Sensitivity of candling as routine method for the detection and recovery of ascaridoids in commercial fish fillets.

Ascaridoids are one of the main parasitic hazards in commercial fish. Candling is the current industrial screening method whereby visible ascaridoid larvae are detected on a light table and manually removed. The aim of this study was to assess the sensitivity (Se) and negative predictive value (NPV) of this method. To make targeted recommendations to the fish industry, the Se was calculated per fish part, larval genus, and fish species. All fish parts (n = 615) were first candled, and larvae were collected, followed by enzymatic digestion to recover the remaining larvae. A fish part was considered positive if at least one larva was detected using candling and/or enzymatic digestion, with both methods combined as reference standard. The overall Se of candling was 31% (95% CI 23-41%) and NPV was 87% (95% CI 85-90%). The Se increased with higher numbers of larvae/100 g infected muscle. A low NPV was found for the belly flaps, therefore we either advise the removal or proper freezing of this part. Lastly, the Se and larval recovery was the highest for the darker and larger Pseudoterranova spp. larvae. Due to the low overall efficacy of candling, further assessment of its cost-benefit and impact on consumers' health risk should be conducted.

The occurrence of Anisakis spp. in Australian waters: past, present, and future trends.

As one of the world's megadiverse countries, Australian biodiversity is vital for global biodiversity. Nematodes belonging to the genus Anisakis (family Anisakidae) are an important part of this biodiversity due to their ability to be repeatedly transmitted among their intermediate hosts before reaching the top of the food pyramid. Therefore, they have a significant impact on the community structures of various ecosystems. In addition, globally, they are known to be of medical and veterinary significance. The aim of this article is to provide an update on the current knowledge about these important parasites in Australia. Since 1916, a total of 234 records of Anisakis spp. from various hosts and localities have been found in Australia. It is estimated that the occurrence of Anisakis spp. and their health impacts in at least 84, 98.5, and 95% of Australian marine mammals, fish, and water birds, respectively, have not been documented yet. The results of this study suggest Australia is perhaps home to the most diverse Anisakis fauna. Available information is dominated by reports of these parasites in fish hosts, many of them among edible fish. Given the popularity of seafood in Australia and the occurrence of infectious stages of Anisakis spp. in edible fish, all stakeholders should be made aware of the occurrence, prevalence, and survival of Anisakis spp. in seafood. Also, as more pet owners feed their pets with a variety of fish and seafood products, it is important for veterinarians to be aware of seafood transmitted Anisakis spp. in pet animals. This study also highlights several important knowledge gaps: (i) The detailed life cycle of Anisakis spp. in Australia is not known. Detecting their first intermediate hosts is important for better management of crustacean zooplankton populations in our waters. (ii) Research on Anisakis spp. in Australia has been restricted to limited taxonomical studies and should extend to other aspects of these important parasites. (iii) The capacity to identify parasite taxa to species is especially important for resolving biological diversity around Australia; however, opportunities to formally train in parasite taxonomy are rare and diminishing. There is a need to train researchers with taxonomy skills. (iv) Given the vast range of biodiversity in Australia and the broad host-specificity of Anisakis spp., particularly in the larval stages, the full range of their intermediate hosts remains unknown. (v) The health impacts of the infection of the intermediate/definitive hosts with Anisakis spp. are not fully understood. Thus, one of the important areas for future studies is investigating the pathogenicity of Anisakis spp. in affected animals. This is a crucial yet unknown factor for the conservation of some endangered species in Australia.

Prevalence and identification of Anisakis nematodes in fish consumed in Marrakesh, Morocco.

Morocco is considered as an important producer of fish with more than one million tons of small pelagic fish caught per year, along more than 3400 km of coastline. Otherwise, few studies have investigated the zoonotic parasites of fish. The purpose of this study is to determine the prevalence of Anisakis nematodes larvae in two fish species, namely sardines Sardina pilchardus and mackerel Scomber scombrus. These two species are widely consumed in Marrakesh due to their availability and their affordable prices. A total of 948 fish, including 546 sardines and 402 mackerel, were purchased from the wholesale market of Marrakesh, from January 2016 to December 2018. Sampling was performed on the days of fish arrival from the fishing areas (Dakhla, Essaouira, Safi and Sidi Ifni). The samples were examined visually for the presence of Anisakis larvae. We obtained a prevalence of 8.4% in mackerel with different rates depending on their origins (Safi: 13.23%; Essaouira: 11.66%; Sidi Ifni: 2.5%; Dakhla: 0%) and the seasons. However, no larvae were detected in the sardines after meticulous visual inspection. The detected larvae were morphologically and genetically identified. We identified the larvae by the PCR-RFLP technique using the primers LSU5-F (TAGGTCGACCCGCTGAAYTTAAGCA) and IR16-R (ATTCACACCCATTGACTCGCG) from the 28S rDNA region. The analysis showed that all larvae belong to Anisakis simplex sensu-stricto (s.s.). According to our results mackerel presents a higher risk of contamination than sardine, while statistical studies show that there is no impact of season and fishing origin on the prevalence.

Species composition and infection levels of Anisakis (Nematoda: Anisakidae) in the skipjack tuna Katsuwonus pelamis (Linnaeus) in the Northwest Pacific.

Parasites can be used as biological tags to assess stock structures in various marine fish species. In the present study, the species composition and infection levels of parasitic nematodes of the genus Anisakis in the skipjack tuna Katsuwonus pelamis were examined in the Northwest Pacific and adjacent seas. A total of 867 third-stage larvae of Anisakis were collected from 112 skipjack tunas captured around Japan and in other subtropical localities. All larvae were identified as A. berlandi, A. pegreffii, A. simplex (s.s.), A. typica, and A. physeteris (s.l.) by the direct sequencing of the mitochondrial cox2 gene and real-time PCR assays targeting the nuclear ITS region. Anisakis species composition differed among northeastern Japan, the Sea of Japan, and other areas (central Japan, the Nansei Islands, and subtropical region), which is largely concordant with previous stock discrimination of skipjack tuna. Molecular phylogenetic analysis resulted in two intraspecific genetic groups in A. simplex (s.s.), one of which occurred almost exclusively in northeastern Japan. This could be a useful indicator for stock discrimination. Skipjack tunas from northeastern Japan were also characterized by a remarkable variety in the intensity of A. simplex (s.s.), suggesting the commingling of individuals with different migration patterns. This idea might be further justified by the geographic distribution of two genetically distinct groups of A. physeteris (s.l.).

Increasing intensities of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae with weight and sea age in returning adult Atlantic salmon, Salmo salar L., of coastal waters of Norway.

Ninety wild Atlantic salmon, Salmo salar L., (1.5-10.3 kg) were caught in the Namsen Fjord near the mouth of River Namsen, mid-Norway, and examined for the presence and distribution of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae by digestion of the viscera and muscles in a pepsin/HCl solution. All salmon were migrating spawners after 1-4 years of feeding in the Atlantic Ocean. All 90 Atlantic salmon had A. simplex larvae in the viscera, and all, except two, had A. simplex larvae in the musculature. The number of A. simplex larvae in each fish varied between 3 and 181, and the total mean number of nematode larvae was 44.5. The intensity of A. simplex larvae was positively correlated with increasing weight and sea age of the host. However, the proportion of larvae in the muscle fillets decreased with increasing host weight and sea age. Atlantic salmon females had more A. simplex larvae than males. In all the fish examined, 70.2% of the A. simplex larvae were found in the viscera and 29.8% in the musculature. The majority (93%) of the larvae in the musculature occurred in the hypaxial sections anterior to the anus. As A. simplex larvae commonly occur in the musculature of wild Atlantic salmon, consumption of unfrozen, raw or semi-raw musculature represents a risk for humans developing anisakiasis.

Genetic identification of Anisakis spp. (Nematoda: Anisakidae) from cetaceans of the Southwestern Atlantic Ocean: ecological and zoogeographical implications.

Adult Anisakis Dujardin, 1845 were found in two specimens of killer whale Orcinus orca and one specimen of franciscana Pontoporia blainvillei stranded from off the coast of Buenos Aires Province, Argentina. Genetic identification of the nematodes (N = 144) was performed by sequence analysis of the mitochondrial (mtDNA cox2) and the nuclear (nas 10 nDNA) gene loci. Anisakis pegreffii and Anisakis berlandi were detected in the two individuals of O. orca, while Anisakis typica and A. pegreffii were identified in P. blainvillei. Morphological and morphometric analysis also carried out on adult specimens of A. pegreffii and A. berlandi has allowed to underlining the usefulness of genetic/molecular markers in their recognition. This represents the first record of A. pegreffii in O. orca and P. blainvillei and of A. berlandi in O. orca. This is also the first sympatric and syntopic occurrence, as adults, of A. pegreffii and A. berlandi from the Austral Region of the Atlantic Ocean waters. These results provide insights into the knowledge of the host ranges and geographical distribution of these parasites in the basin waters of the region. Pontoporia blainvillei showed low abundance values of infection with Anisakis spp., which is the general pattern for coastal dolphins in the area, whereas O. orca harboured higher abundance of Anisakis spp. than those previously recorded among cetacean species in the Argentine Sea. Differences in the Anisakis spp. distribution and their parasitic loads, observed among the three host specimens, are discussed in relation to the oceanographic parameters, as well as to the host ecology. The usefulness of genetic/molecular markers in the recognition of adults of the sibling species A. pegreffii and A. berlandi with considerable overlapping in morphometric and morphological characters was underlined. The distribution of Anisakis species from Southwestern Atlantic waters is discussed in relation to their value as indicators for studies on the zoogeography of their hosts at a regional-scale level.

Severe laryngeal edema caused by Pseudoterranova species: A case report.

RATIONALE: Severe laryngeal edema can cause upper airway obstruction, which is fatal. Pseudoterranova, an uncommon nematode of the family Anisakidae, predominantly invades the stomach after ingestion of the nematodes in raw or undercooked marine fish. There have been a few reports of development of severe laryngeal edema caused by the nematode invading the base of the tongue. PATIENT CONCERNS: A 69-year-old Japanese woman complained of stuffy and scratchy throat for 8 hours and reported eating sashimi, fresh slices of raw jacopever, 4 days before the first visit. DIAGNOSIS: Endoscopy revealed a white-yellowish wriggling worm at the left side of the base of the tongue and severe edema of the larynx. INTERVENTIONS: The worm was extracted using endoscopic forceps. The patient was hospitalized and treated with intravenous injection of an antibiotic and steroid. OUTCOMES: The symptoms and laryngeal edema disappeared the next day. The worm was identified as a 4th-stage larva of Pseudoterranova spp based on morphologic features. The serum Anisakis-specific IgE antibody level was high, at 38.6 UA/mL. LESSONS: Clinicians should be aware of the possibility of severe laryngeal edema due to invasion by anisakid nematodes in the pharyngolaryngeal area in cases involving previous ingestion of raw or uncooked marine fish.

Intrinsic properties of anisakid nematode larvae as a potential tool for the detection in fish products.

Anisakid nematode larvae (NL) in fish products comprise a risk to human health and, if visible, lead to the rejection of these products by consumers. Therefore, great efforts are being made for the identification of these anisakid larvae to estimate the potential consumer health risk as well as to develop effective detection methods in order to prevent the introduction of heavily infected fish products into the market. The tasks of national reference laboratories include the improvement of detection methods and to promote their further development. As a prerequisite for improved detection, it is important to understand the structural properties of anisakid NL and compounds produced during host-parasite interactions. This review provides an overview of the intrinsic properties of anisakid NL and reports the latest detection methods in published literature. First, in order to define the potentially interesting intrinsic properties of anisakid nematodes for their detection, anatomy and compounds involved in host-parasite interactions are summarised. These can be used for various detection approaches, such as in the medical field or for allergen detection in fish products. In addition, fluorescence characteristics and their use as both established and promising candidates for detection methods, especially in the field of optical sensing technologies, are presented. Finally, different detection and identification methods applied by the fish processing industries and by control laboratories are listed. The review intends to highlight trends and provide suggestions for the development of improved detection and identification methods of anisakid NL in fish products.

Analysis of the Prevalence and Species of Anisakis nematode in Sekisaba, Scomber japonicus Caught in Coastal Waters off Saganoseki, Oita in Japan.

Anisakidosis is developed by ingesting Anisakis in marine fish, including the chub mackerel, Scomber japonicus, without proper pre-treatment such as cooking or freezing. Two sibling species of Anisakis are found in S. japonicus from Japanese waters, and the prevalence and species of Anisakis in the fish depend on the sea area. For example, Anisakis simplex sensu stricto (s.s.) is found in the Pacific stock of S. japonicus, whereas A. pegreffii is found in the Tsushima Warm Current stock. S.japonicus caught in the Bungo Channel, off the coast of Saganoseki in Oita Prefecture, which is branded as Sekisaba, inhabits a very limited area; however, the infection states of Anisakis found in Sekisaba remain unclear. In this study, we compared the infection states of Anisakis in Sekisaba with those in S. japonicus caught in the South Oita area and Nagasaki Prefecture. All Anisakis from the Nagasaki Prefecture were A. pegreffii, while most of them found in Sekisaba and fish from the South Oita area were A. simplex s.s. Interestingly, the prevalence of Anisakis in Sekisaba was significantly lower than that in the other two areas. This may reflect the fact that Sekisaba might belong to a distinct stock of S. japonicus, varying from other stocks.

Estimation of the number of Anisakis larvae in commercial fish using a descriptive model based on real-time PCR.

BACKGROUND: Seafood parasitation by Anisakis (Anisakidae) larvae has been reported in most of the oceans and seas worldwide. The presence of these nematodes in commonly consumed fish represents a potential hazard for consumers as they can provoke gastrointestinal symptoms and allergic reactions. In the present work, the capacity of a SYBR Green qPCR protocol to quantify Anisakis larvae in commercial fish was evaluated using experimentally spiked samples with different numbers (0-50) of A. simplex third-stage larvae (L3). To verify the agreement of the obtained results, 25 naturally infected fish specimens of Atlantic blue whiting underwent a parallel visual inspection. RESULTS: The logarithmic behavior of the Cq data obtained from the experimentally spiked samples allowed the development of a descriptive mathematical model that correlates the Cq value with the number of Anisakis larvae (R2 = 0.9908, CV = 2.37%). In the commercial blue whiting specimens there was a high correlation between the results of the molecular technique and the visual inspection (R2 = 0.9912); the Bland-Altman analysis showed that 94% of the differences were within the limits of agreement (-4.98 and 6.68), indicating the reliability of the descriptive mathematical model based on the SYBR Green qPCR technique. CONCLUSION: The descriptive function presented based on the SYBR Green qPCR assay is promising as a sensitive and accurate tool for measuring the Anisakis larval load in commercial fish, with a potential application not only in the food industry but also in prevention programs for public health. © 2020 Society of Chemical Industry.

Anisakis simplex (s.s.) larvae (Nematoda: Anisakidae) hidden in the mantle of European flying squid Todarodes sagittatus (Cephalopoda: Ommastrephidae) in NE Atlantic Ocean: Food safety implications.

Few reports exist upon the occurrence and localization of zoonotic anisakid nematodes in T. sagittatus, especially in the mantle of the squid. The occurrence and site of infection of larval anisakids in 98 T. sagittatus caught West off St. Kilda, NE Atlantic Ocean, were investigated. Squids were examined for anisakids using the UV-Press method. In total, 689 nematodes were detected in the viscera and mantle. According to morphology, all the larvae (L3) were assigned to genus Anisakis. Diagnostic allozymes and mtDNA cox2 sequence analysis permitted to genetically identify all larvae as Anisakis simplex (s.s.) (N = 100). Overall prevalence (P = 81%) and mean intensity (mI = 8.6) of infection with A. simplex are provided. Most of the larvae present in the mantle cavity were embedded in the stomach wall or attached in the outer layer of the stomach and caecum (49%). Over a third of squids (37%) hosted A. simplex (s.s.) larvae in the mantle. A novel schematized representation of larvae distribution in the mantle is provided, showing where they were mostly located. According to the results obtained, the risk of anisakiasis associated with consumption of raw or undercooked T. sagittatus should be considered.

Endoscopic submucosal dissection of colonic anisakiasis

No disponible

A new species of Contracaecum (Nematoda: Anisakidae) found parasitizing Nannopterum brasilianus (Suliformes: Phalacrocoracidae) and Hoplias argentinensis (Characiformes: Erythrinidae) in South America: morphological and molecular characterization of larval and adult stages.

Nematode species of the genus Contracaecum Railliet & Henry, 1912 have been reported around the world in many species of fish-eating birds and seals. Here, Contracaecum jorgei n. sp. is morphologically described using light and scanning electron microscopy for adults and fourth-stage larvae (L4) found in the bird Nannopterum brasilianus and third-stage larvae (L3) found in the freshwater fish Hoplias argentinensis, both from the province of Córdoba, Argentina. Additionally, sequences of cytochrome c oxidase subunit II were obtained from these specimens and molecular phylogenetic analysis was used to determine its relationships within the genus. The present species is distinguished from other species by the number and disposition of cephalic papillae; shape and size of the interlabia; length of the spicules; and number and arrangement of papillae in the posterior end of the male. Furthermore, in the molecular analyses, sequences obtained from adult L4 and L3 specimens of C. jorgei n. sp. were similar and grouped, forming an independent lineage, thus confirming it as a distinct species. Thus, morphological characteristics associated with molecular data support the proposal of a new species.

Investigation of Anisakis larvae in different products of ready-to-eat fish meat and imported frozen fish in Turkey.

Globalization opens new market areas and affects food consumption habits, resulting in rapid and remarkable cultural change. Food habits such as consumption of raw fish meat have become popular, resulting in increased risk of emerging infectious diseases. Anisakis simplex sensu stricto (s.s) and A. pegreffii are the most common and important fish-borne zoonotic nematodes responsible for human anisakiasis, which occurs through the consumption of raw or undercooked fish as well as cooked fish due to their heat-stable allergens. Here, we investigated the prevalence, intensity, and abundance of Anisakis larvae in imported fish and ready-to-eat local fish products in Turkey. A total of 205 ready-to-eat fish products, 100 imported frozen Atlantic salmon (Salmo salar) fillets, and 100 imported frozen whole Atlantic mackerel (Scomber scombrus) were sampled from supermarkets, sushi restaurants, and fish markets. All samples were individually examined using a pepsin digestion technique. In total, 602 Anisakis type I larvae were recovered from 98/100 mackerel. No larvae were found in ready-to-eat products or frozen Atlantic salmon fillets. Overall, 8.8% of the larvae were found in the muscle tissue. The overall mean intensity and abundance of infection in mackerel were 6.14 and 6.02, respectively. The larvae were molecularly identified and their phylogenetic relationships with the relevant Anisakis sequences in GenBank were investigated. For this purpose, a subsample of randomly selected 100 Anisakis larvae were analyzed with PCR-RFLP of the ITS region. The larvae were identified as A. simplex (s.s.) (n = 87) and hybrids (n = 13). ITS and cox2 gene regions of all hybrids and randomly selected 50 A. simplex (s.s.) larvae were sequenced for species confirmation and phylogenetic analyses. No intraspecific nucleotide variation was found among the ITS sequences of either species. Seven and three haplotypes, respectively, were identified for A. simplex (s.s.) and hybrid species according to DNA polymorphism of the cox2 gene. Hybrids in our study clustered within the common A. simplex (s.s.) clade in the cox2 phylogenetic tree indicating the dominance of A. simplex (s.s) in the catching area of Atlantic mackerel. Consequently, our study indicates high occurrence of A. simplex (s.s.) larvae with an overall 98.0% prevalence in imported Atlantic mackerel, and highlights the importance of these fish as potential reservoirs for human allergic anisakiasis in Turkey and possibly in other countries.

Larval ascaridoid nematodes in horned and musky octopus (Eledone cirrhosa and E. moschata) and longfin inshore squid (Doryteuthis pealeii): Safety and quality implications for cephalopod products sold as fresh on the Italian market.

The aim of this study was to evaluate the occurrence, infection level and distribution of ascaridoid larvae in cephalopod products sold in Italy. Data on the species most commonly commercialized as whole and fresh on the Italian market were collected. After comparing commercial and literature data, Eledone spp., comprising E. cirrhosa and E. moschata (horned octopus and musky octopus, respectively) and Doryteuthis pealeii (longfin inshore squid) were selected, as they had been rarely investigated. Overall, 75 Eledone spp. caught in the Mediterranean Sea (FAO area 37) and 70 D. pealeii from the Northwest Atlantic Ocean (FAO area 21) were examined by visual inspection and artificial digestion (viscera and mantle separately). Parasites were submitted to morphological and molecular analysis. Prevalence (P), mean intensity (MI) and mean abundance (MA) were calculated. In D. pealeii, 2 nematode larvae molecularly identified as Anisakis simplex s.s. were found in the viscera and in the mantle of two specimens (P: 2.9% 95% CI: 0-6.8%; MI: 1; MA: 0.028). In Eledone spp. 9 nematode larvae molecularly attributed to Hysterothylacium spp. were found in the mantle of 5 specimens (P: 6.7% 95% CI: 1-12.3%; MI: 1.8; MA: 0.12). This is the first report of A. simplex s.s. in D. pealeii. Considering the zoonotic and allergenic potential of these larvae and their localization also in the edible part (mantle), a potential public health issue exists.

Anisakid nematode larvae in the liver of Atlantic cod Gadus morhua L. from West Greenland.

Anisakid nematode larvae occur frequently in the liver of Atlantic cod, but merely few infection data from cod in waters around Greenland exist. The present study reports the occurrence of third-stage anisakid larvae in the livers of 200 Atlantic cod caught on fishing grounds along the West coast of Greenland (fjord systems of Maniitsoq) in May, June, August and September 2017. Classical and molecular helminthological techniques were used to identify the nematodes. A total of 200 cod livers were examined, and 194 were infected with third-stage nematode larvae (overall prevalence of infection 97%) with a mean intensity of 10.3 (range between 1 and 44 parasites per fish). Prevalences recorded were 96% for Anisakis simplex (s.l.), 55% for Pseudoterranova decipiens (s.l.) and 8% for Contracaecum osculatum (s.l.). Sequencing the mtDNA cox2 from 8 out of 23 these latter larvae conferred these to C. osculatum sp. B. A clear seasonal variation was observed, with a rise in A. simplex (s.l.) and P. decipiens (s.l.) occurrence in June and August and a decline in September. The study may serve as a baseline for future investigations using the three anisakids as biological indicators in Greenland waters.

Occurrence of larval anisakids in horse mackerel (Trachurus trachurus) caught in Portuguese waters.

The third-stage larvae (L3) of the Anisakidae family are parasitic nematodes with zoonotic impact and are frequently encountered in the organs and musculature of various fish intended for human consumption. Since Anisakis simplex (s.s.) and A. pegreffii are the major aetiological agents of human disease, this study aims to combine the morphological and molecular data on the recovered anisakid larvae to contribute to a simplified morphological distinction of those species and conducted a survey of anisakid larvae infection in horse mackerel (Trachurus trachurus). Here, 116 horse mackerel caught in Portuguese waters were analysed for the presence of L3 of anisakids, and 3148 larvae were collected, of which only 30% were retrieved during visual inspection. As such, visual inspection does not appear to be very effective in anisakid detection. A prevalence of 84.5% of infected fish was found, and the mean intensity and mean abundance were 32.1 and 27.1 parasites per fish, respectively. The morphological and molecular analyses of 196 L3 randomly chosen from the total sample of parasites demonstrated the presence of L3 of mostly Anisakis spp., with only one L3 of Hysterothylacium aduncum. Relative frequencies of 62.9% for A. pegreffii and 37.1% for A. simplex (s.s.) were obtained. The morphometry differences between these two sibling species were evaluated, and the results demonstrated significant differences between the length of the ventriculus and the length of the oesophagus. Precisely, A. simplex (s.s.) has a longer oesophagus and ventriculus than A. pegreffii. As such, these differences may be used to distinguish the two species through morphological analysis.