Low detection efficiency of candling as a commonly recommended inspection method for nematode larvae in the flesh of pelagic fish

The third-stage larvae of the parasitic nematode Anisakis simplex commonly occur in most commercially important fish species from the North Atlantic. In this study, the occurrence and site distribution of Anisakis larvae in various size groups of Norwegian spring spawning herring (Clupea harengus), blue whiting (Micromesistius poutassou), and mackerel (Scomber scombrus) from the Northeast Atlantic was investigated. Although most of the larvae were found in the abdominal cavity, the small proportion lodged in the flesh was sufficient to affect food quality and safety. However, the prevalence of Anisakis larvae in fish fillets varied considerably among the size groups of each species: 15 to 60% in herring, 32 to 77% in mackerel, and 89 to 100% in blue whiting. The commonly recommended nematode detection method in the fish processing industry is candling, i.e., a brief visual inspection on a light table. Comparison of successively more accurate detection methods, i.e., candling, enzymatic degradation, and UV illumination, for fillets of each fish species revealed that only 7 to 10% of the nematode larvae present in the fillets were detected by candling. The candling efficiency was apparently independent of fillet thickness. Thus, candling is not sufficient to detect the majority of the nematodes that are actually present in the fillets of pelagic fish from the Northeast Atlantic. These findings emphasize the importance of adequate deep-freezing of any fresh pelagic fish product, especially if it is intended for consumption in a raw or semiraw state.     

Anisakid larvae in the musculature of the Argentinean hake, Merluccius hubbsi

We report the infection levels of third-stage anisakid larva in the muscle of the Argentinean hake, Merluccius hubbsi, in relation to fish size and location in the musculature. The musculature of 42 hake was separated into hypaxial (ventral) and epiaxial (dorsal) parts and surveyed for nematode larvae. Two anisakid species were detected: Anisakis sp. (prevalence, 52.4%; mean +/- SD abundance, 1.2 +/- 1.7) and Pseudoterranova sp. (prevalence, 9.5%; mean +/- SD abundance, 0.2 +/- 0.7). Since the fish were gutted after capture, the occurrence of anisakids in the flesh indicates that the worms had migrated into the muscle before capture. The number of Anisakis sp. in muscle was not correlated with fish length or weight. Therefore, fish size cannot be used as a predictor of parasite loads in the muscle. Only one Anisakis sp. and one Pseudoterranova sp. appeared in the epiaxial musculature. The density of Anisakis sp. in the hypaxial muscles was significantly higher than that in the epiaxial ones. This suggests that removal of the hypaxial musculature can reduce the risk of anisakid-induced allergies and gastrointestinal anisakidoses among consumers.     

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.     

黄海海域鲐鱼感染异尖线虫三期幼虫初步调查

目的 了解异尖线虫三期幼虫在黄海海域鲐鱼体内的感染情况.方法 2010年5月-2011年1月抽取产自黄海海域的鲜活鲐鱼(Pneumatophorus japonicus)113尾(15 ～ 20尾/月),分别检查、记录每个个体的体腔,内脏器官,背部、腹部、尾部鱼肉等感染线虫的情况,将所获线虫经乳酸酚透明液透明后于光镜下进行常规的形态学鉴定,并随机取192条(30 ～35条/月)线虫全基因组进行聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析鉴定.结果 有98尾鲐鱼感染异尖属线虫三期幼虫,感染率为86.7％;共检出4 770条异尖线虫,感染强度为5 ～194条/尾不等,平均感染强度为42.2条/尾;体腔和内脏器官感染虫数占鱼体总感染虫数的88.7％(4 229/4770),腹部肌肉感染虫数占鱼全身肌肉总感染虫数的87.2％ (470/539);感染强度随鱼体重增加而增大;所有检出的异尖线虫依据其形态鉴定均为异尖属线虫I型幼虫;192条异尖属线虫分别为派氏异尖线虫191条、典型异尖线虫1条.结论 黄海海域鲐鱼中异尖属线虫三期幼虫的感染情况比较严重,且感染程度与季节、鲐鱼体重、身体部位有一定关系,检出的异尖属线虫三期幼虫经RFLP分析以派氏异尖线虫为主,这对今后卫生检疫与渔业生产加工出口有一定的提示和指导意义.     

辽宁省海鱼异尖线虫感染调查及分子鉴定

目的了解辽宁省市售海洋鱼类异尖线虫感染情况,并对异尖线虫Ⅲ期幼虫进行分子鉴定,确定感染异尖线虫虫种。方法采用直接剖检法从市售海鱼中检出异尖线虫Ⅲ期幼虫后,在显微镜下观察形态进行初步鉴定;提取异尖线虫虫体总DNA,采用通用引物扩增异尖线虫核糖体DNA内转录间隔区(ITS)序列,并对其ITS1和ITS2序列进行序列比对和进化分析。结果采集222份市售海鱼样品进行剖检,共有70份检出异尖线虫幼虫,检出率为31.53%,其中小黄花鱼和带鱼的检出率较高,感染度最高为233条/份。测序结果经序列比对和进化分析,结果显示检出的异尖线虫幼虫分别为异尖线虫属和宫脂线虫属的5种异尖线虫,包括简单异尖线虫(Anisakis simplex)、派氏异尖线虫(Anisakis pegreffii)、典型异尖线虫(Anisakis typical)、内弯宫脂线虫(Hysterothylcaium aduncum)和厦门宫脂线虫(Hysterothylacium amoyense)。结论辽宁省市售海洋鱼类异尖线虫感染情况较严重,且感染的异尖线虫种类多样,其感染优势种为派氏异尖线虫。     

Molecular identification and population dynamic of Anisakis pegreffii (Nematoda: Anisakidae Dujardin, 1845) isolated from the European anchovy (Engraulis encrasicolus L.) in the Adriatic Sea

Anchovy Engraulis encrasicolus (L.) is a coastal pelagic and euryhaline species that represents the only European species of the family Engraulidae, with a widespread distribution. In Croatia, it is marketed fresh, frozen, salted or marinated and mainly exported to Italy and Spain, however Anisakis sp. larval infection is frequently the reason for border rejection. Since it is known that the prevalence and intensity of Anisakis infection varies with fish species, fishing area and season, the aim of our study was to identify Anisakis sp. parasitizing European anchovy and infer its population dynamic through a 2.5-year period. Larvae were found coiled and encysted on the external wall of intestine (94%) and reproductive organs (6%), rarely in fillets. Prevalence was 76.1% (95% confidence limits 74.51-77.56%), mean abundance 6.59 (bootstrap 95% confidence limits 5.81-7.26) and mean intensity 8.67 (bootstrap 95% confidence limits 7.82-9.35). The partial CO2 mitochondrial DNA sequence of the isolated anisakids confirmed clustering of the anchovy parasite within A. pegreffii sister group. Parasite population structure showed plasticity inferred by fishing ground, sampling year and fish gender and size. Compared to anisakid prevalence/abundance in other fish, the European anchovy in the Adriatic Sea represents a moderately high-infected paratenic host, although in the Mediterranean and Atlantic waters, anchovies have shown strikingly lesser values of prevalence. Since this host represents one of the most attractive Mediterranean fisheries products traditionally consumed without thermal preparation that in any case would not disrupt larval antigenicity and prevent human allergies, and given the high prevalence of the anisakid within the host, it is necessary to include anchovy into more firm risk assessment frames in order to develop measures that will support the safe alimentary production and consumption of seafood.     

Prevalence and mean intensity of Anisakis simplex (sensu stricto) in European sea bass (Dicentrarchus labrax) from Northeast Atlantic Ocean

Viscera and muscle of a total of 40 wild 1-2 kg European sea bass (Dicentrarchus labrax) from Northeast Atlantic (FAO area 27) were examined for Anisakidae larvae detection by digestion method. Extracted parasites were counted and mean intensity was calculated. Parasites were identified by genetic/molecular markers (allozymes and sequences analysis of the mtDNA cox2 gene) as belonging to the species Anisakis simplex (sensu stricto). In viscera, the main localisations of the larvae were under the gastric serosa, where several parasites alive and dead were found, and intestinal serosa. The visceral prevalence was 0.950 and the mean intensity was 96.39. The main localisation of A. simplex (s.s.) in edible parts was in belly muscles, with a prevalence of 0.425 and a mean intensity of 1.9. This is the first record on the prevalence and mean intensity of A. simplex (s.s.) in European sea bass muscle. This finding has an important consequence on epidemiology of anisakiasis and public health risk assessment.     

Molecular identification of Anisakis type I larvae isolated from hairtail fish off the coasts of Taiwan and Japan

Anisakid nematodes are known to cause the zoonotic disease, anisakiasis, through the consumption of raw or undercooked fish. The parasites most frequently associated with the disease in humans are categorized as Anisakis type I, which comprise several species of the genus Anisakis. The larvae show primitive forms and lack the detailed morphological characteristics required for precise species identification. Thus, molecular characterization is necessary for determining the species of Anisakis type I larvae and acquiring important clinical and epidemiological information. In this study, we isolated Anisakis type I larvae from hairtail fish caught off the coasts of Taiwan and Japan. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was sequenced, and restriction fragment length polymorphism (RFLP) analyses using HinfI and HhaI was carried out for species identification. Most larvae isolated from hairtail caught in Taiwan were Anisakis typica (84%), while those isolated from hairtail caught in Japan were almost exclusively identified either as Anisakis simplex sensu stricto (65%) or Anisakis pegreffii (33%). This is the first report of A. typica in fish obtained from Taiwan. Our results shed the light on the epidemiology of Anisakis type I larvae, which is a potential cause of human anisakiasis in Taiwan and Japan.     

Organic and inorganic arsenic and lead in fish from the South Adriatic Sea, Italy.

Total, organic and inorganic arsenic and lead were measured from the muscle of different kinds of fish: skate (Raje spp.), hake (Merluccius merluccius) and blue whiting (Micromesistius poutassou), caught in the South Adriatic Sea (South Italy) in the period June-August 1995. The highest levels of total arsenic were found in skate (Raje spp.) whose values ranged from 14.4 to 61.5 mg/kg ww, followed by blue whiting (M. poutassou) and hake (M. merluccius) that showed lower levels ranging from 8.5 to 21.5 mg/kg ww and from 5.9 to 16.1 mg/kg ww, respectively. In all the species examined most of this element was present as organic compounds while inorganic arsenic was only a very modest percentage (0.47-3.5%) of the total arsenic present. Lead in all species analysed was at small concentrations with mean values ranging from 0.12 to 0.15 mg/kg ww. According to the rules in force in Italy no samples showed concentrations of lead exceeding the peak value of 2 mg/kg.     

Anisakis antigens detected in fish muscle infested with Anisakis simplex L3

Anisakis simplex is a fish parasite that is a public health risk to those consuming raw or poorly cooked marine fish and cephalopods because of the possibility of becoming infested with live larvae. In humans, penetration of the larvae into the gastrointestinal track can cause acute and chronic symptoms and allergic anisakiasis. Excretion and secretion products released by the larvae are thought to play a role in migration through the tissues and induce an immunoglobulin E-mediated immune response. The aim of this preliminary study was to detect parasite antigens and allergens in fish tissues surrounding the migrating larvae. Hake and anchovy fillets were artificially parasitized with Anisakis larvae and stored in chilled conditions for 5 days. Larvae were evaluated for fluorescence, fish muscle tissue was examined with transmission electron microscopy, and immunohistochemical reactions of two rabbit polyclonal antisera against a parasite crude extract and the allergen Ani s 4 were recorded. Larvae immediately migrated into the fish muscle, and no emission of bluish fluorescence was observed. Fish muscle areas in contact with the parasite showed disruptions in the structure and inclusion of granules within sarcomeres. Both parasite antigens and the Ani s 4 allergen were located in areas close to the larvae and where sarcomere structure was preserved. These findings indicate that parasite antigens and allergens are dispersed into the muscle and might cause allergic symptoms such as dyspnea, vomiting, diarrhea, urticaria, angioedema, or anaphylaxis in some individuals sensitive to A. simplex.     

A new procedure for marinating fresh anchovies and ensuring the rapid destruction of Anisakis larvae

The consumption of marinated anchovies is the main route of transmission of anisakiasis in Spain. Because this country is one of the world's major tourist destinations, this traditional food also poses a potential health risk to millions of foreign visitors. Anisakis larvae are not destroyed by the traditional marinating procedure, and alternative methods, such as long-term storage in brine, freezing, or hydrostatic pressure treatment, all present major difficulties. In this study, we used high food-grade acetic acid concentrations (10, 20, 30, and 40% [vol/vol] in line with the quantum satis rule) to destroy these larvae rapidly, and we report data on the survival of Anisakis larvae exposed directly to different marinades and when the larvae are placed under the fish musculature. The percentage of salt and acetic acid in the fish tissue water phase was also determined. A marinating procedure is proposed that ensures the rapid death of Anisakis through the use of strong acetic acid concentrations. Posttreatment washes with water reduce these to levels acceptable to consumers. The sensory characteristics of the product were shown to be satisfactory. The actual selection of an acetic acid concentration for marinating depends on costs and the processing time available. The physiological stress of the larvae exposed to the different marinades was determined by measuring the levels of their stress proteins. The latter are good indicators of injury and might reflect the infectivity of larvae. In addition, we also used a rat model to determine the infectivity of larvae considered microscopically dead.     

Prevalence and Mean Intensity of Anisakidae Parasite in Seafood Caught in the Mediterranean Sea Focusing on Fish Species at Risk of Being Raw-consumed. A Meta Analysis and Systematic Review

Objective: To assess the prevalence and mean intensity of anisakids in seafood caught in the Mediterranean Sea, focusing on fish species at risk of being raw-consumed. Design: Systematic review and meta-analysis of studies published from 1960–2012. Study selection: Main criteria for the inclusion of studies were as follows: Findings of anisakid larvae, in both muscles and viscera; fish species for human consumption caught in the Mediterranean Sea; prevalence and mean intensity data for each species; and sample size equal to or more than 40 fishes. Results: Twelve studies were identified. Among these, four studies considered the following three fish species that are often consumed raw or preserved lightly, or not cooked thoroughly: anchovy, pilchard, and Atlantic mackerel. Data synthesis: All pooled analyses were based on the random-effect model. Anisakids prevalence in fish muscle was 0.64% (P < 0.0001), in viscera it was 1.34% (P < 0.0001), and overall prevalence was 0.95% (P < 0.0001). Mean intensity in muscle was 2.31 (P = 0.0083), in viscera it was 1.55 (P = 0.0174), and overall it was 1.81 (P < 0.0005). Heterogeneity indices (I²) were significantly high with the exception of viscera mean intensity. Conclusions: Anchovy, pilchard, and Atlantic mackerel have a low prevalence and mean intensity of anisakidae larvae in both viscera and muscles. Mean Intensity was also low.     

海产品中异尖线虫危害及其检测控制研究进展

异尖线虫为蛔目异尖科线虫,是一类广泛分布于海洋动物的寄生虫,为国家禁止入境的二类动物寄生虫。人类异尖线虫病是因为食入了含活异尖科第三期幼虫的海鱼和海产软体动物而引起。近些年随着生鲜海产品消费量的增多,我国人民感染异尖线虫病的可能性不断加大,必须予以足够的重视。本文总结了异尖线虫在海产品中的分布情况及致病性,并重点就目前海产品中异尖线虫的检测技术以及危害控制技术,对近年来的相关研究情况进行了综述。     

Anisakis simplex larva killed by high-hydrostatic-pressure processing

Anisakis simplex is a common nematode parasite present in many marine fish, including finfish and squid. It can pose a public health problem if it is not destroyed during food processing. Anisakis larvae were isolated from fish tissue, and their survival of high-pressure treatments in distilled water and physiological isotonic solution was assayed. Treatment at a pressure of 200 MPa for 10 min at a temperature between 0 and 15 degrees C kills all Anisakis larvae, with a lack of motility being used as an indicator of larval death. Lower pressures can be successfully employed down to 140 MPa, but with lower pressures, the treatment time must be increased by up to I h to kill all larvae. Meanwhile, most larvae treated for >10 min at pressures of >120 MPa were dead, with the autofluorescence method being used to determine death. Cycles of compression and decompression increase the destruction of larvae compared with a single pressure treatment for a similar treatment time. Our results indicate that high-pressure treatment is an alternative nonthermal method for killing this nematode. The possible mechanism of death and damage by pressure is discussed, and uses for this treatment in food processing are suggested.     

Survival of Anisakis larvae in marinated herring fillets

The survival of Anisakis sp. larvae in herring fillets marinated in brines with different concentrations of salt (NaCl) and acetic acid was examined. Traditional German and Danish procedures for marinating herring fillets require a storage time of at least 5 and 6 weeks, respectively, for all larvae to be killed. Other concentrations of salt and acetic acid were also studied. A reduction of the salt content in the water phase of the fish tissue from 9% (w/w) to 4.3% (w/w), while keeping the concentration of acetic acid constant at 2.6%, increased the survival time of the nematodes from at least 35 days to more than 119 days. Increases in the acetic acid concentration or the addition of potassium sorbate had little or no effect on the viability of the larvae.     

Seasonal Survey of Contaminants (Cd and Hg) and Micronutrients (Cu and Zn) in Edible Tissues of Cephalopods from Tunisia: Assessment of Risk and Nutritional Benefits

Concentrations of cadmium (Cd), copper (Cu), mercury (Hg), and zinc (Zn) were determined by atomic absorption spectrophotometry in the muscle tissues (arms and mantle) of 3 commercial cephalopods (Loligo vulgaris, Octopus vulgaris, and Sepia officinalis) caught in 3 different Tunisian coastal regions. The highest concentrations found correspond to the essential elements Cu and Zn. Octopuses and cuttlefish showed the highest levels of those elements whereas squid presented with significantly higher values of Hg in both muscular tissues. This may be related to different feeding behavior and detoxification processes among benthic and pelagic cephalopods. Variation of element concentrations between seasons was different between species and seemed to be mostly dependent on the sampling site. From a public health standpoint, average concentrations of Cd, Cu, Hg, and Zn measured in edible tissues of cephalopods from this study did not reveal, in general, any risk for consumers. The estimated target hazard quotients for Cd and Hg for consumers of the selected species were below 1 and within the safety range for human health. Moreover, their consumption could provide in an important contribution to the daily dietary intake of Cu for the Tunisian population, especially regarding the consumption of octopus and cuttlefish muscles.     

Scanning electron microscopy of Anisakis larvae following different treatments

Ingestion of fish parasitized with Anisakis larvae can produce infestation and/or allergy in consumers. Technological and food processing treatments have been applied to parasitized fish in order to kill the larvae and avoid the infestation; however, their influence on allergenicity has not been studied. Four lots of hake (Merluccius merluccius) steaks artificially parasitized with Anisakis larvae were subjected to two storage chilling (5 degrees C +/- 1 degrees C) and freezing (-20 degrees C +/- 1 degrees C) treatments and two food processing treatments of heat (final temperature 86.3 degrees C) and microwave (final temperature 66.9 degrees C) and studied by scanning electron microscopy, environmental scanning electron microscopy (ESEM) (acid [pH = 2] and water preparations), and emission of fluorescence. Anisakis larvae were resistant to acid conditions, remaining alive after treatment. Larvae in the heat- and microwave-treated lots presented coagulated and disrupted zones in the cuticle with release of fluids. The cylindrical shape changed to a dehydrated appearance mainly observed by ESEM. Fluorescence was only noticeable in the frozen larvae. Larvae without apparent changes, together with dehydrated ones, were observed by ESEM in the frozen lot; nevertheless, no disruptions in the cuticle were perceptible. Further studies are needed in order to elucidate if the changes observed in the cuticle reduce the resistance of the parasites to the action of gastric enzymes in the gastrointestinal tract and to determine the release of allergens to the flesh by the live larvae during chilled storage of the fish.     

High hydrostatic pressure treatment of finfish to inactivate Anisakis simplex

High hydrostatic pressure has been demonstrated to be a useful technique for treating food to reduce the number of pathogenic organisms and to extend shelf life. Most research in this area has focused on bacteria. However, a concern in the sashimi (raw fish) industry is that nematode worms such as Anisakis simplex occur naturally in cold-water marine fish. The objectives of this research were to perform a pilot study to determine the effect of high hydrostatic pressure on the viability of Anisakis simplex larvae, commonly found in king salmon and arrowtooth flounder, and to evaluate the effects of high hydrostatic pressure on the color and texture of the fish fillets. Pieces of fish (ca. 100 g per bag) containing 13 to 118 larvae were exposed to pressures of up to 80,000 lb/in2 (552 MPa) for up to 180 s. The times and pressures required to kill 100% of the larvae were as follows: 30 to 60 s at 60,000 lb/in2 (414 MPa), 90 to 180 s at 40,000 lb/in2 (276 MPa), and 180 s at 30,000 lb/in2 (207 MPa). For all salmon treatments that killed 100% of the larvae, a significant increase in the whiteness of the flesh was observed. Although high hydrostatic pressure was effective in killing A. simplex larvae in raw fish fillets, its significant effect on the color and overall appearance of the fillet may limit its application to the processing of fish for raw-fish markets.     

Primary structures of proteinase inhibitors fromPhaseolus vulgaris var.nanus (cv. Borlotto)

The primary structures of two trypsin-chymotrypsininhibitors fromPhaseolus vulgaris var.nanus (bush bean, cv. Borlotto), PVI-3₂ und PVI-4, were derived from automated Edman degradation data, amino acid composition and manual Edman degradation results of enzymatic fragments and homology with other Bowman-Birk type proteinase inhibitors. The highest degrees of homology were observed between PVI-3₂ or PVI-4 and the trypsin-chymotrypsin inhibitors from lima beans (LBI I, IV and IV′, 86%), black-eyed peas (BTCI, 81%), and, in part, adzuki beans (ABI I, II and II′, 74–77%). Similarly, the primary structure of the trypsin-elastase inhibitor from the same source, PVI-3₁, was deduced which showed highest homology with that of the trypsin-elastase inhibitor GBI II from garden beans (92%), followed by GBI II′ from garden beans (86%) and C-II from soybeans (71%). In contrast, homology between PVI-3₂ and PVI-4 on the one hand and PVI-3₁ on the other was relatively low (61%).