Alterations in immunized antigens of Anisakis pegreffii by ampicillin-induced gut microbiome changes in mice.

The gut microbiome plays an essential role in host immune responses, including allergic reactions. However, commensal gut microbiota is extremely sensitive to antibiotics and excessive usage can cause microbial dysbiosis. Herein, we investigated how changes in the gut microbiome induced by ampicillin affected the production of IgG1 and IgG2a antibodies in mice subsequently exposed to Anisakis pegreffii antigens. Ampicillin treatment caused a notable change in the gut microbiome as shown by changes in both alpha and beta diversity indexes. In a 1-dimensional immunoblot using Anisakis-specific anti-mouse IgG1, a 56-kDa band corresponding to an unnamed Anisakis protein was detected using mass spectrometry analysis only in ampicillin-treated mice. In the Anisakis-specific anti-mouse IgG2a-probed immunoblot, a 70-kDa band corresponding to heat shock protein 70 (HSP70) was only detected in ampicillin-treated and Anisakis-immunized mice. A 2-dimensional immunoblot against Anisakis extract with immunized mouse sera demonstrated altered spot patterns in both groups. Our results showed that ampicillin treatment altered the gut microbiome composition in mice, changing the immunization response to antigens from A. pegreffii. This research could serve as a basis for developing vaccines or allergy immunotherapies against parasitic infections.

Anisakis extracellular vesicles elicit immunomodulatory and potentially tumorigenic outcomes on human intestinal organoids.

BACKGROUND: Anisakis spp. are zoonotic nematodes causing mild to severe acute and chronic gastrointestinal infections. Chronic anisakiasis can lead to erosive mucosal ulcers, granulomas and inflammation, potential tumorigenic triggers. How Anisakis exerts its pathogenic potential through extracellular vesicles (EVs) and whether third-stage infective larvae may favor a tumorigenic microenvironment remain unclear. METHODS: Here, we investigated the parasite's tumorigenic and immunomodulatory capabilities using comparative transcriptomics, qRT-PCR and protein analysis with multiplex ELISA on human intestinal organoids exposed to Anisakis EVs. Moreover, EVs were characterized in terms of shape, size and concentration using classic TEM, SEM and NTA analyses and advanced interferometric NTA. RESULTS: Anisakis EVs showed classic shape features and a median average diameter of around 100 nm, according to NTA and iNTA. Moreover, a refractive index of 5-20% of non-water content suggested their effective biological cargo. After treatment of human intestinal organoids with Anisakis EVs, an overall parasitic strategy based on mitigation of the immune and inflammatory response was observed. Anisakis EVs impacted gene expression of main cytokines, cell cycle regulation and protein products. Seven key genes related to cell cycle regulation and apoptosis were differentially expressed in organoids exposed to EVs. In particular, the downregulation of EPHB2 and LEFTY1 and upregulation of NUPR1 genes known to be associated with colorectal cancer were observed, suggesting their involvement in tumorigenic microenvironment. A statistically significant reduction in specific mediators of inflammation and cell-cycle regulation from the polarized epithelium as IL-33R, CD40 and CEACAM1 from the apical chambers and IL-1B, GM-CSF, IL-15 and IL-23 from both chambers were observed. CONCLUSIONS: The results here obtained unravel intestinal epithelium response to Anisakis EVs, impacting host's anthelminthic strategies and revealing for the first time to our knowledge the host-parasite interactions in the niche environment of an emerging accidental zoonosis. Use of an innovative EV characterization approach may also be useful for study of other helminth EVs, since the knowledge in this field is very limited.

Anisakid parasite diversity in a pygmy sperm whale, Kogia breviceps (Cetacea: Kogiidae) stranded at the edge of its distribution range in the Northeast Atlantic Ocean.

Anisakid nematodes are a globally distributed group of marine mammal parasites. Kogiid whales, including the pygmy sperm whale Kogia breviceps, host an assemblage of specific anisakid species. Currently, three species are known to be specific to kogiid hosts, i.e., Skrjabinisakis paggiae, S. brevispiculata, and the less studied Pseudoterranova ceticola. The aim of this study was to investigate the species diversity of anisakid nematodes sampled from a pygmy sperm whale stranded in 2013 at the edge of its distribution range in the Northeast Atlantic, specifically in the North of Scotland. Nematodes were assigned to genus level based on morphology and identified by sequence analysis of the mtDNA cox2 gene and the rDNA ITS region. The present finding represents the first observation of syntopic occurrence of adult stages of S. brevispiculata, S. paggiae, and P. ceticola in a pygmy sperm whale in the Northeast Atlantic, and represent the northernmost record of these species in this area. Skrjabinisakis brevispiculata was the most abundant species, accounting for 55% of the identified nematodes, predominantly in the adult stage. Anisakis simplex (s.s.) was also abundant, with most specimens in the preadult stage, followed by S. paggiae and P. ceticola. The pygmy sperm whale is rarely documented in Scottish waters, and its occurrence in the area could suggest expansion of its geographic range. The presence of S. brevispiculata, S. paggiae, and P. ceticola in this whale species in this region may indicate a shift in the whole host community involved in the life cycle of these parasites in northern waters. However, it is also plausible that these parasites were acquired while the whale was feeding in more southern regions, before migrating northbound.

Title: Diversité des parasites Anisakidae chez un cachalot pygmée, Kogia breviceps (Cetacea : Kogiidae) échoué à la limite de son aire de répartition dans l'Atlantique Nord-Est. Abstract: Les nématodes Anisakidae sont un groupe de parasites de mammifères marins réparti dans le monde entier. Les cétacés Kogiidae, y compris le cachalot pygmée Kogia breviceps, hébergent un assemblage d'espèces d'Anisakidae spécifiques. Actuellement, trois espèces sont connues pour être spécifiques aux hôtes Kogiidae, à savoir Skrjabinisakis paggiae, S. brevispiculata et Pseudoterranova ceticola, la moins étudiée. Le but de cette étude était d'étudier la diversité des espèces de nématodes Anisakidae échantillonnés sur un cachalot pygmée échoué en 2013 à la limite de son aire de répartition dans l'Atlantique Nord-Est, plus précisément au nord de l'Écosse. Les nématodes ont été attribués au niveau du genre en fonction de la morphologie et identifiés par analyse de séquence du gène cox2 de l'ADNmt et de la région ITS de l'ADNr. La présente découverte représente la première observation de l'apparition syntopique de stades adultes de S. brevispiculata, S. paggiae et P. ceticola chez un cachalot pygmée dans l'océan Atlantique Nord-Est, et représente le signalement le plus septentrional de ces espèces dans cette zone. Skrjabinisakis brevispiculata était l'espèce la plus abondante, représentant 55% des nématodes identifiés, principalement au stade adulte. Anisakis simplex (s.s.) était également abondant, la plupart des spécimens étant au stade préadulte, suivi par S. paggiae et P. ceticola. Le cachalot pygmée est rarement documenté dans les eaux écossaises et sa présence dans la région pourrait suggérer une expansion de son aire de répartition géographique. La présence de S. brevispiculata, S. paggiae et P. ceticola chez cette espèce de cachalot dans cette région peut indiquer un changement dans l'ensemble de la communauté hôte impliquée dans le cycle de vie de ces parasites dans les eaux nordiques. Cependant, il est également plausible que ces parasites aient été acquis alors que le cachalot se nourrissait dans des régions plus au sud, avant de migrer vers le nord.

Anisakid larvae in the skipjack tuna Katsuwonus pelamis captured in Japanese waters: Two-year monitoring of infection levels after the outbreak of human anisakiasis in 2018.

In 2018, human anisakiasis caused by the ingestion of the skipjack tuna Katsuwonus pelamis occurred frequently in Japan. This may be attributable to a heavy infection of A. simplex (s.s.) in the host's muscle tissue. In this study, we investigate infection levels of anisakid L3 larvae in skipjack tuna captured in Japanese waters afterward (2019-2020) to contribute to predict and prevent the outbreak of human anisakiasis. A total of 476 larvae were detected from 78 out of 85 skipjack tuna captured at 14 stations of the Pacific and East China Sea. The present parasitological survey suggests that infection levels in 2019-2020 were low, comparing that in 2018; in total only seven larvae were found from the host's muscle tissue. The collected larvae were identified by molecular methods to Anisakis berlandi, A. pegreffii, A. simplex (s.s.), A. typica and Skrjabinisakis physeteris (s.l.). Not only larvae of A. simplex (s.s.) but also those of A. berlandi were found from the muscle tissue and thus the latter species may also be a causative agent of human anisakiasis. In addition, this study confirmed the geographic distribution pattern that A. simplex (s.s.) is abundant in the Pacific, while A. pegreffii is dominant in the East China Sea. Our results contribute to understanding the risk of food poisoning and stock delimitation of host animals.

Ingestion of fishing gear and Anisakis sp. infection in a beached Indo-Pacific finless porpoise (Neophocaena phocaenoides) in the Jeju Island, Republic of Korea: findings from post-mortem computed tomography and necropsy.

BACKGROUND: Human fishing activities have significantly affect environmental concern for marine ecosystems, conservation of marine mammals, and human health. Coastal cetaceans are highly vulnerable to ingestion of fishing gear, bycatching, or entanglement, all of which can be fatal for these animals. In particular, certain coastal dolphins and porpoises are heavily impacted by fishing gear such as angling gear or stownet, as their food often overlap with the target fish species of human fisheries. CASE PRESENTATION: This study presents a case of an Indo-Pacific finless porpoise (Neophocaena phocaenoides) beached on the coast of Jeju Island, Republic of Korea, with ingestion of fishing gear and severe Anisakis infection. Although this species inhabits waters ranging from the Persian Gulf to Taiwan, several stranded carcasses have been reported on Jeju Island in recent years. Post-mortem computed tomography revealed a bundle of four fishing hooks in the forestomach, along with nylon lines and steel lines with connectors, which were assumed to be angling gear for Jeju hairtail (Trichiurus lepturus). Further necroscopic investigation revealed that the forestomach contained a large number of Anisakis spp. (Nematoda: Anisakidae). Histological examination revealed a thickened forestomach wall with pinpoint and volcanic ulcerations, a thickened layer of stratified squamous epithelium, and infiltrated stroma in the squamous epithelium. CONCLUSIONS: This study emphasizes the urgent need to address the impact of fishing activities on marine mammals, marine litter pollution, and the bycatch problem in Korean seawater. In addition, the occurrence of N. phocaenoides in seawater around Jeju Island should be raised in future geographical ecology or veterinary pathology studies and when its distribution is updated.

The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes.

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 µg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

A surgical case of inflammatory pseudotumor by hepatic anisakiasis.

Anisakiasis is a parasitic infection caused by the ingestion of raw or undercooked seafood infected with Anisakis larvae. It generally affects the gastrointestinal tract, particularly the stomach, but very rare cases have been reported in which infection of the liver leads to the formation of inflammatory pseudotumors. We herein report an extremely rare case of an inflammatory pseudotumor induced by hepatic anisakiasis that was laparoscopically resected for the purpose of both diagnosis and treatment. A 51-year-old woman underwent a routine medical checkup by ultrasound examination, which incidentally detected a 15-mm mass on the surface of S6 of the liver. Because a malignant tumor could not be ruled out on several preoperative imaging studies, laparoscopic partial resection of the liver was performed. Histopathological examination revealed Anisakis larva in the inflammatory pseudotumor, suggesting hepatic anisakiasis. This report describes an extremely rare case of an inflammatory pseudotumor induced by hepatic anisakiasis. Because the preoperative diagnosis could not be obtained by several imaging modalities, laparoscopic liver resection with a sufficient margin might be suitable for diagnosis and treatment of this disease.

A novel rhabdovirus detected in Anisakis larvae distributed in the coastal areas of Japan: Viral genome analysis and possible coevolutionary relationship between virus and host nematodes.

In the last decade, it has become evident that various RNA viruses infect helminths including Order Ascaridida. However, there is still no information available for viruses infecting Anisakis. We herewith demonstrate the presence of a novel rhabdovirus from Anisakis larvae detected by next-generation sequencing analysis and following RT-PCR. We determined the nearly all nucleotide sequence (12,376 nucleotides) of the viral genome composed of seven open reading frames, and we designated the virus as Suzukana rhabdo-like virus (SkRV). BLASTx search indicated that SkRV is a novel virus belonging to the subfamily Betanemrhavirus, rhabdovirus infecting parasitic nematodes of the Order Ascaridida. SkRV sequence was detectable only in the total RNA but not in the genomic DNA of Anisakis, ruling out the possibility of SkRV being an endogenous viral element incorporated into the host genomic DNA. When we individually tested Anisakis larvae obtained from Scomber japonicus migrating in the coastal waters of Japan, not all but around 40% were SkRV-positive. In the phylogenetic trees of Betanemrhavirus and of the host Ascaridida nematodes, we observed that evolutional distances of viruses were, to some extent, parallel with that of host nematodes, suggesting that viral evolution could have been correlated with evolution of the host. Although biological significance of SkRV on Anisakis larvae is still remained unknown, it is interesting if SkRV were somehow related to the pathogenesis of anisakiasis, because it is important matter of public health in Japan and European countries consuming raw marine fishes.

Preliminary data on the occurrence of PAH and Anisakis spp. in Moroccan anchovies: Environmental parasitology and human health risk.

This survey focuses on an environmental parasitology study by investigating Polycyclic Aromatic Hydrocarbons (PAH) bioaccumulation and the occurrence of Anisakis spp. on common anchovies collected from Moroccan coasts over 9 months through chemical (PAH) and Biological (Biometrics and parasitic) analysis. Obtained results were statistically analyzed and human health risks from anchovies consumption were assessed. The results obtained highlighted the good biological and chemical status of this fish in all stations of Morocco's coasts. Anisakis spp. was present in only four stations in the Atlantic fringe (maximum prevalence 22.22 %). Results have shown non-significant differences for tissue nature (Muscle, viscera) or spatial variation and were within a low range of concentrations well below the European Commission standards. Results have shown no serious harmful risk for humans from this fish consumption (ILCR and HI), and statistical analysis had shown positive correlations between prevalence and Chr, prevalence and sex-ratio, and prevalence and weight.

A critical review of anisakidosis cases occurring globally.

A review was conducted to identify the most common causative agents of anisakidosis, the methods used for identification of the causative agents, and to summarize the sources of infection, and patients' demographics. A total of 762 cases (409 articles, inclusive of all languages) were found between 1965 and 2022. The age range was 7 months to 85 years old. Out of the 34 countries, Japan, Spain, and South Korea stood out with the highest number of published human cases of anisakidosis, respectively. This raises the question: Why are there few to no reports of anisakidosis cases in other countries, such as Indonesia and Vietnam, where seafood consumption is notably high? Other than the gastrointestinal tract, parasites were frequently found in internal organs such as liver, spleen, pancreas, lung, hiatal and epigastric hernia, and tonsils. There are also reports of the worm being excreted through the nose, rectum, and mouth. Symptoms included sore throat, tumor, bleeding, gastric/epigastric/abdominal/substernal/lower back/testicular pain, nausea, anorexia, vomiting, diarrhea, constipation, intestinal obstruction, intussusception, blood in feces, hematochezia, anemia, and respiratory arrest. These appeared either immediately or up to 2 months after consuming raw/undercooked seafood and lasting up to 10 years. Anisakidosis commonly mimicked symptoms of cancer, pancreatitis, type I/II Kounis syndrome, intussusception, Crohn's disease, ovarian cysts, intestinal endometriosis, epigastralgia, gastritis, gastroesophageal reflux disease, hernia, intestinal obstruction, peritonitis, and appendicitis. In these cases, it was only after surgery that it was found these symptoms/conditions were caused by anisakids. A range of not only mainly marine but also freshwater fish/shellfish were reported as source of infection. There were several reports of infection with >1 nematode (up to >200), more than one species of anisakids in the same patient, and the presence of L4/adult nematodes. The severity of symptoms did not relate to the number of parasites. The number of anisakidosis cases is grossly underestimated globally. Using erroneous taxonomic terms, assumptions, and identifying the parasite as Anisakis (based solely on the Y-shaped lateral cord in crossed section of the parasite) are still common. The Y-shaped lateral cord is not unique to Anisakis spp. Acquiring a history of ingesting raw/undercooked fish/seafood can be a clue to the diagnosis of the condition. This review emphasizes the following key points: insufficient awareness of fish parasites among medical professionals, seafood handlers, and policy makers; limited availability of effective diagnostic methodologies; and inadequate clinical information for optimizing the management of anisakidosis in numerous regions worldwide.

Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae).

Introduction: Anisakis 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. Methods: Genetically 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 discussion: EVs 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.

Anisakis infection in anchovies (Engraulis encrasicolus) from Iberian waters, southwestern Europe: Post-mortem larval migration.

The Anisakis larvae presence in fish for human consumption is a health risk that needs to be monitored. The anchovy is a fish that is highly appreciated by consumers and that can harbour Anisakis. It is thus necessary to periodically evaluate the presence of anisakid larvae in them. So, anchovies from Iberian Peninsula coasts were analysed. Fish examination for macroscopic nematodes showed L3s of both Anisakis type I and Hysterothylacium aduncum. The Anisakis prevalence varies with the catching area and the fish size. The muscle prevalence was 7.45% (mean intensity 1.75; range 1-5). Molecular analysis showed 110 A. simplex s.s. (17 in muscle), 22 A. pegreffii (3) and 7 hybrid genotype individuals (1). Considering that most of the Iberian Peninsula coasts are a sympatry area between these two Anisakis species, it has been observed that A. simplex s.s./A. pegreffii ratio increases from south to north in a clockwise direction. Also, 19 larvae were detected on the fish surface from the Bay of Biscay, indicating the ability of these larvae to migrate after the fish death. The A. simplex s.s./A. pegreffii larvae proportion found on the anchovy surface is similar to the found in viscera and lower than in muscle, suggesting that most of the larvae migrating to the surface must have come from the visceral package. This confirms the importance of removing fish viscera immediately after capture, for those fish species where this is possible. As both species cause anisakiasis/anisakidosis, these data show a real risk to human health, especially in dishes highly prized in Mediterranean countries prepared with raw or semi-raw anchovies.

In vitro culture of the zoonotic nematode Anisakis pegreffii (Nematoda, Anisakidae).

BACKGROUND: Anisakiasis 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. METHODS: Testing 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. RESULTS: Schneider'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. CONCLUSIONS: Closing 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.

Effects of temperature on eggs and larvae of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) and its possible role on their geographic distributions.

Effects of temperature on development of eggs, recently hatched larvae and L3 larvae of the marine parasitic nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were examined in vitro. The eggs of A. simplex s.s. hatched at 3-25 °C and those of A. pegreffii hatched at 3-27 °C. Days before hatching varied between 2 days at 25 °C and 35-36 days at 3 °C in A. simplex s.s. and between 2 and 3 days at 27 °C and 65 days at 3 °C in A. pegreffii. Hatching rates of A. simplex s.s. were maintained high at temperatures between 3 and 25 °C but decreased to 0% at 27 °C. In contrast, those of A. pegreffii were lowest particularly at 3 °C, but also at 27 °C. The mean 50% survivals of hatched larvae ranged from 5.3 days at 25 °C to 82.3 days at 9 °C in A. simplex s.s., while in A. pegreffii it ranged from 1.2 days at 27 °C to 77.2 days at 9 °C. L3 larvae of A. pegreffii exhibited higher survival rates and activity than those of A. simplex s.s., particularly at 20 and 25 °C. These results suggest that the early stages of A. simplex s.s. are more adapted to lower temperatures whereas those of A. pegreffii are more tolerant to warm environments, which may correspond to their distribution patterns in Japan and Europe.

Molecular identification and infection levels of Anisakis species (Nematoda: Anisakidae) in the red scorpionfish Scorpaena scrofa (Scorpaenidae) from the Aegean Sea.

The red scorpionfish Scorpaena scrofa (Scorpaenidae) is a high commercial value marine fish species along the Mediterranean coasts. Anisakiasis is a fish-borne parasitic zoonoses caused by Anisakis larvae in consumers. To date, there are only a few epidemiological studies on the presence and molecular identification of Anisakis larvae infecting S. scrofa. A total of 272 S. scrofa captured from the Gulf of Izmir in the Turkish Aegean coasts (FAO 37.3.1) were examined for Anisakis larvae between March 2019 and March 2020. The prevalence, mean intensity and mean abundance of Anisakis larvae were 9.6% (95% CI 6.5-13.7%), 2.8 (95% CI 1.88-5.19), and 0.27 (95% CI 0.15-0.56), respectively. All Anisakis larvae were collected from the viscera and body cavity of S. scrofa. Anisakis pegreffii, A. typica, and A. ziphidarum were genetically identified by RFLP analysis of the ITS region. These species were also confirmed by cox2 sequence analysis. A weak positive and statistically significant correlation between the total length (ρS 0.204; p = 0.001) and total weight (ρS 0.200; p = 0.001) of S. scrofa and the number of Anisakis larvae was observed. This survey presents the first molecular detection of A. typica and A. ziphidarum in S. scrofa. Thus, this fish species is a new host for A. typica and A. ziphidarum. This is also the first report of the presence of A. ziphidarum in the Aegean Sea.

Anisakid parasites (Nematoda: Anisakidae) in 3 commercially important gadid fish species from the southern Barents Sea, with emphasis on key infection drivers and spatial distribution within the hosts.

Northeast Arctic cod, saithe and haddock are among the most important fisheries resources in Europe, largely shipped to various continental markets. The present study aimed to map the presence and distribution of larvae of parasitic nematodes in the Anisakidae family which are of socioeconomic and public health concern. Fishes were sourced from commercial catches during winter or spring in the southern Barents Sea. Samples of fish were inspected for nematodes using the UV-press method while anisakid species identification relied on sequencing of the mtDNA cox2 gene. Anisakis simplex (s.s.) was the most prevalent and abundant anisakid recorded, occurring at high infection levels in the viscera and flesh of cod and saithe, while being less abundant in haddock. Contracaecum osculatum (s.l.) larvae, not found in the fish flesh, showed moderate-to-high prevalence in saithe, haddock and cod, respectively. Most Pseudoterranova spp. larvae occurred at low-to-moderate prevalence, and low abundance, in the viscera (Pseudoterranova bulbosa) and flesh (Pseudoterranova decipiens (s.s.) and Pseudoterranova krabbei) of cod, only 2 P. decipiens (s.s.) appeared in the flesh of saithe. Body length was the single most important host-related factor to predict overall abundance of anisakid larvae in the fish species. The spatial distribution of Anisakis larvae in the fish flesh showed much higher abundances in the belly flaps than in the dorsal fillet parts. Trimming of the flesh by removing the belly flaps would reduce larval presence in the fillets of these gadid fish species by 86­91%.

Infection process, viability and establishment of Anisakis simplex s.l. L3 in farmed fish; A histopathological study in gilthead seabream.

Anisakis spp. (Nematoda, Anisakidae) are parasites known by their economic and health impacts, as their L3 larval stages infect a variety of fish species, many of them commercial species, sometimes causing zoonotic episodes due to consumption of raw or undercooked fish. The aim of this study is to determine the infection process and the potential impact of A. simplex s.l. L3 on gilthead seabream (Sparus aurata L.), one of the most important fish species in Mediterranean aquaculture, by periodic histological monitoring of the infection process. For this, fish were experimentally infected with A. simplex s.l. L3 and periodically analysed for L3 larvae, collecting samples at different time points (hours post ingestion, hpi): 3, 6, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168 and 192, up to 6 months post infection (mpi). All samples were observed under a stereomicroscope and later fixed for histological examination. A. simplex s.l. L3 were only found on the visceral surface and mesenteric tissue, but never free or encapsulated in muscle. Chronological events were found to occur faster than those reported in previous studies. They were first observed 6 hpi in the coelomic cavity, being present up to 48 hpi. While the earliest evidence of fibrocytes surrounding A. simplex s.l. L3 larvae were observed at 18 hpi, complete spiral encapsulation occurred by 72 hpi. Alive parasites were observed up to 6 mpi. Although the infection of gilthead seabream by Anisakis spp. larvae is feasible, it seems unlikely, especially in aquaculture given the hygienically controlled feeding systems. In the event of infection, the transmission would be unlikely due to the poor condition in which specimens of Anisakis spp. are found. Furthermore, since no larvae were detected in the fish's muscle, human infection seems improbable.

Demographic history and population genetic structure of Anisakis pegreffii in the cutlassfish Trichiurus japonicus along the coast of mainland China and Taiwan.

Studying the genetic diversity of nematode parasite populations is crucial to gaining insight into parasite infection dynamics and informing parasite phylogeography. Anisakiasis is a zoonotic disease caused by the consumption of infectious third-stage larvae (L3) of Anisakis spp. carried by marine fish. In the present study, a total of 206 mitochondrial DNA sequences (cytochrome c oxidase 2, cox2) were used to study the genetic diversity, genetic structure, and historical demography of twelve A. pegreffii populations from Trichiurus japonicas along the coast of mainland China and Taiwan. Two distinct evolutionary lineages of A. pegreffii and no significant genealogical structures corresponding to sampling localities suggested that isolation in the marginal seas shaped their patterns of phylogeographic distribution along the coast of mainland China and Taiwan during glaciation with lower sea levels. Furthermore, pairwise FST values and AMOVA did not indicate any significant genetic differentiation among groups with no relation to the geographic area, which might be attributed to fewer barriers to gene flow as well as large population sizes. The results of the neutrality test, mismatch distribution, and Bayesian skyline plot analyses showed that entire population underwent population expansion during the late Pleistocene. Analysis of the demographic history revealed that A. pegreffii underwent historical lineage diversification and admixture due to secondary contact based on ABC analysis. The present research represents the first definitive population structure and demographic history across sampling locations of A. pegreffii along the coast of mainland China and Taiwan.

Anisakis spp, DNA detection in paraffin-embedded tissue biopsies recovered from patients with gastritis using real-time PCR in Bushehr, Persian Gulf, Iran.

Anisakiasis is a zoonotic fish-born parasitic disease caused by anisakid nematodes. Paraffin-embedded blocks containing biopsy samples taken from patients suffering gastritis with unknown causes were investigated by real-time PCR, in the Bushehr region, Iran; where human anisakiasis has not been reported, so far. A total of 50 paraffin-embedded blocks were randomly selected from 250 archived blocks of the patients with gastritis. A SYBER green-based real-time PCR targeting the ITS1 region was developed for the identification of Anisakis genus. An 86 bp partial fragment of the Anisakis spp. ITS1 gene was amplified successfully. A total of 3 out of 50 samples (6 %) had positive amplification in the samples and their pathology reports showed a significant finding of moderate chronic gastritis with or without ulcers. In conclusion, the developed qPCR could be used for detecting Anisakis spp. larval DNA in human biopsy blocks. This study showed the hidden human cases of anisakiasis in the Bushehr for the first time.

Multigene phylogenetic analysis reveals non-monophyly of Anisakis s.l. and Pseudoterranova (Nematoda: Anisakidae).

The nematode genera Anisakis s.l. and Pseudoterranova (Anisakidae) include causative agents of anisakiasis and pseudoterranovosis, parasitic diseases resulting from eating undercooked or raw fish or squid. Species in both genera have thus attracted considerable attention especially in public health and taxonomic studies. The phylogenetic relationships of these genera within the subfamily Anisakinae, however, remain to be investigated with dense taxonomic sampling. In this study, we collected an anisakid third-stage larva, and identified it morphologically and molecularly as Pseudoterranova ceticola. Phylogeny of 15 anisakine species, including the newly collected specimen of Ps. ceticola, was reconstructed based on sequences of three mitochondrial (cox1, cox2, and 12S rRNA) and two nuclear (ITS and 28S rRNA) regions. The obtained tree suggested the non-monophyly of Anisakis s.l. and Pseudoterranova. Anisakis s.l. was divided into two groups, which are distinguished from each other by the shape of the ventriculus. Based on phylogenetic relationships and morphology, three species with a shorter ventriculus ("A." brevispiculata, "A." paggiae, and "A." physeteris) were assigned to the genus Skrjabinisakis, as recently proposed. Pseudoterranova ceticola was distantly related to the monophyletic Ps. decipiens species complex. Although the phylogenetic position of the type species Ps. kogiae has not been investigated due to a lack of sequence data, this species may morphologically and ecologically resemble Ps. ceticola, inferring a close kinship between the two species.