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.

[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.

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.

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.

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.

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.

Prevalence and abundance of Anisakis larvae in ready-to-eat mackerel products in Japan.

The risk of contracting anisakiasis from consuming ready-to-eat (RTE) mackerel products in Japan was investigated by examining the prevalence and abundance of Anisakis simplex and its sibling species in these products. From 2019 to 2021, a total of 448 RTE mackerel products were purchased in Japan. Anisakis larvae were isolated from 244 of the 448 samples (54 %), and live larvae were isolated from 161 of the 448 samples (36 %). In total, 3170 Anisakis larvae, which included 919 live larvae, were isolated. The isolated Anisakis larvae consisted of 3118 A. simplex (s. s.), 27 A. pegreffii, and 25 hybrid genotype (A. simplex [s. s.] × A. pegreffii) larvae. No A. berlandi larvae were isolated. The prevalence of larvae in samples of mackerel caught in the Southern Japan region and Sea of Japan was much lower than that in mackerel caught in other areas. Both the prevalence of Anisakis larvae in all samples and their abundance in larvae-positive samples exhibited specific seasonal variations, being high in spring.

[Prevalence of Anisakise infections in marine fishes in Eastern Fujian Fishing Ground of Fujian Province].

OBJECTIVE: To investigate the prevalence of Anisakis infection in marine fishes in Eastern Fujian Fishing Ground of Fujian Province, so as to provide insights into the development of the anisakiasis control strategy. METHODS: Marine fish samples were randomly collected from Jiaocheng District, Fuding City and Xiapu County around Eastern Fujian Fishing Ground in Fujian Province from September to December, 2021. All fishes were dissected, and the abdominal contents were collected. Parasites were sampled under a stereomicroscope and the Anisakis species were identified through morphology. The prevalence and intensity of Anisakis infections were calculated. RESULTS: A total of 177 marine fishes belonging to 24 species were dissected, and Anisakis was detected in 73 marine fishes (41.2%) belonging to 16 species (66.7%), with a mean infection intensity of 14.3 parasites per fish. High prevalence of Anisakis infection was found in Ilisha elongata (5/5), Miichthys miiuy (3/3), Plectorhynchus cinctus (2/2), Scomberomorus niphonius (12/13), Trichiurus lepturus (19/23), Pennahia argentata (6/11) and Ditrema temmincki (14/27), with mean infection intensities of 9.2, 2.7, 4.5, 10.9, 39.2, 4.5 parasites per fish and 2.1 parasites per fish. The Anisakis larvae were characterized as Anisakis and Hysterothylacium. CONCLUSIONS: High prevalence of Anisakis infection is detected in marine fishes in Eastern Fujian Fishing Ground of Fujian Province. The health education pertaining to food health is required to be reinforced to prevent the development of human anisakiasis.

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.

Morphological and molecular identification of third-stage larvae of Anisakis typica (Nematoda: Anisakidae) from Red Sea coral trout, Plectropomus areolatus.

Anisakidosis is a foodborne zoonotic infection induced by members of the family Anisakidae via the consumption of raw or undercooked fish such as sushi and sashimi. Identifying anisakid larval species is critical for the epidemiology and diagnosis of diseases caused by them. This study aimed at identifying Anisakis larvae collected from marine fish in Egyptian waters based on morphological characteristics and molecular analysis. Thirty marine fish coral trout, Plectropomus areolatus, were collected from Hurghada, Red Sea, Egypt, to investigate larval nematodes of the genus Anisakis. The larvae were detected encapsulated in the peritoneal cavity and muscle of the fish host. This examination revealed that anisakid larvae naturally infected 19 fish specimens with a prevalence of 63.33% and a mean intensity of 4.1 ± 0.40. Most of them (68 larvae: 71.57%) were found in the musculature. Morphological and morphometric analyses using light and scanning electron microscopy revealed a head region with a prominent boring tooth, inconspicuous lips, and a characteristic protruded cylindrical mucron. All larvae in this study possessed the same morphology as Anisakis Larval type I. Molecular analysis based on ITS region using maximum likelihood and Bayesian phylogenetic methods confirmed them as Anisakis typica. This is the first study to identify A. typica larvae from the commercial fish coral trout P. areolatus in Egyptian waters using morphological and molecular methods.

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.

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.

Anisakidae parasites in frozen fish fillets intended for human consumption. / Parásitos anisákidos en filetes de pescado congelado destinados a consumo humano

INTRODUCTION: Anisakiasis is a human parasitic disease caused by the consumption of raw fish or shellfish containing larvae of the Anisakidae family. It is currently considered an emerging disease of public health interest. OBJECTIVE: To identify the presence of larvae of the Anisakidae family in samples of frozen raw fish fillets intended for human consumption in markets in Medellín and its metropolitan area in Antioquia, Colombia. MATERIALS AND METHODS: A cross-sectional study was carried out, in which larvae of the Anisakidae family were detected and identified in frozen raw fish fillets from three representative markets in Medellín and its metropolitan area. A total of 384 ready for consumption fillets were analyzed (197 sawfish, 137 salmon, 37 tuna, and 13 hake), using the pressing and ultraviolet light method. Taxonomic keys were used to identify the collected parasites and to establish its genus. Conventional PCR and Sanger sequencing was performed to determine the species. RESULTS: Four larvae were found in 4 of the 384 (1.04%) fillets (CI95% 1.04 ± 1.01%). The species of fish in which the larvae were found was sawfish (Scomberomorus spp.) and the genus and species of the larvae was established as Anisakis pegreffii. CONCLUSIONS: According to the study, the presence of Anisakis parasites in frozen raw fish fillets in the influence area is evident.

Introducción. La anisakiasis es una infección producida por parásitos de la familia Anisakidae, transmitida a los humanos por el consumo de pescado o mariscos crudos. En la actualidad, se considera una enfermedad emergente de interés en salud pública. Objetivo. Identificar la presencia de larvas de la familia Anisakidae en muestras de filetes de pescado crudo congelado destinados a consumo humano, en mercados de Medellín y su área metropolitana en Antioquia (Colombia). Materiales y métodos. Se realizó un estudio transversal, en el cual se buscó la presencia de larvas de la familia Anisakidae en filetes de pescado crudo congelado de tres mercados representativos de Medellín y su área metropolitana. Se analizaron 384 filetes listos para el consumo (197 sierras, 137 salmones, 37 atunes y 13 merluzas). Cada filete fue analizado mediante el método de prensado y luz ultravioleta. Los parásitos recolectados se identificaron a partir de claves taxonómicas para establecer el género, así como PCR convencional y posterior secuenciación Sanger, para determinar la especie. Resultados. Se encontraron 4 larvas en 4 de los 384 filetes (1,04 %) (IC95% 1,04 ± 1,01 %). Las larvas encontradas fueron identificadas como Anisakis pegreffi y el tipo de pescado en el cual se encontraron fue la sierra (Scomberomorus spp.). Conclusiones. De acuerdo con el estudio realizado, se evidencia la presencia de parásitos anisákidos en filetes de pescado crudo congelado en el área de influencia.

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%.

Anisakidosis en peces frescos comercializados en tres ciudades de la Región del Bío Bío, Chile / Anisakidosis in fresh fish commercialized in three cities of the Bío Bío Region, Chile

INTRODUCCIÓN: La anisakidosis humana es una zoonosis transmitida por alimentos, causada por la ingestión de carne de pescado parasitada por nematodos anisákidos. Investigaciones sobre la presencia de anisákidos en pescados comercializados, sin cocción o congelación previa, en la región centro sur de Chile son escasas. OBJETIVO: Identificar nematodos anisákidos en catorce especies de peces marinos, obtenidos por pesca artesanal y comercializados en Concepción, Talcahuano y Lebu, ciudades de la Región del Bío Bío, Chile. MÉTODO: Entre los años 2018 y 2020, se examinó en busca de anisákidos a 334 pescados marinos frescos. RESULTADOS: Se identificó larvas de Anisakis spp. y Pseudoterranova sp. en ejemplares de siete y cuatro especies de peces, respectivamente. La mayoría de las larvas se encontraron vivas y ubicadas a nivel visceral. CONCLUSIÓN: La mayoría de las especies de peces que resultaron positivas son reconocidos hospederos intermediarios de estos parásitos. La presencia de anisákidos en la musculatura de estas especies indica que su preparación, en forma ahumada o cruda, es un riesgo para salud pública, recomendándose comprar estos productos en forma eviscerada y/o congelada, junto a promover su cocción.

BACKGROUND: Human anisakidosis is a food-borne zoonosis, caused by the intake of fish meat parasitized by anisakid nematodes. Research of anisakids parasites in commercialized fish, without previous cooking or freezing, in the south central region of Chile is scarce. AIM: To identify anisakid nematodes in fourteen species of marine fish, obtained by artisanal fishing and commercialized in Concepción, Talcahuano and Lebu, cities from Bío Bío Region, Chile. METHODS: During 2018 to 2020, we examinated for anisakids to 334 fresh marine fishes. RESULTS: Larvae of Anisakis spp. and Pseudoterranova sp. were identified in specimens of seven and four fish species, respectively. Most of the larvae were found alive and located at the visceral level. CONCLUSION: Most of the positive fish species are recognized intermediate hosts for these parasites. The presence of anisakids in the musculatura of these species indicates that their preparation, in smoked or raw form, is a risk to public health, and it is recommended to buy these products in gutted and / or frozen form, together with promoting their cooking.

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.

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.

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.

Analysis of potential drivers of spatial and temporal changes in anisakid larvae infection levels in European hake, Merluccius merluccius (L.), from the North-East Atlantic fishing grounds.

We analysed the spatial and temporal variability of Anisakis larvae infection in hake (Merluccius merluccius) from the North-East Atlantic from 1998 to 2020 and the potential drivers (i.e., environmental and host abundance) of such variation. The results showed that hake from separate sea areas in the North Atlantic have marked differences in temporal abundance levels. Hake larger than 60 cm were all parasitized in all ICES (International Council for the Exploration of the Sea) subareas 6, 7, and 8. The belly flaps were the most parasitized parts of the flesh, accounting for 92% of the total. Individuals of Anisakis simplex, Anisakis pegreffii, Anisakis spp. and a hybrid of Anisakis simplex × pegreffii were genetically identified, and Anisakis simplex as the most abundant (88-100%). An ecological niche model of Anisakis occurrence in fishes in the NE Atlantic was built to define the thermal optimum and environmental ranges for salinity, depth, chlorophyll concentration, and diffuse attenuation. The temporal variability of anisakid infection in fishes in the last two decades indicated an increase in the NE Atlantic at an annual rate of 31.7 nematodes per total number of specimens examined per year. This rise in infection levels could be triggered by the increase in intermediate host fish stocks, especially hake in the area.