Cryptosporidium is a protozoan parasite of humans and animals and has a worldwide distribution. The parasite has a unique epidemiology in Middle Eastern countries where the IId subtype family of Cryptosporidium parvum dominates. However, there has been no information on Cryptosporidium species in Yemen. Thus, this study was conducted in Yemen to examine the distribution of Cryptosporidium species and subtype families. Fecal samples were collected from 335 patients who attended hospitals in Sana'a city. Cryptosporidium species were determined by PCR and sequence analysis of the 18 s rRNA gene. Cryptosporidium parvum and C. hominis subtypes were identified based on sequence analysis of the 60 kDa glycoprotein (gp60) gene. Out of 335 samples, 33 (9·9%) were positive for Cryptosporidium. Of them, 97% were identified as C. parvum whilst 1 case (3%) was caused by C. hominis. All 7 C. parvum isolates subtyped belonged to the IIaA15G2R1 subtype. The common occurrence of the zoonotic IIa subtype family of C. parvum highlights the potential occurrence of zoonotic transmission of cryptosporidiosis in Yemen. However, this postulation needs confirmation with future molecular epidemiological studies of cryptosporidiosis in both humans and animals in Yemen.

Species of Marshallagia are abomasal parasites in free-ranging and domesticated ungulates in temperate climatic zones throughout the world. Pervasiveness of these nematodes is significant in various parts of the world. There has been limited research in the area of Marshallagi amarshalli pathogenesis. The aim of this study was to investigate the effects of M. marshalli on the acid secretory capacity of the abomasal mucosa and the morphological changes due to parasitic migration to different parts of abomasal tissue in sheep. Ten lambs, approximately around 6 months old, were allotted to two groups of five (A and B). The sheep from group A were infected orally with a dose of 5000 third-stage larvae (L3) of M. marshalli whereas the sheep of group B were not infected. The results indicated that the development of M. marshalli in the abomasal glands of ruminants causes pathophysiological changes, which include a reduced acidity of the abomasal contents, increased abomasal pH and increased serum pepsinogen concentrations. The reduced acid secretion is explained by a replacement of functional parietal cells by undifferentiated cells. Histology changes include mucosal cell hyperplasia, loss of parietal cells and inflammatory cell infiltration, which includes numerous granulocytes and lymphocytes.

Seroprevalence data illustrate that human exposure to Toxocara is frequent. Environmental contamination with Toxocara spp. eggs is assumed to be the best indicator of human exposure, but increased risk of exposure has also been associated with many other factors. Reported associations are inconsistent, however, and there is still ambiguity regarding the factors driving the onset of Toxocara antibody positivity. The objective of this work was to assess the validity of our current conceptual understanding of the key processes driving human exposure to Toxocara. We constructed an agent-based model predicting Toxocara antibody positivity (as a measure of exposure) in children. Exposure was assumed to depend on the joint probability of 3 parameters: (1) environmental contamination with Toxocara spp. eggs, (2) larvation of these eggs and (3) the age-related contact with these eggs. This joint probability was linked to processes of acquired humoral immunity, influencing the rate of antibody seroreversion. The results of the simulation were validated against published data from 5 different geographical settings. Using simple rules and a stochastic approach with parameter estimates derived from the respective contexts, plausible serological patterns emerged from the model in nearly all settings. Our approach leads to novel insights in the transmission dynamics of Toxocara.

Within southern Africa, the widely distributed four-striped mouse genus (Rhabdomys) is parasitized by, amongst others, the specific ectoparasitic sucking louse, Polyplax arvicanthis. Given the presence of significant geographically structured genetic divergence in Rhabdomys, and the propensity of parasites to harbour cryptic diversity, the molecular systematics of P. arvicanthis was investigated. Representatives of P. arvicanthis were sampled from Rhabdomys at 16 localities throughout southern Africa. Parsimony and Bayesian gene trees were constructed for the mitochondrial COI, 12S rRNA, 16S rRNA and nuclear CAD genes. Our findings support the existence of 2 genetic groups within P. arvicanthis separated by at least 25% COI sequence divergence, which is comparable to that observed among recognized Polyplax species. We therefore propose that these 2 genetic lineages probably represent distinct species and that the apparent absence of clear morphological differences may point to cryptic speciation. The 2 taxa have sympatric distributions throughout most of the sampled host range and also occasionally occur sympatrically on the same host individual. The co-occurrence of these genetically distinct lineages probably resulted from parasite duplication via host-associated allopatric divergence and subsequent reciprocal range expansions of the 2 parasite taxa throughout southern Africa.

The aim of this study was to fractionate and partially characterize the antigenic extract of filariform larvae of Strongyloides venezuelensis in ion-exchange resin diethylaminoethyl sepharose (DEAE), to obtain antigenic fractions potentially applicable in immunoassays. Somatic antigen (SA) and its fractions DEAE S1 and DEAE S2 - which interacted with the resin - were evaluated by 1-dimensional electrophoresis to obtain protein profiles. SA and its fractions were tested in serum samples for IgG detection by ELISA. Serum samples (n = 155) were analysed: 50 from strongyloidiasis patients (G1), 55 from patients with other parasitic infections (G2) and 50 from healthy volunteers. Sensitivity (Se), specificity (Sp), area under curve (AUC) and likelihood ratios (LR) were calculated. The DEAE S2 fraction provided a high diagnostic value for IgG detection (Se 92·0%, Sp 91·4%, AUC 0·981, LR+ 10·75, LR − 0·09). In conclusion, the DEAE S2 fraction would probably be a source of immunodominant polypeptides for IgG detection in human strongyloidiasis serodiagnosis.

The present study compared the miRNA expression profiles of five Toxoplasma gondii strains, namely RH (Type I, ToxoDB10), TgXD (Type I, ToxoDB10), PRU (Type II, ToxoDB1), QHO (Type II, ToxoDB1) and TgC7 (ToxoDB9), by Solexa deep sequencing, bioinformatics analysis and real-time quantitative PCR. A total of 7, 15, 10, 12 and 10 miRNAs were found from RH, TgXD, PRU, QHO and TgC7 strains, respectively. Thirteen miRNAs were shared by three genotypes, with only one miRNA shared by all of the 5 strains and others shared by 2 or more strains. A large number of targets ranging from 1 to 185 were identified for commonly shared miRNAs and strain-specific miRNAs with complete or nearly complete complementarity. Functional prediction showed that these targets were mostly focused on catalytic activity (191 targets) and binding activity (183 targets). Nonetheless, the majority of targets and most of the miRNAs are related to the virulence or invasion proteins of different strains of T. gondii, including ROP and MIC, as well as some other proteins, such as AMA1, GRA and RHO. The present study characterized comparatively the miRNA profiles of 3 different genotypes of T. gondii, identified genotype-shared miRNAs and strain-specific miRNAs.

Blockage of pathogen transmission through water decontamination is considered an important strategy for the prevention of schistosome infection. Many believe that this strategy is feasible, but it has yet to be achieved. Silver has a long history of use as a disinfectant. With the emergence of nanotechnology, silver can be shaped into nanoparticles which have been found to possess superb antimicrobial activities. In this light, we investigated the effects of silver nanoparticles (AgNPs) on Schistosoma japonicum cercariae. AgNPs rapidly induced cercarial tail-shedding, agitated behaviour and a decrease in cercarial secretion in a dose-dependent manner. Prolonged treatment was found to be cercariocidal, which nevertheless might be attributable to AgNP-induced cercarial tail loss rather than to toxicity. Higher concentrations of AgNPs (125 μg mL−1 and above) completely blocked cercarial infectivity. Despite decreased infectivity, cercariae exposed to lower concentrations of AgNPs for 30 min were still found capable of infecting hosts even without their tails, suggesting that tail loss does not necessarily signify a total loss of infective ability. We also found that silver ions (Ag+) were heavily involved in the observed cercarial responses of AgNPs. Our observations provide insight into the interactions between the larvae of helminth parasites and nanoparticles.

Toxoplasma gondii stimulates a potent pro-inflammatory response and neutrophils are involved in early infection. Galectin-3 (Gal-3) is an endogenous modulator of inflammatory processes and anti-infective agents, but its interaction with neutrophils in T. gondii infection is still unclear. Here, we evaluated the role of Gal-3 in peritoneal inflammation, reactive oxygen species (ROS) production by neutrophils and survival, after in vivo T. gondii infection with virulent RH strain, using Gal-3 deficient and wild type mice. Animals were inoculated with thioglycollate or tachyzoites, and peritoneal cells were harvested for analysis of the influx of leukocytes. Neutrophils were isolated from peritoneal exudates from infected mice and stimulated with phorbol myristate acetate (PMA) to evaluate ROS production by luminol-dependent chemiluminescence assay. Our results showed that: (1) Gal-3 upregulates peritoneal inflammation, with enhanced recruitment of neutrophils and lymphocytes after thioglycollate stimulation, but does not influence the enhanced neutrophil influx after early T. gondii infection; (2) Gal-3 upregulates ROS generation by inflammatory peritoneal neutrophils from infected mice, but downregulates its production in non-infected mice and (3) Gal-3 does not influence the survival of mice after infection with the virulent T. gondii strain. In conclusion, Gal-3 is essential for ROS generation by neutrophils in the initial acute phase of T. gondii infection and this phenomenon may constitute an attempt to control parasite growth during in vivo infection with the T. gondii virulent strain.

Detection of taeniasis carriers of Taenia solium is essential for control of cysticercosis in humans and pigs. In the current study, we assessed the positive detection rate of a self-detection tool, stool microscopy with direct smear and coproPCR for taeniasis carriers in endemic Tibetan areas of northwest Sichuan. The self-detection tool through questioning about a history of proglottid expulsion within the previous one year showed an overall positive detection rate of more than 80% for Taenia saginata, T. solium and T. asiatica. The positive detection rate was similar for T. saginata and T. solium. In 132 taeniid tapeworm carriers, 68 (51·5%) were detected by microscopy and 92 (69·7%) were diagnosed by coproPCR. A combination of microscopy and coproPCR increased the positive detection rate to 77·3%. There remained 10 cases (7·6%) coproPCR negative but microscopy positive. Due to the high cost and complicated process, coproPCR is required for the identification of Taenia species only when necessary, though it had a significant higher positive detection rate than microscopy. Combined use of self-detection and stool microscopy are recommended in community-based mass screening for taeniases in this Tibetan area or in other situation-similar endemic regions.

Toxoplasma gondii is a zoonotic pathogen defined by three main clonal lineages (types I, II, III), of which type II is most common in Europe. Very few data exist on the prevalence and genotypes of T. gondii in the UK. Wildlife can act as sentinel species for T. gondii genotypes present in the environment, which may subsequently be transmitted to livestock and humans. DNA was extracted from tissue samples of wild British carnivores, including 99 ferrets, 83 red foxes, 70 polecats, 65 mink, 64 badgers and 9 stoats. Parasite DNA was detected using a nested ITS1 PCR specific for T. gondii, PCR positive samples were subsequently genotyped using five PCR–RFLP markers. Toxoplasma gondii DNA was detected within all these mammal species and prevalence varied from 6·0 to 44·4% depending on the host. PCR–RFLP genotyping identified type II as the predominant lineage, but type III and type I alleles were also identified. No atypical or mixed genotypes were identified within these animals. This study demonstrates the presence of alleles for all three clonal lineages with potential for transmission to cats and livestock. This is the first DNA-based study of T. gondii prevalence and genotypes across a broad range of wild British carnivores.

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio±1·5, P<0·05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

Heliciculture is an excellent alternative to obtain edible snails but its viability is seriously threatened by pathogens. A parasitological survey was conducted in 3 mixed system-based heliciculture farms in Galicia (NW Spain), with the species Tetrahymena rostrata, Tetrahymena limacis, Tetratrichomonas limacis, Cryptobia helicogenae, Brachylaima aspersae (metacercariae and sporocysts), Alloionema appendiculatum, Nemhelix bakeri, and Riccardoella limacum being commonly found infecting Helix aspersa aspersa (petit-gris) snails. With the exception of C. helicogenae, N. bakeri, and B. aspersae sporocysts, all species were also detected in Helix aspersa maxima (gros-gris) snails, although generally with lower parameters. Most monoxenous infections, and consequently multiple parasitism, exhibited a rising trend during the first 2 months of intensive mating, with tendencies being slowed down or even reversed during the third month as a result of accumulated mortality and a sampling-derived reduction in host density. No parasites were vertically transmitted and infections were initially acquired from invading gastropod and micromammal reservoirs during fattening. Finally, artificial hibernation reduced significantly the prevalence of most species. These results confirm the importance of parasites in heliciculture and emphasize the need to prevent the entry of wild reservoirs into the farms and to rapidly remove the carcasses of dead snails from the reproduction units and fattening pens.

There is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy. Trypanosoma lacks the enzymatic machinery for the de novo synthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5′-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinant Trypanosoma brucei IMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculated Km values of 30 and 1300 μm for IMP and NAD, respectively. The obtained Km value of TbIMPDH for NAD is approximately 20–200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies show Ki values of 3·2 μm, 21 nM and 3·3 nM for ribavirin 5′-monophosphate, mycophenolic acid and mizoribine 5′-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

Global aquaculture production of turbot has rapidly increased worldwide in the last decade and it is expected to have even bigger growth in the next years due to new farms operating. The losses caused by pathogen infections have grown at the same time as the production of this species. Parasitological infections are among the main relevant pathologies associated with its culture and produce serious losses in aquaculture, reduce the growth rate in fish and may lead to unmarketable fish due to skeletal muscle abnormalities in cases with high intensity of infection. The microsporidian parasite Tetramicra brevifilum causes severe infections and generates major losses in farmed turbot. Infections are difficult to control due to spore longevity and its direct transmission. To facilitate the infection management, an effective tool for fast detection and identification of T. brevifilum is needed. This study provides a molecular methodology of fast Real-Time PCR for T. brevifilum detection to the aquaculture industry, useful for routine control of T. brevifilum at turbot farms. The method is characterized by its high specificity and sensitivity, and it can be applied to cultured turbot for parasite detection regardless of the life-cycle stage of the pathogen or the infection intensity.

Despite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

Avian trichomonosis, caused by the flagellated protozoan Trichomonas gallinae, is a recently emerged infectious disease of British passerines. The aetiological agent, a clonal epidemic strain of the parasite, has caused unprecedented finch mortality and population-level declines in Britain and has since spread to continental Europe. To better understand the potential origin of this epidemic and to further investigate its host range, T. gallinae DNA extracts were collected from parasite culture and tissue samples from a range of avian species in Britain. Sequence typing at the ITS1/5.8S rRNA/ITS2 region resolved three distinct ITS region types circulating in free-ranging British birds. Subtyping by sequence analyses at the Fe-hydrogenase gene demonstrated further strain variation within these ITS region types. The UK finch epidemic strain was preponderant amongst columbids sampled, however, wide strain diversity was encountered in isolates from a relatively small number of pigeons, suggesting further strains present in columbid populations across the UK are yet to be identified. Fe-hydrogenase gene sequence data in isolates from birds of prey with disease were predominantly identical to the UK finch epidemic strain, demonstrating its presence as a virulent strain in UK birds of prey since at least 2009.

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite.

Class Rudimicrosporea Sprague 1977, with its single family Metchnikovellidae, comprises hyperparasites of gregarines from the guts of marine invertebrates. Metchnikovellids remain poorly studied in spite of their significance to the evolutionary history of microsporidia; their ultrastructure and life cycles require further investigation. Here we present results of the light- and electron-microscopy study of Metchnikovella incurvata Caulleri and Mesnil 1914, isolated from lecudinid gregarines, parasitizing polychaetes Pygospio elegans in the White Sea littoral zone, and yet described only on the light-microscopic level. The life cycle of this microsporidium includes 2 sporogonies: free (FS) and sac-bound (SBS). In FS, sporonts develop into multinuclear cells (sporogonial plasmodia), which generate sporoblasts and free spores residing in direct contact with the host cytoplasm. Electron microscopy revealed their metchnikovellidean structure: a horseshoe-shaped nucleus, short manubrium perpendicular to the long axis of the spore, and a polar cap in a separate membrane container. Merogony was not observed. The earliest stages of SBS were chains of binucleate cells. They underwent a series of nuclear and cell divisions, produced extracellular envelopes, and split into boomerang-shaped spore sacs, containing up to 16 spores each. Ultrastructure and sizes of sac-bounded spores were similar to those of free-living ones. An amended diagnosis of M. incurvata is provided.

Endogenous transplacental transmission (EnTT) of Neospora caninum is the most common route of infection in cattle and occurs as a consequence of a reactivation of N. caninum infection that may lead to abortion or to the birth of congenitally infected calves. The reactivation of N. caninum infection was studied during the gestation of chronically infected dams and, for the first time, in their congenitally infected pups. BALB/c mice were infected with Nc-Spain 7 (Group 1) or Nc-Spain 3H (Group 2), high virulence isolates and low-to-moderate virulence isolates, respectively. The mice were mated after 90 days post-infection, and the morbidity, mortality, vertical transmission and humoral immune responses were recorded for 2 consecutive generations. In the first generation, higher morbidity and mortality rates were observed in G1 before mating than in G2 (P < 0·0001). In the second generation, low vertical transmission rates were observed in both inoculated groups (7·7% and 17·1% in G1 and G2, respectively) and were significantly diminished in the third generation (8·7% in G2 versus 0% in G1). Low rates of reactivation of N. caninum infection were induced in chronically infected mice and decreased in subsequent generations regardless of the isolate employed in the inoculations. Thus, further studies are needed to improve this reactivation mouse model.

Schistosomes are parasitic flatworms that cause schistosomiasis, a neglected tropical disease that affects hundreds of millions worldwide. Treatment and control of schistosomiasis relies almost entirely on the single drug praziquantel (PZQ), making the prospect of emerging drug resistance particularly worrisome. This review will survey reports of PZQ (and other drug) resistance in schistosomes and other platyhelminths, and explore mechanisms by which drug resistance might develop. Newer genomic and post-genomic strategies that offer the promise of better understanding of how drug resistance might arise in these organisms will be discussed. These approaches could also lead to insights into the mode of action of these drugs and potentially provide markers for monitoring the emergence of resistance.