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Parasites & Vectors Jan 2018Larval stages of the sibling species of parasitic nematodes Anisakis simplex (sensu stricto) (s.s.) (AS) and Anisakis pegreffii (AP) are responsible for a fish-borne...
BACKGROUND
Larval stages of the sibling species of parasitic nematodes Anisakis simplex (sensu stricto) (s.s.) (AS) and Anisakis pegreffii (AP) are responsible for a fish-borne zoonosis, known as anisakiasis, that humans aquire via the ingestion of raw or undercooked infected fish or fish-based products. These two species differ in geographical distribution, genetic background and peculiar traits involved in pathogenicity. However, thus far little is known of key molecules potentially involved in host-parasite interactions. Here, high-throughput RNA-Seq and bioinformatics analyses of sequence data were applied to the characterization of the whole sets of transcripts expressed by infective larvae of AS and AP, as well as of their pharyngeal tissues, in a bid to identify transcripts potentially involved in tissue invasion and host-pathogen interplay.
RESULTS
Approximately 34,000,000 single-end reads were generated from cDNA libraries for each species. Transcripts identified in AS and AP encoded 19,403 and 10,424 putative peptides, respectively, and were classified based on homology searches, protein motifs, gene ontology and biological pathway mapping. Differential gene expression analysis yielded 226 and 339 transcripts upregulated in the pharyngeal regions of AS and AP, respectively, compared with their corresponding whole-larvae datasets. These included proteolytic enzymes, molecules encoding anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides.
CONCLUSIONS
This work provides the scientific community with a list of key transcripts expressed by AS and AP pharyngeal tissues and corresponding annotation information which represents a ready-to-use resource for future functional studies of biological pathways specifically involved in host-parasite interplay.
Topics: Animals; Anisakiasis; Anisakis; Computational Biology; DNA, Helminth; Fish Diseases; Fishes; Host-Parasite Interactions; Larva; Peptide Hydrolases; Pharynx; Polymorphism, Restriction Fragment Length; Sequence Analysis, RNA; Species Specificity; Transcriptome; Virulence
PubMed: 29321072
DOI: 10.1186/s13071-017-2585-7 -
Scientific Reports Feb 2021Anisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are...
Anisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO and O) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ℃, 12 ℃, 22 ℃, 37 ℃) at air conditions. Moreover, different combinations of CO and O with N as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ℃ for Anisakis spp. larvae and 37 ℃ for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ℃, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ℃. Larval mobility was not significantly different under the different CO or O conditions at 6 °C and 12 ℃. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO and O did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.
Topics: Animals; Anisakis; Carbon Dioxide; Environment; Fish Diseases; Fishes; Food Parasitology; Larva; Locomotion; Oxygen; Temperature
PubMed: 33608615
DOI: 10.1038/s41598-021-83505-5 -
Journal of Food Protection Oct 2011A study was carried out on the presence of Anisakis and Hysterothylacium larvae in fish and cephalopods caught in Sardinian waters. A total of 369 specimens of 24...
A study was carried out on the presence of Anisakis and Hysterothylacium larvae in fish and cephalopods caught in Sardinian waters. A total of 369 specimens of 24 different species of teleosts and 5 species of cephalopods were collected from different fishing areas of Sardinia. Larvae were detected and isolated by both visual inspection and enzymatic digestion. These methods allowed Anisakis type I and type II third-stage larvae and Hysterothylacium third- and fourth-stage larvae to be detected. The prevalence, mean intensity, and mean abundance were calculated. The results obtained showed the highest prevalence of Anisakidae in Zeus faber (100%) and of Anisakis in Micromesistius poutassou (87.5%). The highest prevalence of Anisakis type I larvae was in M. poutassou (81.2%), and that of Anisakis type II larvae was in Todarodes sagittatus (20%). The highest values for prevalence, mean intensity, and mean abundance for Hysterothylacium were found in Z. faber. These prevalences and the mean intensity and abundance were higher than those reported by different authors in other Mediterranean areas. This may be because the enzymatic digestive method used in this research resulted in higher recovery levels. The data suggest that Sardinia may be a high-risk area for zoonotic diseases and that measures such as information campaigns, aimed at both sanitary service personnel and consumers, should be employed to limit the spread of such zoonosis.
Topics: Animals; Anisakis; Cephalopoda; Consumer Product Safety; Decapodiformes; Fishes; Food Contamination; Gadiformes; Humans; Italy; Larva; Nematoda; Prevalence
PubMed: 22004829
DOI: 10.4315/0362-028X.JFP-10-482 -
Frontiers in Immunology 2018Anisakiasis is a zoonotic disease caused by accidental ingestion of live spp. third-stage larvae present in raw or undercooked seafood. Symptoms of this emerging...
Anisakiasis is a zoonotic disease caused by accidental ingestion of live 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. 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 larvae; and (ii) qualitatively and quantitatively characterize the transcriptomes of affected tissues using RNA-Seq. 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 larvae ( = 5 infected rats per time point) or 1.5 ml of saline (external control = 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. In total, there were 1372 (855 up and 517 down) differentially expressed (DE) genes in the -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 -infected stomach tissue suggests that 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 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. Understanding the cellular and molecular mechanisms underlying host responses to infection is important to elucidate many aspects of the onset of anisakiasis, a disease of growing public health concern.
Topics: Animals; Anisakiasis; Anisakis; Computational Biology; Gastric Mucosa; Gene Expression Profiling; Gene Expression Regulation; Gene Regulatory Networks; Host-Parasite Interactions; Larva; Life Cycle Stages; Male; Rats; Zoonoses
PubMed: 30245697
DOI: 10.3389/fimmu.2018.02055 -
PLoS Neglected Tropical Diseases May 2019Anisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or...
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.
Topics: Animals; Anisakiasis; Anisakis; Cytokines; DNA Methylation; Female; Gastrointestinal Tract; Humans; Interleukin-18; Interleukin-6; Male; MicroRNAs; Rats; Rats, Sprague-Dawley
PubMed: 31091271
DOI: 10.1371/journal.pntd.0007397 -
Journal of Nematology 2020is 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...
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 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. 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 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.
PubMed: 32298057
DOI: 10.21307/jofnem-2020-041 -
PLoS Neglected Tropical Diseases Jan 2020We undertook the first study systematically evaluating the risk of Anisakis-sensitization in Croatian fish-processing workers and potential genetic susceptibility to...
We undertook the first study systematically evaluating the risk of Anisakis-sensitization in Croatian fish-processing workers and potential genetic susceptibility to anisakiasis. Anti-Anisakis IgE seroprevalence and risk factors for 600 employees of Croatian fish processing facilities and 466 blood donor controls, were assessed by indirect ELISA targeted with: recombinant Ani s 1 and Ani s 7 allergens, an Anisakis crude extract, the commercial ImmunoCAP kit, and questionnaires. Genetic susceptibility to anisakiasis was evaluated by genotypisation of human leukocytes alleles (HLA). Anti-Anisakis seropositive and a fraction of negative subjects were also assessed by ELISA and Western Blot (WB) for IgG seroprevalence to Trichinella spp. Overall, the observed anti-Anisakis seroprevalence inferred by indirect ELISA was significantly higher in fish processing workers (1.8%, 95% CI 0.9-3.3%) compared to the controls (0%, 0-0.8%). Seven out of 11 Ani s 1 and Ani s 7-positives and none of selected 65 negative sera, tested positive on whole-Anisakis extract (ImmunoCAP), whereas Anisakis crude extract ELISA detected 3.9% (2.4-6.0%) seropositives in fish processing workers, three (14%) of which showed IgE reactivity to milk proteins. The highest risk associated with Anisakis-sensitization among workers was fishing in the free time, rather than any of attributes related to the occupational exposure. Although no association was observed between anti-Anisakis seropositivity and wearing gloves or protective goggles, the majority of workers (92%) wore protective gloves, minimizing the risk for Anisakis sensitization via skin contact. Six HLA alleles within DRB1 gene were significantly associated with seropositivity under dominant, allelic or recessive models. All sera confirmed negative for anti-Trichinella spp. IgG. The study exhaustively covered almost all marine fish processing workers in Croatia, reflecting real-time Anisakis sensitization status within the industry, already under the influence of wide array of allergens.
Topics: Animals; Anisakis; Antibodies, Helminth; Antigens, Helminth; Croatia; Eye Protective Devices; Fishes; Food Handling; Gloves, Protective; Helminth Proteins; Humans; Hypersensitivity; Occupational Exposure; Risk Factors; Trichinella
PubMed: 31986138
DOI: 10.1371/journal.pntd.0008038 -
Pathogens (Basel, Switzerland) Nov 2022Spotted flounder ( L.) caught in the Gulf of Cadiz (area FAO 27 ICES IXa) were examined for larvae and to assess the possible risk of anisakiasis in humans through...
Spotted flounder ( L.) caught in the Gulf of Cadiz (area FAO 27 ICES IXa) were examined for larvae and to assess the possible risk of anisakiasis in humans through consumption of this fish. Larvae of the genera and were identified in the analysis of 128 purchased fish specimens. All larvae corresponded to type I. Molecular analysis showed the presence of , s.s., and recombinant genotype between the two. The prevalence of was 9.4% with a mean intensity of 1.42, while for the values were 12.5% and 1.06. The length and weight of the fish, but not Fulton's condition factor, varied significantly between infected and uninfected fish. The prevalence of increased with fish length, with no fish parasitized with measuring less than 15.5 cm (2-2.5 years old), which is probably related to the reported dietary change of these fish at around 2 years of age. Fish not parasitized with any of these nematodes showed positive allometric growth, while those parasitized only with showed negative allometric growth. When comparing both groups including only fish ≥ 15.5 cm (the smallest size of -infected fish), the difference is shown to be statistically significant ( = 0.01), suggesting that infection of spotted flounder negatively affects fish growth even when parasite intensity is low, which may have important economic repercussions. Finally, the low prevalence and, above all, intensity of in these fish, as well as the habit of consuming this fish fried in oil in our geographical area, means that the risk of acquiring anisakiasis through consumption of this fish is low.
PubMed: 36558766
DOI: 10.3390/pathogens11121432 -
The Journal of Parasitology Dec 2005Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic...
Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.
Topics: Animals; Anisakiasis; Anisakis; Ascaridida Infections; Ascaridoidea; Base Sequence; California; Caniformia; Cetacea; DNA, Ribosomal; DNA, Ribosomal Spacer; Likelihood Functions; Molecular Sequence Data; Otters; Pacific Ocean; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 28S; RNA, Ribosomal, 5.8S; Sequence Alignment
PubMed: 16539026
DOI: 10.1645/GE-522R.1 -
The Korean Journal of Parasitology Feb 2020The third stage larvae (L3) of Anisakis typica were detected in 2 species of threadfin bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand, and were...
The third stage larvae (L3) of Anisakis typica were detected in 2 species of threadfin bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand, and were morphologically and molecularly characterized. Total 100 threadfin breams, 50 Nemipterus hexodon and 50 N. japonicus, were examined with naked eyes after the opening of abdominal cavity with scissors. Almost all infected larvae remained alive and active even the fish were transported for 1-2 days. Anisakid larvae were exclusively distributed in the body cavity and rarely in the liver. The prevalence of A. typica L3 were 68.0% and 60.0% in N. hexodon and N. japonicus and their infection intensities were 3.5 and 4.2 per fish infected each. Morphological and morphometric analysis were performed by viewing specimens under both a light microscope and a scanning electron microscope. Interestingly, the protruded mucron of Anisakis typica under SEM showed a distinct cylindrical shape that differed from the cone shape of A. simplex. The protruded mucron could be used to identify A. typica L3 larvae in the future. A comparison of the ITS1-5.8S-ITS2 rDNA nucleotide sequences of these species revealed high blast scores with A. typica. Conclusively, it was confirmed that A. typica L3 are prevalent in threadfin breams from the Gulf of Thailand, and their morphological and molecular characters are something different from those of other anisakid larvae, including A. simplex and A. pegreffii.
Topics: Animals; Anisakis; Fishes; Larva; Microscopy, Electron, Scanning; Thailand
PubMed: 32145723
DOI: 10.3347/kjp.2020.58.1.15