De novo transcriptome assembly and annotation of the third stage larvae of the zoonotic parasite Anisakis pegreffii.

OBJECTIVES: Anisakis pegreffii is a zoonotic parasite requiring marine organisms to complete its life-history. Human infection (anisakiasis) occurs when the third stage larvae (L3) are accidentally ingested with raw or undercooked infected fish or squids. A new de novo transcriptome of A. pegreffii was here generated aiming to provide a robust bulk of data to be used for a comprehensive "ready-to-use" resource for detecting functional studies on genes and gene products of A. pegreffii involved in the molecular mechanisms of parasite-host interaction. DATA DESCRIPTION: A RNA-seq library of A. pegreffii L3 was here newly generated by using Illumina TruSeq platform. It was combined with other five RNA-seq datasets previously gathered from L3 of the same species stored in SRA of NCBI. The final dataset was analyzed by launching three assembler programs and two validation tools. The use of a robust pipeline produced a high-confidence protein-coding transcriptome of A. pegreffii. These data represent a more robust and complete transcriptome of this species with respect to the actually existing resources. This is of importance for understanding the involved adaptive and immunomodulatory genes implicated in the "cross talk" between the parasite and its hosts, including the accidental one (humans).

Molecular and evolutionary basis for survival, its failure, and virulence factors of the zoonotic nematode Anisakis pegreffii.

Parasitism is a highly successful life strategy and a driving force in genetic diversity that has evolved many times over. Accidental infections of non-targeted hosts represent an opportunity for lateral host switches and parasite niche expansion. However, if directed toward organisms that are phylogenetically distant from parasite's natural host, such as humans, it may present a dead-end environment where the parasite fails to mature or is even killed by host immunity. One example are nematodes of Anisakidae family, genus Anisakis, that through evolution have lost the ability to propagate in terrestrial hosts, but can survive for a limited time in humans causing anisakiasis. To scrutinize versatility of Anisakis to infect an evolutionary-distant host, we performed transcriptomic profiling of larvae successfully migrating through the rat, a representative model of accidental human infection and compared it to that of larvae infecting an evolutionary-familiar, paratenic host (fish). In a homeothermic accidental host Anisakis upregulated ribosome-related genes, cell division, cuticle constituents, oxidative phosphorylation, in an unsuccessful attempt to molt to the next stage. In contrast, in the paratenic poikilothermic host where metabolic pathways were moderately upregulated or silenced, larvae prepared for dormancy by triggering autophagy and longevity pathways. Identified differences and the modelling of handful of shared transcripts, provide the first insights into evolution of larval nematode virulence, warranting their further investigation as potential drug therapy targets.

Comparative Proteomics Analysis of Anisakis simplex s.s.-Evaluation of the Response of Invasive Larvae to Ivermectin.

Ivermectin (IVM), an antiparasitic drug, has a positive effect against Anisakis simplex s.s. infection and has been used for the treatment and prevention of anisakiasis in humans. However, the molecular mechanism of action of IVM on A. simplex s.s. remains unknown. Herein, tandem mass tag (TMT) labeling and extensive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis were used to identify the effect of IVM on the proteome of A. simplex s.s. in vitro. During the study, 3433 proteins, of which 1247 had at least two protein unique peptides, were identified. Comparative proteomics analysis revealed that 59 proteins were differentially regulated (DRPs) in IVM-treated larvae, of which 14 proteins were upregulated and 38 were downregulated after 12 h of culture, but after 24 h, 12 proteins were upregulated and 22 were downregulated. The transcription level of five randomly selected DRPs was determined by real-time PCR as a supplement to the proteomic data. The functional enrichment analysis showed that most of the DRPs were involved in oxidoreductase activity, immunogenicity, protein degradation, and other biological processes. This study has, for the first time, provided comprehensive proteomics data on A. simplex s.s. response to IVM and might deliver new insight into the molecular mechanism by which IVM acts on invasive larvae of A. simplex s.s.

Mass Spectrometry Based-Proteomic Analysis of Anisakis spp.: A Preliminary Study towards a New Diagnostic Tool.

Anisakiasis is nowadays a well-known infection, mainly caused by the accidental ingestion of Anisakis larvae, following the consumption of raw or undercooked fishes and cephalopods. Due to the similarity of symptoms with those of common gastrointestinal disorders, this infection is often underestimated, and the need for new specific diagnostic tools is becoming crucial. Given the remarkable impact that MALDI-TOF MS biotyping had in the last decade in clinical routine practice for the recognition of bacterial and fungi strains, a similar scenario could be foreseen for the identification of parasites, such as nematodes. In this work, a MALDI-TOF MS profiling of Anisakis proteome was pursued with a view to constructing a first spectral library for the diagnosis of Anisakis infections. At the same time, a shotgun proteomics approach by LC-ESI-MS/MS was performed on the two main fractions obtained from protein extraction, to evaluate the protein species enriched by the protocol. A set of MALDI-TOF MS signals associated with proteins originating in the ribosomal fraction of the nematode extract was selected as a potential diagnostic tool for the identification of Anisakis spp.

Immunoreactive Proteins in the Esophageal Gland Cells of Anisakis Simplex Sensu Stricto Detected by MALDI-TOF/TOF Analysis.

In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus Anisakis. Taking into account the increase of anisakiasis and allergies related to these nematodes, immunoreactive properties of gland cell proteins were investigated. Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining which revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp.-allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, with 82 spots revealed in 2D. The corresponding protein bands and spots were excised from the silver-stained gel and protein assignation was made by MALDI-TOF/TOF. A total of 13 (including proteoforms) were unambiguously identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin and one is an Anisakis simplex allergen.

Leukocyte Nucleolus and Anisakis pegreffii-When Falling Apart Means Falling in Place.

The view of the nucleolus as a mere ribosomal factory has been recently expanded, highlighting its essential role in immune and stress-related signalling and orchestrating. It has been shown that the nucleolus structure, formed around nucleolus organiser regions (NORs) and attributed Cajal bodies, is prone to disassembly and reassembly correlated to various physiological and pathological stimuli. To evaluate the effect of parasite stimulus on the structure of the leukocyte nucleolus, we exposed rat peripheral blood mononuclear cells (PBMC) to the crude extract of the nematode A. pegreffii (Anisakidae), and compared the observed changes to the effect of control (RPMI-1640 media), immunosuppressive (MPA) and immunostimulant treatment (bacterial lipopolysaccharide (LPS) and viral analogue polyinosinic:polycytidylic acid (poly I:C)) by confocal microscopy. Poly I:C triggered the most accentuated changes such as nucleolar fragmentation and structural unravelling, LPS induced nucleolus thickening reminiscent of cell activation, while MPA induced disassembly of dense fibrillar and granular components. A. pegreffii crude extract triggered nucleolar segregation, expectedly more enhanced in treatment with a higher dose. This is the first evidence that leukocyte nucleoli already undergo structural changes 12 h post-parasitic stimuli, although these are likely to subside after successful cell activation.

Differences in Gene Expression Profiles of Seven Target Proteins in Third-Stage Larvae of Anisakis simplex (Sensu Stricto) by Sites of Infection in Blue Whiting (Micromesistius poutassou).

The third-stage larvae of the parasitic nematode genus Anisakis tend to encapsulate in different tissues including the musculature of fish. Host tissue penetration and degradation involve both mechanic processes and the production of proteins encoded by an array of genes. Investigating larval gene profiles during the fish infection has relevance in understanding biological traits in the parasite's adaptive ability to cope with the fish hosts' defense responses. The present study aimed to investigate the gene expression levels of some proteins in L3 of A. simplex (s.s.) infecting different tissues of blue whiting Micromesistius poutassou, a common fish host of the parasite in the NE Atlantic. The following genes encoding for Anisakis spp. proteins were studied: Kunitz-type trypsin inhibitor (TI), hemoglobin (hb), glycoprotein (GP), trehalase (treh), zinc metallopeptidase 13 (nas 13), ubiquitin-protein ligase (hyd) and sideroflexin 2 (sfxn 2). Significant differences in gene transcripts (by quantitative real-time PCR, qPCR) were observed in larvae located in various tissues of the fish host, with respect to the control. ANOVA analysis showed that relative gene expression levels of the seven target genes in the larvae are linked to the infection site in the fish host. Genes encoding some of the target proteins seem to be involved in the host tissue migration and survival of the parasite in the hostile target tissues of the fish host.

Interplay between proinflammatory cytokines, miRNA, and tissue lesions in Anisakis-infected Sprague-Dawley rats.

BACKGROUND: Anisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to characterize the early cellular (6-72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM); ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, iNOS+, and S100A8/A9+ cells. The greatest number of iNOS+ and S100A8/A9+ cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls. CONCLUSIONS/SIGNIFICANCE: Anisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans.

Molecular and Cellular Response to Experimental Anisakis pegreffii (Nematoda, Anisakidae) Third-Stage Larval Infection in Rats.

Background: Anisakiasis is a zoonotic disease caused by accidental ingestion of live Anisakis spp. third-stage larvae present in raw or undercooked seafood. Symptoms of this emerging infectious disease include mild-to-severe abdominal pain, nausea, and diarrhea. Some patients experience significant allergic reactions. Aims: In order to better understand the onset of anisakiasis, we aimed to: (i) histopathologically describe severe inflammatory/hemorrhagic infection site lesions in Sprague-Dawley rats experimentally infected with Anisakis pegreffii larvae; and (ii) qualitatively and quantitatively characterize the transcriptomes of affected tissues using RNA-Seq. Methodology: The experiment was performed on 35 male rats, sacrificed at 5 time points (6, 10, 18, 24, and 32 h post-infection). Gastric intubation was performed with 10 A. pegreffii larvae (N = 5 infected rats per time point) or 1.5 ml of saline (external control N = 2 rats). 16 pools, seven for muscle tissues and nine for stomach tissues, were created to obtain robust samples for estimation of gene expression changes depicting common signatures of affected versus unaffected tissues. Illumina NextSeq 500 was used for paired-end sequencing, while edgeR was used for count data and differential expression analyses. Results: In total, there were 1372 (855 up and 517 down) differentially expressed (DE) genes in the Anisakis-infected rat stomach tissues, and 1633 (1230 up and 403 down) DE genes in the muscle tissues. Elicited strong local proinflammatory reaction seems to favor the activation of the interleukin 17 signaling pathway and the development of the T helper 17-type response. The number of DE ribosomal genes in the Anisakis-infected stomach tissue suggests that A. pegreffii larvae might induce ribosomal stress in the early infection stage. However, the downstream pathways and post-infection responses require further study. Histopathology revealed severe inflammatory/hemorrhagic lesions caused by Anisakis infection in the rat stomach and muscle tissues in the first 32 h. The lesion sites showed infiltration by polymorphonuclear leukocytes (predominantly neutrophils and occasional eosinophils), and to a lesser extent, macrophages. Conclusion: Understanding the cellular and molecular mechanisms underlying host responses to Anisakis infection is important to elucidate many aspects of the onset of anisakiasis, a disease of growing public health concern.

Exploring tumourigenic potential of the parasite Anisakis: a pilot study.

Anisakiasis is a global disease caused by consumption of raw or lightly cooked fish parasitised with Anisakis spp. third-stage larvae. Cases in the literature show colocalised anisakiasis and colorectal cancer, and the incidental finding of Anisakis larvae at the tumour site was reported. Data from our group suggested an epidemiological link between previous infection and gastrointestinal cancer. Furthermore, it has recently been reported that Anisakis products lead to inflammation and DNA damage. Based on these facts, the aim was to investigate whether Anisakis antigens are able to induce changes in the proliferation of epithelial cells in vitro or in the expression of serum microRNA (miRNA) in Sprague-Dawley rats. Anisakis complete extract (CE) induced increases in cell proliferation and decreases in apoptosis compared with nontreated cells, which resulted in a significant increase in the absolute number of viable cells at 48 h of exposure (P < .05). Furthermore, the miRNAs mmu-miR-1b-5p and mmu-miR-10b-5p (a cancer-related miRNA) were significantly decreased (P < .05) in sera from the rats inoculated with Anisakis CE, compared with control rats inoculated with saline. Additionally, based on their relative quantification values, four other cancer-related miRNAs were considered to be differently expressed, rno-miR-218a-5p and mmu-miR-224-5p (decreased) and rno-miR-125a-3p and rno-miR-200c-3p (increased). Anisakis CE was able to induce changes both in epithelial cells in vitro and in an animal model. The results obtained with Anisakis CE, in terms of increasing cell proliferation, decreasing apoptosis and inducing changes in the expression of serum cancer-related miRNAs in rats, suggest that Anisakis could have tumourigenic potential.

Expression of immune relevant genes in rainbow trout following exposure to live Anisakis simplex larvae.

Basic immune response mechanisms in vertebrates against helminths are still poorly understood. Fish-nematode models may prove valuable for elucidation of this question. In this study we orally challenged rainbow trout (Oncorhynchus mykiss) with larvae of Anisakis simplex (Nematoda: Anisakidae) and subsequently investigated the expression of 18 immune relevant genes in spleen and liver 1, 4 and 8days post infection (d.p.i.). Gene expression data were analysed with regard to the infection status of the challenged rainbow trout at the time of necropsy; "worms rejected" (÷worms), "worms present" (+worms) and a combined group consisting of samples pooled from both previous groups (÷/+worms). No significant regulation of cytokine genes was recorded but fish which had rejected worms up-regulated the CD4 gene (6.1-fold change, 8d.p.i.) in liver. The gene encoding CD8 was significantly down-regulated 24h post challenge in livers in fish still carrying worms (2.7-fold change) but not in the worm-free group. The immunoglobulin gene IgM was significantly down-regulated (2.9-fold change, 8d.p.i.) in liver samples from the +worms group. Complement factor C3 and precerebellin genes were significantly up-regulated twofold in liver samples from infected fish 4d.p.i. Significant up-regulation of the acute-phase protein SAA was observed in all three groups and in both tissues. To our knowledge, this is the first study to describe the expression of immune genes in a fish host challenged with live nematode larvae.

Experimental comparison of pathogenic potential of two sibling species Anisakis simplex s.s. and Anisakis pegreffii in Wistar rat.

OBJECTIVES: There are little data available on the pathology caused by the sibling species Anisakis simplex s.s. and Anisakis pegreffii. The differences shown in their ability to penetrate the muscle of fish may also be manifested in humans. The purpose of this study is to confirm possible differences in pathogenicity between A. simplex s.s. and A. pegreffii using an experimental model which simulates infection in humans. METHODS: Female Wistar rats were infected with 190 Anisakis type I L3 larvae from the Iberian coastline. After the animal was sacrificed, these L3 larvae were then recovered and identified via PCR-RFLP of the ITS1-5.8S-ITS2. A logistic regression analysis was performed searching for association between experimental pathogenic potential and species. RESULTS: The distribution of A. simplex s.s. and A. pegreffii between Atlantic and Mediterranean waters of the Iberian Peninsula showed statistically significant differences (P < 0.001) which were not observed in the hybrid genotypes (P > 0.3). 21.6% showed pathogenic potential, interpreted as the capacity of the larvae to cause lesions, stick to the gastrointestinal wall or penetrate it. The species variable showed association with the pathogenic role of the larva (P = 0.008). Taking A. simplex s.s. as our reference, the OR for A. pegreffii is 0.351 (P = 0.028). CONCLUSIONS: Despite this difference, A. pegreffii is also capable of causing anisakiasis, being responsible for 14.3% of the penetrations of the gastric mucosa found in rats, which justifies both species being considered aetiologic agents of this parasitic disorder.

Identification and genetic characterization of Anisakis larvae from marine fishes in the South China Sea using an electrophoretic-guided approach.

From 35 species of marine fishes (n = 327) from the South China Sea, 237 nematode larvae were collected and identified morphologically as Anisakis. Genomic DNA was isolated from each larva and subjected to PCR-based RFLP and targeted sequencing of a nuclear ribosomal DNA region between the 3'-end of the small subunit and 5'-end of the large subunit of the rRNA genes (= internal transcribed spacers, ITS+). Four different RFLP profile combinations (sets) were detected for all restriction endonucleases (HinfI, HhaI, and TaqI), of which three were characteristic of Anisakis typica, A. pegreffii, and A. physeteris, respectively. One profile set (for sample CA-2012) was linked to an ITS+ sequence that was identical to a previously published sequence of Anisakis sp. (sample HC-2005; originating from the African shelf) and another sequence (PH-2010; Madeira, Portugal). Phylogenetic analysis was carried out using the ITS+ sequence data from this study and reference sequences from the GenBank database. Neighbor joining and maximum parsimony trees displayed three clades. Clades I and II included nine described species of Anisakis, including all type I and type II larvae; clade III represented some undescribed species of Anisakis. Morphological comparison showed that Anisakis sp. CA-2012 was distinct from type I and type II larvae based on its tail shape and ratio of tail length to body length. The phylogenetic analysis and morphological characters suggest that Anisakis sp. CA-2012 represents a new record, now called Anisakis type III larvae.

Prevalence and mean intensity of Anisakis simplex (sensu stricto) in European sea bass (Dicentrarchus labrax) from Northeast Atlantic Ocean.

Viscera and muscle of a total of 40 wild 1-2kg European sea bass (Dicentrarchus labrax) from Northeast Atlantic (FAO area 27) were examined for Anisakidae larvae detection by digestion method. Extracted parasites were counted and mean intensity was calculated. Parasites were identified by genetic/molecular markers (allozymes and sequences analysis of the mtDNA cox2 gene) as belonging to the species Anisakis simplex (sensu stricto). In viscera, the main localisations of the larvae were under the gastric serosa, where several parasites alive and dead were found, and intestinal serosa. The visceral prevalence was 0.950 and the mean intensity was 96.39. The main localisation of A. simplex (s.s.) in edible parts was in belly muscles, with a prevalence of 0.425 and a mean intensity of 1.9. This is the first record on the prevalence and mean intensity of A. simplex (s.s.) in European sea bass muscle. This finding has an important consequence on epidemiology of anisakiasis and public health risk assessment.

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.

Allozyme and PCR-RFLP markers in anisakid nematodes, aethiological agents of human anisakidosis.

In the framework of the researches granted by MURST COFIN97, studies on anisakid nematodes, aethiological agents of human anisakidosis, were carried out. The project was aimed to implement the knowledge on the systematics, genetics, ecology and epizootiology of species of the genera Anisakis and Pseudoterranova by applying genetic markers obtained from multilocus allozyme electrophoresis and from PCR-based techniques. The results obtained by allozyme studies allowed to extend the geographic distribution and to detect new definitive and intermediate/paratenic hosts of two sibling species of the A. simplex complex, i.e. A. simplex s.s. and A. pegreffii and to characterise the species A. simplex C, a new sibling species within the A. simplex complex as well as a new species belonging to the genus Anisakis, A. ziphidarum. Combined allozyme and morphological analyses provided markers for the identification of the four sibling species of the Pseudoterranova decipiens complex and their nomenclatural designations. New markers based on PCR-RFLP analysis were used for the identification of sibling species of the Anisakis simplex complex and of another four species of the genus. Genetic markers based on three diagnostic restriction enzymes allowed the identification as A. pegreffii of a larva obtained by endoscopy in a case of human anisakidosis in Southern Italy.