Anisakiasis and gastroallergic reactions associated with Anisakis pegreffii infection, Italy.
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ABSTRACT:
SUBMITTER: Mattiucci S
PROVIDER: S-EPMC3647659 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus Anisakis. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of Anisakis are detected. Thus, molecular-based diagnostic approaches are warranted. METHOD:We have developed a PCR method that amplifies the DNA of Anisakis spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA cox2 gene were used and sequence analysis was performed according to the procedures already established for species of Anisakis. RESULTS:The sequence obtained (629 bp) was compared with those of the other species of Anisakis which have so far been genetically characterized and with sequences obtained from larval stages of Anisakis collected from the Mediterranean fish Engraulis encrasicolus. This enabled the genetic identification of the larva in the human tissue as A. pegreffii. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin. CONCLUSION:The case of human anisakiasis presented reinforces the pathological significance of the species A. pegreffii to humans. The molecular/genetic methodological approach based on mtDNA cox2 sequence analysis, described here, can allow easy and rapid identification of Anisakis spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.
| S-EPMC3080813 | biostudies-other
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: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:The aim of the present study was to describe gastric granuloma caused by Anisakis pegreffii in Sphyraena viridensis caught in the central Mediterranean Sea. Sixty-eight S. viridensis specimens were collected from different fish markets on the east coast of Sicily. Coelomic organs were observed both macroscopically and with the aid of stereomicroscope. Parasite specimens and lesioned tissues were collected for identification, histological and molecular analyses. Twelve specimens (p = 17.6%) were positive for the presence of nematode larvae, morphologically identified as larvae of Anisakis sp., with values of mean abundance and mean intensity of 0.9 and 4.8, respectively. One large female specimen showed massive parasite infection associated with nodular lesions of the gastric wall. By histology, several nematode larvae encysted through the gastric wall were found. The parasite bodies were surrounded by a granulomatous reaction made up of macrophages, epithelioid cells, some lymphocytes and an external connective sheet. Molecular analysis of 18S rRNA and cox2 genes from Anisakis sp. collected larvae, identified them as A. pegreffii. The lesions here described, though macroscopically superimposable on human eosinophilic granuloma, microscopically showed significant differences in the inflammatory cells involved and in the type of immune reaction.
| S-EPMC8697904 | 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-other
Project description:Ascaridoid nematodes are widespread in marine fishes. Despite their major socioeconomic importance, mechanisms associated to the fish-borne zoonotic disease anisakiasis are still obscure. RNA-Seq and de-novo assembly were herein applied to RNA extracted from larvae and dissected pharynx of Hysterothylacium aduncum (HA), a non-pathogenic nematode. Assembled transcripts in HA were annotated and compared to the transcriptomes of the zoonotic species Anisakis simplex sensu stricto (AS) and Anisakis pegreffii (AP). Approximately 60,000,000 single-end reads were generated for HA, AS and AP. Transcripts in HA encoded for 30,254 putative peptides while AS and AP encoded for 20,574 and 20,840 putative peptides, respectively. Differential gene expression analyses yielded 471, 612 and 526 transcripts up regulated in the pharynx of HA, AS and AP. The transcriptomes of larvae and pharynx of HA were enriched in transcripts encoding collagen, peptidases, ribosomal proteins and in heat-shock motifs. Transcripts encoding proteolytic enzymes, anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides were up-regulated in AS and AP pharynx. This study represents the first transcriptomic characterization of a marine parasitic nematode commonly recovered in fish and probably of negligible concern for public health.
| S-EPMC7140869 | biostudies-literature
Project description: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.
| S-EPMC5763927 | biostudies-literature
Project description:BACKGROUND:Anisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood. RESULTS:Third stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes. CONCLUSIONS:A. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses.
| S-EPMC6080401 | biostudies-literature
Project description:Anisakis simplex sensu stricto (s.s.), Anisakis pegreffii, Anisakis berlandi (=A. simplex sp. C), and Anisakis typica are the 4 major species of Anisakis type I larvae. In the Republic of Korea (Korea), A. pegreffii, A. berlandi, and A. typica larvae in fish hosts has seldom been documented. In this study, molecular analysis was performed on Anisakis larvae from the sea eels (Astroconger myriaster), the major source of human anisakiasis in Korea, collected from Tongyeong City, a southern coastal area of Korea. All 20 sea eels examined were infected with Anisakis type I larvae (160 larvae; 8 per fish). Their species were analyzed using PCR-RFLP patterns and nucleotide sequences of internal transcribed spacers (ITS1, 5.8 subunit gene, and ITS2) and mitochondrial cytochrome c oxidase 2 (cox2). Most (86.8%; 112/129) of the Anisakis type I larvae were A. pegreffii, and 7.8% (10/129) were A. typica. The remaining 5.4% (7/129) was not identified. Thus, A. pegreffii is the major species of anisakid larvae in sea eels of the southern coast of Korea.
| S-EPMC4510688 | biostudies-literature
Project description:BackgroundInferring the microbiota diversity of helminths enables depiction of evolutionarily established ecological and pathological traits that characterize a particular parasite-host interaction. In turn, these traits could provide valuable information for the development of parasitosis control and mitigation strategy. The parasite Anisakis pegreffii (Nematoda: Anisakidae) realizes the final stage of its life-cycle within gastric chambers of aquatic mammals, causing mild-to-moderate granulomatous gastritis with eosinophilic infiltrate, to severe ulcerative gastritis with mixed inflammatory infiltrate, often associated with bacterial colonies. However, its interaction with the host microbiota remains unknown, and might reveal important aspects of parasite colonization and propagation within the final host.MethodsMySeq Illumina sequencing was performed for the 16S rRNA gene from microbiota isolated from larvae, and uterus and gut of adult A. pegreffii parasitizing stranded striped dolphins (Stenella coeruleoalba). To assess the potential presence of Brucella ceti within isolated microbiota, Brucella-targeted real-time PCR was undertaken. In addition, TEM of the gastrointestinal tract of the infective third-stage (L3) and transitioning fourth-stage larvae (L4) was performed to characterize the morphological differences and the level of larval feeding activity.ResultsIn total, 230 distinct operational taxonomic units (OTUs) were identified across all samples (n = 20). The number of shared taxa was lower than the number of taxa found specifically in each parasite stage or organ. The dominant taxon was Mycoplasmataceae (genus Mycoplasma) in the gut and uterus of adult A. pegreffii, whereas Fusobacteriaceae (genus Cetobacterium) was the most abundant in 40% of larvae, alongside Mycoplasmataceae. No B. ceti DNA was detected in any of the microbiota isolates. TEM revealed differences in gut ultrastructure between L3 and L4, reflecting a feeble, most likely passive, level of feeding activity in L3.ConclusionsMicrobiota from L3 was more related to that of the gut rather than the uterus of adult A. pegreffii. Taxa of the larval microbiota showed qualitative and quantitative perturbations, likely reflecting the propagation through different environments during its life-cycle. This suggests an ontogenetic shift in the alpha and beta diversity of microbial communities from uterus-derived towards cetacean-derived microbiota. Although TEM did not reveal active L3 feeding, microbiota of the latter showed similarity to that of an actively feeding adult nematode.
| S-EPMC6668197 | biostudies-literature