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

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

[Investigation on the risk of of Anisakis infection among high - risk populations along the coastal areas of Jiangsu Province].

OBJECTIVE: To investigate the risk of Anisakis infections among high-risk populations along the coastal areas of Jiangsu Province, so as to develop the strategy for the prevention and control of anisakiasis in the province. METHODS: Three counties along the coastal areas of Jiangsu Province were selected as the study sites in 2018, including Rudong County in Nantong City, Haizhou District in Lianyungang City and Dongtai City in Yancheng City. The knowledge, attitude and practice (KAP) of anisakiasis prevention and control, and the prevalence of serum specific IgG antibody against Anisakis were investigated among high-risk populations among these three study sites, including fishermen, fish seller and people who liked eating fresh and live marine fish. Factors affecting the prevalence of the specific IgG antibody against Anisakis were identified using a multiple logistic regression model. In addition, Anisakis larvae infections were detected in fresh and live marine fish samples collected from local markets, and the prevalence and intensity of Anisakis infections were estimated. RESULTS: A total of 625 high-risk populations were investigated, including 349 men (55.8%). Only 13.0% of the subjects heard about anisakiasis, and a low awareness rate of anisakiasis prevention and control knowledge was seen among these three types of high-risk populations. There were 21.6% of the subjects eating raw or half-cooked marine fish, 5.8% eating undercooked marine fish, 3.2% presenting vomiting, nausea and diarrhea after eating marine fish, 5.1% developing systemic allergic symptoms, and 65.6% using the same chopping board for raw and cooked food. The sero-prevalence of the anti-Anisakis IgG antibody was 7.0% among the study subjects. Multiple logistic regression analysis identified education level [OR = 0.687, 95% CI (0.478, 0.987)] and development of systemic allergic symptoms [OR = 4.641, 95% CI(1.411, 15.268)]as factors affecting the positive anti-Anisakis IgG antibody among the study subjects. Among 494 fresh and live marine fish detected, the prevalence and intensity of Anisakis larvae infection was 64.0% and 8.1 larvae per fish, with high prevalence seen in Trichiurus haumela and Pneumatophorus japonicas. CONCLUSIONS: The awareness of anisakiasis prevention and control knowledge is low among the high-risk populations living along the coastal areas of Jiangsu Province, and there are high-risk behaviors, such as eating raw or half-cooked food, using the same chopping board for raw and cooked food. In addition, the prevalence of Anisakis infections is high in the marine fish in these areas. Therefore, the health education and health promotion for anisakiasis prevention and control should be intensified.

Multidisciplinary approach in study of the zoonotic Anisakis larval infection in the blue mackerel (Scomber australasicus) and the largehead hairtail (Trichiurus lepturus) in Northern Taiwan.

BACKGROUND: Anisakid larvae are the food-borne pathogen highly prevalent among numerous marine fishes. Accidental consumption of infected raw or poorly cooked fish fillets may cause anisakiasis. METHODS: This study used the multidisciplinary approach to investigate the occurrence of Anisakis nematodes in commonly consumed fish species, Scomber australasicus and Trichiurus lepturus purchased in Taipei Xinyi traditional fish market. RESULTS: All the Anisakis larvae collected herein were identified morphologically as Anisakis type I or Anisakis type II. The prevalence and the mean intensity of Anisakis larvae collected from S. australasicus was 80.77%, 26.8 (10-32) and 100%, 49.0 (27-70) for T. lepturus. Using molecular analysis, 83.33% (180/216) were identified as Anisakis pegreffii, 6.05% (13/216) as Ascaris typica, 1.85% (4/216) as Ascaris physeteris and 8.80% (19/216) as hybrid genotype (A. pegreffii + Anisakis simplex) in S. australasicus. In T. lepturus, 86.31% (290/336) were identified as A. pegreffii, 2.38% (8/336) as A. typica, and 11.31% (38/336) as hybrid genotype (A. pegreffii + A. simplex [s.s]). The molecular phylogenetic analysis shows two cluster clades, one group includes A. pegreffii complex and the other include Ascaris paggiae, Ascaris brevispiculata, and A. physeteris. CONCLUSION: Thus, A. pegreffii is the most abundant species and may be the potential causes of human infection.

Molecular Epidemiology of Anisakis and Anisakiasis: An Ecological and Evolutionary Road Map.

This review addresses the biodiversity, biology, distribution, ecology, epidemiology, and consumer health significance of the so far known species of Anisakis, both in their natural hosts and in human accidental host populations, worldwide. These key aspects of the Anisakis species' biology are highlighted, since we consider them as main driving forces behind which most of the research in this field has been carried out over the past decade. From a public health perspective, the human disease caused by Anisakis species (anisakiasis) appears to be considerably underreported and underestimated in many countries or regions around the globe. Indeed, when considering the importance of marine fish species as part of the everyday diet in many coastal communities around the globe, there still exist significant knowledge gaps as to local epidemiological and ecological drivers of the transmission of Anisakis spp. to humans. We further identify some key knowledge gaps related to Anisakis species epidemiology in both natural and accidental hosts, to be filled in light of new 'omic' technologies yet to be fully developed. Moreover, we suggest that future Anisakis research takes a 'holistic' approach by integrating genetic, ecological, immunobiological, and environmental factors, thus allowing proper assessment of the epidemiology of Anisakis spp. in their natural hosts, in human populations, and in the marine ecosystem, in both space and time.

Validation of a commercial kit aimed to the detection of pathogenic anisakid nematodes in fish products.

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005.

Visceral larvae as a predictive index of the overall level of fish batch infection in European anchovies (Engraulis encrasicolus): A rapid procedure for Food Business Operators to assess marketability.

The European anchovy (Engraulis encrasicolus), one of the most important pelagic fish resources in the Mediterranean Sea, is frequently infected by anisakid larvae. Food Business Operators (FBOs) should use appropriate sampling plans and analytical methods to avoid commercialization of massively infected batches and reduce the risk of transmission of viable zoonotic larvae. In this study, performed at FishLab (Department of Veterinary Sciences of the University of Pisa) during 2016, an official sampling plan was associated with a digestion protocol for the inspection of anchovies. Considering that anisakid larvae are usually located in the fish visceral cavity and in the adjacent muscles (VM), this part was analyzed. In particular, we assessed the reliability of the digestion of a subsample of 150g (±30g) of VM, randomly collected from 29 specimens, in estimating the marketability of the anchovies' batch. Fifty-seven samples of 29 anchovies were collected. Each anchovy was sectioned to separate VM. All the subsamples were digested, and visible larvae counted. A high correlation between the number of larvae in VM regions and in the total batch was observed, indicating a very significant contribution of the VM region on total number of parasites. The Mean Abundance (MA) was used to assess the batch marketability according to a threshold calculated on the basis of the maximum number of nematodes tolerated per sample. Considering that the MA can be calculated only when the number of examined specimens is known, the number of visible Larvae per gram of tissue (LpG) was calculated on 150g (±30g) of VM subsamples. A LpG marketability threshold was calculated dividing the maximum number of tolerated nematodes by the average weight of a sample of 29 anchovies calculated considering data available in literature. To evaluate the diagnostic performance of the LpG threshold, the marketability of 57 batches assessed on the basis of the MA threshold was assumed as the gold standard. The proposed LpG showed very high Specificity and Sensitivity. These findings suggest that the analysis of VM is representative of the overall infestation of the batch, both when considering the absolute number of parasites and the LpG, and may represent a valid alternative to the whole anchovy digestion. In particular, the use of an automated digestive method, coupled with the aforesaid sampling plan, could allow the procedure to be used by FBOs in operational conditions.

Assessing the risk of an emerging zoonosis of worldwide concern: anisakiasis.

Anisakiasis is an emerging zoonosis caused by the fish parasitic nematode Anisakis. Spain appears to have the highest reported incidence in Europe and marinated anchovies are recognised as the main food vehicle. Using data on fishery landings, fish infection rates and consumption habits of the Spanish population from questionnaires, we developed a quantitative risk assessment (QRA) model for the anchovy value chain. Spaniards were estimated to consume on average 0.66 Anisakis per untreated (non-frozen) raw or marinated anchovy meal. A dose-response relationship was generated and the probability of anisakiasis was calculated to be 9.56 × 10-5 per meal, and the number of annual anisakiasis cases requiring medical attention was predicted between 7,700 and 8,320. Monte Carlo simulations estimated post-mortem migration of Anisakis from viscera to flesh increases the disease burden by >1000% whilst an education campaign to freeze anchovy before consumption may reduce cases by 80%. However, most of the questionnaire respondents who ate untreated meals knew how to prevent Anisakis infection. The QRA suggests that previously reported figures of 500 anisakiasis per year in Europe is a considerable underestimate. The QRA tool can be used by policy makers and informs industry, health professionals and consumers about this underdiagnosed zoonosis.

Environmental variables and definitive host distribution: a habitat suitability modelling for endohelminth parasites in the marine realm.

Marine nematodes of the genus Anisakis are common parasites of a wide range of aquatic organisms. Public interest is primarily based on their importance as zoonotic agents of the human Anisakiasis, a severe infection of the gastro-intestinal tract as result of consuming live larvae in insufficiently cooked fish dishes. The diverse nature of external impacts unequally influencing larval and adult stages of marine endohelminth parasites requires the consideration of both abiotic and biotic factors. Whereas abiotic factors are generally more relevant for early life stages and might also be linked to intermediate hosts, definitive hosts are indispensable for a parasite's reproduction. In order to better understand the uneven occurrence of parasites in fish species, we here use the maximum entropy approach (Maxent) to model the habitat suitability for nine Anisakis species accounting for abiotic parameters as well as biotic data (definitive hosts). The modelled habitat suitability reflects the observed distribution quite well for all Anisakis species, however, in some cases, habitat suitability exceeded the known geographical distribution, suggesting a wider distribution than presently recorded. We suggest that integrative modelling combining abiotic and biotic parameters is a valid approach for habitat suitability assessments of Anisakis, and potentially other marine parasite species.

Scavenging hagfish as a transport host of Anisakid nematodes.

Hagfish are the most primitive craniates and scavengers, feeding on dead organisms as well as fish and invertebrates. Hagfish play an important ecological role in recycling nutrients, helping to recycle biomass from the upper water column. We investigated 265 specimens of four hagfish species, including Eptatretus burgeri, Eptatretus yangi, Eptatretus sheni and Eptatretus taiwanae from northeastern Taiwanese waters of the northwestern Pacific from November 2013 to June 2014. Eight species of Anisakid nematodes were identified: Anisakis pegreffii, Anisakis simplex s.s., a recombinant genotype of A. pegreffii and A. simplex s.s., Anisakis typica, Anisakis sp., Anisakis brevispiculata, Anisakis physeteris and Hysterothylacium amoyense. Anisakis sp. and H. amoyense represented new locality records. The prevalence, mean intensity and mean abundance of anisakid nematodes for all specimens were 21.51%, 5.39 larvae per fish and 1.16 larvae per fish, respectively. A. pegreffii was the most frequent species in E. burgeri, E. yangi and E. taiwanae, whereas in E. sheni, the dominant species was Anisakis sp. The number of nematodes was significantly related to the host length for E. burgeri and E. sheni, but was not related to the sex of the four hagfish species. This report of scavenging hagfish infected with Anisakid larvae suggests hagfish as a transport/paratenic host between cetaceans and piscivores. Anisakiasis may be caused by the consumption of infectious third-stage larvae in raw or undercooked hagfish.

Occurrence of anisakid nematode larvae in chub mackerel (Scomber japonicus) caught off Korea.

Chub mackerel (Scomber japonicus) is a pelagic fish species widely distributing in the Indo-Pacific and a commercially important fish species in Korea. It is known to harbor anisakid nematodes larvae, and ingesting the raw or undercooked fish can accidentally cause human infection. In this study, we isolated the nematode larvae in 417 chub mackerel caught from 7 sampling locations around the Korean Peninsula in 2011 and 2012, and identified them by PCR-RFLP of the ITS (internal transcribed spacer) of ribosomal DNA and the direct sequencing of the mitochondrial DNA cox2 gene. The prevalence of infection was 55.4% (231/417) and the mean intensity was 7.0 (1628/231). Most of the nematodes (1523/1628; 93.6%) were found in the body cavity, while 5.5% (89/1628) were found in the gastrointestinal tract. Four different species were identified by PCR-RFLP and direct sequencing. Most of the nematodes (1535/1628; 94.3%) were identified as Anisakis pegreffii, and 2.8% (46/1628) were identified as Hysterothylacium sp. A hybrid genotype (Anisakis simplex sensu stricto×A. pegreffii) and A. simplex sensu stricto were 2.5% (41/1628) and 0.4% (6/1628) of the identified nematodes, respectively. The anisakid nematode assemblage of chub mackerel in Korea was similar to that of chub mackerel from the Tsushima Current stock in Japan, in that A. pegreffii was the dominant species. Since most of the anisakid nematodes were found in the body cavity and most of them were identified as A. pegreffii or Hysterothylacium sp. by PCR-RFLP and direct sequencing, chub mackerel may not greatly contribute to human anisakidosis in Korea. Alternately, A. pegreffii may be responsible for human anisakidosis in Korea, in addition to A. simplex sensu stricto. Further studies, such as the molecular diagnosis of human anisakidosis, are necessary for assessing the epidemiological role of chub mackerel in Korea.

A technique for the intra-gastric administration of live larvae of Anisakis simplex in mice.

To understand the mechanisms of infection and to attempt to simulate human infection by the Anisakidae family, many in vivo experimental approaches have been developed. The aim was to develop and present a technique for the induction of an oral infection through the use of an intra-gastric gavage of live Anisakis simplex in mice. A commercial pediatric gastric tube (No. 4) was cut longitudinally to produce a 3-cm slit at the distal end where the larva was placed to then be administered to the stomach of the mouse. There were no abnormal clinical complications before, during or after the procedure. In conclusion oral infection through the direct delivery of larvae in the stomach is simple and effective.

Anisakis simplex s.l. parasitization in mackerel (Scomber japonicus) caught in the North of Morocco--prevalence and analysis of risk factors.

Consumption of mackerel (Scomber japonicus) is very widespread in Morocco, where its production is an important economic pillar. We investigated the prevalence of infection by Anisakis spp. in mackerel caught in Moroccan waters, analyzing infection risk factors. The prevalence was generally higher in fish from the Atlantic (67.9%) than from the Mediterranean (57.0%), but they did not differ in the mean abundance, intensity, or prevalence of muscle parasitization. A. pegreffii was the predominant species (82.6% of larvae) followed by the hybrid A. simplex s.s./A. pegreffii (16.3%). Only one L3 of A. simplex s.s. was found in a specimen of S. japonicus from the Atlantic. Mackerel infection was associated with total fish weight, gonad weight, catch area, and catch season. However, muscle infection was associated solely with total weight and parasite loads. The consumption of lower-weight mackerel may be a good prophylactic measure against human anisakiasis.

Anisakis pegreffi etiological agent of gastric infections in two Italian women.

Two cases of gastric anisakiasis have been documented in two Italian women who had consumed raw anchovies (Engraulis encrasicolus). The first patient was a 49-year-old woman presenting with epigastric pain and bloody vomiting after ingestion of marinated (vinegar) raw anchovies. During the esophagogastroduodenoscopy (EGDS) a white color worm was detected and extracted from cardia by means of biopsy forceps. The second patient was a 59-year-old woman with irritable bowel syndrome and gastritis, who underwent to periodical EGDSs. In the course of the last EGDS, a white color round worm on antrum and a small polyp on the fundus of the stomach were observed. The two nematodes have been identified as L3 larvae of the genus Anisakis by a light microscope, and as Anisakis pegreffi by polymerase chain reaction-restriction fragment length polymorphism. The molecular identification of the etiological agent at the species level allows to identify what Anisakidae species play a zoonotic role and which are the fish host species.

[Pseudoterranovosis and sushi]. / Pseudoterranovosis y sushi.

A case of accidental ingestión of Pseudoterranova decipiens is reported, on account of this patient we review main concepts related to this emerging pathology in Chile. Consumption of raw fish in different preparations such as sushi or sashimi, cebiche and seafood is becoming more and more popular. Through intermediary hosts used in the preparation, one can acquire the third stage larval of Anisakis simplex or P. decipiens. These parasites frequently cause gastritis, acutely after the consumption of raw fish, or more rarely intestinal involvement. It may also cause allergic symptoms such as urticaria, angioedema and anaphylactic shock. The larvae is eliminated spontaneously in most cases; others need to be removed by upper gastrointestinal endoscopio examination. In Chile, the most common species is P. decipiens, which are generally no invasive, rarely migrate beyond the stomach and cause mild or no symptoms. Prevention strategies include avoid eating raw fish or undercooked seafood containing the parasite, to cook food with high temperatures or to freeze raw fish prior to preparation of meals, which inactivates the larvae.

Pseudoterranovosis y sushi / Pseudoterranovosis and sushi

Se comunica un caso de ingestión accidental de Pseudoterranova decipiens. A raíz de esta paciente se revisan los principales conceptos relativos a esta naciente patología en Chile. El consumo de platos preparados con pescados crudos como el sushi, sashimi, cebiche y mariscal es cada vez más popular. A través de la ingesta de hospederos intermediarios usados en la preparación, se puede adquirir la larva de tercer estadio de Anisakis simplex o P. decipiens. Estos parásitos pueden provocar gastritis aguda que se manifiesta inmediatamente después de consumido el pescado crudo. En ocasiones puede haber compromiso intestinal e invasión del tracto gastrointestinal con manifestaciones en otros órganos. Se asocia a manifestaciones alérgicas como urticaria, angioedema y shock anafiláctico. La larva se elimina en forma espontánea en la mayoría de los casos, pero en otros debe removerse por vía endoscópica. En Chile, es más frecuente la presencia de P. decipiens, que por lo general no migra a la mucosa gástrica, no invade y produce pocos síntomas. Las medidas de prevención son no comer pescados crudos, cocinarlos a temperaturas elevadas o congelación previa a la preparación de los alimentos, para inactivar las larvas.

A case of accidental ingestión of Pseudoterranova decipiens is reported, on account of this patient we review main concepts related to this emerging pathology in Chile. Consumption of raw fish in different preparations such as sushi or sashimi, cebiche and seafood is becoming more and more popular. Through intermediary hosts used in the preparation, one can acquire the third stage larval of Anisakis simplex or P. decipiens. These parasites frequently cause gastritis, acutely after the consumption of raw fish, or more rarely intestinal involvement. It may also cause allergic symptoms such as urticaria, angioedema and anaphylactic shock. The larvae is eliminated spontaneously in most cases; others need to be removed by upper gastrointestinal endoscopio examination. In Chile, the most common species is P. decipiens, which are generally no invasive, rarely migrate beyond the stomach and cause mild or no symptoms. Prevention strategies include avoid eating raw fish or undercooked seafood containing the parasite, to cook food with high temperatures or to freeze raw fish prior to preparation of meals, which inactivates the larvae.