The complete mitochondrial genome of Anisakis pegreffii Campana-Rouget & Biocca, 1955, (Nematoda, Chromadorea, Rhabditida, Anisakidae) - clarification of mitogenome sequences of the Anisakis simplex species complex.
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SUBMITTER: Yamada A
PROVIDER: S-EPMC7800010 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAnisakiasis is a foodborne disease caused by the third-stage larvae (L3) of two species belonging to the genus Anisakis: Anisakis pegreffii and Anisakis simplex sensu stricto. Both species have been the subject of different -omics studies undertaken in the past decade, but a reliable in vitro culture protocol that would enable a more versatile approach to functional studies has never been devised. In nature, A. pegreffii shows a polyxenous life-cycle. It reproduces in toothed whales (final host) and disseminates embryonated eggs via cetacean faeces in the water column. In the environment, a first- (L1) and second-stage larva (L2) develops inside the egg, and subsequently hatched L2 is ingested by a planktonic crustacean or small fish (intermediate host). In the crustacean pseudocoelom, the larva moults to the third stage (L3) and grows until the host is eaten by a fish or cephalopod (paratenic host). Infective L3 migrates into the visceral cavity of its paratenic host and remains in the state of paratenesis until a final host preys on the former. Once in the final host's gastric chambers, L3 attaches to mucosa, moults in the fourth stage (L4) and closes its life-cycle by becoming reproductively mature.MethodsTesting two commercially available media (RPMI 1640, Schneider's Drosophila) in combination with each of the six different heat-inactivated sera, namely foetal bovine, rabbit, chicken, donkey, porcine and human serum, we have obtained the first reliable, fast and simple in vitro cultivation protocol for A. pegreffii.ResultsSchneider's Drosophila insect media supplemented with 10% chicken serum allowed high reproducibility and survival of adult A. pegreffii. The maturity was reached already at the beginning of the third week in culture. From collected eggs, hatched L2 were maintained in culture for 2 weeks. The protocol also enabled the description of undocumented morphological and ultrastructural features of the parasite developmental stages.ConclusionsClosing of the A. pegreffii life-cycle from L3 to reproducing adults is an important step from many research perspectives (e.g., vaccine and drug-target research, transgenesis, pathogenesis), but further effort is necessary to optimise the efficient moulting of L2 to infective L3.
| S-EPMC9896804 | biostudies-literature
Project description:Genetic markers (ribosomal DNA and mitochondrial DNA) were used for molecular dissection of the Anisakis simplex sensu lato (s.l). complex populations. Host fish were caught off Moroccan coasts, where only Anisakis pegreffii is present, the sympatric area comprising Spanish coasts, and the Little Sole Bank fishing area from Nordeast Atlantic Ocean where the only present species is A. simplex sensu stricto(s.s.). Sequence variations in the amplification products were then assessed indirectly by digestion with restriction endonucleases or directly by sequencing for 623 L3 larvae. The sequences were used to infer the relationships between the two species under study using various methodological approaches. We reveal the high genetic diversity of Anisakis simplex s.s. and A. pegreffii in both mitochondrial and nuclear genes. We detected 10 and 2 fixed differences between A. simplex s.s and A. pegreffii in the Cox2 and ITS1, respectively. We found a proportion of putative hybrids below 20% with similar figures on the Atlantic and Mediterranean coasts. Moroccan hybrids were more similar to A. pegreffii reflecting backcrosses between these mixed genotypes and his ancestor A. pegreffii. We discuss the possible interpretation of these putative hybrids.
| S-EPMC5991843 | biostudies-literature
Project description:In countries with elevated prevalence of zoonotic anisakiasis and high awareness of this parasitosis, a considerable number of cases that associate Anisakis sp. (Nematoda, Anisakidae) and different bowel carcinomas have been described. Although neoplasia and embedded larvae were observed sharing the common site affected by chronic inflammation, no association between the nematode and malignancy were directly proved. Similarly, no data are available about the effect of secretory and excretory products of infecting larvae at the host's cellular level, except in respect to allergenic interaction.To test the mechanisms by which human non-immune cells respond to the larvae, we exposed the fibroblast cell line HS-68 to two Anisakis products (ES, excretory/secretory products; and EC, crude extract) and evaluated molecular markers related to stress response, oxidative stress, inflammation and apoptosis, such as p53, HSP70, TNF-?, c-jun and c-fos, employing cell viability assay, spectrophotometry, immunoblotting and qPCR.Both Anisakis products led to increased production of reactive oxygen species (ROS), especially in EC-treated cells. While the ES treatment induces activation of kinases suggesting inflammation and cell proliferation (or inhibition of apoptosis), in EC-treated cells, other signaling pathways indicate the inhibition of apoptosis, marked by strong upregulation of Hsp70. Elevated induction of p53 in fibroblasts treated by both Anisakis products, suggests a significantly negative effect on the host DNA.This study shows that in vitro cell response to Anisakis products can result in at least two different scenarios, which in both cases lead to inflammation and DNA damage. Although these preliminary results are far from proving a relationship between the parasite and cancer, they are the first to support the existence of conditions where such changes are feasible.
| S-EPMC5124272 | biostudies-literature
Project description: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.
| S-EPMC6137129 | biostudies-literature
Project description:Anisakis pegreffii is known as one of the causes of a fish-borne zoonosis, anisakidosis. Despite its significant public health and food hygiene impacts, little is known of the pathogenesis, genetic background of this parasite, at least partly due to the lack of genome and transcriptome information. In this study, RNA-seq and de novo assembly were conducted to obtain transcriptome profiles of the A. pegreffii third and fourth larvae. The third stage larvae (APL3) were collected from chub mackerel and the fourth stage larvae (APL4) were obtained by in vitro culture. In total, 47,243 and 43,660 unigenes were expressed in APL3 and APL4 transcriptomes. Of them, 18,753 were known and 28,490 were novel for APL3, while 18,996 were known and 24,664 were novel for APL4. The most abundantly expressed genes in APL3 were mitochondrial enzymes (COI, COII, COIII) and polyubiquitins (UBB, UBIQP_XENLA). Collagen-related genes (col-145, col-34, col-138, Bm1_54705, col-40) were the most abundantly expressed in APL4. Mitochondrial enzyme genes (COIII, COI) were also highly expressed in APL4. Among the transcripts, 614 were up-regulated in APL3, while 1,309 were up-regulated in APL4. Several protease and protein biosynthesis-related genes were highly expressed in APL3, all of which are thought to be crucial for invading host tissues. Collagen synthesis-related genes were highly expressed in APL4, reflecting active biosynthesis of collagens occurs during moulting process of APL4. Of these differentially expressed genes, several genes (SI, nas-13, EF-TSMT, SFXN2, dhs-27) were validated to highly transcribed in APL3, while other genes (col-40, F09E10.7, pept-1, col-34, VIT) in APL4. The biological roles of these genes in vivo will be deciphered when the reference genome sequences are available, together with in vitro experiments.Anisakis pegreffii is known as one of the causes of a fish-borne zoonosis, anisakidosis. Despite its significant public health and food hygiene impacts, little is known of the pathogenesis, genetic background of this parasite, at least partly due to the lack of genome and transcriptome information. In this study, RNA-seq and de novo assembly were conducted to obtain transcriptome profiles of the A. pegreffii third and fourth larvae. The third stage larvae (APL3) were collected from chub mackerel and the fourth stage larvae (APL4) were obtained by in vitro culture. In total, 47,243 and 43,660 unigenes were expressed in APL3 and APL4 transcriptomes. Of them, 18,753 were known and 28,490 were novel for APL3, while 18,996 were known and 24,664 were novel for APL4. The most abundantly expressed genes in APL3 were mitochondrial enzymes (COI, COII, COIII) and polyubiquitins (UBB, UBIQP_XENLA). Collagen-related genes (col-145, col-34, col-138, Bm1_54705, col-40) were the most abundantly expressed in APL4. Mitochondrial enzyme genes (COIII, COI) were also highly expressed in APL4. Among the transcripts, 614 were up-regulated in APL3, while 1,309 were up-regulated in APL4. Several protease and protein biosynthesis-related genes were highly expressed in APL3, all of which are thought to be crucial for invading host tissues. Collagen synthesis-related genes were highly expressed in APL4, reflecting active biosynthesis of collagens occurs during moulting process of APL4. Of these differentially expressed genes, several genes (SI, nas-13, EF-TSMT, SFXN2, dhs-27) were validated to highly transcribed in APL3, while other genes (col-40, F09E10.7, pept-1, col-34, VIT) in APL4. The biological roles of these genes in vivo will be deciphered when the reference genome sequences are available, together with in vitro experiments.
| S-EPMC7266050 | biostudies-literature
Project description:BackgroundThis is the first report of anisakiasis in a Croatian patient, evidenced from an archival paraffin-embedded and hematoxylin-eosin stained tissue section. Anisakiasis has been only suspected in the country based on previously detected anti-Anisakis IgE seroprevalence in the healthy coastal population, as well as an acute case where pathohistological and serological findings suggested the diseases, but the migrating larva has not been retrieved.Case presentationSeventy years-old female, operated in 1998 for pulmonary carcinoma, was admitted to the General hospital Šibenik, Croatia in 2003, because of gastric pain and nausea that lasted for couple of days. She was showing good general condition, full mobility and lucidity, subfebrile status. Abdominal palpation inferred acute pain in paraumbilical and ileocecal region. Exploratory right pararectal laparotomy revealed a hardened, 5 cm-long structure, located intraluminally in the sigmoid colon, not perforating colon serosa. The process has been dissected and sent for patohistological diagnosis. Results showed a 2 mm-long whitish nematode spiralised in muscular layer of colon mucosa surrounded by granulomatous inflammation.ConclusionAfter genomic DNA isolation of the nematode from the histological section, and amplification at the mitochondrial cytochrome oxidase 2 locus, etiological agent has been identified as Anisakis pegreffii. Used methodology suggests that screening of archival suspicious sections is feasible in order to study epidemiology of this zoonotic disease poorly recognised in Croatia.
| S-EPMC4736626 | biostudies-literature
Project description:Infective third-stage larvae (L3) of the marine nematode Anisakis pegreffii cause inflammation and clinical symptoms in humans, their accidental host, that subside and self-resolve in a couple of weeks after L3 die. To characterise the differences in an early immune response of a marine vs. terrestrial host, we stimulated peripheral blood leukocytes (PBLs) of fish (paratenic host) and rat (accidental, human-model host) with A. pegreffii crude extract and analysed PBL transcriptomes 1 and 12 h post-stimulation. Fish and rat PBLs differentially expressed 712 and 493 transcripts, respectively, between 1 and 12 h post-stimulation (false discovery rate, FDR <0.001, logFC >2). While there was a difference in the highest upregulated transcripts between two time-points, the same Gene Ontologies, biological processes (intracellular signal transduction, DNA-dependent transcription, and DNA-regulated regulation of transcription), and molecular functions (ATP and metal ion binding) were enriched in the two hosts, showing an incrementing dynamic between 1 and 12 h. This suggests that the two distinct hosts employ qualitatively different transcript cascades only to achieve the same effect, at least during an early innate immunity response. Activation of later immunity elements and/or a combination of other host's intrinsic conditions may contribute to the death of L3 in the terrestrial host.
| S-EPMC9797851 | biostudies-literature
Project description:BackgroundAnisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen.MethodsParasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied.ResultsAccording to the mtDNA cox2 and the EF1 α - 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases. In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like.ConclusionThe Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis.
| S-EPMC5539894 | biostudies-literature
Project description:IntroductionAnisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized.MethodsGenetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis.Results and discussionEVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.
| S-EPMC10050594 | biostudies-literature
Project description:The prevalence of infection and the identification of anisakid larvae in European hake Merluccius merluccius lessepsianus from Hurghada City, Red Sea Governorate, Egypt, were investigated. Fish samples were collected during the period of February and November 2014. Twenty-two (36.66%) out of sixty examined fish specimens were found to be naturally infected with Anisakis type I larvae mostly found as encapsulated larvae in visceral organs. There was a positive relationship between host length/weight and prevalence of infection. Based on morphological, morphometric, and molecular analyses, these nematodes were identified as third-stage larvae of Anisakis simplex. The present study was considered as the first report of anisakid larvae from European hake in the Egyptian water.
| S-EPMC4746349 | biostudies-literature