The present study addresses the effect of varying temperature and host species on the size and shape of the opisthaptoral hard-parts in isogenic strains of Gyrodactylus salaris and G. thymalli. Variation in shape was examined using geometric morphometrics. Since the opisthaptoral hard-parts of Gyrodactylus have few specific landmarks, their shape information mostly being represented by outlines and surfaces, a method based on sliding semi-landmarks was applied. The ventral bars of G. salaris did not follow the previously postulated negative correlation between size and temperature, and the largest hamuli and marginal hooks from G. salaris and the smallest from G. thymalli clearly overlapped in size. Consistent shape differences with temperature were detected for the hard-parts from G. thymalli but not from G. salaris. The hard-parts of G. salaris were similar in size but significantly different in shape when grown on secondary hosts rather than the primary host.

The invasive blood stage of malaria parasites, merozoites, are complex entities specialized for the capture and entry of red blood cells. Their potential for vaccination and other anti-malaria strategies have attracted much research attention over the last 40 years, and there is now a considerable body of data relating to their biology. In this article some of the major advances over this period and remaining challenges are reviewed.

We have recently proposed revival of the name Entamoeba nuttalliCastellani, 1908 for a virulent amoeba (P19-061405 strain) isolated from a rhesus monkey (Macaca mulatta) and located phylogenetically between E. histolytica and E. dispar. In this study, E. nuttalli was isolated from feces of captive Japanese macaques (M. fuscata) in an open-air corral in Japan. The sequence of the 18S rRNA gene in the isolates differed from the P19-061405 strain in 2 nucleotide positions, but was identical to the EHMfas1 strain isolated previously from a cynomolgus monkey (M. fascicularis). One of the E. nuttalli isolates from Japanese macaques, named the NASA6 strain, was axenized and cloned. In isoenzyme analysis, the mobilities of hexokinase and phosphate glucose isomerase in the NASA6 strain were identical to those in the P19-061405 and EHMfas1 strains, but the mobility of phosphoglucomutase was different. These results were supported by gene analyses of these enzymes. Inoculation of NASA6 strain trophozoites into the liver of hamsters led to formation of an amoebic liver abscess. The liver lesions were characterized by extensive necrosis associated with inflammatory reactions. These results demonstrate that the NASA6 strain is potentially virulent and that E. nuttalli should be recognized as a common parasite in macaques.

Individual animals are often infected not only by different parasite species, but also by multiple genotypes of the same parasite species. Genetic relatedness among parasites sharing a host is expected to modulate their strategies of resource exploitation, growth and virulence. We experimentally examined the effects that genetic diversity and infection intensity had on host mortality, infectivity and growth of the marine trematode Maritrema novaezealandensis in amphipod hosts. The presence of 2 versus 1 parasite genotype during infection did not influence subsequent host mortality, had different effects on infectivity among genotypes and did not influence growth or variation in parasite growth. Density-dependent growth reductions revealed that the number of parasites infecting a host was more important than their genetic relatedness. Temperature, host size, and host sex influenced the degree to which density-dependent factors affected parasite growth. Our results suggest that the effects of parasite relatedness vary among parasite genotypes in this trematode species, and reveal that many factors play an important role during parasite development and transmission.

Following a period characterized by severe epidemics of sleeping sickness, restoration of effective control and surveillance systems has raised the question of eliminating the disease from sub-Saharan Africa. Given sufficient political and financial support, elimination is now considered a reasonable aim in countries reporting zero or less than 100 cases per year. This success may lead health authorities across the affected region to downgrade the disease from ‘neglected’ to simply being ignored. In view of the significant levels of under-reporting of sleeping sickness mortality in rural communities, this could be a short-sighted policy. Loss of capacity to deal with new epidemics, which can arise as a consequence of loss of commitment or civil upheaval, would have serious consequences. The present period should be seen as a clear opportunity for public-private partnerships to develop simpler and more cost-effective tools and strategies for sustainable sleeping sickness control and surveillance, including diagnostics, treatment and vector control.

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0·9% at the 18S rDNA and 10·7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38·2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0·5 of 0·1 μm and a Hill number (nH)=2·8±0·4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7·0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0·25±0·19 μm and Km2=29·6±6·8 μm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.

Gall bladders from 273 otter carcasses, collected throughout England and Wales, were screened to assess the status of gall bladder parasites in the Eurasian otter, Lutra lutra. The digenean Pseudamphistomum truncatum had previously been found in UK otters collected between 2000 and 2007. The parasite was established in Somerset and Dorset but its distribution elsewhere in the UK was largely unknown. In the current study, P. truncatum was also found to be abundant in south Wales, with occasional cases elsewhere, but appears to be absent from the north of England. Overall, 11·7% of otters were infected with 1–238 P. truncatum. A second digenean, Metorchis albidus, previously unreported in British otters, was found in the biliary system of 6·6% of otters. M. albidus appears well established in Suffolk, Norfolk and north Essex but was recorded elsewhere rarely. Both parasites are associated with pathological damage to the otter gall bladder. The recent discovery of these two non-native parasites provides a unique opportunity to assess their impact on native British fauna.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We characterized 2 novel GPx genes from a lung fluke, Paragonimus westermani (designated PwGPx1 and PwGPx2). These single copy genes spanned 6559 and 12 371 bp, respectively, and contained each of 5 intervening introns. The PwGPx2 harboured a codon for Sec and a Sec insertion sequence motif. Proteins encoded by the Paragonimus genes demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic and glutathione-binding domains and absence of the subunit interaction domain. Expression of PwGPx1 increased gradually as the parasite matured, whereas that of PwGPx2 was temporally regulated. PwGPx2 was expressed at the basal level from the metacercariae to the 3-week-old juveniles; however, the expression was significantly induced in the 7-week-old immature worms and reached a plateau in the 12-week-old adults and eggs. PwGPx1 and PwGPx2 were largely localized in vitellocytes within vitelline glands and eggs. Oxidative stress-inducible paraquat, juglone and H2O2 substantially augmented the PwGPx1 and PwGPx2 expressions in viable worms by 1·5- to 11-fold. Our results strongly suggested that PwGPxs may actively participate in detoxification of oxidative hazards in P. westermani.

Although there is an increasing understanding of the role of parasites in their host dynamics, accurate, quantitative estimates of parasite caused morbidity in wild animals are rare. Here, we examine the possible impact of 2 tick species (Ixodes ricinus, I. hexagonus) on the condition of the European hedgehog (Erinaceus europaeus). For this, we tested for correlations between blood parameters of 36 adult hedgehogs from an experimental population enclosed in a natural habitat and their tick infestation over a period of 8 months (March–October 2007). We found correlations between the tick infestation and the concentration of red blood cells, haemoglobin, haematocrit, MCH, MCHC, thrombocytes, lymphocytes and neutrophils. These results indicate that ticks can induce anaemia in the hedgehog. The peripheral blood characteristics and the erythrocyte indices characterize this anaemia as haemorrhagic and regenerative. During the course of our study the hedgehogs of our population showed below normal mortality but morbidity was found to be high resulting from the blood loss caused by the feeding activity of the ticks.

Renal infections by parasitic ciliates were studied in adult snails of Helix aspersa aspersa and Helix aspersa maxima collected from 2 mixed rearing system-based heliciculture farms located in Galicia (NW Spain). The occurrence of ciliates was also examined in slugs (Deroceras reticulatum) invading the greenhouses where first growing and fattening of snails is carried out. Histological examinations revealed a severe destruction of the renal epithelium in heavily infected hosts. Three ciliate isolates, one from each host species, were obtained and grown in axenic cultures. Cultured and parasitic ciliates were characterized morphologically and morphometrically. In addition, the encystment behaviour, the occurrence of autogamy, and the sequences of the mitochondrial cytochrome-c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (SSU rRNA) genes were also studied in the 3 isolates. A polymorphic life cycle involving resting and reproductive cysts, together with the morphological and morphometrical characteristics and the confirmation that autogamy occurs within cysts, demonstrate that our ciliates belong to the species Tetrahymena rostrata (Kahl, 1926) Corliss, 1952. The 3 isolates formed a well-supported clade using both genetic markers, and were clearly separate from the strain ATCC® 30770™, which has been identified as Tetrahymena rostrata. We argue that our Spanish isolates should be regarded as Tetrahymena rostrata, and that the ATCC isolate should be regarded as a misidentification as neither cytological nor cytogenetical support for its identity has been presented.

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.

Many complex life cycle parasites exhibit seasonal transmission between hosts. Expression of parasite traits related to transmission, such as the manipulation of host phenotype, may peak in seasons when transmission is optimal. The acanthocephalan Acanthocephalus lucii is primarily transmitted to its fish definitive host in spring. We assessed whether the parasitic alteration of 2 traits (hiding behaviour and coloration) in the isopod intermediate host was more pronounced at this time of year. Refuge use by infected isopods was lower, relative to uninfected isopods, in spring than in summer or fall. Infected isopods had darker abdomens than uninfected isopods, but this difference did not vary between seasons. The level of host alteration was unaffected by exposing isopods to different light and temperature regimes. In a group of infected isopods kept at 4°C, refuge use decreased from November to May, indicating that reduced hiding in spring develops during winter. Keeping isopods at 16°C instead of 4°C resulted in higher mortality but not accelerated changes in host behaviour. Our results suggest that changes in host and/or parasite age, not environmental conditions, underlie the seasonal alteration of host behaviour, but further work is necessary to determine if this is an adaptive parasite strategy to be transmitted in a particular season.

Hosts in nature will often acquire infections by different helminth species over their lifetime. This presents the potential for new infections to be affected (particularly via the host immune response) by a host's history of previous con- or hetero-specific infection. Here we have used an experimental rat model to investigate the consequences of a history of primary infection with either Nippostrongylus brasiliensis, Strongyloides venezuelensis or S. ratti on the fitness of, and immunological response to, secondary infections of S. ratti. We found that a history of con-specific, but not hetero-specific, infection reduced the survivorship of S. ratti; the fecundity of S. ratti was not affected by a history of either con- or hetero-specific infections. We also found that a history of con-specific infection promoted Th2-type responses, as shown by increased concentrations of total IgE, S. ratti-specific IgG1, rat mast cell protease II (RMCPII), IL4 (but decreased concentrations of IFNγ) produced by mesenteric lymph node cells in response to S. ratti antigen. Additionally, S. ratti-specific IgG1 was positively related to the intensity of both primary and secondary infections of S. ratti. Hetero-specific primary infections were only observed to affect the concentration of total IgE and RMCPII. The overall conclusion of these experiments is that the major immunological effect acting against an infection is induced by the infection itself and that there is little effect of prior infections of the host.

Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in β-tubulin genes have been associated with BZ-resistance. In the present study, we investigated β-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000–10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the ‘egg hatch test’, and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.

Third-stage larvae of Pseudoterranova decipiens commonly infect whitefish such as cod, and the parasite can be transferred to humans through lightly prepared (sushi) meals. Because little is known about the nematode's cold tolerance capacity, we examined the nematode's ability to supercool, and whether or not cold acclimation could induce physiological changes that might increase its ability to tolerate freezing conditions. Even if third-stage Pseudoterranova decipiens larvae have some supercooling ability, they show no potential for freezing avoidance because they are not able to withstand inoculative freezing. Still, they have the ability to survive freezing at high subzero temperatures, something which suggests that these nematodes have a moderate freeze tolerance. We also show that acclimation to high temperatures triggers trehalose accumulation to an even greater extent than cold acclimation. Trehalose is a potential cryoprotectant which has been shown to play a vital role in the freeze tolerance of nematodes. We suggest that the trehalose accumulation observed for the cold acclimation is a general response to thermal stress, and that the nematode's moderate freeze tolerance may be acquired through adaptation to heat rather than coldness.

Little is known of the prevalence and life-cycle of trypanosomes in mammals native to Australia. Native Australian trypanosomes have previously been identified in marsupials in the eastern states of Australia, with one recent report in brush-tailed bettongs (Bettongia penicillata), or woylie in Western Australia in 2008. This study reports a novel Trypanosoma sp. identified in blood smears, from 7 critically endangered Gilbert's potoroos (Potorous gilbertii) and 3 quokkas (Setonix brachyurus) in Western Australia. Trypanosomes were successfully cultured in vitro and showed morphological characteristics similar to members of the subgenus Herpetosoma. Phylogenetic analysis of 18S rRNA gene sequences identified 2 different novel genotypes A and B that are closely related to trypanosomes previously isolated from a common wombat (Vombatus ursinus) in Victoria, Australia. The new species is proposed to be named Trypanosoma copemani n. sp.

Over the last 30 years, evidence has been gathered suggesting that merozoite surface protein 1 (MSP1) is a target of protective immunity against malaria. In a variety of experimental approaches using in vitro methodology, animal models and sero-epidemiological techniques, the importance of antibody against MSP1 has been established but we are still finding out what are the mechanisms involved. Now that clinical trials of MSP1 vaccines are underway and the early results have been disappointing, it is increasingly clear that we need to know more about the mechanisms of immunity, because a better understanding will highlight the limitations of our current assays and identify the improvements required. Understanding the structure of MSP1 will help us design and engineer better antigens that are more effective than the first generation of vaccine candidates. This review is focused on the carboxy-terminus of MSP1.

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.