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.

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.

Anguilla anguilla intestinal immune response to natural infection with Contracaecum rudolphii A larvae.

The European eel, Anguilla anguilla, is a major warm-water fish species cultured in North and South Europe. Seventy-one A. anguilla collected between 2010 and 2015 from the Comacchio lagoons were examined. Fish were infected and damaged by larvae (L3) of the nematode Contracaecum rudolphii A, which were encapsulated within the thickness of the intestinal wall and within the external visceral peritoneum (serosa). Conspicuous granulomas, visible at sites of infection, were arranged in a trilayer, formed by a series of concentric whorls. The cells involved in the immune response and their distribution in the granuloma layers were assessed by immunohistochemical, immunofluorescence, and ultrastructural techniques. The outer part of the granuloma contained macrophages, macrophage aggregates, and mast cells (MCs) scattered among fibroblasts. This layer was vascularized, with degranulation of MCs occurring in close proximity to the capillaries. The middle layer was rich in MCs and fibroblasts. The inner layer, closest to the parasite larva, consisted mainly of dark epithelioid cells, some of which were necrotic. Non-necrotic epithelioid cells formed desmosomes between themselves or with fibroblasts. Within the granulomas, numerous cells of different types were positive to proliferative cell nuclear antigen antibody, indicating a high degree of cellular proliferation around the larvae.

Detection and isolation of the α-proteobacterium Asaia in Culex mosquitoes.

Investigations of microbiota within mosquitoes continue to widen the spectrum of possible symbiont-based applications against vector-borne diseases. In this context, α-proteobacteria of the genus Asaia (Rhodospirillales: Acetobacteraceae) are emerging as possible endosymbiotic candidates, particularly in paratransgenic approaches aimed at interrupting pathogen transmission. Previous studies have shown that Asaia spp. distribution among Anopheles gambiae and Anopheles stephensi (Diptera: Culicidae) mosquitoes displayed positive rates of infection in isolated midguts, salivary glands and reproductive tissues. Similarly, Asaia has been detected in Aedes albopictus (Stegomyia albopicta) and Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) populations. Within the Culex pipiens complex (Diptera: Culicidae), Asaia infection is still largely unexplored. Here, we summarize a preliminary survey of laboratory-reared Cx. pipiens complex and field-collected Culex quinquefasciatus for the presence of Asaia spp., and present the first identification of Asaia in some of the members of the Cx. pipiens complex and the first description in West African populations of Cx. quinquefasciatus.

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.

Genetic variability of Haemonchus contortus (Nematoda: Trichostrongyloidea) in alpine ruminant host species.

Genetic variability of the ovine parasite Haemonchus contortus from the Alpine area was investigated using mitochondrial DNA (nd4 gene), internal transcribed spacers 1 and 2 and microsatellites, in order to assess whether cross-transmission between domestic and wild ruminants occurs. The dataset was composed of 78 individual adult male H. contortus collected from chamois (Rupicapra r. rupicapra), roe deer (Capreolus capreolus), alpine ibex (Capra ibex ibex), domestic goat (Capra hircus) and sheep (Ovis aries) from different alpine areas. The data obtained show low host specificity and high genetic variation within H. contortus populations. The analyses indicate the presence of two mitochondrial haplotype clusters among host species and the absence of cryptic parasite species, confirming H. contortus as a generalist nematode and suggesting that parasite transmission between populations of domestic and wild ruminants normally occurs.

Cystic echinococcosis in Turkey: genetic variability and first record of the pig strain (G7) in the country.

A sample of 22 Echinococcus granulosus isolates collected from 12 sheep and ten humans from a focus of cystic echinococcosis in western Turkey was examined by DNA sequencing of four mitochondrial genes (cox1, atp6, nad1, rrnS). Results demonstrated the presence of two species of E. granulosus complex, E. granulosus sensu stricto and E. canadensis. Of E. granulosus sensu stricto, the G1 genotype (including three microvariants) was found in 17 isolates from humans and sheep, the G3 genotype and an intermediate form G1/G3 in one isolate each (both from sheep). Of E. canadensis, the pig strain G7 was found in three isolates from sheep and human. This is the first report of this strain in Turkey. Its presence has implications for local control programs due to its shorter maturation rate in dogs compared with E. granulosus sensu stricto. Goat and/or wild boar are likely reservoirs for G7 in the region. We provided further data on the pattern and frequency of nucleotide substitutions within the G1/G3 cluster. Based on our results and GenBank records, G2 (Tasmanian sheep strain) is not considered as a discrete genotypic unit, as its sequences at polymorphic sites conform to microvariants of both G1 and (more often) G3.

Acanthamoeba T4 and T15 genotypes associated with keratitis infections in Italy.

Thus far there is little data available concerning Acanthamoeba associated amoebic keratitis (AK) from Italy. In order to understand the incidence of Acanthamoeba in patients with ocular infections and to characterize the isolates at the molecular level, ocular specimens and contact lenses or lens case solutions from 140 patients were analysed by culture and by an 18S rRNA (Rns) gene-based PCR method. Nineteen (13.6%) patients showed Acanthamoeba culture positive samples. Eleven out of the 14 genetically characterized isolates were assigned to the T4 genotype. Three isolates, two of them from patients with keratitis responding to specific anti-Acanthamoeba therapy, were identified as belonging to the T15 genotype. This finding represents the first association between the T15 genotype and human amoebic keratitis. PCR amplification of the 18S ribosomal DNA proved to be a sensitive method, potentially able to detect Acanthamoeba without the need of long culture incubation, and thus considerably useful for clinical applications.

Molecular and morphological evidence for the occurrence of Anisakis sp. A (Nematoda, Anisakidae) in the Blainville's beaked whale Mesoplodon densirostris.

Twenty-three adults (only one male) and two fourth-stage larvae of Anisakis, recovered from the stomach of a Blainville's beaked whale (Mesoplodon densirostris) stranded in Galicia (NW Spain), were studied morphologically and molecularly. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing studies carried out on the ribosomal DNA spacers and on the cox2 mitochondrial gene confirm that these nematodes belong to the taxon Anisakis sp. A, which was previously detected as a larval stage in fishes from Madeiran waters. In addition, our molecular studies demonstrate that Anisakis sp. A and the taxon Anisakis sp., previously found in other Mesoplodon spp., are the same species. The adults of Anisakis sp. A are morphologically similar to A. ziphidarum but with a shorter body length and longer spicules (right spicule 2.42 mm, left spicule 2.30 mm). Since the poor condition of the male tail cuticle prevented a proper description of the caudal plates and the pattern of caudal papillae, we propose to retain the name Anisakis sp. A until new males are described correctly.

Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois.

The major histocompatibility complex (MHC) harbours some of the most polymorphic loci in vertebrate genomes. MHC genes are thought to be subject to some form of balancing selection, most likely pathogen-mediated selection. Hence, MHC genes are excellent candidates for exploring adaptive processes. In this study, we investigated the genetic variation at exon 2 of the DRB class II MHC locus in 191 alpine chamois (Rupicapra rupicapra) from 10 populations in the eastern Alps of Italy. In particular, we were interested in distinguishing and estimating the relative impact of selective and demographic factors, while taking into account the confounding effect of recombination. The extremely high d(n)/d(s) ratio and the presence of trans-species polymorphisms suggest that a strong long-term balancing selection effect has been operating at this locus throughout the evolutionary history of this species. We analysed patterns of genetic variation within and between populations, and the mitochondrial D-loop polymorphism patterns were analysed to provide a baseline indicator of the effects of demographic processes. These analyses showed that (i) the chamois experienced a demographic decline in the last 5000-30 000 years, most likely related to the postglacial elevation in temperature; (ii) this demographic process can explain the results of neutrality tests applied to MHC variation within populations, but cannot justify the much weaker divergence between populations implied by MHC as opposed to mitochondrial DNA; (iii) similar sets of divergent alleles are probably maintained with similar frequencies by balancing selection in different populations, and this mechanism is also operating in small isolated populations, which are strongly affected by drift.

Genetic variation within and between G1 and G3 genotypes of Echinococcus granulosus in Italy revealed by multilocus DNA sequencing.

Numerous studies have provided evidence that Echinococcus granulosus exists as a complex of different strains, that differ in a wide variety of criteria that have an impact on the epidemiology, pathology and control of cystic hydatid disease (CHD) and, to date, 10 distinct genotypes (G1-G10) have been identified. In Italy, sequence analysis of the mitochondrial cox1 and nad1 genes showed the occurrence of the G1 genotype, the common sheep strain, the G3 genotype, the buffalo strain and of one isolate identified as G2 genotype, the Tasmanian sheep strain. In the present work, we have analysed E. granulosus strains in Italy, by genotyping a large sample of isolates and by checking out the genetic differentiation within and among the G1 and G3 genotypes using an additional mitochondrial gene as marker, the rrnS gene. Sequencing of the rrnS gene revealed a significant genetic differentiation between isolates identified as belonging to the G1 and G3 genotypes, with fixed nucleotide substitutions. This study provides further evidence of the occurrence of the E. granulosus G3 buffalo strain in Italy, a strain previously thought to be confined to the Indian region.

Proliferative peritoneal and pleural cestodiasis in a cat caused by metacestodes of Mesocestoides sp. Anatomohistopathological findings and genetic identification.

A 10-year-old female cat was brought to Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana for post-mortem examination. The animal used to live, together with 26 other cats, in the big terrace of an apartment at the 8th floor in Rome; and was always fed with industrial pet food. Anamnesis referred balance troubles, vomit and convulsions, during a couple of days, followed by sudden death. At necropsy, the cat presented mucoid rhinitis, purulent tracheitis, small areas of pneumonia, dark spots in the liver, catarrhal-hemorrhagic gastritis, fibrinous enteritis and meningeal hyperemia. Thoracic and abdominal cavities were completely invaded by hundreds of larval stages of cestodes. The same parasites were also included in nodules in pancreatic, lung and kidney parenchyma. Microscopic examination of parasites allowed their identification as larval stages (metacestodes) of cestodes of the genus Mesocestoides. The molecular genotyping of the metacestodes indicates a close relationship with members of the genus Mesocestoides, although a significant variation was found with respect to the available sequences of other species of the genus.

Genetic characterization of members of the genus Contracaecum (Nematoda: Anisakidae) from fish-eating birds from west-central Florida, USA, with evidence of new species.

Specimens of Contracaecum spp. from Phalacrocorax auritus and Pelecanus occidentalis from Florida were characterized by sequencing of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) and by PCR-based RFLP analysis of the same gene and of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Analyses of the rrnS sequence data using the MP and UPGMA approaches yielded trees with similar topologies, delineating 3 main clusters. Specimens from Ph. auritus, morphologically assigned to C. rudolphii (s.l.), were part of the cluster comprising also the other 2 species of the C. rudolphii complex (A and B), but representing a genetically distinct group, potentially corresponding to a distinct lineage within the complex, provisionally named as C. rudolphii C. The second cluster comprised 5 individuals from P. occidentalis, which formed a genetically relatively homogeneous group. The rrnS data indicate that these specimens (indicated as Contracaecum sp. 1) are clearly genetically different from the morphologically most closely related species, i.e. C. rudolphii (s.l.) and C. microcephalum, and could represent a new species. The third cluster comprised 7 specimens from P. occidentalis morphologically assigned to C. multipapillatum (s.l.). These were shown to be genetically homogeneous and related to but quite distinct from C. multipapillatum from Greece, although additional studies are needed to assess their status. PCR-RFLP based markers for the quick identification of these taxa are provided.

Practical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea: Anisakidae) using genetic markers in nuclear ribosomal DNA.

Using genetic markers defined previously in the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA), PCR-coupled restriction fragment length polymorphism (PCR-RFLP) and specific PCR assays were established for the specific detection of each of two morphologically indistinguishable operational taxonomic units (Contracaecum rudolphii A and Contracaecum rudolphii B) within Contracaecum rudolphii (s.l.) and their differentiation from Contracaecum septentrionale, a closely related congener. Application of these tools to C. rudolphii (s.l.) adults from Phalacrocorax carbo sinensis (the Eurasian subspecies of the great cormorant) from Qinghai Lake in China, revealed C. rudolphii B to infect this host. This is the first report of C. rudolphii B in P. carbo sinensis outside of Europe (where it was originally detected), supporting the proposal that this species has a broad geographical distribution. Together with other methods, each of these molecular tools will be useful for investigating the ecology of C. rudolphii A and C. rudolphii B as well as C. septentrionale.

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing.

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.

Molecular identification of Anisakis species from Pleuronectiformes off the Portuguese coast.

Anisakid nematodes belonging to the Anisakis simplex complex are highly prevalent in several fish species off the coast of Portugal and are an important zoonotic problem in the Iberian Peninsula. Two reproductively isolated sibling species of the Anisakis simplex complex were identified from Pleuronectiformes inhabiting the Portuguese coast using restriction fragment length polymorphism (RFLP). Recombinant genotypes corresponding to presumptive Anisakis simplex sensu stricto and Anisakis pegreffii hybrids were also detected by this technique, as well as the species Anisakis typica. Although 25 species of Pleuronectiformes were investigated, Anisakis spp. larvae were only found in seven: Arnoglossus imperialis, Arnoglossus laterna, Lepidorhombus boscii, Citharus linguatula, Platichthys flesus, Dicologlossa cuneata and Solea senegalensis. The occurrence of hybrids in relatively sedentary fishes such as the Pleuronectiformes suggests that the Portuguese coast may constitute an area of hybridization and, therefore, is of particular interest for the study of the process of hybridization and speciation for these anisakids.

Microsatellite analysis of olive fly populations in the Mediterranean indicates a westward expansion of the species.

Bactrocera oleae is the major insect pest of the olive fruit. Twelve microsatellite loci isolated from the genome of this insect were used in a Mediterranean-wide population analysis. These loci were highly polymorphic with a mean number of alleles per locus of 10.42 and a mean effective number of alleles of 2.76. The analysis was performed on a sample of 671 flies collected from nineteen locations around the European part of the Mediterranean basin. Despite the high level of gene flow across the Mediterranean, results support the notion of a differentiation of three subpopulations: one of the Iberian Peninsula, one of Greece and Italy and one of Cyprus. In addition, the gradual decrease of heterozygosity from the Eastern to the Western part of the Mediterranean indicates a westward expansion of the species.