Mesomycetozoean-induced infections (order Dermocystida, genus Amphibiocystidium) in European and North American amphibians are causing alarm. To date, the pathogenicity of these parasites in field conditions has been poorly studied, and demographic consequences on amphibian populations have not been explored. In this study, an Amphibiocystidium sp. infection is reported in a natural population of the Italian stream frog (Rana italica) of Central Italy, over a 7-year period from 2008 to 2014. Light and electron microscope examinations, as well as partial 18S rDNA sequence analysis were used to characterize the parasite. Moreover, a capture-mark-recapture study was conducted to assess the frog demographics in response to infection. Negative effects of amphibiocystidiosis on individual survival and population fitness were absent throughout the sampling period, despite the high estimates of disease prevalence. This might have been due to resistance and/or tolerance strategies developed by the frogs in response to the persistence of Amphibiocystidium infection in this system. We hypothesized that in the examined R. italica population, amphibiocystidiosis is an ongoing endemic/epidemic infection. However, ecological and host-specific factors, interacting in a synergistic fashion, might be responsible for variations in the susceptibility to Amphibiocystidium infection of both conspecific populations and heterospecific individuals of R. italica.

The objective of this study was to identify species of Angiostrongylus spp. infecting wild carnivores in Southern Brazil, as well as to describe gross and histopathological findings associated with the infection. Necropsy was conducted in 16 wild carnivores parasitized by Angiostrongylus spp. Analysed lungs revealed multifocal dark-red areas of consolidation; in one case, multifocal firm white nodules spread in all pulmonary lobes were observed. In one animal, a focally extensive area of malacia associated with haemorrhage was noted in the encephalon. Histologically, multifocal granulomatous pneumonia or bronchopneumonia, associated with eggs and larvae in blood vessels, lung interstitium, alveoli, and sometimes in bronchi and bronchioles was observed. Adult nematodes were seen within blood vessels. The lesion observed in the brain was characterized as a focally extensive area of malacia associated with gitter cells, haemorrhage, thrombosis and a free intralesional larva. Through molecular techniques, seven positive samples of Angiostrongylus cantonensis were obtained, including the brain sample, and a positive sample of Angiostrongylus vasorum-like, all in Cerdocyon thous. The positive sample for A. vasorum showed 97% similarity with sequences deposited in GenBank, suggesting a new species or subspecies of Angiostrongylus sp. Infection of Lycalopex gymnocercus by Angiostrongylus spp. was confirmed by histological evaluation.

Periodicity in nematode egg excretion may be of evolutionary origin as it can favour dispersal of the eggs in the environment. We investigated whether egg excretion by Heterakis gallinarum shows a repeatable pattern of periodicity. The faecal egg concentration and total number of eggs excreted within 4-h intervals were significantly affected by the sampling time within 1 day, but remained unaffected by the sampling day or interaction effects. By contrast, the total number of eggs excreted within 24 h did not differ among the 4 days of the study, collectively indicating repeatable egg excretion patterns. Both host feces and parasite egg excretion increased from night to late afternoon, followed by a decrease in the evening, resulting in higher egg excretion during daytime than the dark period. Feces excretion and worm fecundity showed overlapping diurnal rhythms with similarly timed phases, suggesting the existence of synchronicity between the host feces and nematode egg excretion patterns. We conclude that egg excretion by H. gallinarum is synchronized with host feces excretion and is higher during the daytime than during the dark period. This overlaps with the maximum activity of the day-active host and allows a maximal dispersal of the eggs in the environment.

Host shifts of parasites are often causing devastating effects in the new hosts. The Varroa genus is known for a lineage of Varroa destructor that shifted to the Western honey bee, Apis mellifera, with disastrous effects on wild populations and the beekeeping industry. Despite this, the biology of Varroa spp. remains poorly understood in its native distribution range, where it naturally parasitizes the Eastern honey bee, Apis cerana. Here, we combined mitochondrial and nuclear DNA analyses with the assessment of mite reproduction to determine the population structure and host specificity of V. destructor and Varroa jacobsonii in Thailand, where both hosts and several Varroa species and haplotypes are sympatric. Our data confirm previously described mite haplogroups, and show three novel haplotypes. Multiple infestations of single host colonies by both mite species and introgression of alleles between V. destructor and V. jacobsonii suggest that hybridization occurs between the two species. Our results indicate that host specificity and population genetic structure in the genus Varroa is more labile than previously thought. The ability of the host shifted V. destructor haplotype to spillback to A. cerana and to hybridize with V. jacobsonii could threaten honey bee populations of Asia and beyond.

Mullets inhabit a wide range of habitats from tropical to temperate regions and play a critical role in their ecosystems. This commercially important fish group constitutes a significant source of food in several geographic regions, and the production of some species for consumption is an increasing trend. About 64 myxosporean species have been reported in mullets, some of which are cryptic, as is the case of Myxobolus exiguus, and M. muelleri. This paper provides, for the first time, a detailed and critical revision of the data available for myxobolids reported in mullets, determining the species that have bona fide mugiliform fish hosts, in accordance with the original species descriptions, the available molecular data and the currently accepted taxonomic and phylogenetic criteria. Phylogenetic analyses using Bayesian inference and maximum-likelihood methodologies suggest that the evolutionary history of myxobolids with bona fide mugiliform fish hosts reflects that of its vertebrate hosts, while reinforcing known evolutionary factors and old systematic issues of the clade of myxobolids. A comprehensive morphological, ultrastructural and molecular redescription is also provided for the cryptic species M. exiguus, from infections in the visceral peritoneum of the thinlip-grey mullet Chelon ramada in the River Minho, Portugal.

Hepatocytes constitute the majority of hepatic cells, and play a key role in controlling systemic innate immunity, via pattern-recognition receptors (PRRs) and by synthesizing complement and acute phase proteins. Leishmania infantum, a protozoan parasite that causes human and canine leishmaniasis, infects liver by establishing inside the Kupffer cells. The current study proposes the elucidation of the immune response generated by dog hepatocytes when exposed to L. infantum. Additionally, the impact of adding leishmanicidal compound, meglumine antimoniate (MgA), to parasite-exposed hepatocytes was also addressed. L. infantum presents a high tropism to hepatocytes, establishing strong membrane interactions. The possibility of L. infantum internalization by hepatocytes was raised, but not confirmed. Hepatocytes were able to recognize parasite presence, inducing PRRs [nucleotide oligomerization domain (NOD)1, NOD2 and Toll-like receptor (TLR)2] gene expression and generating a mix pro- and anti-inflammatory cytokine response. Reduction of cytochrome P 450s enzyme activity was also observed concomitant with the inflammatory response. Addition of MgA increased NOD2, TLR4 and interleukin 10 gene expression, indicating an immunomodulatory role for MgA. Hepatocytes seem to have a major role in coordinating liver's innate immune response against L. infantum infection, activating inflammatory mechanisms, but always balancing the inflammatory response in order to avoid cell damage.

Haemosporidian parasites belonging to Haemoproteus cause avian diseases, however, vectors remain unidentified for the majority of described species. We used the laboratory-reared biting midges Culicoides nubeculosus to determine if the sporogonic development of three widespread Haemoproteus parasites completes in this insect. The midges were reared and fed on one common blackbird, white wagtail and thrush nightingale naturally infected with Haemoproteus minutus, Haemoproteus motacillae and Haemoproteus attenuatus, respectively. The engorged females were dissected in order to follow their sporogonic development. Microscopic examination was used to identify sporogonic stages. Bayesian phylogeny based on partial cytochrome b gene was constructed in order to determine phylogenetic relationships among Culicoides species-transmitted haemoproteids. All three parasites completed sporogony. Phylogenetic analysis placed Culicoides species transmitted haemoproteids in one well-supported clade, proving that such analysis readily indicates groups of dipteran insects transmitting avian haemoproteids. Available data show that 11 species of Culicoides have been proved to support complete sporogony of 18 species of avian haemoproteids. The majority of Culicoides species can act as vectors for many Haemoproteus parasites, indicating the low specificity of these parasites to biting midges, whose are globally distributed. This calls for control of haemoproteid infections during geographical translocation of infected birds.

The 5-nitroimidazole drug metronidazole has remained the drug of choice in the treatment of anaerobic infections, parasitic as well as bacterial, ever since its development in 1959. In contrast to most other antimicrobials, it has a pleiotropic mode of action and reacts with a large number of molecules. Importantly, metronidazole, which is strictly speaking a prodrug, needs to be reduced at its nitro group in order to become toxic. Reduction of metronidazole, however, only takes place under very low concentrations of oxygen, explaining why metronidazole is exclusively toxic to microaerophilic and anaerobic microorganisms. In general, resistance rates amongst the pathogens treated with metronidazole have remained low until the present day. Nevertheless, metronidazole resistance does occur, and for the treatment of some pathogens, especially Helicobacter pylori, metronidazole has become almost useless in some parts of the world. This review will give an account on the current status of research on metronidazole's mode of action, metronidazole resistance in eukaryotes and prokaryotes, and on other 5-nitroimidazoles in use.

C-mannosylation was recently identified in the thrombospondin-related anonymous protein (TRAP) from Plasmodium falciparum salivary gland sporozoites. A candidate P. falciparum C-mannosyltransferase (PfDPY-19) was demonstrated to modify thrombospondin type 1 repeat (TSR) domains in vitro, exhibiting a different acceptor specificity than their mammalian counterparts. According to the described minimal acceptor of PfDPY19, several TSR domain-containing proteins of P. falciparum could be C-mannosylated in vivo. However, the relevance of this protein modification for the parasite viability remains unknown. In the present study, we used CRISPR/Cas9 technology to generate a PfDPY19 null mutant, demonstrating that this glycosyltransferase is not essential for the asexual blood development of the parasite. PfDPY19 gene disruption was not associated with a growth phenotype, not even under endoplasmic reticulum-stressing conditions that could impair protein folding. The data presented in this work strongly suggest that PfDPY19 is unlikely to play a critical role in the asexual blood stages of the parasite, at least under in vitro conditions.

A prevailing hypothesis for the evolution of parasitism posits that the fitness benefits gained from parasitic activity results in selection for and fixation of parasitic strategies. Despite the potential fitness advantage of parasitism, facultative parasites continue to exhibit genetic variation in parasitic behaviour in nature. We hypothesized that evolutionary trade-offs associated with parasitic host-attachment behaviour maintain natural variation observed in attachment behaviour. In this study, we used replicate lines of a facultatively parasitic mite, previously selected for increased host-attachment behaviour to test whether increased attachment trades off with mite fecundity and longevity, as well as the phenotypic plasticity of attachment. We also tested for potential correlated changes in mite morphology. To test for context-dependent trade-offs, mite fecundity and longevity were assayed in the presence or absence of a host. Our results show that selected and control mites exhibited similar fecundities, longevities, attachment plasticities and morphologies, which did not provide evidence for life history trade-offs associated with increased attachment. Surprisingly, phenotypic plasticity in attachment was maintained despite directional selection on the trait, which suggests that phenotypic plasticity likely plays an important role in maintaining attachment variation in natural populations of this facultative parasite.

Levamisole (Lms) is an anthelminthic drug with immunomodulatory activity. Chagas disease (CD) is caused by Trypanosoma cruzi and there is very low access to the drugs available, benznidazole (Bz) and nifurtimox, both far from ideal. In a drug-repurposing strategy to test potential activity as antiparasitic and immunomodulatory agent for CD, Lms was assayed on acute T. cruzi murine infection, alone and in co-administration with Bz. During protocol standardization, 100 and 10 mpk of Bz given for five consecutive days resulted in parasitaemia suppression and 100% animal survival only with the highest dose. Flow cytometry showed that both optimal (100 mpk) and suboptimal (10 mpk) doses of Bz equally decreased the plasma levels of cytokines commonly elevated in this acute infection model. Lms alone (10–0.5 mpk) did not decrease parasitaemia nor mortality rates. Co-administration was investigated using the suboptimal dose of Bz and different doses of Lms. While Bz 10 mpk did not alter parasitaemia, the combo partially reduced it but only slightly promoted animal survival. This effect could be related to Th1-response modulation since interleukin-6 and interferon-γ were higher after treatment with the combo.

The genetic variation and taxonomic status of the four morphologically-defined species of Macropostrongyloides in Australian macropodid and vombatid marsupials were examined using sequence data of the ITS+ region (=first and second internal transcribed spacers, and the 5.8S rRNA gene) of the nuclear ribosomal DNA. The results of the phylogenetic analyses revealed that Ma. baylisi was a species complex consisting of four genetically distinct groups, some of which are host-specific. In addition, Ma. lasiorhini in the common wombat (Vombatus ursinus) did not form a monophyletic clade with Ma. lasiorhini from the southern hairy-nosed wombat (Lasiorhinus latifrons), suggesting the possibility of cryptic (genetically distinct but morphologically similar) species. There was also some genetic divergence between Ma. dissimilis in swamp wallabies (Wallabia bicolor) from different geographical regions. In contrast, there was no genetic divergence among specimens of Ma. yamagutii across its broad geographical range or between host species (i.e. Macropus fuliginosus and M. giganteus). Macropostrongyloides dissimilis represented the sister taxon to Ma. baylisi, Ma. yamagutii and Ma. lasiorhini. Further morphological and molecular studies are required to assess the species complex of Ma. baylisi.

Parasite transmission can be altered via the removal of parasites by the ambient communities in which parasite–host interactions take place. However, the mechanisms driving parasite removal remain poorly understood. Using marine trematode cercariae as a model system, we investigated the effects of consumer and host body size on parasite removal rates. Laboratory experiments revealed that consumer or host body size significantly affected cercarial removal rates in crabs, oysters and cockles but not in shrimps. In general, cercarial removal rates increased with consumer (crabs and oysters) and host (cockles) body size. For the filter feeding oysters and cockles, the effects probably relate to their feeding activity which is known to correlate with bivalve size. Low infection levels found in cockle hosts suggest that parasite removal by hosts also leads to significant mortality of infective stages. The size effects of crab and shrimp predators on cercarial removal rates were more complex and did not show an expected size match-mismatch between predators and their cercarial prey, suggesting that parasite removal rates in predators are species-specific. We conclude that to have a comprehensive understanding of parasite removal by ambient communities, more research into the various mechanisms of cercarial removal is required.

This study aimed to set up methodology to monitor parasite-specific T-cell activation in vitro using Eimeria tenella-infected chickens. A sonicated E. tenella sporozoite protein preparation was used for the activation of chicken spleen cell cultures. Proliferation assessed by 3H-thymidin incorporation or blast transformation of T-cells assessed by immunofluorescence labelling and flow cytometry were used as read-outs for activation. Results showed that E. tenella-specific proliferation was detected in cultures of spleen cells collected in a ‘window’ between 8 and 14 days after primary infection. However, due to high variation in proliferative responses between individuals and to high background proliferation, large numbers of observations were needed to obtain significant results. Moreover, the outcome was not improved by increasing the infection dose to chickens or by depletion of T-cell receptor (TCR) γ/δ expressing cells from cultures. An E. tenella-specific blast transformation response was observed for TCRα/β expressing cells within the same ‘window’, confirming the identity of the responding cells as classic T-cells. Thus, it is possible to study the kinetics of E. tenella-specific T-cell responses in vitro. However, more in-depth phenotypic identification of the responding T-cells could improve the methodology.

Leishmaniasis is considered a parasitic disease that still causes serious consequences for mankind, because it presents a high mortality rate worldwide. Considered multi-hosts, the parasites of the genus Leishmania are able of infecting a wide variety of animal species. The dog was considered the main source of infection of visceral leishmaniasis (VL), in the urban area. However, the role of other animal species in the epidemiological cycle of the disease, such as cattle, remains unclear. Therefore, the aim of the present study was to evaluate the occurrence of Leishmania spp. in 100 bovines (Bos taurus) from an area endemic for canine VL, using blood culture and molecular analysis. By the sequencing analysis, one sample showed 100% similarity with Leishmania infantum. The results provide the first case of L. infantum isolation in one bovine from the periurban areas of Bauru, state of São Paulo, Brazil.

Trichomonas vaginalis is responsible for the most common non-viral, sexually transmitted infection, human trichomoniasis, and is associated with an increased susceptibility to HIV. An escalation in resistance (2.5–10%) to the clinical drug, metronidazole (MTZ), has been detected and this compound also has adverse side-effects. Therefore, new treatment options are urgently required. Herein, we investigate the possible anti-T. vaginalis activity of 1,10-phenanthroline-5,6-dione (phendione) and its metal complexes, [Ag(phendione)2]ClO4 and [Cu(phendione)3](ClO4)2·4H2O. Minimum inhibitory concentration (MIC) against T. vaginalis ATCC 30236 and three fresh clinical isolates and mammalian cells were performed using serial dilution generating IC50 and CC50 values. Drugs combinations with MTZ were evaluated by chequerboard assay. A strong anti-T. vaginalis activity was found for all test compounds. IC50 values obtained for [Cu(phendione)3](ClO4)2·4H2O were similar or lower than those obtained for MTZ. In vitro assays with normal cells showed low cytotoxicity and [Cu(phendione)3](ClO4)2·4H2O presented a high selectivity index (SI) for fibroblasts (SI = 11.39) and erythrocytes (SI > 57.47). Chequerboard assay demonstrated that the combination of [Cu(phendione)3](ClO4)2·4H2O with MTZ leads to synergistic interaction, which suggests distinct mechanisms of action of the copper–phendione complex and avoiding the MTZ resistance pathways. Our results highlight the importance of phendione-based drugs as potential molecules of pharmaceutical interest.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Cerebral malaria (CM) is the severe neurological complication causing acute non-traumatic encephalopathy in tropical countries. The mechanisms underlying the fatal cerebral complications are still not fully understood. Glutamate, a major excitatory neurotransmitter in the central nervous system of the mammalian brain, plays a key role in the development of neuronal cells, motor function, synaptic plasticity, learning and memory processes under normal physiological conditions. The subtypes of ionotropic glutamate receptor are N-methyl-D-aspartate receptors (NMDARs) which are involved in cellular mechanisms of learning and memory, synaptic plasticity and also mediate excitotoxic neuronal injury. In the present study, we found that glutamate level in synaptosomes, as well as expression of NMDAR, was elevated during the extreme condition of CM in C57BL6 mice. Arteether at 50 mg kg−1 × 1, 25 mg kg−1 × 2, days decreased the NMDAR expression and increased the overall survival of the experimental CM mice.

Infection is a significant cause of morbidity and mortality in patients with chronic kidney disease, especially who were under dialysis due to their depressed immunity. Toxoplasma gondii is a ubiquitous parasite that causes severe manifestations in immunocompromised patients. This case-control study was conducted to the immunodiagnosis and molecular validation of T. gondii infection among patients with end-stage renal disease undergoing haemodialysis. The study population consisted of 260 haemodialysis patients and 259 healthy controls referred to the main dialysis centres of Tehran, Iran during 2016. Anti-T. gondii immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies were assessed using enzyme-linked immunosorbent assay. As well, the T. gondii genomic DNA in whole blood samples of IgM-positive patients and healthy controls was evaluated using GRA6-polymerase chain reaction (PCR) and SAG1-loop-mediated isothermal amplification (LAMP) assays. The anti-T. gondii IgG and IgM antibodies were detected in 175 (67.3%) and 18 (7%) of haemodialysis patients and 122 (47%) and 4 (1.5%) of controls, respectively. Two of the 18 blood samples from IgM-positive patients and none of the IgM-positive control subjects were positive by GRA6-PCR. Whereas, nine and two blood samples of IgM-positive patients and controls were positive for Toxoplasma DNA by a SAG1-LAMP technique respectively. The seropositivity of the Toxoplasma IgM antibody was significantly different between haemodialysis patients and healthy controls which was confirmed by PCR and LAMP. The higher prevalence of T. gondii infection in haemodialysis patients compared with the controls proposes that these patients can be a group at risk for toxoplasmosis and screening for toxoplasmosis before dialysis is necessary for the patients.

The biogeographic histories of parasites and pathogens are infrequently compared with those of free-living species, including their hosts. Documenting the frequency with which parasites and pathogens disperse across geographic regions contributes to understanding not only their evolution, but also the likelihood that they may become emerging infectious diseases. Haemosporidian parasites of birds (parasite genera Plasmodium, Haemoproteus and Leucocytozoon) are globally distributed, dipteran-vectored parasites. To date, over 2000 avian haemosporidian lineages have been designated by molecular barcoding methods. To achieve their current distributions, some lineages must have dispersed long distances, often over water. Here we quantify such events using the global avian haemosporidian database MalAvi and additional records primarily from the Americas. We scored lineages as belonging to one or more global biogeographic regions based on infection records. Most lineages were restricted to a single region but some were globally distributed. We also used part of the cytochrome b gene to create genus-level parasite phylogenies and scored well-supported nodes as having descendant lineages in regional sympatry or allopatry. Descendant sister lineages of Plasmodium, Haemoproteus and Leucocytozoon were distributed in allopatry in 11, 16 and 15% of investigated nodes, respectively. Although a small but significant fraction of the molecular variance in cytochrome b of all three genera could be explained by biogeographic region, global parasite dispersal likely contributed to the majority of the unexplained variance. Our results suggest that avian haemosporidian parasites have faced few geographic barriers to dispersal over their evolutionary history.