more efficient one based mainly on oxidative phosphorylation using L-proline. Procyclic form trypanosomes grow faster and to higher density in D-glucose-depleted medium than in D-glucose-rich medium. The ability of trypanosomes to use L-proline as an energy source can be regulated depending upon the availability of D-glucose and here we show that this regulation is a graded response to D-glucose availability and determined by the overall metabolic state of the cell. It appears, therefore, that the growth stimulatory effect of GlcNAc in vitro relates to the switch from D-glucose to L-proline metabolism. In tsetse flies, however, it seems probable that the effect of GlcNAc is independent of this switch as pre-adaptation to growth in proline had no effect on tsetse infection rate.

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2·4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies†. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

We investigated whether host specificity is linked to variability within species of Lamellodiscus monogeneans, which are gill ectoparasites of the Sparidae. We sampled fish parasites in the northeastern part of the Mediterranean Sea: 4 specialist species, using 1 single host species, and 3 generalist species, using 2 distinct host species. Intraspecific variability was assessed from 2 different datasets. Morphometric variability of the attachment organ, called the haptor, was estimated first from measurements of several sclerified haptoral parts on 102 individuals. Genetic variability was calculated based on comparisons of sequences derived from the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA of 62 individuals. Morphometric variances in the specialist versus generalist species were compared via principal component analysis and F-tests, and uncorrected genetic distances (p-distances) were estimated within each species. We showed that the inter-individual variance of morphometric characters, as well as p-distances, are clearly greater within generalist species than specialist ones. These findings suggest that a relative increase in morphological and molecular variability enhances the possibility to colonize new host species in monogeneans, and supports the hypothesis that intraspecific variability could be a potential determinant of host specificity.

Trophically-transmitted parasites frequently alter multiple aspects of their host's phenotype. Correlations between modified characteristics may suggest how different traits are mechanistically related, but these potential relationships remain unexplored. We recorded 5 traits from individual isopods infected with an acanthocephalan (Acanthocephalus lucii): hiding, activity, substrate colour preference, body (pereon) coloration, and abdominal (pleon) coloration. Infected isopods hid less and had darker abdominal coloration than uninfected isopods. However, in 3 different experiments measuring hiding behaviour (time-scales of observation: 1 h, 8 h, 8 weeks), these two modified traits were not correlated, suggesting they may arise via independent mechanisms. For the shorter experiments (1 h and 8 h), confidence in this null correlation was undermined by low experimental repeatability, i.e. individuals did not behave similarly in repeated trials of the experiment. However, in the 8-week experiment, hiding behaviour was relatively consistent within individuals, so the null correlation at this scale indicates, less equivocally, that hiding and coloration are unrelated. Furthermore, the difference between the hiding behaviour of infected and uninfected isopods varied over 8 weeks, suggesting that the effect of A. lucii infection on host behaviour changes over time. We emphasize the importance of carefully designed protocols for investigating multidimensionality in host manipulation.

Phenotypic alterations induced by parasites in their intermediate hosts often result in enhanced trophic transmission to appropriate final hosts. However, such alterations may also increase the vulnerability of intermediate hosts to predation by non-host species. We studied the influence of both infection with 3 different acanthocephalan parasites (Pomphorhynchus laevis, P. tereticollis, and Polymorphus minutus) and the availability of refuges on the susceptibility of the amphipod Gammarus pulex to predation by 2 non-host predators in microcosms. Only infection with P. laevis increased the vulnerability of amphipods to predation by crayfish, Orconectes limosus. In contrast, in the absence of refuges, the selectivity of water scorpions, Nepa cinerea, for infected prey was significant and did not differ according to parasite species. When a refuge was available for infected prey, however, water scorpion selectivity for infected prey differed between parasite species. Both P. tereticollis- and P. laevis-infected gammarids were more vulnerable than uninfected ones, whereas the reverse was true of P. minutus-infected gammarids. These results suggest that the true consequences of phenotypic changes associated with parasitic infection in terms of increased trophic transmission of parasites deserve further assessment.

The original host of the swimbladder nematode Anguillicola crassus, the Japanese eel (Anguilla japonica) and the recently colonized European eel (Anguilla anguilla) were immunized with 40 irradiated (500 Gy) 3rd-stage larvae (L3) of this parasite and challenged with an infection of 40 normal L3. The immunization induced a significant reduction of the number of adult worms developing from the challenge infection in A. japonica, but not in A. anguilla. The induced resistance (calculated using the relation of the number of adult worms in immunized eels and in non-immunized control eels) in A. japonica was 87·3%±30·4%. Following a single infection, the percentage of adult worms found in A. japonica was lower as compared to A. anguilla, and the few adult worms were much smaller, revealing a lower susceptibility of A. japonica to A. crassus in comparison to A. anguilla. Both eel species developed an antibody response against A. crassus, but the level of antibody responses was not positively correlated with the protection against infection, suggesting that the antibody response is not a key element in resistance of eels against A. crassus. This study suggests that the original host of A. crassus is able to mount efficient protective immune responses against its parasite, whereas the newly acquired host seems to lack this ability.

Taenia saginata cysticercosis causes financial losses to the beef industry and farmers, and represents a significant source for human infection in many countries. A case-control study was conducted to identify risk factors for bovine cysticercosis on farms in Switzerland. The case group (n=119) consisted of farms with infected cattle identified at slaughter in 2005 and 2006. Infections were confirmed by morphological or molecular diagnosis. The control group (n=66) comprised randomly selected farms with cattle slaughtered in the same period but with no evidence or history of infection. In personal structured interviews with the farmers, information regarding local surroundings and farm management was collected. Logistic regression revealed the following 5 factors as being positively associated with the occurrence of bovine cysticercosis: the presence of a railway line or a car park close to areas grazed by cattle, leisure activities around these areas, use of purchased roughage and organized public activities on farms attracting visitors. This information is considered useful for government authorities to direct control strategies as well as for farmers to take measures tailored to local situations.

In this study, we investigated the presence of the larval stage of the tapeworm Echinococcus granulosus in wild ungulates in Estonia, genetically characterized E. granulosus isolates using mitochondrial gene sequences and used the sequence data, together with those available in a public database, to infer the phylogenic relationships of E. granulosus ‘genotypes’ G5-G10. While 0·8% of the 2038 moose (Alces alces) examined were found to be infected with E. granulosus, the parasite was not detected in other wild ungulates, such as roe deer (Capreolus capreolus: 1044 specimens examined) and wild boar (Sus scrofa: 442 specimens). Genetic analyses of concatenated atp6, nad1 and cox1 gene (1028 bp) sequences revealed that 2 novel E. granulosus haplotypes, namely E8 (11 samples: 69%) and E10 (5 samples: 31%), grouped with E. granulosus G8 and G10, respectively, are present in Estonia. This is the first record of an E. granulosus G8 in Eurasia. Phylogenetic analyses, using 4 different methods, demonstrated with considerable statistical support that E. granulosus G6/7 forms a subgroup together with G10, whereas G8 is a sister taxon to G6/7-G10.