Occurrence and abundance of zoonotic nematodes in snapper Chrysophrys auratus, a popular table fish from Australian and New Zealand waters.
ABSTRACT: In Australia and New Zealand (NZ), snapper Chrysophrys auratus is known for delicate mild flavoured flesh and is a favoured species to serve raw as sashimi or in sushi. The diet of snapper includes a variety of intermediate hosts of larval nematodes, and as a result, snapper has potential to become highly infected with zoonotic/non-zoonotic nematodes. The aims of this study were to survey nematodes in snapper from Australia and New Zealand waters and to identify nematode species using combined morphological and molecular methods. The zoonotic potential of nematodes identified in this study are discussed. A total of 112 snapper were purchased from the Sydney fish market, New South Wales, Australia. Fish were dissected and only the visceral content and digestive tract were examined for nematode infection. Parasites were initially identified by the microscopic method as four different types belonging to the families Anisakidae (Anisakis types I & III, and Terranova type II) and Cucullanidae (Dichelyne spp.). All Anisakidae nematodes were at infective stages. Species-level identification was actualised through sequencing of the internal transcribed spacer (ITS-1, 5.8S, ITS-2) regions. The Anisakis types I & III were confirmed as Anisakis pegreffii and A. brevispiculata, respectively of which A. pegreffii is considered globally as a zoonotic nematode. The specific identification of Terranova type II and Dichelyne spp. was not possible as no comparable sequence data were available in GenBank. The phylogenetic tree clustered Anisakis types I & III with A. pegreffii and A. brevispiculata, respectively; Terranova type II sequences as a separate clade with previously identified larval and adult Terranova and Pseudoterranova species. Based on phylogenetic analyses the present Cucullanid specimens were assigned herein as Dichelyne cf. pleuronectidis, and an unknown species Dichelyne sp. 1. This study represents the first host record globally for zoonotic Anisakid nematodes in this popularly consumed table fish and a new region record for D. cf. pleuronectidis and Dichelyne sp. 1. Further investigation is required, using more comprehensive parasite detection and recovery methods, to assess the health risk these nematodes may pose to human and fish health in Australia/NZ.
Project description:Anisakidosis is a zoonotic parasitosis induced by members of the family Anisakidae. The anisakid genera includes <i>Anisakis, Pseudoterranova, Hysterothylacium</i> and <i>Contracaecum</i>. The final definitive hosts of these nematodes are marine mammals with a complex life cycle. These nematode parasites use different crustaceans and fish species as intermediate or paratenic hosts and humans are accidental hosts. Human anisakiasis, the infections caused by members of the genus <i>Anisakis,</i> occurs<i>,</i> when seafoods, particularly fish, contaminated with the infective stage (third stage larvae [L3]) of this parasite, are consumed. Pseudoterranovosis, on the other hand is induced by members of the genus <i>Pseudoterranova.</i> These two genera of anisakids have been implicated in human disease globally. There is a rise in reports of gastro-intestinal infections accompanied by allergic reactions caused by <i>Anisakis simplex</i> and <i>Anisakis pegreffii</i>. This review provides an update on current knowledge on <i>Anisakis</i> as a food-borne parasite with special focus on the increasingly reported diversity of fish and crustacean hosts, allergens and immunological cross-reactivity with invertebrate proteins rendering this parasite a significant public health issue.
Project description:In this work a total of 949 fish samples were analysed for the identification of nematode larvae belonging to the Anisakidae family. Biomolecular application for the identification of Anisakidae larvae can be an optimal instrument for the traceability of fish products, described on the Reg. EC 178/2002. Results confirm a correlation between geographical distribution of fishes and presence of specific Anisakid larvae. FAO 37 zone (Mediterranean sea) showed a prevailing distribution of Anisakis pegreffii and a minimal presence of A. simplex s.s. in hybrid form with Anisakis pegreffii. FAO 27 zone showed a prevailing distribution of A. simplex s.s. in fish like Brosme (Brosme brosme) and infestation prevalence of Pseudoterranova krabbei and P. decipiens s.s. in Gadus morhua. Obtained results validate the hypothesis that molecular biology methods for identifying Anisakidae larvae are effective traceability markers of fish products.
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.
Project description:Alive anisakids cause acute gastrointestinal diseases, and dead worms contained in food can provoke sensibilization and allergic reactions in humans. Detected in the purchased minced salmon <i>Oncorhynchus nerka</i> nematodes were identified as <i>Anisakis simplex sensu stricto</i> (Anisakidae). We found that recently published phylogenetic trees (reconstructed using different ribosomal and mitochondrial genetic markers) showed independent clusterization of species recognized in the <i>A. simplex sensu lato</i> species complex. This prompted us to undertake this full-fledged molecular genetics study of anisakids from Kamchatka with phylogenetic reconstructions (NJ/ML) and calculated ranges of interspecific and intergeneric p-distances using ITS1-5.8S-ITS2 sequences. We confirmed that molecular markers based on the ITS region of rDNA were able to recognize 'pure' specimens belonging to the cryptic species. We offer new insights into the systematics of anisakids. The genus <i>Anisakis sensu stricto</i> should include <i>Anisakis simplex sensu stricto</i>, <i>Anisakis pegreffii</i>, <i>Anisakis berlandi</i>, <i>Anisakis ziphidarum</i>, and <i>Anisakis nascettii</i>. Presumably, two genera should be restored in the structure of the subfamily Anisakinae: <i>Skrjabinisakis</i> for the species <i>Anisakis paggiae</i>, <i>Anisakis brevispiculata</i>, and <i>Anisakis physeteris</i>; and <i>Peritrachelius</i> for the species <i>Anisakis typica</i>. In addition, we provide the short annotated list of some genera of the family Anisakidae, including their diagnoses.
Project description:Anisakiasis is a zoonotic disease induced by anisakid nematodes, and endoscopic inspection is used for a diagnosis or remedy for it. Anisakis simplex, Anisakis physeteris, and Pseudoterranova decipiens had been reported to be the major species causing human infections, particularly, in Japan. However, in Korea, recent studies strongly suggested that Anisakis pegreffii is the major species of human infections. To support this suggestion, we collected anisakid larvae (n=20) from 20 human patients who were undergone gastrointestinal endoscopy at a health check-up center in Korea, and molecular identification was performed on the larvae using PCR-RFLP analysis and gene sequencing of rDNA ITS regions and mtDNA cox2. In addition, anisakid larvae (n=53) collected from the sea eel (Astroconger myriaster) were also examined for comparison with those extracted from humans. The results showed that all human samples (100%) were identified as A. pegreffii, whereas 90.7% of the samples from the sea eel were A. pegreffii with the remaining 9.3% being Hysterothylacium aduncum. Our study confirmed that A. pegreffii is the predominant species causing human anisakiasis in Korea, and this seems to be due to the predominance of this larval type in the fish (sea eels) popularly consumed by the Korean people. The possibility of human infection with H. aduncum in Korea is also suggested.
Project description:Consumption of raw or thermally inadequately treated fishery products represents a public health risk, with the possibility of propagation of live Anisakis larvae, the causative agent of the zoonotic disease anisakidosis, or anisakiasis. We investigated the population dynamics of Anisakis spp. in commercially important fish-anchovies (Anisakis), sardines (Sardina pilchardus), European hake (Merluccius merluccius), whiting (Merlangius merlangus), chub mackerel (Scomber japonicus), and Atlantic bluefin tuna (Thunnus thynnus)-captured in the main Adriatic Sea fishing ground. We observed a significant difference in the numbers of parasite larvae (1 to 32) in individual hosts and between species, with most fish showing high or very high Anisakis population indices. Phylogenetic analysis confirmed that commercial fish in the Adriatic Sea are parasitized by Anisakis pegreffii (95.95%) and Anisakis simplex sensu stricto (4.05%). The genetic structure of A. pegreffii in demersal, pelagic, and top predator hosts was unstructured, and the highest frequency of haplotype sharing (n = 10) was between demersal and pelagic fish.
Project description:Anisakiosis is a fish-borne disease with gastrointestinal and/or allergic symptoms caused by the consumption of raw or undercooked fish parasitized with nematode larvae of the genus Anisakis. In Europe, Anisakis pegreffii has been detected as the causative agent, although the sibling species Anisakis simplex sensu stricto (s.s.) is also known to cause the disease in other parts of the world, and discrepancies exist regarding their respective pathogenic potential. In Spain a high number of cases has been recorded, with marinated anchovies being the main source of infection, although no specific diagnosis has been documented in humans. In this study, we analyzed three cases of anisakiosis in patients from Barcelona (Spain) who had consumed undercooked hake. All patients described epigastric pain and several larval nematodes were removed endoscopically from their stomachs. Larvae were morphologically characterized as third-stage larvae of Anisakis simplex sensu lato (s.l.) and molecularly identified as A. simplex (s.s.) by means of PCR RFLP of the ITS region of the rDNA and sequencing of the elongation factor1 alpha1 (EF1 ?-1) nDNA gen. This study represents the first specific identification of Anisakis larvae in clinical cases of anisakiosis reported in Spain. Specific molecular diagnosis is of crucial importance for assessing the health risk of Anisakis sibling species. Hake consumption stands out as a risk factor for anisakiosis, since this fish species can be highly parasitized.
Project description:Human cases of gastric anisakiasis caused by the zoonotic parasite Anisakis pegreffii are increasing in Italy. The disease is caused by ingestion of larval nematodes in lightly cooked or raw seafood. Because symptoms are vague and serodiagnosis is difficult, the disease is often misdiagnosed and cases are understimated.
Project description:BACKGROUND: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. CONCLUSION: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.
Project description:The whitespotted conger Conger myriaster (Brevoort) (Anguilliformes: Congridae) is an extremely marketable food fish, commonly consumed as sashimi or sushi in some Asian countries (i.e. Japan, Korea and China). Conger myriaster is also suspected as being an extremely important source of human anisakidosis. However, there is currently very little information on the levels of infection with ascaridoid nematode parasites in this economically important marine fish. The aims of the present study are to determine the species composition, prevalence and mean intensity of ascaridoid parasites of C. myriaster caught in the Zhoushan Fishery.A total of 1142 third-stage ascaridoid larvae were isolated from 204 C. myriaster. The overall prevalence of infection was 100% (mean intensity 5.6). Nine species of such larvae were accurately identified using integrative taxonomic techniques involving both morphological and genetic data; these included Anisakis pegreffii, A. typica and A. simplex (sensu stricto) × A. pegreffii, Hysterothylacium fabri, H. aduncum, H. sinense, H. amoyense, H. zhoushanense and Raphidascaris lophii. Although high levels of infection and species richness were revealed in C. myriaster, most of the ascaridoid parasites (1135 individuals) were collected from the body cavity and visceral organs of the fish and only seven individuals of A. pegreffii were found in the musculature.This study represents the first report C. myriaster from the Zhoushan Fishery being heavily infected with third-stage ascaridoid larvae. Among the ascaridoid larvae parasitic in this fish, an important etiological agent of human anisakidosis, A. pegreffii (L3), represents the predominant species. The genus Hysterothylacium has the highest species richness, with H. fabri (L3) being the most prevalent species. This high level of infection of A. pegreffii (L3) in C. myriaster suggests a high risk of anisakidosis or associated allergies for people consuming raw or poorly cooked fish originating from this marine area. These findings provide important basic information on the occurrence and infection parameters of ascaridoid nematodes in this economically important marine fish. They also have significant implications for the prevention and control of human anisakidosis when conger eels from the Zhoushan Fishery are consumed.