Risk Analysis of Human Anisakidosis Through the Consumption of the Blue Whiting,Micromesistius poutassou,Sold at Spanish Supermarkets

Abstract

European legislation directed at the catering industry concerning the prevention of anisakidosis proposes efficient measures to avoid human infestation, but this legislation does not directly address the consumer at the household level. Assessing the anisakidosis risk for consumers who buy fresh fish at supermarkets in Spain, 284 blue whiting, Micromesistius poutassou, specimens, originating from two fishing zones and seasons of capture, sold at five nationwide Spanish supermarket chains, were examined to identify the presence of anisakid species and analyze their prevalence and abundance in viscera and flesh. The potential influence of intrinsic (length and weight) and extrinsic (origin, season, and days after catch) factors was statistically analyzed. The only two species detected were Anisakis simplex complex and Hysterothylacium sp. Total anisakid prevalence was 55.6%, with A. simplex being more prevalent (53.9%) and abundant (3.9 helminths/fish), and also carrying the biggest risk for consumers. Origin (Atlantic and Mediterranean) and season (spring and autumn) were the most influential factors among those considered. Results suggest that specimens originating from the Atlantic, of greater weight and length, and caught in spring pose the highest transmission risk of anisakidosis. Moreover, the days passed between the catch and consumption should be considered as a risk factor. Thus, in addition to the recommendation of freezing fish at −20°C for at least 24 h (in case the fish is consumed raw or poorly cooked), new and easy-to-follow recommendations are being proposed to avoid anisakidosis at home, which emphasize the importance of the information provided on fish crate labels.

Introduction

N ematodes of the family Anisakidae have an aquatic triheteroxen biological cycle, with cetacean and pinniped mammals as their main definitive hosts, while a great variety of fish and cephalopods may act as secondary intermediate or paratenic hosts. Humans may be involved in this cycle as accidental hosts through the ingestion of raw or undercooked secondary intermediate or paratenic hosts harboring infective L₃ larvae. However, in this case, the parasite does not develop and the cycle is discontinued (Smith and Wootten, 1978; Cheng, 1982).

Similar to the legislation in other European Union countries, the Spanish Royal Decree 1437/1992 lays down preventive measures requiring the visual control of captured fish and prohibits the sale of highly parasitized fish. Moreover, Royal Decree 1420/2006 obliges establishments serving raw fish to freeze it prior to consumption; the same applies to cold-smoked, pickled, or salted fish. With regard to the catering industry, these measures have decreased the number of anisakidosis cases in Spain. However, the high percentage of fish prepared in Spanish homes presents an obstacle to the correct application of these regulations at the consumer level (Domínguez and Martínez-Corcera, 2000).

Anisakis simplex is the species that carries the greatest risk for consumers due to its wide geographic distribution and its frequent presence in fish habitually consumed in Spain (Valero et al., 2000; Abollo et al., 2001; Fernández et al., 2005; Quiazon et al., 2008).

Spain is the second highest fish-consuming country per capita in the world (Navarro Cantarero et al., 2005), and the blue whiting, Micromesistius poutassou, is among the most popular species with Spanish consumers, but it is also among those with the highest anisakid prevalence (Viu et al., 1996; De la Torre Molina et al., 2000; Fernández et al., 2005; Cruz et al., 2007). These studies show that parasitization risk is elevated for consumers, especially when considering that this fish, originating from either the Atlantic or the Mediterranean, can be consumed over the entire year and that supermarkets offer it fresh and uneviscerated. Recent surveys have shown that 33% of Spanish consumers regularly buy fresh fish at super- or hypermarkets, and that almost 75% of those who consume this fish do so after frying or grilling it, which are inadequate methods to eliminate anisakids.

These high prevalences also have economic implications, as the presence of larvae in the flesh diminishes the fish's quality, with ensuing rejection by consumers. Swift evisceration of the fish is crucial to avoid the migration of helminths to the flesh, yet supermarkets and fishmongers sell the blue whiting, as well as other fish of small size, such as the small hake, the sardine, or the anchovy, among others, uneviscerated. Moreover, in the case of the blue whiting, freezing is not recommended as it changes the organoleptic characteristics of its delicate flesh. Consequently, the main preventive measure to avoid anisakid infestation would be adequate cooking of the blue whiting, yet allergic reactions (Audicana and Kennedy, 2008; Jofre et al., 2008) and gastrointestinal symptoms (Clavel et al., 1993) have been described even after this process.

Due to its flesh fragility, the surplus of blue whiting is used as feed for aquaculture (e.g., for turbot), and given its high prevalence of parasitization, the likelihood of transmission increases if the surplus is not treated adequately before it is turned into fish flour to be fed to other fish.

The present study assesses the risk of human anisakidosis through the consumption of the blue whiting sold at nationwide Spanish supermarket chains, by means of the identification of the anisakids found, and the analysis of intrinsic as well as extrinsic factors that may influence the prevalence and abundance of helminths.

Materials and Methods

Two hundred eighty-four specimens of the blue whiting purchased at branches of five nationwide Spanish supermarket chains located in València and its metropolitan area, originating from the north Atlantic (Food and Agriculture Organization of the United Nations [FAO] zone 27) or from the Mediterranean (FAO zone 37.1), between autumn 2007 and spring 2011 were analyzed (Table 1).

Table 1.

Origin and Season of Capture of the Blue Whiting Sample

	Atlantic	Mediterranean	Total
Autumn	94	40	134
Spring	75	75	150
Total	169	115	284

The specimens were chosen at random, but different weights (total range 17.58–145.20 g), divided into two groups, W₁<50 g (n=130) and W₂≥50 g (n=154), various lengths (total range 14.50–28.60 cm), also divided into two groups, L₁<20 cm (n=146) and L₂≥20 cm (n=138), as well as days passed since the date of capture and the date of the analysis, which also corresponds to the date of consumption (1–7 days), were selected. At the moment of purchase, the fish were offered fresh and uneviscerated. At the selling point, information about the date of capture and origin, as well as the port of discharge and the preservation method were obtained. Before the helminthological analysis, each specimen was weighed and its total length measured.

Each specimen was dissected, its viscera were placed in a petri dish containing distilled water to allow the release of larvae, and the interior of the viscera were examined under a stereoscopic microscope. Anisakid larvae obtained, as well as other potential helminths found, were individually preserved in jars, containing 70% ethanol. The flesh of each specimen underwent artificial pepsic digestion following the classic method proposed by Huang (1990), which simulates natural digestive conditions. The digested flesh was recovered in a sieve of small diameter in order to get rid of liquid and retain any potential larvae present. The product resulting from the digestion was transferred to a petri dish and examined under a stereoscopic microscope. The helminths encountered in the flesh were also preserved in individual jars.

Each larva found was placed between a slide and cover glass with a few drops of lactophenol for adequate clarification, which in turn makes it possible to visualize the internal organography through optic microscopy. Identification of each specimen was based on the morphological characteristics described by Berland (1989) and Grabda (1991), considering, mainly, the size, the characteristics of the esophagic ventricle, presence or absence of the intestinal caecum and/or ventricular appendix, characteristics of the anterior and posterior end, and the position of the excretory pore. However, molecular diagnosis of each specimen was not carried out, as the aim of this study was not the identification of anisakids at a specific level, but the risk assessment for consumers derived from the prevalence and abundance of A. simplex complex as a whole.

For the total of all anisakids found, as well as for each of the identified species, and also the microhabitat (viscera and flesh), origin, and season of capture, the following parameters were analyzed: the number of parasitized hosts, the range of parasitization, prevalence, and mean abundance, according to Bush et al. (1997).

The helminthoecological study was carried out solely for A. simplex complex, as it was the only species that remained alive after digestion and that consequently posed any infestation risk. Statistical methods were not applied to Hysterothylacium sp. as its prevalence was low and live specimens were not encountered in the flesh, but also because there are only a few references in the literature implicating it as a human parasite (Yagi et al., 1996).

Statistical analysis

The analysis was completed by the comparison of prevalences, using the χ² test, and abundances, applying the Mann-Whitney (U) and the Wilcoxon (Z) tests. Moreover, to analyze the potential influence on the two dependent variables of intrinsic (weight and length) and extrinsic factors (origin, season, and days after capture), binary logistic regression for prevalences, and analysis of variance (ANOVA; F) and the Spearman test (Rho) for abundances were applied. For the ANOVA analysis, as the number of helminths was highly aggregated, the helminth abundance of each infrapopulation was normalized by ln (x+1). In the binary logistic regression analysis, to know the influence of each factor included in the models, the odds ratio (OR) has been incorporated taking as reference categories W2, L2, Mediterranean, and spring, when dichotomic independent variables are considered. Moreover, when considering parasitized hosts only, the potential influence of the days after capture on the presence of A. simplex complex in the flesh was also analyzed. Statistical significance was established at p<0.05.

Results

A total of 55.6% (51.1% in viscera and 28.5% in the flesh) of the blue whiting specimens analyzed were parasitized by L₃ larvae of anisakids. The only two species found were classified as A. simplex complex and Hysterothylacium sp.

Hysterothylacium sp.

A total of 12.0% of the blue whiting specimens was parasitized by Hysterothylacium sp. larvae, 8.5% in viscera and 6.0% in the flesh (Table 2), and mean abundance for the total was 0.64 (Table 3).

Table 2.

Prevalence (P) of Hysterothylacium sp. in the Blue Whiting, with Respect to Its Geographic Origin and Season of Capture

	Viscera		Flesh		Total
Sample	No.	P (95% CI)	No.	P (95% CI)	No.	P (95% CI)
Atlantic	16	10 (6–16)	8	5 (2–9)	21	12 (8–18)
Mediterranean	8	7 (3–13)	9	8 (4–15)	13	11 (6–18)
Autumn	—	—	1	1 (0–5)	1	1 (0–5)
Spring	24	16 (11–23)	16	11 (7–17)	33	22 (16–29)

CI, confidence interval; No., number of infected fish.

Table 3.

Mean Abundance (mA) and Intensity Range of Hysterothylacium sp. in the Blue Whiting, with Respect to Its Geographic Origin and Season of Capture

	Viscera		Flesh		Total
Sample	mA (SE)	Range	mA (SE)	Range	mA (SE)	Range
Atlantic	0.2 (0.1)	1–8	0.2 (0.1)	1–17	0.3 (0.2)	1–25
Mediterranean	0.5 (0.4)	1–51	0.6 (0.3)	1–30	1.1 (0.7)	1–76
Autumn	—	—	0.1 (0.01)	1	0.1 (0.01)	1
Spring	0.6 (0.3)	1–51	0.6 (0.3)	1–30	1.2 (0.6)	1–76

SE, standard error.

No differences were found in prevalences and mean abundances when viscera and flesh were compared.

Anisakis simplex complex

A total of 53.9% of the blue whiting specimens were parasitized by A. simplex complex larvae, 49.6% in viscera and 26.1% in the flesh. Prevalences were higher in viscera (χ²=32.600; p<0.0001) and in the Atlantic (χ²=91.537; p<0.0001), but no statistical significant differences were found between autumn and spring (Table 4).

Table 4.

Prevalence (P) of Anisakis simplex Complex in the Blue Whiting, with Respect to Its Geographic Origin and Season of Capture

	Viscera		Flesh		Total
Helminth species	No.	P (95% CI)	No.	P (95% CI)	No.	P (95% CI)
Atlantic	124	73 (66–80)	65	39 (32–47)	131	78 (72–84)
Mediterranean	17	15 (9–23)	8	7 (3–13)	22	19 (12–27)
Autumn	60	45 (37–54)	31	23 (16–31)	64	48 (39–57)
Spring	81	54 (46–62)	43	29 (22–37)	89	59 (51–70)

CI, confidence interval; No., number of infected fish.

Mean abundance for the total sample was 3.90 helminths/fish, being higher in the specimens originating from the Atlantic (U=3375; p<0.0001), as well as in the group with a greater length (U=6524; p<0.0001) and weight (U=7622; p<0.0001). Moreover, mean abundance was also higher in viscera than in the flesh, if they originated from the Atlantic (Z=7.448; p<0.0001), as well as for the two seasons, autumn (Z=5.097; p<0.0001) and spring (Z=5.720; p<0.0001) (Table 5).

Table 5.

Mean Abundance (mA) and Intensity Range of Anisakis simplex Complex in the Blue Whiting, with Respect to Its Geographic Origin and Season of Capture

	Viscera		Flesh		Total
Helminth species	mA (SE)	Range	mA (SE)	Range	mA (SE)	Range
Atlantic	4.0 (0.8)	1–90	0.8 (0.1)	1–12	4.8 (0.9)	1–95
Mediterranean	1.9 (1.6)	1–184	0.6 (0.4)	1–35	2.5 (1.9)	1–219
Autumn	3.8 (1.1)	1–90	0.6 (0.1)	1–12	4.4 (1.1)	1–95
Spring	2.6 (1.2)	1–184	0.8 (0.3)	1–35	3.4 (1.5)	1–219

SE, standard error.

Influence of intrinsic and extrinsic factors on Anisakis simplex complex

The results of the binary logistic regression show that the prevalence of A. simplex complex is influenced by intrinsic factors (χ²=135.490; p<0.0001), mainly individual weight (OR=2.760), as well as extrinsic factors, namely origin (OR=0.027) and season of capture (OR=8.747). Moreover, and taking into account only parasitized hosts, a significant statistical relation was found between the presence of parasites in the flesh and the number of days passed between the catch and the analysis of the specimens, both for the total of the analyzed sample (χ²=5.119; p=0.024; OR=1.183) as well as for those originating from the Atlantic (χ²=4.100; p=0.043; OR=1.168) and spring (χ²=4.829; p=0.028; OR=1.1229).

Intrinsic factors, length (Rho=0.382; p<0.0001) and weight (Rho=0.251; p<0.0001), as well as extrinsic factors, origin (F=6.012; p=0.015), and season (F=21.207; p<0.0001), influenced the abundance of A. simplex complex in the total of the analyzed sample.

Discussion

The results obtained in this regional study from València can easily be extrapolated to the whole of Spain, as the nationwide supermarket chains surveyed sell fish originating from almost the same fishery areas. The high prevalence of parasitization reported herein confirms the results obtained concerning the blue whiting by other studies carried out in the Iberian Peninsula: 69.9% in Granada (Ruíz-Valero et al., 1992), 85.5% in Saragossa (Viu et al., 1996), 63.6% in Castile-Leon (Pereira Bueno and Ferré Pérez, 1997), 42.0% in Cordoba (De la Torre Molina et al., 2000), and 54.0% in Cadiz (Ojeda-Torrejón et al., 2001).

For Hysterothylacium sp., in general, the highest prevalences have been detected in viscera and in the specimens originating from the Atlantic; the same goes for A. simplex complex.

The high A. simplex complex prevalence in fish originating from the Atlantic has also been reported by other authors (Sanmartín et al., 1994; Abollo et al., 2001; Fernández et al., 2005; Cruz et al., 2007) and may be due to various factors, namely the higher abundance of definitive and intermediate hosts in this area (Arthur et al., 1982; Boily and Marcogliese, 1995), the lower temperature of the Atlantic waters when compared to the Mediterranean (Højgaard, 1998), or the diet of intermediate and definitive hosts (Rello-Yubero et al., 2004).

The high prevalence of anisakids in the blue whiting caught in spring has also been reported by De la Torre Molina et al. (2000) for fish sold in Cordoba, and by Strømnes and Andersen (2000) for Norwegian waters, which may be due to the increased migration of whales in this season, and consequently a greater presence of anisakid eggs, as well as a higher proliferation of zooplankton, permitting the continuity of the biological cycle (Boily and Marcogliese, 1995; Køie and Fagerholm, 1995; Køie et al., 1995).

The influence of intrinsic factors such as weight and length on the A. simplex complex prevalence and abundance, which are higher in longer and heavier specimens, may be due to the accumulation of helminths as a consequence of the larger amount of intermediate hosts ingested. Strømnes and Andersen (2000) suggested that a fish of a larger size—and thus being nearer the top of the trophic chain—has a greater likelihood of ingesting infected intermediate hosts.

The higher prevalence and abundance found in viscera when compared to the flesh were expected considering the fact that viscera are the usual microhabitat of anisakid larvae. However, the days between the catch and the sale influenced the A. simplex complex presence in the flesh of parasitized fish. The blue whiting and other fish that mainly feed on euphausiids tend to accumulate anisakid larvae in their viscera, although larvae are more abundant in the hypoaxial musculature of piscivorous fish such as cod. Some authors have suggested that fatty species (such as anchovies, and blue fish in general) present lower parasitic prevalences but a greater migration to the flesh, whereas less fatty fish (such as the blue whiting) present higher prevalences but a lower migration, preferably to the liver (Smith, 1984). The relation between the presence of anisakids in the flesh and the number of days passed between the capture and the sale is of particular importance considering that one larva alone may already cause anisakidosis. Consequently, the infestation risk is significant and increases along with the number of days passed between the catch and its consumption.

In general, it can be affirmed that the main parasite species encountered in the blue whiting was A. simplex complex. Its high prevalence also coincides with the fact that this anisakid has shown to be resistant to digestion, contrary to Hysterothylacium sp., and thus poses the greatest risk for the consumer.

In the prevention of anisakidosis, as required by European as well as Spanish legislation, adequate health education of consumers with regard to fish preparation at home is of utmost importance so that consumers become aware of the danger that the consumption of raw or undercooked fish, without prior freezing, poses and that simple—yet effective—prevention measures have to be applied. Additionally, as confirmed by the results of the present study, the increase of the transmission risk through a prolonged time lag between the catch and the consumption, especially in the blue whiting of the Atlantic origin, caught in spring and with a larger size and weight, has to be considered.

Thus, the following recommendations, and verified by the results of this study, should be adhered to: preferably consume the blue whiting from the Mediterranean, choosing, moreover, the smallest specimens (not only because of their lower prevalence but also because the recommended prevention measures can be applied more easily as their small size allows for a homogeneous temperature whilst being cooked to inactive the parasite in the entirety of the fish), and above all consume the fish fresh and previously eviscerated, preferably straight after the catch. Information concerning the place and the date of capture, preservation method, as well as the scientific and the common name of the fish, included on the crate, should be easily visible for the consumers, who are thus informed about the most important variables likely to increase the risk of anisakidosis.

Footnotes

Authors' Contributions

Elena Madrid gathered and analyzed the data. Màrius V. Fuentes and M. Teresa Galán-Puchades designed the study and the analysis. All authors were involved in the writing of the manuscript, and all of them read and approved its final version.

Disclosure Statement

No competing financial interests exist.

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