Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is currently being used for rapid and reproducible identification of bacteria, viruses and fungi in clinical microbiological laboratories. However, some studies have also reported the use of MALDI-TOF MS for identification of parasites, like Leishmania, Giardia, Cryptosporidium, Entamoeba, ticks and fleas. The present review collates all the information available on the use of this technique for parasites, in an effort to assess its applicability and the constraints for identification/diagnosis of parasites and diseases caused by them. Though MALDI-TOF MS-based identification of parasites is currently done by reference laboratories only, in future, this promising technology might surely replace/augment molecular methods in clinical parasitology laboratories.

Over a hundred years since their first description in 1913, the sparsely described malaria parasites (genus Plasmodium) of ungulates have been rediscovered using molecular typing techniques. In the span of weeks, three studies have appeared describing the genetic characterization and phylogenetic analyses of malaria parasites from African antelope (Cephalophus spp.) and goat (Capra aegagrus hircus), Asian water buffalo (Bubalus bubalis), and North American white-tailed deer (Odocoileus virginianus). Here we unify the contributions from those studies with the literature on pre-molecular characterizations of ungulate malaria parasites, which are largely based on surveys of Giemsa-reagent stained blood smears. We present a phylogenetic tree generated from all available ungulate malaria parasite sequence data, and show that parasites from African duiker antelope and goat, Asian water buffalo and New World white-tailed deer group together in a clade, which branches early in Plasmodium evolution. Anopheline mosquitoes appear to be the dominant, if not sole vectors for parasite transmission. We pose questions for future phylogenetic studies, and discuss topics that we hope will spur further molecular and cellular studies of ungulate malaria parasites.

In reproducing ewes, a periparturient breakdown of immunity is often observed to result in increased fecal egg excretion, making them the main source of infection for their immunologically naive lambs. In this study, we expanded a simulation model previously developed for growing lambs to explore the impact of the genotype (performance and resistance traits) and host nutrition on the performance and parasitism of both growing lambs and reproducing ewes naturally infected with Teladorsagia circumcincta. Our model accounted for nutrient-demanding phases, such as gestation and lactation, and included a supplementary module to manage the age structure of the ewe flock. The model was validated by comparison with published data. Because model parameters were unknown or poorly estimated, detailed sensitivity analysis of the model was performed for the sheep mortality and the level of infection, following a preliminary screening step. The parameters with the greatest effect on parasite-related outputs were those driving animal growth and milk yield. Our model enables different parasite-control strategies (host nutrition, breeding for resistance and anthelmintic treatments) to be assessed on the long term in a sheep flock. To optimize in silico exploration, the parameters highlighted by the sensitivity analysis should be refined with real data.

The cat flea, Ctenocephalides felis, is a major pest species on companion animals thus of significant importance to the animal health industry. The aim of this study was to develop sampling and storage protocols and identify stable reference genes for gene expression studies to fully utilize the growing body of molecular knowledge of C. felis. RNA integrity was assessed in adult and larvae samples, which were either pierced or not pierced and stored in RNAlater at ambient temperature. RNA quality was maintained best in pierced samples, with negligible degradation evident after 10 days. RNA quality from non-pierced samples was poor within 3 days. Ten candidate reference genes were evaluated for their stability across four group comparisons (developmental stages, genders, feeding statuses and insecticide-treatment statuses). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), 60S ribosomal protein L19 (RPL19) and elongation factor-1α (Ef) were ranked highly in all stability comparisons, thus are recommended as reference genes under similar conditions. Employing just two of these three stable reference genes was sufficient for accurate normalization. Our results make a significant contribution to the future of gene expression studies in C. felis, describing validated sample preparation procedures and reference genes for use in this common pest.

We report the pharmacological activity of organoruthenium complexes containing chloroquine (CQ) as a chelating ligand. The complexes displayed intraerythrocytic activity against CQ-sensitive 3D7 and CQ-resistant W2 strains of Plasmodium falciparum, with potency and selectivity indexes similar to those of CQ. Complexes displayed activity against all intraerythrocytic stages, but moderate activity against Plasmodium berghei liver stages. However, unlike CQ, organoruthenium complexes impaired gametocyte viability and exhibited fast parasiticidal activity against trophozoites for P. falciparum. This functional property results from the ability of complexes to quickly induce oxidative stress. The parasitaemia of P. berghei-infected mice was reduced by treatment with the complex. Our findings demonstrated that using chloroquine for the synthesis of organoruthenium complexes retains potency and selectivity while leading to an increase in the spectrum of action and parasite killing rate relative to CQ.

Cerebral malaria (CM) shows lethality rate of 15–25% despite effective antimalarial chemotherapy. The effective adjunct treatment to counteract the CM pathogenesis is urgently required. In murine CM model, most interventions studied till date are administered before the onset of CM symptoms, which belittle its translational value to human. We studied intramuscular arteether–vitamin D (ART–VD) combination treatment for CM outcome improvement after the onset of neurological symptoms. The intramuscular dose of 50 µg kg−1 VD for 3 days combined with a loading dose of 25 mg kg−1α/β arteether followed by 12·5 mg kg−1 dose for two consecutive days led to significant improvement in survival (73% in combination group vs 29 and 0% in arteether and VD monotherapy, respectively) and clinical recovery. The treatment in all the groups partially restored the blood–brain barrier integrity and reduced the level of serum proinflammatory cytokines tumour necrosis factor-α and interferon-γ. The brain transcripts of inflammatory chemokines viz. CXCL10, CXCL9, CCL4 and CCL5 and T cell migration in the brain microvasculature were significantly diminished in all the treatment groups. ART–VD treatment significantly reduced intercellular cell adhesion molecule-1 expression. Taken together, our findings show that coordinated actions of ART–VD improve the outcome of experimental CM.

Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4’−6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

We studied the monogenean communities of 34 species of butterflyfish from the tropical Indo-West Pacific, identifying 13 dactylogyrid species (including two species that are presently undescribed). Monogenean assemblages differed significantly between host species in terms of taxonomic structure, intensity and prevalence. Parasite richness ranged from 0 (Chaetodon lunulatus) to 11 (C. auriga, C. citrinellus and C. lunula). Host specificity varied between the dactylogyrids species, being found on 2–29 of the 34 chaetodontid species examined. Sympatric butterflyfish species were typically parasitized by different combinations of dactylogyrid species, suggesting the existence of complex host–parasite interactions. We identified six clusters of butterflyfish species based on the similarities of their dactylogyrid communities. Dactylogyrid richness and diversity were not related to host size, diet specialization, depth range or phylogeny of butterflyfish species. However, there was a weak positive correlation between monogenean richness and diversity and host geographical range. Most communities of dactylogyrids were dominated by Haliotrema aurigae and H. angelopterum, indicating the importance of the genus Haliotrema in shaping monogenean communities of butterflyfishes. This study casts light on the structure of the monogenean communities of butterflyfishes, suggesting that the diversity and complexity of community structures arises from a combination of host species-specific parameters.

The Renicolidae are digenean parasites of piscivorous and molluscivorous birds. Although they exhibit few morphological autapomorphies and are highly variable, the numerous suggested re-classifications within the family have never been supported by any molecular analyses. We address the possible synonymization of species within the Renicola pinguis complex suggested previously by Odening. We provide and analyse sequences of two nuclear (ITS2, 28S rDNA) and two mitochondrial (CO1, ND1) DNA loci of central European species of the Renicolidae, namely Renicola lari, Renicola pinguis and Renicola sternae sp. n., and we also provide first sequences of Renicola sloanei. The combined molecular and comparative morphological analysis confirms the previously questioned validity of the three Renicola spp. of highly similar morphology, which display strict niche separation in terms of host specificity and selectivity. We identify two previously unreported clades within the genus Renicola; however, only one of them is supported by the analysis of adult worms. We also provide comparative measurements of the three examined closely related central European renicolids, and describe the newly proposed tern-specialized species Renicola sternae sp. n., which was previously repeatedly misidentified as Renicola paraquinta. Based on the extensive dataset collected in 1962–2015, we update the host spectrum of Renicolidae parasitizing central European birds (Renicola bretensis, R. lari, Renicola mediovitellata, R. pinguis, Renicola secunda and R. sternae sp. n.) and discuss their host-specific prevalence and intensity of infections.

The presence of introduced hosts can increase or decrease infections of co-introduced parasites in native species of conservation concern. In this study, we compared parasite abundance, intensity, and prevalence between native Awaous stamineus and introduced poeciliid fishes by a co-introduced nematode parasite (Camallanus cotti) in 42 watersheds across the Hawaiian Islands. We found that parasite abundance, intensity and prevalence were greater in native than introduced hosts. Parasite abundance, intensity and prevalence within A. stamineus varied between years, which largely reflected a transient spike in infection in three remote watersheds on Molokai. At each site we measured host factors (length, density of native host, density of introduced host) and environmental factors (per cent agricultural and urban land use, water chemistry, watershed area and precipitation) hypothesized to influence C. cotti abundance, intensity and prevalence. Factors associated with parasitism differed between native and introduced hosts. Notably, parasitism of native hosts was higher in streams with lower water quality, whereas parasitism of introduced hosts was lower in streams with lower water quality. We also found that parasite burdens were lower in both native and introduced hosts when coincident. Evidence of a mutual dilution effect indicates that introduced hosts can ameliorate parasitism of native fishes by co-introduced parasites, which raises questions about the value of remediation actions, such as the removal of introduced hosts, in stemming the rise of infectious disease in species of conservation concern.

Synthesized lipophilic tyrosyl ester derivatives with increasing lipophilicity were effective against Leishmania (L.) major and Leishmania infantum species in vitro. These findings prompted us to test in vivo leishmanicidal properties of these molecules and their potential effect on the modulation of immune responses. The experimental BALB/c model of cutaneous leishmaniasis was used in this study. Mice were infected with L. major parasites and treated with three in vitro active tyrosyl esters derivatives.

Among these tested tyrosylcaprate (TyC) compounds, only TyC10 exhibited an in vivo anti-leishmanial activity, when injected sub-cutaneously (s.c.). TyC10 treatment of L. major-infected BALB/c mice resulted in a decrease of lesion development and parasite load. TyC10 s.c. treatment of non-infected mice induced an imbalance in interferon γ/interleukin 4 (IFN-γ/IL-4) ratio cytokines towards a Th1 response. Our results indicate that TyC10 s.c. treatment improves lesions’ healing and parasite clearance and may act on the cytokine balance towards a Th1 protective response by decreasing IL-4 and increasing IFN-γ transcripts. TyC10 is worthy of further investigation to uncover its mechanism of action that could lead to consider this molecule as a potential drug candidate.

In this study, the prevalence of piroplasms in dogs was assessed using polymerase chain reaction (PCR) to identify Babesia and Theileria species in 200 dogs from Northern and Central Tunisia between spring and autumn 2014. The overall molecular prevalence for piroplasms was 14·5% ± 0·05 (29/200); PCR detected 2 species, namely Babesia vogeli and Theileria annulata with an overall prevalence of 12·5 ± 0·04 and 2% ± 0·02, respectively. No differences in the molecular prevalences of B. vogeli were revealed for age and sex (P > 0·05). The molecular prevalence of B. vogeli was significantly higher in central Tunisia (26·5% ± 0·01) compared with the North (9·6% ± 0·04) (P < 0·05). More working and companion dogs were infected by B. vogeli (25·8 ± 0·15 and 21·1% ± 0·13, respectively) in comparison with guarding dogs (1·8% ± 0·03) (P < 0·05). Concerning T. annulata, no significant variation was observed for all studied risk factors (P > 0·05). Comparison of the partial sequences of 18S rRNA and Tams 1 genes confirmed the presence of 2 novel B. vogeli and T. annulata genotypes. This is the first molecular detection of T. annulata and genetic characterization of dogs’ piroplasms in Tunisia. Further studies are needed to better assess the epidemiological feature of piroplasms infection in North Africa.

Interleukin (IL)-13-associated signal pathway plays an important role in schistosomiasis hepatic fibrosis. In this study we tried to investigate the effects of corilagin to ameliorate schistosomiasis hepatic fibrosis through regulating IL-13-associated signal pathway in vitro and in vivo. Cellular model was set up with hepatic stellate cells-T6 cells stimulated by rIL-13 and male Balb/c mice were infected with Schistosoma japonicum cercariaeas as animal model. Liver histological changes were observed with haematoxylin and eosin staining. Masson staining was employed to observe the change of egg granulomas. Expression of Col (collagen) and Col III were examined with Immunohistochemistry. Western bolt was employed to detect the JAK-1 and IL13Rα1 proteins. The mRNA expression of Col I, Col III, IL-13, JAK-1 and IL13Rα1 were tested by quantitative polymerase chain reaction. As a result, less inflammatory changes were found in all corilagin groups compared with model group and praziquantel group. The mRNA levels of Col I, Col III, IL-13, JAK-1 and IL13Rα1 were significantly decreased after corilagin intervention (P < 0·01). JAK-1 and IL-13Rα1 protein levels were also greatly decreased in the corilagin groups (P < 0·01). In conclusion, corilagin could ameliorate schistosomiasis hepatic fibrosis by down-regulating the expression of IL-13 and signal molecules in IL-13 pathway.

Paragonimus westermani is one of the most medically important lung flukes and is widely distributed in Asia. It exhibits considerable variation in morphological, genetic and biological features. In central provinces of Vietnam, a high prevalence of metacercariae of this species has been reported from the crab intermediate host, Vietopotamon aluoiense. In this study, we detected P. westermani metacercariae in two additional crab hosts, Donopotamon haii in Quang Tri Province, central Vietnam and Indochinamon tannanti in Yen Bai Province in the north. The latter is a new locality for P. westermani in a northern region of Vietnam where P. heterotremus is the only species currently known to cause human paragonimiasis. Paragonimus westermani metacercariae found in Vietnam showed considerable morphological variation but slight genetic variation based on DNA sequences from the nuclear ribosomal ITS2 region and the mitochondrial 16S gene. Co-infection of the same individual crabs with P. westermani and P. heterotremus and/or some other Paragonimus species was found frequently, suggesting potential for co-infection in humans. The findings of the present study emphasize the need for highly specific molecular and immunodiagnostic methods to differentially diagnose between P. westermani and P. heterotremus infections.

Obesity is the main causal factor for metabolic syndrome and chronic systemic inflammation, which impacts on immune function and increases susceptibility to pathogens. Here, we investigated the effect of obesity on the outcome of visceral leishmaniasis caused by Leishmaniasis infantum chagasi. C57BL/6 mice fed with high-sugar and butter diet (HSB) showed a significant increase in body weight, adiposity index and morphological changes in adipocyte. To investigate the consequences of obesity on the specific immunity against Leishmania, both control and HSB diet groups were infected with 107L. infantum chagasi promastigotes in the eighth-week after diet started and euthanized 4 weeks later. HSB-diet fed mice exhibited a significantly higher parasite burden in both liver and spleen compared with control- diet group. Gonadal adipocyte tissue from HSB-diet mice showed increased TNF-α, IL-6 and leptin and diminished IL-10 production compared with control. Cytokines production analysis in the spleen and liver from these animals also demonstrated higher production of IFN-γ, TNF-α, IL-6 and nitric oxide and diminished production of IL-10 and TGF-β, which correlate with inflammatory foci and the cell hyperplasia observed. Taken together, obesity can interfere with responses to pathogen-derived signals and impair the development of protective anti-Leishmania immunity.

Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29–130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29–130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29–130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

Effective control of gastrointestinal parasites is necessary in sheep production. The development of anthelmintics resistance is causing the available chemically based anthelmintics to become less effective. Biological control strategies present an alternative to this problem. In the current study, we tested the larvicidal effects of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus larvae. Bacterial suspensions [2 × 108 colony-forming units (CFU) g−1 of the feces] of B. thuringiensis var. israelensis and recombinant Escherichia coli expressing Cry11Aa toxin were added to naturally H. contortus egg-contaminated feces. The larvae were quantified, and significant reductions of 62 and 81% (P < 0·001) were, respectively observed, compared with the control group. A 30 mL bacterial suspension (1 × 108 CFU mL−1) of B. thuringiensis var. israelensis and recombinant E. coli expressing Cry11Aa toxin were then orally administered to lambs naturally infected with H. contortus. Twelve hours after administration, feces were collected and submitted to coprocultures. Significant larvae reductions (P < 0·001) of 79 and 90% were observed respectively compared with the control group. The results suggest that the Cry11Aa toxin of B. thuringiensis var. israelensis is a promising new class of biological anthelmintics for treating sheep against H. contortus.

Malaria remains one of the most devastating diseases. Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection resulting in high mortality and morbidity worldwide. Analysis of precise mechanisms of CM in humans is difficult for ethical reasons and animal models of CM have been employed to study malaria pathogenesis. Here, we describe a new experimental cerebral malaria (ECM) model with Plasmodium berghei ANKA infection in KunMing (KM) mice. KM mice developed ECM after blood-stage or sporozoites infection, and the development of ECM in KM mice has a dose-dependent relationship with sporozoites inoculums. Histopathological findings revealed important features associated with ECM, including accumulation of mononuclear cells and red blood cells in brain microvascular, and brain parenchymal haemorrhages. Blood–brain barrier (BBB) examination showed that BBB disruption was present in infected KM mice when displaying clinical signs of CM. In vivo bioluminescent imaging experiment indicated that parasitized red blood cells accumulated in most vital organs including heart, lung, spleen, kidney, liver and brain. The levels of inflammatory cytokines interferon-gamma, tumour necrosis factor-alpha, interleukin (IL)-17, IL-12, IL-6 and IL-10 were all remarkably increased in KM mice infected with P. berghei ANKA. This study indicates that P. berghei ANKA infection in KM mice can be used as ECM model to extend further research on genetic, pharmacological and vaccine studies of CM.