This study analyses the anti-proliferative effect of lemongrass essential oil and its main constituent (citral) on all 3 evolutive forms of Trypanosoma cruzi. Steam distillation was used to obtain lemongrass essential oil, with chemical composition determined by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). The IC50/24 h (concentration that reduced the parasite population by 50%) of the oil and of citral upon T. cruzi was determined by cell counting in a Neubauer chamber, while morphological alterations were visualized by scanning and transmission electron microscopy. Treatment with the essential oil resulted in epimastigote growth inhibition with IC50=126·5 μg/ml, while the IC50 for trypomastigote lysis was 15·5 μg/ml. The IC50/48 h for the Association Index (% macrophage infection×number of amastigotes per cell) was 5·1 μg/ml, with a strong inhibition of intracellular amastigote proliferation. Ultrastructural analysis demonstrated cytoplasmic and nuclear extraction, while the plasma membrane remained morphologically preserved. Our data show that lemongrass essential oil is effective against T. cruzi trypomastigotes and amastigotes, and that its main component, citral, is responsible for the trypanocidal activity. These results indicate that essential oils can be promising anti-parasitic agents, opening perspectives to the discovery of more effective drugs of vegetal origin for treatment of parasitic diseases. However, additional cytotoxicity experiments on different cell lines and tests in a T. cruzi-mouse model are needed to support these data.

We examined the relative importance of abiotic and biotic factors on the ability of adult Dermacentor variabilis ticks to attach and engorge with blood across 10 populations of free-ranging raccoons (Procyon lotor). We developed a priori models that represented explicit hypotheses based on the literature and tested the ability of these models to explain non-replete and replete (fully engorged with blood) tick infestation using generalized linear models and Akaike's Information Criterion. Abiotic models that included month and site of collection clearly provided a better fit for non-replete tick abundance data, while biotic models with host age and sex covariates best fit the replete tick data. Abiotic models of non-replete abundance were superior to biotic models because of large seasonal and site fluctuations in non-replete abundance that masked differences due to host characteristics. Conversely, best-fitting models of replete tick abundance included only age and sex and suggest that once a tick has reached a host, host-parasite interactions are the primary determinant of engorgement by female ticks. Host population structure may have a large influence on potential cohort size of ticks by reducing or increasing the total number and proportion that can become engorged and moult or lay eggs.

In this study, the proteome of axenically grown Entamoeba histolytica parasites was explored by two-dimensional gel electrophoresis (2-DE), employing a practical and effective procedure for the solubilization of E. histolytica proteins. Approximately 900 protein species in the pH range between 4 and 7 were detected by Coomassie Blue staining. Ninety-five spots were excised, trypsinated and subjected to mass spectrometry. The resultant data from peptide mass fingerprints were compared with those available in the E. histolytica genome and the (non-redundant) National Center for Biotechnology Information (NCBI) databases for the identification and categorization of proteins. Sixty-three of the proteins identified were predicted to relate to the cytoskeleton, surface, glycolysis, RNA/DNA metabolism, the ubiquitin-proteasome system, vesicular trafficking and signal transduction. The present study demonstrates, for the first time, that corresponding genes are indeed expressed in E. histolytica and provides a foundation for further proteomic studies of this parasite.

Cyclic GMP (cGMP) is responsible for various cellular functions including signal pathways and it acts as a mediator for nitric oxide (NO). In order to evaluate the anthelmintic efficacy of the plant-derived isoflavones, the crude peel extract of Flemingia vestita and pure genistein were tested with respect to the activity of nitric oxide synthase (NOS), NO efflux and the cGMP concentration in Rallietina echinobothrida, the cestode parasite of domestic fowl. For comparison, the parasites were also treated with genistein (the major isoflavone present in the crude peel extract), sodium nitroprusside (SNP), a known NO donor, and praziquantel (PZQ), the reference drug. At the time of onset of paralysis in the parasite, the activity of NOS showed a significant increase (35–46%) and a 2-fold increase of NO efflux into the incubation medium in the treated worms in comparison to the respective controls. The cGMP concentration in the parasite tissue increased by 46–84% in the treated test worms in comparison to the controls. The results show that the isoflavones, genistein in particular, from the crude peel extract of F. vestita influence the cGMP concentration in the parasite tissue, which plays a major role in the downstream signal pathways.

Neobenedenia girellae, a capsalid monogenean, is a significant pathogen due to both its ability to cause high mortality in fishes and its low host specificity. Established control methods have both advantages and disadvantages. Biological control measures with no unfavourable effects on the environment should be incorporated into the control strategy. The response of N. girellae oncomiracidia to brightness and black-and-white contrast was investigated to search for an alternative approach of disease prevention or control. Japanese flounder, Paralichthys olivaceus (Paralichthyidae), were exposed to oncomiracidia in an aquarium divided into areas of different brightness (∼1·3, 41·3 and 138·0 lux). The number of parasites on the fish group reared in 138·0 lux was significantly higher than on those reared in the lower brightness levels. Thus, the fish tended to be more vulnerable to infection by N. girellae under brighter conditions. Challenge trials using host fish mucus and whole live fish were established to detect the response by oncomiracidia to black-and-white contrast on a white versus a black background. Markedly more N. girellae oncomiracidia attached to black-painted areas and dark-coloured fish (normal spotted halibut, Verasper variegatus (Pleuronectidae) compared with white-painted areas and light-coloured fish (mal-coloured V. variegatus) on a white-coloured background. On a black-coloured background, more N. girellae oncomiracidia tended to attach to white-painted areas and light-coloured fish. Thus, black-and-white contrast is considered important for host finding by N. girellae oncomiracidia. The simplicity of the positive phototactic behaviour and the response to black-and-white contrast may lead to the development of a simple, practical and inexpensive method to control N. girellae outbreaks.

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.

Given that numerous amphibians are suffering population declines, it is becoming increasingly important to examine the relationship between disease and environmental disturbance. Indeed, while many studies relate anthropogenic activity to changes in the parasitism of snails and fishes, little is known of the impact on the parasites of amphibians, particularly from agriculture. For 2 years, the parasite communities of metamorphic northern leopard frogs from 7 agricultural wetlands were compared with those from 2 reference wetlands to study differences in parasite community diversity and abundance of various species under pristine conditions and 3 categories of disturbance: only agricultural landscape, only pesticides, and agricultural landscape with pesticides. Agricultural (and urban) area was negatively related to species richness, and associated with the near absence of adult parasites and species that infect birds or mammals. We suggest that agriculture and urbanization may hinder parasite transmission to frogs by limiting access of other vertebrate hosts of their parasites to wetlands. The only parasite found at all localities was an unidentified echinostome infecting the kidneys. This parasite dominated communities in localities surrounded by the most agricultural land, suggesting generalist parasites may persist in disrupted habitats. Community composition was associated with dissolved organic carbon and conductivity, but few links were found with pesticides. Pollution effects may be masked by a strong impact of land use on parasite transmission.

A multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers for Echinococcus multilocularis (amplicon size 395 bp) were species-specific as assessed by in silico analysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA from E. granulosus or Taenia spp. was not possible. The primers designed for E. granulosus also amplified DNA (117 bp) from E. vogeli, and those designed for Taenia spp. amplified products (267 bp) from species of Mesocestoides, Dipylidium and Diphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the described E. granulosus genotypes. Taenia spp. can be identified by direct sequencing of the 267 bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.

Genetic variation was examined in the anoplocephalid cestode Progamotaenia festiva, from Australian marsupials, in order to test the hypothesis that P. festiva, is a complex of sibling species and to assess the extent of host switching reported previously based on multilocus enzyme electrophoresis (MEE). Polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) was used for the analysis of sequence variation in the cytochrome c oxidase subunit 1 (cox1) gene among 179 specimens of P. festiva (identified based on morphology and predilection site in the host) from 13 different host species, followed by selective DNA sequencing. Fifty-three distinct sequence types (haplotypes) representing all specimens were defined. Phylogenetic analyses of these sequence data (utilizing maximum parsimony and neighbour-joining methods) revealed 12 distinct clades. Other heterologous species, P. ewersi and P. macropodis, were used as outgroups and the remaining bile-duct inhabiting species, P. diaphana and P. effigia, were included in the analysis for comparative purposes. The latter 2 species were nested within the clades representing P. festiva. Most clades of P. festiva identified were restricted to a single host species; one clade primarily in Macropus robustus was also found in the related host species M. antilopinus in an area of host sympatry; another clade occurring primarily in M. robustus occurred also in additional kangaroo species, M. rufus and M. dorsalis. High levels of genetic divergence, the existence of distinct clades and their occurrence in sympatry provide support for the hypothesis that P. festiva represents a complex of numerous species, most of which, but not all, are host specific. Three distinct clades of cestodes were found within a single host, M. robustus, but there was no evidence of within-host speciation.

Didymobothrium rudolphii (Cestoda: Spathebothriidea) was collected seasonally from the sand sole, Solea lascaris, off the northern, central and southern areas of the Portuguese coast. Morphological and molecular analyses were conducted in order to examine the possible existence of cryptic species and to facilitate the circumscription of their morphological boundaries. Data were compared between D. rudolphii specimens from each of the 3 geographical areas and 4 seasons, and principal components analysis of 18 morphological characters was used to detect differences. Two distinct genotypes were present with sequence divergences of 1·9% and 2·1% in the large subunit (lsrDNA) and second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA), respectively. The less common ‘central’ genotype was present only off the central area from summer to winter, whereas the ‘common’ genotype was present throughout the year off the northern and southern areas, but only during spring in the central area. No sequence variation was found within each genotype. The presence of 2 distinct genetic entities was supported by morphological analyses, which showed the ‘central’ genotype specimens to be more slender and elongate, although morphometric ranges overlapped considerably for most characters. Molecular phylogenetic analysis of 4 of the 5 known genera of the Spathebothriidea showed Spathebothrium to be the earliest branching lineage and the 2 genotypes of Didymobothrium formed a sister group to Cyathocephalus. The concordance of genetic differences with variation in host diet according to season and locality could account for sympatric speciation occurring in the central region of the Portuguese coast.

The aim of the present study was to investigate the potential benefits of grazing lactating ewes and their lambs on chicory (Cichorium intybus). Fifty-six certified organic twin-rearing ewes were either drenched with an anthelmintic or not, within 2 days after parturition and were grazed upon either grass/clover or chicory pastures. Around 12 weeks after parturition a subset of 12 lambs per treatment was slaughtered for worm number and parasite species determination. The faecal egg counts of lambs from undrenched ewes grazing on chicory were significantly lower than those of lambs from undrenched ewes grazing on grass. Lambs grazing on chicory had similar abomasal worm counts as those grazing on grass at 12 weeks of age; the predominant species was Teladorsagia circumcincta. There was no difference between the intestinal worm counts in lambs grazing on grass or chicory, with Trichostrongylus vitrinus being the predominant species. Liveweight gains over the 126-day experimental period were significantly higher in lambs from drenched than those from undrenched ewes. Lambs from undrenched ewes grazing on chicory had higher liveweight gains compared to those from undrenched ewes grazing on grass. Although chicory grazing did not affect ewe nematode egg excretion, it resulted in lower egg counts in lambs and improved their liveweight gains to the same level as those deriving from drenched ewes.

Stepek, G., Lowe A. E., Buttle D. J., Duce I. R. and Behnke J. M. (2007). Anthelmintic action of plant cysteine proteinases against the rodent stomach nematode, Protospirura muricola, in vitro and in vivo. Parasitology134, 103–112 (Published online 11 October 2006. doi:10.1017/S0031182006001302)

The authors of the above article regret that several errors appear in their published paper:

On Page 105: In the methods section entitled “In vivo assessment of anthelmintic efficacy of plant cysteine proteinases”, the sentence:

‘Five grams of papaya latex were mixed with 8 ml of sterile distilled water (dH2O), filtered, and the amount of active enzyme present was measured, by active-site titration, to be 331 nmol’

should read:

‘Five grams of papaya latex were mixed with 8 ml of sterile distilled water (dH2O), filtered, and the amount of active enzyme present was measured, by active-site titration, to be 13·24 micromol’

And the sentence:

‘Each group of mice received a different treatment: 0·2 ml of papaya latex alone (containing 8 nmol active enzyme), …’

should read:

‘Each group of mice received a different treatment: 0·2 ml of papaya latex alone (containing 331 nmol active enzyme), …’

On Page 110: In column 2, line 5, the sentence:

‘The amount of active enzyme administered in each dose (8 nmol) is based …’

should read:

‘The amount of active enzyme administered in each dose (331 nmol) is based …’

And on lines 10–15, the sentence:

‘Assuming the volume of a mouse stomach to be 1 ml and the enzyme to be present throughout the stomach at equal dilution, the concentration of enzyme would be in the order of 8 microM, which is somewhat lower than the in vitro concentrations.’

Should read:

Assuming the volume of a mouse stomach to be 1 ml and the enzyme to be present throughout the stomach at equal dilution, the concentration of enzyme would be in the order of 330 microM, which is about an order of magnitude higher than effective in vitro concentrations. A possible reason for the …’

A parasitological survey in the Bay of Fundy, Canada, resulted in the recovery of mature specimens from 5 species of phyllobothriid tapeworms (Cestoda: Tetraphyllidea) from 4 rajid skates: Echeneibothrium canadensis and E. dubium abyssorum specimens from Amblyraja radiata; E. vernetae and Pseudanthobothrium n.sp. from Leucoraja erinacea and L. ocellata; and P. hanseni from A. radiata and Malacoraja senta. Partial sequence data of a variable region (D2) from the large subunit ribosomal DNA (LSU) were used here to determine the host distribution of immature specimens for 4 of these 5 species (E. d. abyssorum was not included in the analyses). Immature specimens from both Pseudanthobothrium spp. were identified in the same hosts as recorded previously for mature specimens, thus suggesting that there are mechanisms that prevent the attachment of the parasite in an ‘unsuitable’ host species. Immature E. canadensis specimens were recovered exclusively from A. radiata, whereas immature E. vernetae specimens were recovered from L. erinacea and A. radiata, despite the latter host species not harbouring mature E. vernetae specimens. Their presence in the latter host species may be explained by host restriction or resistance, which allows the attachment of the parasites in the ‘wrong’ host species, but not establishment or development.

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.

Ascariasis is an important infection in humans (Ascaris lumbricoides) and pigs (Ascaris suum) and individuals appear to be predisposed to either heavy or light worm burdens. These extremes of susceptibility and resistance are represented in a mouse model by 2 strains of mice, CBA mice showing high resistance to infection and C57BL/6 which are highly susceptible, as reflected in worm burdens in the lungs 6–7 days after infection. In an attempt to identify the point at which the difference between these 2 strains is first manifested, we quantified worm burdens at key stages during infection leading up to the pulmonary stage of development. Thus mice were inoculated with fully embryonated A. suum eggs and larval burdens were enumerated in the large intestine and rectum, liver and lungs of the 2 strains at 6 h post-inoculation (p.i.) and on each of days 1–8 p.i. inclusively. A higher percentage of the total inoculum was recovered from the intestine/rectum of C57BL/6j mice in contrast to CBA/Ca mice at 6 h p.i. Larvae were recovered from the intestinal contents and also whilst actively migrating through the large intestinal wall. The number of larvae recovered was significantly reduced in CBA/Ca mice in contrast to C57BL/6j mice between the phase of migration from the liver and arrival in the lungs. The combined results of the inoculation of mice with corticosteroids and the examination of the change in profile and number of leukocytes present in bronchoalveolar lavage fluid suggested that the pulmonary inflammatory immune response was not prominently involved in primary protection of mice to A. suum infection in the latter days of infection in the lungs. The susceptible C57BL/6j mice produced a BAL response almost twice as intense as that of resistant CBA/Ca mice with stronger neutrophil, lymphocyte and eosinophil but not macrophage components, suggesting that the difference in worm burdens between the strains was generated earlier in the course of infection. These results were further corroborated by a histological examination of the lung tissues which showed that the passage of the larval stages of A. suum through the mouse lungs was associated with a marked inflammatory response in both strains. Again, C57BL/6j mice exhibited increased inflammation relative to CBA/Ca mice. Hence some hepatic/post-hepatic factor that varies between the 2 strains, but exerts its effect before the lung phase plays a critical role in determining the success of larvae through the host tissues. The possible sites of this host defence are reviewed.