[Investigation of Anisakis infections in coastal marine fishes and awareness of anisakiasis control knowledge among residents in Yantai City in 2021].

OBJECTIVE: To investigate the prevalence of Anisakis infections in coastal marine fishes and awareness of anisakiasis control knowledge among local residents in Yantai City, Shandong Province in 2021, so as to provide insights into formulation of anisakiasis control interventions. METHODS: Marine fishes were purchased from Shunxin Port, Yantai City, Shandong Province in November 2021, and the presence of Anisakis was detected in different species of fishes and different fish sites. The correlations between body length and weight of marine fish and intensity of Anisakis infections were examined using Spearman's rank correlation analysis, and the dietary habits and anisakiasis control knowledge were investigated using questionnaire surveys among local residents. RESULTS: A total of 201 marine fishes belonging to 20 species were dissected, and Anisakis was detected in 77 marine fishes (38.31%) belonging to 11 species (55.00%), with a mean infection intensity of 45.04 parasites per fish (3 468/77). Spearman's rank correlation analysis revealed that the body length (rs = 0.74, P < 0.05) and weight (rs = 0.79, P < 0.01) of the monkfish correlated positively with the intensity of Anisakis infections, and the body length (rs = 0.68, P < 0.05) of the flatfish correlated positively with the intensity of Anisakis infections, while no correlations were examined between the body length or weight of other marine fishes and the intensity of Anisakis infections. Of all respondents, 53.38% men and 56.67% women did not know anisakiasis control knowledge at all, and there was a significant difference in the proportion of respondents using separate chopping boards for raw and cooked food from different villages (χ2 = 17.89, P < 0.01), while there was an age-specific proportion of respondents with habitats of eating raw or semi-raw seafood (χ2 = 28.27, P < 0.01). CONCLUSIONS: The prevalence and intensity of Anisakis infections were high in coastal marine fishes in Yantai City in 2021, and the awareness of anisakiasis control knowledge was low among local residents. Intensified health education pertaining to anisakiasis control knowledge is recommended to reduce the risk of Anisakis infections.

Anti-Anisakis antibodies in colon cancer patients and their relationship with γδ T-cells.

Many pathogens are related to carcinogenesis. Chronic inflammation, as a result of persistent infection, leads to DNA damage, higher expression of oncogenes, decreased apoptosis and immunosuppression, which are some of the reasons for cancer induction. Among parasites, Schistosoma, Opistorchis and Clonorchis are recognised as infectious agents which contribute to cancer. A relationship between Anisakis and cancer was hypothesised because cellular responses to Anisakis products could result in inflammation and DNA damage. Previous research has shown a decrease in CD8+ γδ T-cells and an increase in αß and γδ T-cell apoptosis in colon cancer (CC) samples. Ninety-two CC patients and 60 healthy subjects were recruited. γδ and αß T-cells were analysed, and their apoptosis was evaluated. Anti-Anisakis antibodies were tested in sera from CC patients and controls. Anti-Anisakis IgG, IgM, IgA and IgE antibodies were significantly higher in CC patients. A significant increase in anti-Anisakis IgA levels was observed in patients with angiolymphatic invasion. The number of all γδ T-cells, as well as CD3+ CD4+ αß T-cells, was significantly lower in CC patients. The apoptosis of all T-cells was significantly increased in patients with CC. We observed a significantly higher percentage of anti-Anisakis IgE positive patients having a deficit of CD3+ γδ T-cells. Our results suggest a relationship between Anisakis and CC.

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.

Trends in Anisakis simplex Global Research: A Bibliometric Analysis Study.

Objective: Anisakis simplex is a fish-borne zoonotic parasite. Although this parasite has been known since the 19th century, publications on anisakiasis have increased in recent years. Despite this, this subject has yet to be well studied by bibliometric analysis. This study was conducted to show the research points and trends of A. simplex. Methods: The Web of Science Core Collection (WoSCC) was mined for articles on A. simplex. The VOSviewer software visually evaluated countries, institutions, authors, references, and keywords in this field. Results: A total of 1362 publications were included in this bibliometric analysis. The included publications were published between 1970 and 2022 from 79 countries, mainly from Spain (n=456, 33.48%). The most prolific year was 2020 (n=74). The research area that attracted the most publications was parasitology (n=452), while the most productive author in this area was Cuellar C (n=53). "Anisakis simplex", "Anisakis" and "anisakiasis" were the most used three keywords. Conclusion: The number of publications on anisakiasis has been increasing over time, suggesting that A. simplex is becoming an increasingly important disease worldwide. Research cooperation should be established between researchers from developed and developing countries to determine effective control strategies for anisakiasis.

Agarose Hydrogel-Boosted One-Tube RPA-CRISPR/Cas12a Assay for Robust Point-of-Care Detection of Zoonotic Nematode Anisakis.

Rapid and accurate detection of the zoonotic nematode Anisakis is poised to control its epidemic. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated assay shows great potential in the detection of pathogenic microorganisms. The one-tube method integrated the CRISPR system with the recombinase polymerase amplification (RPA) system to avoid the risk of aerosol pollution; however, it suffers from low sensitivity due to the incompatibility of the two systems and additional manual operations. Therefore, in the present study, the agarose hydrogel boosted one-tube RPA-CRISPR/Cas12a assay was constructed by adding the CRISPR system to the agarose hydrogel, which avoided the initially low amplification efficiency of RPA caused by the cleavage of Cas12a and achieved reaction continuity. The sensitivity was 10-fold higher than that of the one-tube RPA-CRISPR/Cas12a system. This method was used for Anisakis detection within 80 min from the sample to result, achieving point-of-care testing (POCT) through a smartphone and a portable device. This study provided a novel toolbox for POCT with significant application value in preventing Anisakis infection.

Serum IgA contributes to the comprehension of Anisakis simplex associated chronic urticaria.

The phenotype of allergic diseases associated with Anisakis determines the pattern of cytokines related to antibody production. However, the role of serum IgA and the immunomodulatory mechanisms exerted by active infection of L3 or passive mucosal contact with A. simplex specific antigens has not been studied before. We measured serum cytokine by flow cytometry (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, TGF-ß1) and antibody levels (IgE, IgG4, IgA) by ELISA against total and excretory-secretory (ES) antigens, Ani s 3,and the group of major allergens Ani s 1, Ani s 7, and Ani s 13 in sera from 10 patients with gastro-allergic anisakiasis (GAA), 11 Anisakis sensitization associated chronic urticaria (CU+) as well as 17 non-Anisakis-sensitized patients with chronic urticaria (CU-), compared with the urticaria control group (18 subjects). Specific IgE, IgG4 and IgA were high in the GAA, but IgA levels were significantly higher in the CU+ with respect the CONTROL group. We observed higher levels of the ratio IgA/IgG4 in CU+ than GAA group for Ani s 1, Ani s 7, Ani s 13 and ES. Furthermore, chronic urticaria (CU) patients showed significant lower levels of IL-10, IFN-γ and IL-17A than patients without CU. The anti-Ani s 13 IgA/IgG4 ratio correlated positively with pro-inflammatory cytokines and ratios (TNF-α, IL-17A, Th17/Th2, Type1/Type2 and TNF-α/IL-10) in CONTROL group. In general, Anti-Anisakis IgA/G4 ratio was high in CU patients. In conclusion, this study demonstrates the importance of serum IgA because it is associated with chronic urticaria independently of Anisakis sensitization.

Variation in the levels of anisakid infection in the European anchovy Engraulis encrasicolus (Linnaeus) from the Bay of Biscay during the period 2000-2023 (ICES Subarea 8).

The European anchovy Engraulis encrasicolus is one of the most important commercial species in the Bay of Biscay (ICES Subarea 8), and our analysis focused on the analysis of the temporal mean abundance, prevalence, and intensity of Anisakis spp. larvae species in anchovies from ICES Subarea 8 in the years 2000, 2001, 2014-2016, and 2019-2023. Prevalence in adult individuals of anchovy was only 1% in 2000 but increased to 90% in 2014. Since 2015, the prevalence has decreased, and the number of individuals affected in 2023 accounted for 17.6%. The mean abundance showed a similar trend, with a peak of 3.79 nematodes/anchovy in 2014, falling to 0.21 in 2023. The species A. simplex sensu stricto and A. pegreffii were identified by PCR/SANGER sequencing and PCR/RLFP techniques in 2019 and 2020. Anisakis simplex (s.s.) was the most abundant species and, according to the results returned by these two techniques, it accounted for an average of 62.4% and 52.1% of total nematodes in 2019 and 2020, respectively. The results of studies monitoring infection levels in anchovies showed that the mean abundance and prevalence changed over the course of the study period and that the proportion of different species of Anisakis is also subject to variation from year to year.

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.

The involvement of Pseudoterranova decipiens fish infestation on the shelf-life of fresh Atlantic cod (Gadus morhua) fillet.

Zoonotic nematodes of the family Anisakidae are highly common in many marine fish species, which act as paratenic hosts for the third larval stage. In the fish, these parasites may migrate from the fish's gastro-intestinal tract (GI-tract) further to the coelomic cavity and muscles, making them a possible contamination source of bacteria they carry on their cuticle and in their GI-tract. A previous study revealed no apparent effect of Anisakis simplex on spoilage of fish, but the equally common anisakid species Pseudoterranova decipiens has a larger body surface potentially increasing the bacterial load brought into the fish muscle upon migration. As the presence of shelf-life reducing spoilage bacteria in the microbiome of this anisakid species has been demonstrated, the objective of the present study was to assess the potential shelf-life reducing effect of P. decipiens in fresh fish fillets stored in a domestic refrigerator. Atlantic cod was used as a model since members of the cod family are the third most consumed marine fish globally and it has the highest prevalence of P. decipiens infections. Infected and non-infected codfish fillet portions were collected and microbiologically analyzed at day 0 and day 4 of storage in a domestic fridge. Three isolation media were used to enhance maximum bacterial recovery and isolates were identified using MALDI-TOF MS and 16S rRNA gene sequencing. In parallel to the microbiological examination, sensory analysis was performed daily on the cod fillets to evaluate the freshness of the fish. Results revealed the presence of typical spoilage bacteria (e.g., Pseudomonas sp., Photobacterium sp.) in all fish, but based on the total viable counts, total H2S-producing bacteria, and sensory analysis, there were no objective indications to assume an increased fish spoilage rate by the presence and migration P. decipiens. Additionally, a beta-diversity comparison revealed no significant differences in microbiota composition between infected and non-infected fish parts, though individual heterogeneity in microbiome composition among Atlantic codfish individuals was found. As total viable counts did, however, exceed the guideline limits for fresh fish, further research should now focus on the role of the candling step as a potential source of post-harvest contamination. As such, anisakid infection might still accelerate fish spoilage, though now in an indirect way.

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.

[COMPONENT ANALYSIS OF THREE ANISAKIASIS ALLERGY CASES IN OUR DEPARTMENT].

Reactivity to an anisakis allergen component was examined in three patients with a history of an anisakiasis anaphylaxis. Case 1, a 38-year-old man, allergic symptoms appeared 0.5 hours after ingestion, and the component Ani s 1 and 3 were positive. Case 2, a 44-year-old woman, allergic symptoms appeared 4 hours after ingestion, and components Ani s 3 and 12 were positive. Case 3, a 36-year-old woman, developed allergic symptoms 7 hours after ingestion of fish and shellfish, and tested positive for Ani s 1, 4, and 12. Case 3 reacted strongly to both heated and unheated Anisakis extract, while cases 1 and 2 reacted weakly to heated Anisakis extract. The most common allergen was Ani s 12, followed by Ani s 1, when analyzed in conjunction with existing reports on 10 cases. Anisakis IgE was class 3 or higher in all cases. Analysis of 13 cases showed 2 cases sensitized to Ani s 4 and moderate or higher anaphylaxis, while Ani s 4-sensitized patients were reported to be more likely to develop severe disease. It is possible that the patients sensitized to Ani s 4 need to be careful about the severity of their allergic symptoms.

Morphological identification of Skrjabinisakis Mozgovoi, 1951 (Nematoda: Anisakidae) in Kogia sima (Cetacea: Kogiidae) from Brazilian waters.

New morphological, morphometric and scanning electron microscopy data of a nematode of the family Anisakidae, recovered from a specimen of Kogia sima, a cetacean that died off the northern coast of Brazil, are presented in this paper. Morphological features such as the violin-shaped ventricle and short and equal spicules, as well as the distribution of post-cloacal papillae and specificity for the definitive host (Kogiidae cetaceans) demonstrate similarity to Skrjabinisakis paggiae. This research records Kogia sima and S. paggiae on the estuarine coast of Pará, northern Brazil.

Zoonotic Marine Nematode Infection of Fish Products in Landlocked Country, Slovakia.

Fish products in Slovakia have been heavily infected with Anisakis spp. larvae, which causes human anisakiasis. We found larvae in all tested samples of frozen Atlantic herring. Anisakid allergen t-Ani s7 testing revealed 2 positive cases in humans, signaling need for health authorities to closely monitor zoonotic marine parasites, even in inland areas.

Anisakiasis in the Upper Esophagus: A Case Report.

Anisakiasis is caused by consuming raw fish contaminated with Anisakis sp. larvae and is extremely rare, especially when originating in the esophagus. We present a case of esophageal anisakiasis in a 61-year-old male who experienced severe precordial pain and radiating discomfort to the neck after consuming raw fish sashimi. Upper gastrointestinal endoscopy revealed the presence of a larva in the upper esophagus. On the basis of anatomo-morphological features, the worm was provisionally identified as Anisakis sp. and was easily extracted with forceps, which led to a prompt improvement in the patient's symptoms. This case highlights the importance of considering anisakiasis as a differential diagnosis in patients with gastrointestinal symptoms and a history of consuming raw fish.

Does Anisakis spp. infestation affect the proximate composition, fatty acids, and minerals contents of its host Merluccius merlucccius?

Fish play an important role in human nutrition. They are not only a great source of protein and healthy fats, but also a unique source of essential nutrients such as omega-3 fatty acids. Moreover, most fish are parasitized, and some of these parasites are able to influence the reallocation of resources in their favor and thus reduce the nutritional quality of the fish. The present study was conducted to investigate the impact of the third stage larvae (L3) of Anisakis spp. on the proximate composition, macro-minerals (potassium, calcium, and sodium), and fatty acids of European hake (Merluccius merluccius Linnaeus, 1758). In parasitized female group, our results revealed a decrease (p< 0.005) in the amount of carbohydrate by 6.5%, of calcium by 17%, and of 2 polyunsaturated fatty acids (arachidonic acid (C20: 4w-6), and eicosapentaenoic acid (C20: 5w-3) with 33% and 15% respectively. Simultaneously, an increase by 25% in the level of a single saturated fatty acid C10:0 was noticed. According to the principal component analysis, the parasitized female was wealthy of saturated fatty acids and monounsaturated fatty acids and contains less of polyunsaturated fatty acids, omega-3 fatty acids, and omega-6 fatty acids than the unparasitized female and male. No significant changes were observed in the biochemical composition of male hake, probably due to the low mean intensity of L3 larvae of Anisakis spp. in this group.

Infection levels of Gnathostomatidae (Nematoda) larvae in commercial fishes in north-eastern Australian waters and related food safety concerns.

The majority of research on the safety of marine edible fish has primarily focused on anisakid nematodes, neglecting the potential risks posed by other parasites, including those belonging to the family Gnathostomatidae. In Australia, there have been few reported cases of human infections with gnathostomatid parasites since 2011. However, due to the absence of a standardized diagnostic test in the country, it is believed that the actual number of infections is higher than reported. This study aimed to assess the occurrence and prevalence of infectious gnathostomatid parasites in selected commercial fish species in Australia. A total of 1947 marine fish from northern Australia, representing 9 families, 16 genera, and 30 species, were examined for gnathostomatid nematode infections. Overall, 12.3 % of the fish were found to be infected with at least one gnathostomatid larva. Among the species examined, the yellow-dabbled flounder (Branchypleura novaezeelandiae) exhibited the highest prevalence (83.3 %; n = 6) and the largest number of gnathostomatid larvae. The identification of the gnathostomatid larvae was confirmed as belonging to the genus Echinocephalus based on both morphological characteristics and sequence data. No significant correlation was observed between the prevalence, mean abundance, and mean intensity of infection with the length or weight of the examined fish species. Notably, several of the infected fish species are considered popular choices in the Australian market. Hence, it is imperative to raise awareness among relevant food safety authorities regarding the occurrence of these parasites. The findings from this study should be taken into consideration for the revision of current seafood safety protocols in the country.

Prevalence of Anisakis larvae in cultured mackerel Scomber japonicas in Japan and the relationship between the intensity of Anisakis infection in cultured mackerel and fish fatness.

Incidences of food poisoning caused by Anisakis have increased in Japan, and a significant number of anisakiasis cases in Tokyo attributed to the consumption of mackerel (Scomber japonicus) have been reported. There are two types of cultured mackerel in Japan: those cultured fully from controlled parent fish eggs and those cultured from wild juveniles collected from the sea. In this study, we aimed to investigate the prevalence of Anisakis larvae in cultured mackerel (184 fish) in 15 products and identified the species using molecular analysis to evaluate the risk of food poisoning. In total, 1567 Anisakis larvae were detected in 70 of 130 mackerel in 10 products; however, Anisakis larvae were not detected in 54 mackerel using artificially reared juveniles in 5 products. Moreover, 277 larvae were detected in fish muscle, and 98.6 % (273/277 larvae) were molecularly identified as Anisakis simplex sensu stricto (A. simplex). Conversely, 1043 Anisakis pegreffii larvae were identified genetically and/or morphologically but only 2 larvae were identified in the muscle. There was no significant relationship between the host coefficient of fatness and the infection intensity of Anisakis larvae in individual fish (Spearman's rank correlation coefficient test, P > 0.05). Based on the results of the analysis of the cytochrome c oxidase subunit2 (cox2) gene of A. simplex and A. pegreffii detected in this study, we attempted to estimate the catch area of the juveniles (Pacific stock and Tsushima Warm Current stock). The clusters on the phylogenetic tree of the cox2 gene of A. pegreffii from the mackerel presumed to be the two above mentioned geographic distributions were not separated and these geographic origins could not be estimated. This study revealed that mackerel cultured using wild juveniles are likely to be contaminated with Anisakis larvae, which can be detected not only in the visceral organs, but also in the muscle. Anisakis infection in cultured mackerel did not influence fish growth and evaluating the intensity of Anisakis based on the fatness level of the mackerel was complicated. To prevent anisakiasis caused by the consumption of mackerel cultured using wild juveniles, it is important to steadily control Anisakis through heating and freezing.

Phylogenetic and phylogeographic analyses of Anisakis simplex sensu stricto (Nematoda: Anisakidae) from the common minke whale in Korean waters.

The genus Anisakis is among the most significant parasites to public health, as it causes anisakiasis, a parasitic infection in humans resulting from consuming raw or undercooked seafood. Although the infection status of i>Anisakis in second intermediate hosts, such as marine fishes and cephalopods, and humans have been severally reported in Korea, no information about the definitive host in Korean waters is available. In 2014, 2 adult gastric nematodes were collected from a common minke whale (Balaenoptera acutorostrata) found in the East Sea, Korea. These worms were identified as A. simplex sensu stricto (s.s.) by comparing the mitochondrial COX2 marker with previously deposited sequences. Phylogenetic and phylogeographic analyses of A. simplex (s.s.) worldwide revealed 2 distinct populations: the Pacific population and the European waters population. This is the first report on adult i>Anisakis and its definitive host species in Korea. Further studies on Anisakis infection in other cetacean species and marine mammals in Korean seas are warrantedi>Anisakis.