Entamoeba moshkovskii and Entamoeba dispar are microscopically indistinguishable from the pathogenic species Entamoeba histolytica. Although sporadic cases of human infection with E. moshkovskii have been reported, the amoeba is still considered primarily as a free-living amoeba. A cross-sectional study was carried out among Orang Asli communities in 3 different states of Peninsular Malaysia. Fecal samples were examined by formalin-ether sedimentation and trichrome staining techniques and then single-round PCR assay was used to detect E. moshkovskii. Out of 500 fecal samples examined microscopically, 93 (18·6%) samples were positive for E. histolytica/E. dispar/E. moshkovskii complex cysts and/or trophozoites. PCR products were detected in 106 fecal samples. E. moshkovskii isolates were detected in 13 (12·3%) fecal samples. Of the 13 E. moshkovskii-positive samples, 5 were of single isolation of E. moshkovskii, 6 were also positive for E. dispar, and only 2 samples were positive for E. dispar and E. histolytica. Moreover, 3 E. moshkovskii-positive samples were collected from symptomatic individuals while the remaining 10 samples were from asymptomatic subjects. This is the first report on the identification of E. moshkovskii in Malaysia. Further studies are needed to confirm the pathogenicity of E. moshkovskii infection and determine the epidemiology among Orang Asli communities in Malaysia.

Significant mortalities of the Pacific oyster, Crassostrea gigas, have been reported worldwide since the 1950s. The impact these re-occurring mortality events have had on the C. gigas industry has highlighted the necessity to determine the factors that may be causing these mortalities. This study investigated the possible role of ostreid herpes virus (OsHV-1) in C. gigas mortalities over 2 successive summers at 2 study areas in Ireland. A single sample of adult C. gigas, which had been experiencing mortalities at one of the sites was screened. Successive cohorts of C. gigas spat obtained from a hatchery outside Ireland was relayed to both sites in 2003 and in 2004. Spat were screened each year prior to relaying. Samples were collected every 2 weeks and mortality counts were recorded and observed at both sites. Polymerase chain reaction (PCR) analysis and subsequent sequencing indicated that a previously undocumented variant genotype of OsHV-1 was present in the single cohort of adult C. gigas and in seed and juveniles at both sites, in both years. Analysis suggests that the Irish OsHV-1 μvar variant genotype is closely related to OsHV-1 μvar, first described in France in 2008.

Bile salts have been identified as an important trigger for excystation of Cryptosporidium oocysts but the hierarchy or synergism of this signal in relation to other triggers involved in excystation is poorly understood. In addition to excystation, bile salts have also been reported to increase the invasiveness of sporozoites within in vitro culture, possibly by affecting the secretory pathway via modification of intracellular calcium signalling. Nevertheless, incorporation of bile or bile salts into in vitro assays is not universal, with recent reports of negative effects on parasite growth. Here we report that bile and sodium taurocholate significantly affect both excystation rate and parasite in vitro growth. We demonstrate that their effect on excystation is dose, time and pre-treatment temperature dependent, while increases in parasite replication appear to be associated with modulation of parasite intracellular calcium and increased host cell susceptibility to infection. Notably, we illustrate that bile has a significant effect on host cells and can be cytotoxic at concentrations not much higher than those currently used for in vitro assays. This work should assist with more rational design of in vitro culture systems, with significant considerations for assay format when incorporating bile or bile salts as an excystation trigger.

The group of haemosporidian parasites is of general interest to basic and applied science, since several species infect mammals, leading to malaria and associated disease symptoms. Although the great majority of haemosporidian parasites appear in bird hosts, as in the case of Leucocytozoon buteonis, there is little genomic information about genetic aspects of their co-evolution with hosts. Consequently, there is a high need for parasite-enrichment strategies enabling further analyses of the genomes, namely without exposure to DNA-intercalating dyes. Here, we used flow cytometry without an additional labelling step to enrich L. buteonis from infected buzzard blood. A specific, defined area of two-dimensional scattergramms was sorted and the fraction was further analysed. The successful enrichment of L. buteonis in the sorted fraction was demonstrated by Giemsa-staining and qPCR revealing a clear increase of parasite-specific genes, while host-specific genes were significantly decreased. This is the first report describing a labelling-free enrichment approach of L. buteonis from infected buzzard blood. The enrichment of parasites presented here is free of nucleic acid-intercalating dyes which may interfere with fluorescence-based methods or subsequent sequencing approaches.

Eimeria tenella is recognized worldwide as a significant pathogen in the poultry industry. However, a lack of methods for isolating developing schizonts has hindered the use of transcriptome analyses to discover novel and developmentally regulated genes. In the present study, we characterized the long-term successive development of E. tenella in infected chicken caeca and assessed the utility of laser microdissection (LMD) for the isolation of schizont RNA. Developmental stages, including those of the first, second, and third-generation schizonts and gametocytes, were synchronous. Using LMD, only the mature second-generation schizonts were successfully excised from the lamina propria, and non-degraded RNA was purified from the schizonts. E. tenella-specific genes were amplified by reverse transcription polymerase chain reaction (RT-PCR). These results augment our understanding of the E. tenella life cycle, and reveal LMD as a potentially useful tool for gene expression analyses of the intracellular stages of E. tenella.

LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in the protozoan of the genus Leishmania, which is associated with the immunopathogenesis and susceptibility of BALB/c mice to Leishmania major infection. We previously demonstrated that intranasal immunization with a plasmid DNA encoding the p36/LACK leishmanial antigen (pCI-neo-LACK) followed by challenge 7 days after a booster dose effectively protects BALB/c mice against both cutaneous and visceral leishmaniasis. In the present study, the correlation between systemic mRNA expression after nasal DNA uptake, and the duration of protective immunity was addressed. LACK mRNA transcripts were detected in the spleen, brain, cervical lymph nodes and popliteal lymph nodes as early as 7 days, lasting 3 months after vaccination with pCI-neo-LACK. The kinetics of transcript expression correlated with enhanced cutaneous hypersensitivity against parasite antigens. Leishmania chagasi infection at 7 days or 3 months, but not 6 months after vaccination resulted in significantly lower parasite loads as compared with non-vaccinated controls. Protection also correlated with enhanced spleen cell responsiveness to parasite antigens leading to increased IFN- γ and IL-4 and decreased IL-10 production. Together, these data demonstrate that the protection conferred by the intranasal DNA vaccine lasts at least 3 months and is associated with expression of vaccine mRNA in peripheral organs.

We assessed the distribution of Trypanosoma cruzi Discrete Typing Units (DTUs) in domestic and peridomestic Triatoma infestans and Triatoma sordida specimens collected in a well-defined rural area in Pampa del Indio, northeastern Argentina. Microscopically-positive bugs were randomly selected with a multi-level sampling design, and DTUs were identified using direct PCR strategies. TcVI predominated in 61% of 69 T. infestans and in 56% of 9 T. sordida. TcV was the secondary DTU in T. infestans (16%) and was found in 1 T. sordida specimen (11%). Three T. sordida (33%) were found infected with TcI, a DTU also identified in local Didelphis albiventris opossums. Mixed DTU infections occurred rarely (5%) and were detected both directly from the bugs' rectal ampoule and parasite cultures. The identified DTUs and bug collection sites of T. infestans were significantly associated. Bugs infected with TcV were almost exclusively captured in domiciles whereas those with TcVI were found similarly in domiciles and peridomiciles. All mixed infections occurred in domiciles. TcV-infected bugs fed more often on humans than on dogs, whereas TcVI-infected bugs showed the reverse pattern. T. sordida is a probable sylvatic vector of TcI linked to D. albiventris, and could represent a secondary vector of TcVI and TcV in the domestic/peridomestic cycle.

Paraiotonchium autumnale (Nickle) (Tylenchida: Iotonchiidae) causes parasitic sterilization in female Musca autumnalis DeGeer (Diptera: Muscidae). In addition to sterilization, P. autumnale causes physiological and behavioural changes within its host. However, there have been no records of reduced host lifespan in this system. Studies were conducted in 2009 and 2010 with wild M. autumnalis collected as larvae from cow dung pats from Browns Valley, California, USA. Field-collected larvae were reared to adulthood and flies of the same eclosion dates were caged together, regardless of whether or not they were parasitized. Dead flies were collected daily, and parasitism status was confirmed by dissection. Due to the very different effects of P. autumnale-parasitism on male versus female face flies, flies were analysed separately by sex. Kaplan-Meier analysis revealed a non-significant difference in longevity between non-parasitized and parasitized flies for female and male flies in 2009. In 2010, however, significant differences (P < 0·05) were found between parasitized and non-parasitized female and male flies. In 2010, the median time to death for non-parasitized female flies was 20 days and 15 days for parasitized females. The 2010 median survival time for non-parasitized male flies was 33 days and 15 days for parasitized males. This is a parasite-mediated cost to the host that has not been recorded previously. Based on our results and results from another published study we suggest that the potential for sterilizing parasites to alter host longevity is condition dependent.

The sanitary control of threatened wild animals is of pivotal interest for their conservation. This task, however, is highly complex in wildlife/livestock systems. In this paper we report findings from a 2-year cross-sectional study of the epidemiology and attempted control of a Sarcoptes mite infestation in the threatened cheetah population in Masai Mara (Kenya), and discuss its interaction with sympatric wild (lion, wildebeest and Thomson's gazelle) and domestic (dog, cattle and sheep) animals. Sarcoptes scabiei was isolated from cheetahs, Thomson's gazelles, wildebeests, lions, cattle, goats and dogs; Psoroptes ovis, on the other hand, was only isolated from sheep. The prevalence study revealed 12·77% infection rates in cheetahs, 4·7% in dogs, 0·8% in Thomson's gazelles, 0·8% in sheep, 0·09% in cattle, and 0·09% in goats, while it opportunistically affected lions and wildebeest. Our study revealed that prevalence of Sarcoptes mite in cheetah population was not associated with the studied geographical blocks, animal sex or the presence of affected domestic animals. Cheetah infection with S. scabiei was associated with the climatic conditions (dry more than wet season) and the balancing between the total number of Thomson's gazelles and the prevalence of infected individuals. Apparently the high prevalence of mangy gazelles has a negative effect on cheetah; this negative effect was reduced when the number of healthy gazelles was increased. Treatment with injectable ivermectin of the clinically affected wild and domestic animals during the first year of this study was associated with much lower incidence of sarcoptic mange during the second year.

Correlations between host phenotype and vulnerability to parasites can clarify the processes that enhance rates of parasitism, and the effects of parasites on their hosts. We studied an invasive parasite (the pentastome Raillietiella frenatus, subclass Pentastomida, order Cephalobaenida) infecting a new host (the invasive cane toad Rhinella marina), in tropical Australia. We dissected toads over a 27-month period to investigate seasonal changes in pentastome population dynamics and establish which aspects of host phenotype are related to infection. Pentastome prevalence and intensity varied seasonally; male toads were 4 times more likely to be infected than were females; and prevalence was highest in hosts of intermediate body size. The strong sex effect may reflect habitat or dietary divergence between the sexes, resulting in males encountering parasites more often. The relationship between pentastome prevalence and host size likely reflects a role for acquired immunity in preventing re-infection. Infection did not influence host body condition (fatbody size), suggesting that R. frenatus does not impose high energy costs in cane toads. Infected toads had heavier spleens (likely an immune response to infection) and larger testes (perhaps since reproductively active hosts have altered microhabitat use and/or immunocompetence) than did uninfected conspecifics. Although experimental studies are required to identify the causal bases of such patterns, our data confirm that infection status within a population can be strongly linked to host phenotypic traits.

Aggregation of parasites amongst hosts is important for the epidemiology of vector-borne diseases because hosts that support the majority of the vector population are responsible for the majority of pathogen transmission. Ixodes ricinus ticks transmit numerous pathogens of medical importance including Borrelia burgdorferi s.l. and tick-borne encephalitis virus. One transmission route involved is ‘co-feeding transmission’, where larvae become infected via feeding alongside infected nymphs. The aggregation of ticks on hosts leads to an increase in the number of larvae feeding alongside nymphs, increasing the transmission potential via this route. The basic reproduction number, R0, can be used to identify whether a pathogen will become established if introduced. In the current study we use previously published tick, and pathogen, specific data to parameterize an R0 model to investigate how the degree of aggregation of ticks on hosts affects pathogen persistence. The coincident aggregated distribution permitted the establishment of tick-borne encephalitis virus but did not influence whether B. burgdorferi s.l. became established. The relationship between the k-exponent of the negative binomial distribution and R0 was also defined. Therefore, the degree of aggregation of ticks on small mammal hosts has important implications for the risk to human health in a given area.

Lice are obligate parasites of mammals and birds and have become an important model for studies of host-parasite co-evolution and co-phylogenetics. Population genetic and phylogeographic studies represent an important bridge between microevolution and co-phylogenetic patterns. We examine co-phylogeographic patterns in sika deer and their parasitic lice. Co-phylogeographic patterns in deer and lice were evaluated using homologous regions of mitochondrial COI sequences. The phylogeographic breaks recovered for deer populations matched those of previous studies. Comparisons of the phylogeographic tree topology for deer lice with that of their hosts revealed a significant level of congruence. However, comparisons of genetic distances between deer and lice suggested that one of the estimated co-divergence events is more likely a recent host switch. Taking into account genetic divergence, there is not strong evidence for complete phylogeographic co-divergence between deer and their parasitic lice. However, mitochondrial phylogenies only track genetic structure of female lineages, and the incongruence between deer and louse phylogeography may be explained by louse migration mediated by male deer. Morphological analysis of head shape variation based on an elliptic Fourier descriptor showed that overall morphological variation contained phylogenetic signal, suggesting that in general morphology of these lice evolves congruent to population history.

Notocotylus malhamensis n. sp. is described from the caecum of the bank vole (Myodes glareolus) and the field vole (Microtus agrestis) from Malham Tarn Nature Reserve in North Yorkshire, UK. In total, 581 specimens were collected from rodents trapped at a wetland site (Tarn Fen) between July 2010 and October 2011 with a prevalence of 66·7% and mean intensity of 94·6 in the bank vole and 50% prevalence and a mean intensity of 4·3 in the field vole. This species appears to be most closely related to other previously described Notocotylus species infecting rodents in Europe but differs principally by the metraterm to cirrus sac ratio (1:1·5–1:1·2) in combination with a densely spinulated cirrus, simple caeca and a greater number of ventral glands in the lateral rows (14–17). The use of molecular differentiation was of limited use in this study due to a paucity of relevant information in the DNA sequence databases. However, the complete ITS1-5.8S rDNA-ITS2 and partial 28S gene sequences have been generated to provide a definitive tool for identification of this species in future studies. As far as we know this is the first report of a notocotylid infection in M. glareolus in the UK.

Miracidia are short-lived, non-feeding (lecithotrophic) free-living stages of trematodes, whose survival is potentially influenced by temperature. Climate change may result in elevated temperatures affecting trematode transmission. Therefore understanding their thermobiology forms an important step in determining the future dynamics of parasite populations. An empirical relationship exists between the mean expected life span of lecithotrophic larvae and the half life of their population (t0·5) and therefore t0·5 is a good indicator of glycogen utilization. In this study experimental data on the effects of temperature on miracidial survival were compiled from the scientific literature and evaluated in terms of metabolism using Q10 and Arrhenius activation energy (E* or μ). Temperature poorly influenced survival/metabolism with all miracidia having distinct zone(s) of thermostability. Overall there were few differences in Q10 and E* values between most species temperature ranges whilst there were only limited strain-specific variations in thermal responses of laboratory-maintained Schistosoma mansoni. Miracidia demonstrated a trend of greater thermal resistance than cercariae. In particular, comparative studies on 4 strains of the same species of miracidia and cercariae showed little correlation in thermal biology between the 2 life-history stages. The importance of these results for trematode transmission under global climate change is discussed.

Freshwater fish helminths, the most well known Mexican vertebrate parasites, include approximately 260 species (platyhelminthes, acanthocephalans, nematodes, and hirudineans). The distribution patterns of adult helminth diversity (throughout parasite and host groups and hydrological regions) are described and the effects of host traits and environmental and geographical factors on diversity are evaluated. Adult helminths include 160 species, parasitizing 149 fish species of 23 families distributed in 21 regions. Nematoda was the most species-rich (>50 species). Cichlidae harboured rich helminth assemblages, with widespread parasites. By contrast, Atherinopsidae and Goodeidae showed relatively poor helminth assemblages, including specific parasites with narrow distribution. Helminth richness in southeastern Mexico was higher than northern or central regions. Non-parametric richness estimators were used to avoid confusion in comparisons with unequal sampling efforts. Bootstrap values, the method with the best performance, indicated that estimated richness shows the same distribution pattern that observed richness. Non-phylogenetic and phylogenetic analyses were used to determine the role of different factors in the parasite diversification. The distribution range was the most important richness predictor (widespread fishes harbour richer parasite assemblages), although interactions between this variable and others such as trophic level, latitude, habitat temperature and precipitation are also important. Likewise, biogeographical factors can also affect parasite diversity.

Although results from field surveys have linked parasites to oxidative stress in their fish hosts, direct evidence involving experimentally infected hosts is lacking. We evaluated the effects of experimental infections with larval trematodes on induction of oxidative stress in fathead minnows, Pimephales promelas. Juvenile fish were exposed in the laboratory to the larvae (cercariae) of 2 species of trematode: Ornithodiplostomum sp. that develops in the liver, and O. ptychocheilus that develops in the brain. For Ornithodiplostomum sp., lipid peroxidation concentration in liver tissue increased 5 days after exposure and remained higher than controls until the end of the experiment at 28 days. For O. ptychocheilus, liver lipid peroxidation concentration was higher than controls at 5 days, but not thereafter. Sustained elevation in lipid peroxidation concentration for the liver trematode may be explained by direct tissue damage caused by developing larvae in the liver, or by an immune response. These experimental results support those from field studies, indicating that the lipid peroxidation assay may be an effective biomonitor for parasite-induced oxidative stress in fish, and that the nature of the oxidative stress response is species and/or tissue specific.