Anisakis L3 Research Articles

Occurrence of Anisakis larvae in Atlantic horse mackerel (Trachurus trachurus) sold in the city of Marrakesh, Morocco

Human anisakidosis is a parasitic infection caused by the ingestion of nematode larvae of the Anisakidae family contained in various organs of fish and cephalopods consumed raw or undercooked. The aim of this work was to detect and identify morphologically and genetically (PCR and sequencing) the nematode larvae present in both flesh and abdominal cavity of horse mackerel (Trachurus trachurus) which are widely consumed by the population of Marrakesh. For this purpose, 299 fish were purchased at the wholesale market in refrigerated conditions. They were originated from seven different landing ports along the Atlantic coast (Agadir, Dakhla, El Jadida, Essaouira, Mohammedia, Safi and Sidi Ifni). The fishes were analyzed from November 2017 to December 2019, and 4815 larvae were detected out of 294 positive horse mackerel. This resulted in a prevalence of 98.33% and a mean intensity of 16.38 larvae per fish. Horse mackerel from Mohammedia, El Jadida, Safi, Essaouira, Agadir and Sidi Ifni were more infested than those from Dakhla with a mean intensity of 25.29, 21.33, 18.88, 16.15, 15.90 and 13.86 larvae per fish, respectively, while that from Dakhla was the least infested with a mean intensity of 11.83. These results could be explained by the availability of primary intermediate hosts of Anisakis spp., such as crustaceans (shrimps), which are more abundant in the northern Moroccan Atlantic coast. Furthermore, we have recorded a higher infection in fish during warm seasons (spring and summer) than during cold ones. We also have noticed that as the distance from the fishing area increased, the larvae had more time to migrate from the abdominal cavity to the muscles. All detected larvae were identified morphologically as Anisakis type I and genetically as Anisakis simplex sensu stricto. Statistical analysis showed that the intensity of infection by Anisakis L3 larvae was positively correlated with the weight of horse mackerel and that origin had no impact on fish parasitism.

Modeling survival curves of Anisakis L3 after isothermal heat treatments at lethal temperatures

Even though anisakiasis is considered, nowadays, a significant threat to public health all over the world, no attempt has been made up to date to mathematically describe the thermal susceptibility of Anisakis larvae in the third stage (L3). To fill this gap, in this paper, more than 10,000 free (non-encysted) Anisakis L3 were individually heat treated in a thermal cycler at temperatures between 44 °C and 61 °C for different exposure times. After heat exposition, viability was assessed in each larva, survival curves at isothermal conditions were derived, and the effectiveness of four kinetic models (fundamental kinetic model, Mafart model, and probit and logit models) in describing these curves was tested. Evaluation of larvae viability after heat exposition revealed sigmoidal survival curves that increased their steepness with temperature. Of the four models tested, the Mafart model was the one that best fitted the data only differing from the observed survival ratios by 0.12 units on average. Validation experiments performed at temperatures different to those used to create the model corroborated its predictive capacity. Future efforts should be focused in predicting larvae viability at non-isothermal conditions as those occurring during fish cooking.

Inflammatory Response in Caco-2 Cells Stimulated with Anisakis Messengers of Pathogenicity.

Background: Anisakis spp. third-stage larvae (L3) are the causative agents of human zoonosis called anisakiasis. The accidental ingestion of L3 can cause acute and chronic inflammation at the gastric, intestinal, or ectopic levels. Despite its relevance in public health, studies on pathogenetic mechanisms and parasite-human interplay are scarce. The aim of this study was to investigate the human inflammatory response to different Anisakis vehicles of pathogenicity. Methods: Human colorectal adenocarcinoma (Caco-2) cells were exposed to Anisakis L3 (the initial contact with the host), extracellular vesicles (EVs, Anisakis–host communication), and crude extract (CE, the larval dying). The protein quantity and gene expression of two pro-inflammatory cytokines (IL-6 and IL-8) were investigated using an ELISA test (6 h and 24 h) and a qReal-Time PCR (1 h, 6 h, and 24 h), respectively. Results: The L3 and EVs induced a downregulation in both the Il-6 and Il-8 gene expression and protein quantity. On the contrary, the CE stimulated IL-6 gene expression and its protein release, not affecting IL-8. Conclusions: The Caco-2 cells seemed to not react to the exposure to the L3 and EVs, suggesting a parasite’s immunomodulating action to remain alive in an inhospitable niche. Conversely, the dying larva (CE) could induce strong activation of the immune strategy of the host that, in vivo, would lead to parasite expulsion, eosinophilia, and/or granuloma formation.

Chemical Composition, Cytotoxicity and Larvicidal Activity of Essential Oils of Three Medicinal Plants of Ethiopian Flora Against Anisakis L3 Larvae

Background and objectives: Uvaria scheffleri Diels (Annonaceae), Zanthoxylum chalybeum Engl. (Rutaceae), and Vepris dainelli (Pichi-Serm.) Kokwaro (Rutaceae) are medicinal plants traditionally used in Ethiopia against pathogenic infections. In the present study, the chemical composition, larvicidal activity, and cytotoxic effect of essential oils were investigated. Methods: Hydrodistillation technique was used to extract essential oils. In vitro larvicidal activity against Anisakis L3 larvae was tested in marinated solutions. MTT assay was used to assess the cytotoxicity. Results: The yields (v/w) of essential oils obtained from U. scheffleri roots, Z. chalybeum, and V. dainelli fruits were 0.5, 2.7, and 2.0 %, respectively. Gas chromatography-mass spectrometry analysis of essential oils revealed a total of 58, 18, and 20 chemical constituents, representing 97.6, 99.6, and 98.8 % of the oil contents, respectively. Tricyclo [5.3.0.0(3, 9)] decane was identified to be the principal constituent in the essential oils of Z. chalybeum (82.8%) and V. dainelli (69.8%), reported herein for the first time. Essential oils of Z. chalybeum, U. scheffleri, and V. dainelli displayed a dose-dependent larvicidal activity with LT100 values of 3 h, 5 h, and 5 h for 5% concentrations, respectively. The cytotoxicity study of essential oils on VERO cells showed moderate toxicity with IC50 values of 65.46 µg/mL, 83.88 µg/mL, and 96.82 µg/mL, respectively. Conclusion: The results obtained revealed that the studied essential oils could serve as larvicidal agents in treating human anisakidosis. The observed weak cytotoxicity at low concentrations points out the possibility of developing effective and safe botanical larvicides.

Thermal patterns of heat treated Anisakis L3-infected fishery products allow separation into low, intermediate and high risk groups of potential use in risk management

Anisakis third-stage larvae (L3) is moderately tolerant to heat and, to mitigate the risk of live L3 intake in cooked seafood, it is important to define with precision at which point after heat treatment the parasite is no longer infective. We aimed to find thermal patterns that allowed to classify fish sandwiches spiked with Anisakis L3 into “low” (100% probability of mortality), “intermediate” and “high” risk groups. For that, experiments with varying set temperatures and heating times have been performed in conditions of different external heating temperatures. Decision points to classify the samples into terminal nodes associated to different risk groups have been obtained with decision tree analyses and then confirmed with linear discriminant analysis. Separation into two (i.e. low vs high + intermediate risk) or three (i.e. low, intermediate, and high risk) distinct thermal patterns (98% and 95.9% correct classifications by cross-validation respectively) was achieved. These results refine heating conditions reported in the EU Regulation, since reaching 60 °C for 1 min in the thermal centre is not sufficient to kill all L3. However, when factors such as relative temperature of heating or time to reach the set temperature are taken into account, other thermal conditions are found that are equally safe in terms of Anisakis L3 inactivation. This, together with the description of “intermediate” and “high” risk groups can help in the risk identification and management, as well as in providing clearer recommendations to consumers.

Freezing kinetic parameters influence allergenic and infective potential of Anisakis simplex L3 present in fish muscle

The effect of freezing rate, temperature and storage time of Anisakis L3 infecting fish mince in controlled conditions were studied in terms of larval viability and parasite antigen release. In order to determine the potential infectivity of larvae that might accidentally survive a freezing process, their agar penetration ability, survival in artificial gastric juice, and respiratory control were analysed in infected hake muscle in conditions that produced various survival rates. For that a series of experiments was conducted with 10–30 L3 per 75 g of mince, frozen and stored to provide a variety of freezing kinetic conditions and storage times. The results highlight the importance of a number of kinetic parameters in the survival of Anisakis larvae, not only final temperatures or storage times. High freezing rates, preferred in order to maintain optimum fish eating quality, are able to kill Anisakis larvae faster, but may cause a higher release of antigens to the surrounding medium. The characteristics of the surviving L3 were impaired as compared to the controls. No relationship between the latter and the percentage of viability due to different freezing conditions was found, which suggest that any larva which shows any sign of movement after freezing should be treated as equally infective.

Evaluation of the effects of weak oscillating magnetic fields applied during freezing on systems of different complexity

Abstract The effects of weak oscillating magnetic fields (OMFs ≤7 mT at 50 Hz) on freezing were studied in three systems of different complexity. To do so, ferric chloride solutions, lactate dehydrogenase (LDH), and minced hake muscle experimentally infected with Anisakis L3 were frozen with and without OMF application. OMFs did not affect freezing kinetics of either ferric chloride solutions or minced hake muscle. LDH activity, Anisakis mortality, and water-holding capacity of the hake muscle after thawing were not affected by OMF either. Further studies are needed to evaluate the effectiveness of stronger OMFs in a wider frequency range.

Pathogenic potential of Anisakis L3 after freezing in domestic freezers

Anisakis L3 were subjected to freezing in domestic freezers and the risk posed by those larvae that might possibly survive freezing was analysed by determining their agar penetration ability, survival in gastric juice, allergenic potential and oxygen consumption rate. Anisakis L3 gradually became non-viable as the temperature fell from −1 °C to −28 °C. The faster the freezing rate, the greater the decrease in survival, but viability was not found to be affected by the proportions of Anisakis species per batch (A. simplex s.s. 83–100%, A. pegreffii 0–17%, and their heterozygote genotypes at the ITS region of rDNA 0–10%). Surviving larvae after freezing presented a drastically reduced ability to penetrate into a layer of solid agar, as compared to controls, but still 5% of them penetrated into this medium. About 80% of surviving larvae died within 24 h under conditions simulating those of gastric fluid, whereas up to 85% of the controls were able to resist for 24 h. Allergen release during freezing and after freezing was higher than in controls, and this release was compatible with a contribution of both passive (by rupture of the cuticle) and active liberation of excretion/secretion allergens during and after freezing, suggesting that the pathogenic potential of these frozen and thawed larvae cannot be discarded. These results could be of use for more precise risk assessment and providing guidelines for consumers.

Occurrence and prevalence of fish-borne Anisakis larvae in the spotted mackerel Scomber australasicus from Taiwanese waters.

Anisakid nematodes have been found in a variety of marine fishes throughout the world and they are known to cause anisakiasis in human hosts. The present study investigated the prevalence of potentially zoonotic anisakid larvae in spotted mackerel caught from Taiwanese waters where fish represents an important food sources. Anisakis third-stage larvae (L3, n=502) were isolated from 250 spotted mackerel Scomber australasicus. Anisakis L3 larvae were divided morphologically into two types, Anisakis type I larvae had a longer ventriculus and mucron while type II larvae had a shorter ventriculus and no mucron. Anisakis species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer (ITS) regions of ribosomal DNA and direct sequencing. A simple molecular taxonomic key, utilizing RFLP by two restriction enzymes HinfI and HhaI, enabled the differentiation of the genus Anisakis. The prevalence, mean intensity and mean abundance of Anisakis nematodes recorded for the total specimens were 72.8%, 2.8 (1-15) and 2.0 (0-15), respectively. Anisakis pegreffii was determined to be the dominant species (prevalence=57.2%) and important agent of human anisakiasis. A recombinant genotype (Anisakis simplex sensu stricto × A. pegreffii) was identified as the subdominant species (25.3%) followed by Anisakis typica (10%), Anisakis physeteris (4.0%), Anisakis paggiae (3.0%) and Anisakis brevispiculata (0.5%). The topology of the maximum likelihood and neighbor-joining trees show two well supported clades: one includes the species of A. pegreffii and the other includes A. paggiae, A. physeteris and A. brevispiculata, while A. typica has basal position to all other Anisakis spp. analyzed. This study advances our knowledge of the prevalence of different Anisakis spp. in the spotted mackerel from Taiwanese waters, which is helpful for monitoring the fish populations throughout a diverse array of aquatic ecosystems. More importantly, we provide the concise characterization of multiple Anisakis spp. by PCR-RFLP, which could also be applicable for the rapid diagnosis of human anisakiasis.

Molecular analysis of Anisakis type I larvae in marine fish from three different sea areas in Korea.

Anisakiasis, a human infection of Anisakis L3 larvae, is one of the common foodborne parasitic diseases in Korea. Studies on the identification of anisakid larvae have been performed in the country, but most of them have been focused on morphological identification of the larvae. In this study, we analyzed the molecular characteristics of 174 Anisakis type I larvae collected from 10 species of fish caught in 3 different sea areas in Korea. PCR-RFLP and sequence analyses of rDNA ITS and mtDNA cox1 revealed that the larvae showed interesting distribution patterns depending on fish species and geographical locations. Anisakis pegreffii was predominant in fish from the Yellow Sea and the South Sea. Meanwhile, both A. pegreffii and A. simplex sensu stricto (A. simplex s.str.) larvae were identified in fish from the East Sea, depending on fish species infected. These results suggested that A. pegreffii was primarily distributed in a diverse species of fish in 3 sea areas around Korea, but A. simplex s.str. was dominantly identified in Oncorhynchus spp. in the East Sea.

Activity of Matricaria chamomilla essential oil against anisakiasis

The increase in diagnosed cases of anisakiasis and the virtual absence of effective treatments have prompted the search for new active compounds against Anisakis L3 larvae. The biocidal efficacy against different pathogens shown by various essential oils (EO) led us to study the Matricaria chamomilla EO and two of its main components (chamazulene and α-bisabolol) against the L3 larvae of Anisakis type I. The activity of M. chamomilla EO, chamazulene and α-bisabolol was established by in vitro and in vivo experiments. The EO (125μg/ml) caused the death of all nematodes, which showed cuticle changes and intestinal wall rupture. In the in vivo assays, only 2.2%±1.8 of infected rats treated with M. chamomilla EO showed gastric wall lesions in comparison to 93.3%±3.9 of control. Chamazulene was ineffective, while α-bisabolol showed a high activity to that of the EO in vitro tests but proved less active in vivo. These findings suggest that the larvicidal activity may result from the synergistic action of different compounds of M. chamomilla EO. Neither of the tested products induces irritative damage in the intestinal tissues. In conclusion, M. chamomilla EO is a good candidate for further investigation as a biocidal agent against Anisakis type I.

Morphological and molecular characterization of Anisakis larvae (Nematoda: Anisakidae) in Beryx splendens from Japanese waters

The third-stage (L3) larvae of Anisakis, which are the etiological agents of human anisakiasis, have been categorized morphologically into Anisakis Type I larvae and Anisakis Type II larvae. Genetic analysis has allowed easy identification of these larvae: Anisakis Type I larvae include the species Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis simplex C, Anisakis typica, Anisakis ziphidarum, and Anisakis nascettii, whereas Anisakis Type II larvae include the species Anisakis physeteris, Anisakis brevispiculata, and Anisakis paggiae. Since human consumption of raw fish and squid is common in Japan, we investigated Anisakis L3 larvae in 44 specimens of Beryx splendens from Japanese waters. A total of 730 Anisakis L3 larvae collected from B. splendens were divided morphologically into 4 types: Type I, Type II, and 2 other types that were similar to Anisakis Type III and Type IV described by Shiraki (1974). Anisakis Type II, Type III, and Type IV larvae all had a short ventriculus, but their tails were morphologically different. In addition, data from genetic analysis indicated that Anisakis Type II, Type III, and Type IV larvae could be identified as A. physeteris, A. brevispiculata, and A. paggiae, respectively. Therefore, A. physeteris, A. brevispiculata, and A. paggiae can be readily differentiated not only by genetic analysis but also by morphological characteristics of L3 larvae.

A study of the infection and physicochemical characteristics of the marine fish infected by Anisakis L₃ caught in Zhoushan Fishery

The aim of this research was to investigate the third phase Anisakis simplex larvae (Anisakis L₃) infection in marine fish caught in Zhoushan Fishery and to find out its physicochemical and biological characteristics. A total of 444 fish belonging to 29 species were dissected to isolate anisakis larvae which were then morphologically identified. The survival tolerance of Anisakis L₃ were observed in various conditions, such as in different temperature and medium. A total of 218 fish from 21 species were infected by Anisakis simplex larvae, yielding an overall infection rate of 49.10% (218/444). Trichiurus haumela, pneumatophorus japonicus, miichthys miiuy, argyrosomus argentatus and anguilliformes had high infection rate and had an average infection intensity of 15.28 per fish. 3332 Anisakis larvae were detected in 218 fish, among which Anisakis L₃ and Pseudoterranova larvae accounted for 99.46% (3314/3332) and 0.54% (18/3332) respectively. Anisakis L₃ was highly resistant to common condiment. We found the liquor with high concentration of alcohol showed better insecticidal effect than that with low concentration of alcohol (t = 4.105, P < 0.05) and low concentration mebendazole composite was not only more effective than high concentration mebendazole composite (F = 45.198, P < 0.01) but also more effective than other drugs, such as albendazole and mebendazole. Anisakis L₃ could live up to 9 h and 12 h at -20°C, -10°C respectively, however they were very sensitive to high temperature. It has been shown that they could only survive for less than 11 s and 1 s at 50°C and 60°C respectively. The observed Anisakis L₃ infection rate in the marine fish found in Zhoushan Fishery was very high. Anisakis L₃ showed high resistance to low temperature but not to high temperature.

Parasitic infection levels by &lt;em&gt;Anisakis&lt;/em&gt; spp. larvae (Nematoda: Anisakidae) in the black scabbardfish &lt;em&gt;Aphanopus carbo&lt;/em&gt; (Osteichthyes: Trichiuridae) from Portuguese waters

Aphanopus carbo, black scabbardfish, specimens from 3 Portuguese regions (the mainland, Madeira and the Azores) during four (mainland and Madeira) or two (Azores) seasons of the year were examined for the presence of Anisakis. All the fish examined (n=287) were infected with Anisakis L3 larvae (prevalence = 100%). Significant geographical differences in intensity were found: the Azores showed the lowest mean intensity value (53.7) and Madeira the highest one (253.9). The intensity of infection was positively correlated with the host length in specimens of Sesimbra and Madeira. Significant seasonal differences in intensity were found in the studied regions. The very high values in prevalence and intensity strongly suggest that the consumption of raw or undercooked black scabbard fish is a potential risk for human health.

Atlantic codGadus morhuaL.: Visceral organ topography and the asymetrical distribution of larval ascaridoid nematodes in the musculature

Abstract Visceral organ topography (VOT) in marine teleosts has been defined as the “spatial arrangement and contiguity of organs within the body cavity”, and it has been suggested that VOT may influence the habitat(s) occupied by the third-stage larvae (L3) of ascaridoid nematodes. Reports of an asymmetrical distribution of Anisakis (“whaleworm”) L3 and Pseudoterranova (“sealworm”) L3 in Atlantic cod Gadus morhuaL. prompted studies of VOT in this host. The left liver lobe dominates the left sagittal half of the body cavity and is largely contiguous with the stomach wall. Examination of ã 630 cod confirmed that more L3 occur in the left than in the right body musculature. Two hypotheses discussed in relation to the asymmetry both pertain to the liver. Feeding and growth of larval ascaridoids in fish are discussed briefly.