Anisakiasis: The Importance of Prevention and the Role of Diet Therapy in Allergic Patients.

INTRODUCTION: Anisakiasis is a zoonosis of parasitic origin whose diffusion seems to be continuously increasing. OBJECTIVE: The aim of this study was to evaluate the benefits of a fish-free diet in patients allergic to Anisakis simplex as well as underlining the importance of awareness and prevention. Furthermore, we aimed to investigate the critical issues related to the spread of anisakiasis in relation to eating habits. METHODS: Patients were assessed by means of skin prick tests (SPTs) and targeted laboratory testing, with an 18-month-long fish-free diet being recommended in cases of severe sensitization. The degree of awareness about anisakiasis was evaluated from interviews. Patients were subjected to follow-up visits after 18 months. RESULTS: A total of 70 cases of sensitization to A. simplex were evaluated. The Interview answers highlighted a general state of misinformation among patients and healthy subjects along with a remarkable underestimation of anisakiasis-related risks. An overall lack of care regarding eating habits and diet plans also emerged. In 21 patients affected by severe sensitization, clinical and laboratory evaluations were repeated after 18 months of the subjects being on a fish-free diet. There was a remarkable improvement in serum IgE levels and clinical symptoms. CONCLUSION: Data analysis proved the need to implement new and more effective awareness-raising and prevention campaigns in order to reduce the incidence of anisakiasis. It is crucial to establish an adequate diet therapy for sensitized patients. Evaluation of cytokine patterns suggests how a polyphenol-rich regime can activate regulatory T cell function and possibly reduce the allergic and inflammatory components of the disease.

An Overview of Fish-borne Nematodiases among Returned Travelers for Recent 25 Years- Unexpected Diseases Sometimes Far Away from the Origin.

Along with globalization of traveling and trading, fish-borne nematodiases seems to be increasing in number. However, apart from occasional and sporadic case reports or mini-reviews of particular diseases in particular countries, an overview of fish-borne nematodiasis among travelers have never been performed. In this review, we gathered fishborne nematodiasis among travelers for recent 25 years by an extensive global literature survey using appropriate keywords, e.g. travelers diseases, human infection, anisakiasis, gnathostomiasis, capillariasis, sushi, sashimi, ceviche, Gnathostoma, Pseudoterranova, Anisakis, Capillaria, etc., as well as various combinations of these key words. The Internet search engines PubMed, Medline, Google and Googler Scholar were used as much as possible, and the references of every paper were checked in order to identify useful and reliable publications. The results showed unexpectedly high incidence of gnathostomiasis and low incidence of anisakidosis. The different incidence values of the infection with several fish-borne zoonotic nematode species are discussed, as well as some epidemiological aspects of the infections. The difficulties of differential diagnosis in non-endemic countries are emphasized. It is concluded that travelers must avoid risky behaviors which can lead to infection and that physicians and health authorities must advice travelers on the risks of eating behaviors during travel.

Evaluation of different Mediterranean essential oils as prophylactic agents in anisakidosis.

CONTEXT: Anisakis Dujardin 1845 (Anisakidae) nematodes can cause gastrointestinal and allergic diseases when humans eat raw or undercooked seafood containing larvae. There is currently no drug available in the market against this parasitic disease, and the study of plant-derived molecules could be useful in the discovery of effective compounds. OBJECTIVE: This research assesses the biocidal activity of a range of essential oils (EOs) from some Mediterranean plants against larvae found in the musculature of fresh fish. MATERIALS AND METHODS: EOs composition was analyzed by gas chromatography-mass spectroscopy. All the EOs were diluted at 5% v/v in olive oil to cover the fish with the solutions for 24 h. The larvae that abandoned the muscle and the larvae obtained from the artificial digestion of the fish were collected. Controls were carried out in parallel. Furthermore, Wistar rats were infected with the live larvae collected from the in vitro trials in order to find any larvae that may have penetrated the gastrointestinal wall. RESULTS: Between 60.8% and 87.6% of parasites treated with EOs abandoned the fish muscle, and the highest in vitro mortality rate was achieved with oregano EO (53.9%). Rats previously treated with oregano, cumin and Spanish lavender EOs showed no detectable lesions in the digestive tract due to the infection with larvae. CONCLUSIONS: Oregano (Origanum vulgare L. Lamiaceae), cumin (Cuminum cyminum L. Apiaceae) and Spanish lavender (Lavender stoechas L. Lamiaceae) EOs could be used as promising ingredients in the development of products for the control of anisakiasis.

Is the quality of sushi ruined by freezing raw fish and squid? A randomized double-blind trial with sensory evaluation using discrimination testing.

BACKGROUND: Sushi is a traditional Japanese cuisine enjoyed worldwide. However, using raw fish to make sushi may pose risk of certain parasitic infections, such as anisakidosis, which is most reported in Japan. This risk of infection can be eliminated by freezing fish; however, Japanese people are hesitant to freeze fish because it is believed that freezing ruins sushi's taste. METHODS: A randomized double-blind trial with discrimination testing was conducted to examine the ability of Japanese individuals to distinguish between frozen and unfrozen sushi. A pair of mackerel and squid sushi, one once frozen and the other not, was provided to the participants, and they were asked to answer which one tasted better. RESULTS: Among 120 rounds of discrimination testing involving the consumption of 240 pieces of mackerel sushi, unfrozen sushi was believed to taste better in 42.5% (51 dishes) of cases, frozen sushi was thought to taste better in 49.2% (59 dishes), and the participants felt the taste was the same in 8.3% (10 dishes). The odds ratio for selecting unfrozen sushi as "tastes better" over frozen sushi was 0.86 (95% confidence interval [CI], .59-1.26; P = .45). For squid, unfrozen sushi was believed to be superior 48.3% of the time (58 dishes), and frozen sushi, 35.0% of the time (42 dishes). They were felt to be the same in 16.7% (20 dishes) (odds ratio, 1.38; 95% CI, .93-2.05; P = .11). CONCLUSIONS: Freezing raw fish did not ruin sushi's taste. These findings may encourage the practice of freezing fish before using it in sushi, helping to decrease the incidence of anisakidosis.

Methods for inactivation of seafood Anisakis larvae and prevention of human anisakiasis: a mini-review.

Anisakiasis is an arising zoonosis induced by parasitic nematodes belonging to the family Anisakidae. Anisakiasis is often caused by the ingestion of larval nematodes in uncooked or minimally processed seafood dishes, which are regularly consumed by humans. Significant potential sources of infection are raw fish (e.g., sushi and sashimi) that can be found in traditional Japanese cuisine and can be part of the culinary tradition of consumption of raw or marinated fish that is particularly diffused in European countries. During the last five decades, the global prevalence of human anisakiasis has been rising, becoming an emergent major public health problem. Thus, there is an unmet need for well-defined and cost-effective methods aimed at killing Anisakis larvae, thus reducing the incidence of anisakiasis. In this mini-review, we discuss the clinical features of anisakiasis as well as the effectiveness and mechanisms of action of the main methods employed for increasing seafood safety and killing Anisakis larvae, including freezing, heating, use of high hydrostatic pressure, salting process, pepsin digestion method and use of garlic oil.

Intrinsic properties of anisakid nematode larvae as a potential tool for the detection in fish products.

Anisakid nematode larvae (NL) in fish products comprise a risk to human health and, if visible, lead to the rejection of these products by consumers. Therefore, great efforts are being made for the identification of these anisakid larvae to estimate the potential consumer health risk as well as to develop effective detection methods in order to prevent the introduction of heavily infected fish products into the market. The tasks of national reference laboratories include the improvement of detection methods and to promote their further development. As a prerequisite for improved detection, it is important to understand the structural properties of anisakid NL and compounds produced during host-parasite interactions. This review provides an overview of the intrinsic properties of anisakid NL and reports the latest detection methods in published literature. First, in order to define the potentially interesting intrinsic properties of anisakid nematodes for their detection, anatomy and compounds involved in host-parasite interactions are summarised. These can be used for various detection approaches, such as in the medical field or for allergen detection in fish products. In addition, fluorescence characteristics and their use as both established and promising candidates for detection methods, especially in the field of optical sensing technologies, are presented. Finally, different detection and identification methods applied by the fish processing industries and by control laboratories are listed. The review intends to highlight trends and provide suggestions for the development of improved detection and identification methods of anisakid NL in fish products.

The oil of garlic, Allium sativum L. (Amaryllidaceae), as a potential protectant against Anisakis spp. Type II (L3) (Nematoda) infection in Wistar rats.

The consumption of inadequately thermally treated fish is a public health risk due to the possible propagation of Anisakis larvae. The present study demonstrated the physiological and histopathological changes that accompanied an oral inoculation of crude extracts from fresh and thermally treated Anisakis Type II (L3) in rats. Worms were isolated from a marine fish and examined and identified using light and scanning electron microscopy. The study was performed in 6 rat groups: control (I), garlic oil (GO) inoculated (II), fresh L3 inoculated (III), thermally treated L3 inoculated (IV), fresh L3 + GO inoculated (V), and a thermally treated L3 + GO inoculated (VI) groups. Rats inoculated with fresh and thermally treated L3 showed abnormal liver and kidney functions associated with the destruction of normal architecture. GO produced a protective effect in rat groups inoculated with L3 extracts + GO via the amelioration of liver and kidney functions, which was confirmed by the marked normal structure on histology. Cooking of L3-infected fish induced severe alterations compared to uncooked fish. The administration of garlic before and after fish eating is recommended to avoid the dangerous effect of anisakids, even if they are cooked.

[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.

Anisakis simplex s.l.: Larvicidal activity of various monoterpenic derivatives of natural origin against L3 larvae in vitro and in vivo.

This paper describes the activity against Anisakis simplex s.l. L(3) larvae of six monoterpenic derivatives obtained from different essential oils, (alpha-pinene, beta-pinene, ocimene, myrcene, geranyl acetate, and cineole). In in vitro assays, alpha-pinene, ocimene and cineole showed high activity at a concentration of 125 microg/mL (48h) but only alpha-pinene and ocimene were active at 62.5 microg/mL. In in vivo assays, L(3) larvae and study compounds were simultaneously administered per os to Wistar rats. The most active compound was alpha-pinene, finding lesions in only 20% of treated rats versus 98% of controls. Further in vivo studies are required to investigate whether addition of these compounds to food could have a prophylactic effect, reducing the pathogenicity of A. simplex s.l. L(3) in humans, and to explore any possible synergy among compounds.

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.

Radioresistance of Anisakis simplex third-stage larvae and the possible role of superoxide dismutase.

The radioresistance of Anisakis simplex third-stage larvae and the possible role of sublethal radiation on superoxide dismutase (SOD) were investigated. Larvae were isolated from the viscera of the sea eel Anago anago; irradiated with 10, 100, 200, 500, or 1,000 Gy; and then given orally to rats. Worms were recovered at 16 hr postinoculation. Most larvae were found to have invaded the gastric wall, omentum, and abdominal cavity, suggesting that their viability and infectivity were not controlled by irradiation with the doses used. To determine the relationship between SOD activities in parasites and their radiosensitivities, the larvae of A. simplex and the metacercariae of Neodiplostomum seoulense (a radiosensitive control) were irradiated with 0, 30, 100, or 500 Gy, and parasite SOD levels were measured. In nonirradiated A. simplex larvae, the average SOD level was 38.9 U/mg, and this increased to 51.3 U/mg at 500 Gy. However, at all radiation doses applied, SOD activities of N. seoulense metacercariae were significantly (P < 0.05) lower than those of A. simplex larvae. Our results demonstrate that A. simplex third-stage larvae are radioresistant, and suggest that SOD plays a role in this radioresistance.

[Parasitic dead-end: update]. / Impasses parasitaires: quoi de neuf?

Parasitic dead-ends occur when a parasite is unable to establish a permanent interaction in an unnatural host. Although the likelihood of successful reproduction by the pathogenic agent is nul, parasitic dead-end heralds capture of new parasites and therefore expansion of the host range. Angiostrongyliasis due to A. cantonensis or A. costaricensis, anisakiasis, Ancylostoma caninum infection, gnathostomiasis and sparganosis are undoubtedly emerging zoonoses of particular medical interest. Prevention of these diseases relies on abstinence from eating raw meat from invertebrates or cold-blooded (poikilotherm) vertebrates (e.g. used in exotic dishes). These guidelines must be included in recommendations to travelers.

Inactivation of Anisakis simplex L3 in the flesh of white spotted conger (Conger myriaster) by high hydrostatic pressure and its effect on quality.

Koreans consume much seafood; the country is surrounded on the east, west and south by the sea. Koreans have eaten raw sashimi for a long time. However, a concern in the raw sea food industry is that the parasitic nematode Anisakis simplex L3 occurs naturally in marine fish. Thus, the fishery industry needs a non-thermal processing method. High hydrostatic pressure (HPP) has been demonstrated to be effective. White spotted conger flesh containing 20 live larvae was exposed to different pressures (150 and 200 MPa for 1 and 5 min; 250 and 300 MPa each for 1 min). The viability of A. simplex L3 was significantly (p < 0.05) reduced in the flesh of white spotted conger by the stepwise increase of high pressure and time. The conditions required to eliminate A. simplex L3 were as follows: 200 MPa for 5 min or 300 MPa for 1 min. The flesh of the white spotted conger treated at 300 MPa for 1 min was whiter and yellower than untreated controls or that treated at 200 MPa for 5 min. No significant changes (p > 0.05) in any of the Hunter colour ('L', 'a' and 'b') values were found after HPP at 200 MPa for 5 min. The fresh treated at 300 MPa for 1 min scored < 4.0 (the defect limit of quality) of flavour, texture and overall acceptability in untrained sensory evaluation using a seven-point hedonic scale. However, the flesh treated at 200 MPa for 5 min scored > 5.0 ('like') for all sensory parameters. This study suggested that HPP at 200 MPa for 5 min could potentially be used for the inactivation of A. simplex L3 in raw fishery food products without any concomitant changes in their colour or sensory qualities.

The oil of garlic, Allium sativum L. (Amaryllidaceae), as a potential protectant against Anisakis spp. Type II (L3) (Nematoda) infection in Wistar rats / O óleo de alho, Allium sativum L. (Amaryllidaceae), como potencial protetor contra Anisakis spp. Infecção tipo II (L3) (Nematoda) em ratos Wistar

Abstract The consumption of inadequately thermally treated fish is a public health risk due to the possible propagation of Anisakis larvae. The present study demonstrated the physiological and histopathological changes that accompanied an oral inoculation of crude extracts from fresh and thermally treated Anisakis Type II (L3) in rats. Worms were isolated from a marine fish and examined and identified using light and scanning electron microscopy. The study was performed in 6 rat groups: control (I), garlic oil (GO) inoculated (II), fresh L3 inoculated (III), thermally treated L3 inoculated (IV), fresh L3 + GO inoculated (V), and a thermally treated L3 + GO inoculated (VI) groups. Rats inoculated with fresh and thermally treated L3 showed abnormal liver and kidney functions associated with the destruction of normal architecture. GO produced a protective effect in rat groups inoculated with L3 extracts + GO via the amelioration of liver and kidney functions, which was confirmed by the marked normal structure on histology. Cooking of L3-infected fish induced severe alterations compared to uncooked fish. The administration of garlic before and after fish eating is recommended to avoid the dangerous effect of anisakids, even if they are cooked.

Resumo O consumo de peixe inadequadamente tratado termicamente representa um risco para a saúde pública, com a possibilidade da propagação de larvas de Anisakis. O presente estudo demonstrou as alterações fisiológicas e histopatológicas acompanhadas de inoculação oral de extractos brutos de Anisakis tipo II (L3) frescos e termicamente tratados em ratos. Os vermes foram isolados de um peixe marinho, examinados e identificados por microscopia de luz e eletrônica de varredura. O estudo foi conduzido em 6 grupos de ratos: controle (I), óleo de alho (GO) inoculado (II), L3 fresco inoculado (III), L3 tratado termicamente inoculado (IV), L3 fresco + GO inoculado (V), e um grupo L3 + GO tratado termicamente inoculado (VI). Observou-se que ratos inoculados com L3 fresco e tratados termicamente mostraram funções hepáticas e renais anormais, associadas à destruição da sua arquitetura normal. GO produziu um efeito protector em grupos de ratos inoculados com extractos L3 + GO através da melhoria das funções do fígado e dos rins, o que foi confirmado pela estrutura normal marcada da sua histologia. A cozedura de peixes infectados com L3 induziu alterações mais graves do que os peixes não cozidos. Recomenda-se a administração de alho antes e depois do consumo de peixe, para evitar o efeito perigoso dos anisakids, mesmo que sejam cozidos.

Food safety considerations in relation to Anisakis pegreffii in anchovies (Engraulis encrasicolus) and sardines (Sardina pilchardus) fished off the Ligurian Coast (Cinque Terre National Park, NW Mediterranean).

Engraulis encrasicolus and Sardina pilchardus are pelagic fishes of notable economic and gastronomic importance in the northwest Mediterranean (Ligurian Sea, Italy). The consumption of thermally unprocessed or lightly processed, marinated or salted anchovies and sardines presents a potential risk to acquire anisakiasis, a fish-borne parasitic disease in humans. Prevalence and abundance of Anisakis larvae in Engraulis encrasicolus and Sardina pilchardus from the Monterosso fishing grounds (Cinque Terre National Park, Ligurian Sea, Italy) were assessed, and the larvae were identified by morphological and PCR-RFLP methods. Anisakis larvae, all belonging to Anisakis pegreffii spp. were found in the visceral mass of 1050 anchovies (0.8% overall prevalence), whereas no Anisakis larvae were found in the 750 sardines examined. According to these data, the risk of acquiring anisakiasis from the consumption of raw or undercooked anchovies and sardines caught in the fishing area we investigated is very low.

El ciclo evolutivo de parásitos de la familia anisakidae. Hacia una prospectiva de medidas sanitarias de control / The evolutionary cycle of parasites of the anisakidae family. towards a prospective of sanitary control measures

Las larvas de nematodos en los peces, generalmente corresponde a especies de la familia Anisakidae, son gusanos redondos, sin segmentación; con hospedadores intermediarios y definitivos y un huésped accidental que es el humano, en quién va producir una enfermedad parasitaria del tacto digestivo, mundialmente conocida como anisakiasis, asociada a reacciones de hipersensibilidad debido a los alérgenos del parásito. La presencia de estos parásitos en los productos de la pesca, deberá ser controlada con medidas preventivas establecidas en la legislación sanitaria de países; estableciendo profilaxis, medidas higiénicas como consumir el pescado cocido, o sometido previamente a congelación a -20°C por 48 a 72 horas, para prevenir la enfermedad. Cuando enfocamos hacia una prospectiva de medidas sanitarias de control preventivo, se plantea el establecimiento de un plan de requerimientos de alimentos seguros, basado en el análisis de riesgos y control de puntos críticos (HACCP), constituido por un paquete de documentos escritos basados en los principios de seguridad de alimentos; que contendrá: el análisis de riesgos; controles preventivos; programas en la cadena de suministros; delineamiento de los procedimientos para ser seguidos por monitoreo, acciones correctivas y verificación. Estará basado en las obligaciones que se puedan establecer bajo jurisdicción regulatoria para el pescado, en cuanto a la pesca o captura, manipulación a bordo o en la orilla y mecanismos de conservación hasta el expendio; que dé lugar a un manual de buenas prácticas de manufactura, análisis de riesgo y controles preventivos de riesgos para la regulación de alimentos de origen marino para consumo humano

The larvae of nematodes in fish generally correspond to species of the Anisakidae family, they are round worms, without segmentation; with intermediate and definitive hosts and an accidental host that is human, in whom it will produce a parasitic disease of the digestive tract, worldwide known as anisakiasis, associated with hypersensitivity reactions due to parasite allergens. The presence of these Parasites in fishery products should be controlled with preventive measures established in the sanitary legislation of countries; establishing prophylaxis, hygienic measures such as consuming the cooked fish, or previously subjected to freezing at -20 ° C for 48 to 72 hours to prevent the disease. When we focus on a prospective of preventive control sanitary measures, the establishment of a plan for safe food requirements based on risk analysis and control of critical points (HACCP), constituted by a package of written documents based on the principles, is considered food safety; which will contain: risk analysis; preventive controls; programs in the supply chain; delineation of the procedures to be followed by monitoring, corrective actions and verification. It will be based on the obligations that can be established under the regulatory jurisdiction for fish, in terms of fishing or capture, handling on board or on the shore and conservation mechanisms up to the sale; that results in a manual of good manufacturing practices, risk analysis and preventive risk controls for the regulation of food of marine origin for human consumption