A miRNAs catalogue from third-stage larvae and extracellular vesicles of Anisakis pegreffii provides new clues for host-parasite interplay.

Anisakids are widespread marine parasites of medical, veterinary and economic relevance. They infect marine natural hosts but humans can accidentally acquire the fish-borne zoonosis anisakiasis by ingesting infected raw fishes or mollusks. Among the several species described, Anisakis pegreffii is one of the main etiological agent of the disease, in particular in the Mediterranean area. Despite the growing evidence of miRNAs involvement in host-parasite interplay, and the emerging role of exosomal microvesicles in shuttling them between different cell types (and sometime across species), no information on miRNAs from any Anisakis species is presently available. In this study we isolated extracellular vesicles (EVs) released by Anisakis pegreffii infective third-stage larvae (L3) and analyzed by RNA-seq small RNAs from both L3 and EVs. We showed by nanoparticle tracking analysis that L3 release in culture medium particles of size compatible with the one of extracellular vesicles. A catalogue of 156 miRNAs from A. pegreffii was compiled by sequence comparison to evolutionary close species and miRNA prediction software. Using differential expression analysis, we identified a small number of highly abundant miRNAs in larvae and extracellular vesicles fractions whose potential biological relevance may deserve future investigation. Finally, A. pegreffii miRNAs were compared to those described in other parasitic helminths and predicted targets among human genes were searched, suggesting their potential involvement during infection.

Toxoplasmosis and knowledge: what do the Italian women know about?

Toxoplasmosis is a worldwide zoonotic infectious disease caused by Toxoplasma gondii. This infection is estimated to affect about a third of the world's population. The aim of this study was to evaluate the knowledge of Italian women about toxoplasmosis and its forms of transmission, clinical manifestations, diagnosis and prevention through two different modalities (e-research and traditional research). In a cross-sectional study, 808 Italian women were interviewed, using a self-administered questionnaire, through two different modalities: an e-research or web survey and a traditional paper research and 84% reported to have heard about toxoplasmosis, but from most of the sample, it resulted that the knowledge of the protozoan disease was superficial and incomplete.The assessment of the dimensionality related to the toxoplasmosis knowledge's instrument showed that the scale is composed by two stable and reliable factors which explain 58.6% of the variance: (a) the basic knowledge (α = 0.83), which explains the 45.2% of the variance and (b) the specialist knowledge (α = 0.71), which explains the 13.4% of the variance. The variance and the multiple linear regression data analysis showed significant predictors of correct basic knowledge of toxoplasmosis: the highest age, the highest degree of study, to have previously contracted illness or to know someone who had contracted it, to be working or to be housewives. In conclusion, this study showed limited awareness of toxoplasmosis and suggested the implementation of effective education and learning programs. The results also showed that online data collection, in academic research, might be a valid alternative to more traditional (paper-and-pencil) surveys.

Dirofilaria Immitis in Bulgaria: The First Genetic Baseline Data and an Overview of the Current Status.

Dirofilaria immitis, the agent of canine dirofilariosis, is a common parasite of domestic and wild carnivores with zoonotic potential and worldwide distribution, being endemic in many countries. Bulgaria is among European countries recognized as endemic for this heartworm parasite. In the present study, D. immitis adults recovered from pulmonary arteries of domestic dog and golden jackal originating from the Pazardzhik region in southern Bulgaria, and from red fox originating from the Plovdiv region in central-southern Bulgaria, were genetically analyzed in nuclear targets. The first PCR amplification of the internal transcribed region 2 (ITS2) of the ribosomal DNA with previously published D. immitis-specific primers yielded single fragments in size of 302 bp that is characteristic for these heartworms. PCR products of three isolates, resulted from the second amplification of the 5.8S-ITS2 region (235 bp) with pan-filarioid primers, were subjected to direct DNA sequencing. Identical nucleotide composition was detected across the screened target region for these Bulgarian isolates. When the 5.8S-ITS2 sequences were phylogenetically compared to the GenBank-retrieved D. immitis sequences in a worldwide context, the neighbor-joining analysis has shown three discrete clades. The first clade was composed of D. immitis isolates from Europe (including the studied Bulgarian samples), Asia and South America, in the second clade samples from Asia and South America were placed, whereas the third clade was formed by two Brazilian dog isolates originated from the north and southeast part of the country. The purpose of the present study was to verify the taxonomic characterization of D. immitis nematodes from Bulgaria based on morphology and compare their genetic structure with filariae obtained from the different world regions using molecular assays. It also summarizes previous epidemiological and ecological studies on the parasite distribution and prevalences in different hosts and regions undertaken so far in Bulgaria.

The fox tapeworm, Echinococcus multilocularis, in grey wolves and dogs in Slovakia: epidemiology and genetic analysis.

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is an important emerging parasite in the northern hemisphere. In epidemiological studies, the highest attention is being paid to foxes as the main reservoir hosts responsible for geographic expansion from multiple focal populations and the invasion of urban habitats, but little information is available on the parasite distribution in other carnivores. Hence, the study was designed to obtain updated information about the occurrence and genetic diversity of E. multilocularis in grey wolves and dogs in Slovakia. Faecal samples of wolves were collected from three locations under a certain level of environmental protection in the central and eastern parts of the country, and the presence of the parasite DNA was detected in 35.7% of 112 samples, with the highest rate (51.2%) recorded in the Poloniny National Park in north-eastern Slovakia. Among 110 faecal dog samples, E. multilocularis was detected in three faeces from segregated Roma settlements in the eastern part of the country, which accounted for an overall positivity of 2.7%. Sequence analysis of two mitochondrial genes, 12S rRNA and NADH dehydrogenase subunit 1, revealed four haplotypes in 13 isolates from wolves and dogs originating from four sites in eastern and central Slovakia, with all samples bearing a European-type pattern of E. multilocularis. The more than one-third positivity rate of E. multilocularis in wolf faecal samples dispersed over a large part of the country has corroborated the extensive circulation of the parasite in wildlife and confirmed the need to improve intervention control strategies.

The Mouse Bile Duct Tapeworm, Hymenolepis Microstoma in Free-living Small Mammals in Slovakia: Occurrence and Genetic Analysis.

The mouse bile duct tapeworm Hymenolepis microstoma, is a potentially zoonotic species with a wide variety of reported definitive hosts of rodent genera. In the present study the occurrence of H. microstoma in free-living small mammals in selected areas of Slovakia and the retrospective analysis of epidemiological data published in Slovakia were performed. Hymenolepis microstoma was detected in two animal species, the common shrew (Sorex araneus) and the European hamster (Cricetus cricetus) of 186 small mammals examined from two ecosystems, urban and natural ecosystem of national park. No mention about the presence of this parasite in Slovakia in the past was found following a bibliographical search. Partial sequences of the nuclear paramyosin gene showed the shrew isolate placed in a subclade together with H. microstoma from Portugal, with high bootstrap value for its differentiation from the sister species Hymenolepis nana. Similarly, the analysis of the nuclear ribosomal ITS region placed the hamster isolate in the cluster composed of H. microstoma from Australia, Spain and Portugal. The Slovak isolate was the most distinctive sample among available H. microstoma, differing in 1.4 - 1.9% of nucleotides from the remaining isolates. The difference (seven of 17 nucleotide positions) was partially due to indel polymorphisms associated with two and five nucleotides. To our knowledge, these are the first reports of H. microstoma in Central Europe and also the first record of infection in the common shrew. A recently indicated zoonotic potential of H. microstoma along with a possibility of its direct transmission between animals and/or humans without the need of intermediate hosts pose a public health concern in contaminated areas of Slovakia. The use of molecular techniques may substantially facilitate more thorough understanding of the epidemiological situation of H. microstoma and related tapeworms in various ecosystems of the country.

Comparative Study Among Lactophenol Blue, Lactophenol Solution and Proteinase-K Lytic Solution for Rostellar Hooks Morphometry of Echinococcus Granulosus Protoscolices.

Echinococcus granulosus is a tapeworm whose life cycle includes dogs and other canines as final hosts, while domestic and wild ungulates act as intermediate hosts for the tissue-invading larval stage (metacestode). E. granulosus has a worldwide geographical distribution. Protoscolices and rostellar hooks of E. granulosus are useful for diagnosis and rostellar hook morphometric features may be useful to discriminate E. granulosus and related species. The present study was aimed to determine a more suitable lytic solution and to obtain a clearest vision for performing morphometric studies on the rostellar hooks of E. granulosus protoscolices. Five fertile hydatid cyst samples were collected from sheep in Kirkuk slaughterhouse, Iraq, during June of 2015. According to the results of the present study, proteinase-K lytic solution is the best approach in morphometric analysis to get a clear vision of rostellar hooks and a safer usage in comparison with solutions containing lactophenol (lactophenol, lactophenol blue).

Occurrence of Anisakis and Hysterothylacium Nematodes in Atlantic Chub Mackerels from Libyan Coasts.

The occurrence of zoonotic parasitic nematodes in Atlantic chub mackerels (Scomber colias syn. Scomber japonicus) from Libyan waters was investigated, using epizootiological estimations and molecular specific characterization of larvae. Nematodes belonging to Anisakis spp., the main etiological agent of anisakiasis in Mediterranean waters, and to Hysterothylacium spp. so far considered not pathogenic to humans, were detected. Prevalence values were generally high in visceral cavities (over 40 % for both parasites) while were low for Anisakis (around 1 %) and null for Hysterothylacium in muscles. Moreover, the level of infections was associated with seasons, a feature potentially useful to plan fishing captures and to elaborate risk mitigation strategies for anisakiasis. Species molecular identification performed on a subsample described the presence of Hysterothylacium aduncum as the predominant species, along with Anisakis pegreffii and the hybrids (A. pegreffii and A. simplex sensu stricto), thus posing a concrete zoonotic risk following the consumption of such fish species as a raw preparation.

Molecular identification of Mesocestoides sp. metacestodes in a captive gold-handed tamarin (Saguinus midas).

Mesocestoides spp. are tapeworms harbored in the intestine of many domestic and wild carnivores, birds of prey and, rarely, of humans. The life cycle is assumed to involve three hosts and the metacestode juvenile stage (tetrathyridium) may reproduce asexually, provoking severe systemic infections. In the present study, a case of a peritoneal infection by Mesocestoides sp. occurred in a captive gold-handed tamarin (Saguinus midas) is described. Phylogenetic positions based on CO1 and 12S loci were analysed to describe in details this unusual finding of Mesocestoides in a callitrichid monkey. The phylogenetic analyses has not clearly resolve relationships between the metacestode forms from the tamarin and any of the described Mesocestoides species available so far, including those described from non-human primates, thus supporting the hypothesis of a new taxon within the genus.

Survey of Anisakis sp. and Hysterothylacium sp. in sardines and anchovies from the North Adriatic Sea.

The occurrence of larval Anisakidae and Raphidascarididae in anchovies and sardines from the North Adriatic Sea has been estimated. Anisakis pegreffii and Hysterothylacium aduncum were reported, with low prevalence values. In brief, a total amount of 7650 fish specimens collected between September 2011 and 2012 were analysed using three different inspection analyses: a visual inspection of the coelomic cavity, an examination of the viscera exploiting the positive hydro-tropism of the larvae (modified Baermann technique) and enzymatic digestion of muscular tissue pools. Low level of infestation was reported for Anisakis sp. in both in anchovies and sardines, while higher values were reported for Hysterothylacium sp. Subsamples of nematodes collected were characterized at species level using the molecular diagnostic key based on ITS nuclear ribosomal region, and A. pegreffii and H. aduncum were identified. The low prevalence of Anisakis sp. in sardines and anchovies from the North Adriatic Sea could be related to the peculiar distribution of cetaceans and carnivorous zooplankton in the investigated region and could be used as a potential tag to define oily fishes from this specific fishing area as at low-risk for anisakiasis.

Putative hybrids between two Anisakis cryptic species: molecular genotyping using High Resolution Melting.

The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended.

Molecular identification of Anisakis spp. from fishes collected in the Tyrrhenian Sea (NW Mediterranean).

The accurate identification of anisakid nematodes at any life cycle stage is important both to deepen the knowledge on their taxonomy, ecology, epidemiology and for diagnosis and control, as larval stages cause a clinical disease in humans known as anisakidosis. With the aim to investigate the presence of anisakid larvae, specimens of horse mackerel, Trachurus trachurus (Linnaeus, 1758), silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), European anchovy, Engraulis encrasicolus (Linnaeus, 1758) and opah fish, Lampris guttatus (Brunnich, 1788), were collected by trawling at depths ranging from 50 to 400 m. A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region was used to identify Anisakis spp. larvae recovered in fish samples. Restriction profiles showed three banding patterns, corresponding to Anisakis pegreffii, Anisakis physeteris and to heterozygote pattern between A. pegreffii and Anisakis simplex s.s. Specimens showing the heterozygote restriction pattern were also analyzed by sequencing of the entire ITS region, to confirm the heterozygote status.

Two new species of Contracaecum Railliet & Henry, 1912 (Nematoda: Anisakidae), C. fagerholmi n. sp. and C. rudolphii F from the brown pelican Pelecanus occidentalis in the northern Gulf of Mexico.

DNA sequencing of the nuclear ribosomal DNA internal transcribed spacers (ITS) and mitochondrial rrnS and cox2 genes, and analysis of polymorphisms in restriction profiles in the ITS and rrnS, were used to characterise anisakid nematodes belonging to Contracaecum Railliet & Henry, 1912 infecting the brown pelican Pelecanus occidentalis (L.) in Galveston Bay, Texas and Sarasota Bay, Florida. Molecular data led to the detection of two new species: Contracaecum fagerholmi n. sp., which was also supported by clear morphological evidence, and Contracaecum rudolphii F, a new cryptic species within the Contracaecum rudolphii Hartwich, 1964 complex. Bayesian phylogenetic analysis demonstrated that C. fagerholmi and C. rudolphii F form two well-separated clusters, with C. fagerholmi being closely related to Contracaecum bioccai Mattiucci et al., 2008 and C. rudolphii F being included in the C. rudolphii complex. C. fagerholmi can be readily differentiated morphologically from all of its congeners, other than C. microcephalum (Rudolphii 1809) and the five currently recognised members of the C. rudolphii complex (C. rudolphii A, B, C, D and E). C. fagerholmi differs from C. microcephalum in the length of the spicules and the shape of the distal tip of the spicules, and from C. rudolphii (sensu lato) in the shape and size of the ventro-lateral and dorsal lips and by having interlabia which are not distally bifurcate. Further studies are needed to determine which morphological characteristics can be used to distinguish the cryptic species of the C. rudolphii complex in order to assign them with formal names. The recovery of a third species, C. bioccai, from the brown pelican confirms its occurrence in this host and extends its known geographical distribution.

Acute sodium overload produces renal tubulointerstitial inflammation in normal rats.

The aim of the present study was to determine whether acute sodium overload could trigger an inflammatory reaction in the tubulointerstitial (TI) compartment in normal rats. Four groups of Sprague-Dawley rats received increasing NaCl concentrations by intravenous infusion. Control (C): Na+ 0.15 M; G1: Na+ 0.5 M; G2: Na+ 1.0 M; and G3: Na+ 1.5 M. Creatinine clearance, mean arterial pressure (MAP), renal blood flow (RBF), and sodium fractional excretion were determined. Transforming growth factor beta1 (TGF-beta1), alpha-smooth muscle actin (alpha-SMA), RANTES, transcription factor nuclear factor-kappa B (NF-kappaB), and angiotensin II (ANG II) were evaluated in kidneys by immunohistochemistry. Animals with NaCl overload showed normal glomerular function without MAP and RBF modifications and exhibited a concentration-dependent natriuretic response. Plasmatic sodium increased in G2 (P < 0.01) and G3 (P < 0.001). Light microscopy did not show renal morphological damage. Immunohistochemistry revealed an increased number of ANG II-positive tubular cells in G2 and G3, and positive immunostaining for NF-kappaB only in G3 (P < 0.01). Increased staining of alpha-SMA in the interstitium (P < 0.01), TGF-beta1 in tubular cells (P < 0.01), and a significant percentage (P < 0.01) of positive immunostaining for RANTES in tubular epithelium and in glomerular and peritubular endothelium were detected in G3 > G2 > C group. These results suggest that an acute sodium overload is able 'per se' to initiate TI endothelial inflammatory reaction (glomerular and peritubular) and incipient fibrosis in normal rats, independently of hemodynamic modifications. Furthermore, these findings are consistent with the possibility that activation of NF-kappaB and local ANG II may be involved in the pathway of this inflammatory process.

Fructose overload modifies vascular morphology and prostaglandin production in rats.

1. A fructose (Fru)-enriched diet induces a mild increase in blood pressure associated with hyperglycaemia, hypertriglyceridaemia, and insulin resistance, resembling the human 'syndrome X', being an useful model to study hypertension and type 2 diabetes. 2. A sustained elevation of blood pressure is associated with cardiovascular structural modifications such as left ventricular hypertrophy and increased wall thickness:lumen diameter ratio in blood vessels. 3. Prostanoids (PR), metabolites of arachidonic acid through the cyclooxygenase pathway, include vasoactive substances synthesized and released by the vessel walls. 4. The aim of the present study was to analyse, in Fru-treated rats: (i) the morphology of mesenteric vessels and; (ii) the PR production in aorta and mesenteric vessels, in order to assess whether these parameters are related with the haemodynamic alterations observed in this experimental model. 5. Blood pressure, glycaemia and triglyceridaemia, were significantly elevated in both (4 and 22 weeks) Fru-treated groups. Meanwhile body and heart weight as well as insulinaemia were similar between experimental animals and controls. 6. The mesenteric vessels of Fru-treated rats (22 weeks) showed an increased thickness and area of the media when compared with the controls; meanwhile, the lumen diameter was similar in both groups. 7. The Fru treatment for 4 weeks did not modify PR production in aorta, whereas in the mesenteric bed it diminished prostaglandin (PG) E(2) release significantly compared with the controls. However, in the group treated for 22 weeks, Fru reduced PGI(2) production in the aorta, as assessed by 6-keto-PGF(1)alpha measurements. Meanwhile, in the mesenteric bed, the chronic Fru treatment decreased PGE(2) release but, rather surprisingly, increased the output of PGI(2) when compared with its corresponding controls. 8. In conclusion, the present study shows the existence of an alteration in the morphology of mesenteric vessels in Fru-treated rats, which could be related to an increase in peripheral resistance and the consequent mild hypertension observed in this model. However, a diminished release of vasodilator PRs, such as PGE(2) in mesenteric vessels at 4 and 22 weeks and PGI(2) in aorta at 22 weeks could further impair the vessel response. The increase in PGI(2) observed in the chronic group in mesenteric vessels could be attributed to a compensatory mechanism.

Impaired response to insulin associated with protein kinase C in chronic fructose-induced hypertension.

A fructose-enriched diet induces an increase in blood pressure associated with metabolic alterations in rats. Our hypothesis was that an increase in protein kinase C (PKC) activation, reported in the acute period of fructose overload, and an impaired vessel's response to vasoactive substances contribute to maintain elevated blood pressure levels in the chronic period. The aims of this study were to investigate in this animal model of hypertension: (1) if the increase in PKC activation was also found in the chronic stage; (2) the involvement of nitric oxide and insulin in the vessel's response; and plasma atrial natriuretic factor and nitrites/nitrates (nitric oxide metabolites) behavior. We evaluated the effects of: PKC-stimulator 12,13-phorbol dibutyrate, phenylephrine, insulin, nitric oxide synthase-inhibitor NG-nitro-L-arginine methyl esther (L-NAME) and PKC-inhibitor Calphostin C on aortic rings responses of Sprague-Dawley rats: fructose-fed and control. The fructose-fed group showed higher contractility to 12,13-phorbol dibutyrate than the control group in aortic rings pre-incubated with insulin, and this difference disappeared with L-NAME. The response to phenylephrine in rings pre-incubated with Calphostin C was decreased in the fructose-fed group and increased with Calphostin C plus L-NAME. Fructose-fed rats showed higher levels of plasma atrial natriuretic factor and nitrites/nitrates than controls. In conclusion, chronic fructose feeding seems to develop an impaired response to insulin, dependent on nitric oxide, suggesting a PKC alteration. Vasorelaxant agents, such as atrial natriuretic factor and nitric oxide, would behave as compensatory mechanisms in response to high blood pressure.