Abstract

Introduction

The consumption of marine fish has increased over recent decades, raising the number of cases of human anisakidosis. The main agent responsible for this parasitosis is the third larval stage (L3) of Anisakis Dujardin, 1845 (Anisakidae), nematodes which are widely found in fish and cephalopods, with high mean prevalence and intensity rates in many species of commercial interest (Chía et al. 2010; Pekmezci 2014; Klapper et al. 2015). Human beings get infected when they consume raw or undercooked fish or cephalopods. According to various authors, these nematodes are highly resistant to the conditions created by traditional marinating methods; they are able to survive for periods of a few days up to several weeks, depending on the concentration of salt, acetic acid and marinating times (Karl et al. 1995; Sánchez-Monsalvez et al. 2005; AESAN 2007; Giarratana et al. 2012; Anastasio et al. 2015). All these circumstances lead to frequent reports of anisakidosis all over the world, with Japan at the top of the list with more than 2000 cases a year, due to high raw fish consumption in this country. When larvae invade the human gastrointestinal tract, they are usually found in the gastric cavity, they can also be located in various different areas of the intestine. Clinical symptoms range from digestive problems, which vary depending on the location of the larvae, to allergic reactions of varying severity, and sometimes both at the same time. In terms of prevention, various different prophylactic measures were proposed shortly after the first cases were diagnosed in humans. The European Union and WHO, now aware of the problem, stress the importance of freezing fish and seafood products destined for salting, marinating and cold smoking, at a temperature of −20°C or lower for at least 24h, or heating the fish to over 60°C. For its part, the FDA advises freezing the fish at −20°C for seven days or at −35°C for 15h in order to kill any larvae. Nevertheless, it would be of interest to have other alternative measures at our disposal in daily practices that guarantee the destruction of the larvae. To fulfil this aim, various studies reporting on the larvicidal effect of different natural products have been published, especially the essential oils and/or their components (Hierro et al. 2004, 2006; Navarro et al. 2008; Romero et al. 2012, 2014; Giarratana et al. 2014, 2015a,b; Gómez-Rincón et al. 2014; Valero et al. 2015). In this study, various essential oils (EOs) extracted from plant species present in the Mediterranean region have been evaluated. The specific purpose of this research was to study the potential action of these EOs on Anisakis larvae in the musculature of fish and later on to assess their infectivity on laboratory animals.

Materials and methods

Results

In vitro studies

In the trials conducted with the parasitized fish treated with the Eos, a displacement of the larvae was generally observed from the parasitized tissues to the container in which the test was conducted. This migration was above 60% in all cases. Moreover, all the EOs tested produced a drop in the survival rate of the larvae. The highest total mortality rate was detected for oregano EO (53.9%), with the larvae showing severe oesophageal and intestinal deterioration (Figure 1). Cumin, thyme and Spanish lavender EOs proved less lethal than the oregano one (20.3, 11.4 and 3.5% mortality rates, respectively). For the lavender, marjoram and rosemary EOs the survival rate was 100%. For the larvae that remained alive, a reduction in spontaneous movement was detected, with the maximum reduction (65.8%) corresponding to larvae treated with cumin EO and the minimum (12.3%) to Spanish lavender EO; results in between these values were obtained for thyme (53.4%), lavender (31.7%) and oregano (31.4%) EOs. None of the larvae treated with marjoram and rosemary EOs required stimulation to be actively mobile. In the controls, all the larvae displayed great mobility. Table 2 displays data related to the larvae that abandoned the fish muscle or remained in it, and their survival rates and mobility following exposure to the different EOs.

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Figure 1.

Larva L3 of Anisakis after being treated with essential oil of oregano in vitro. It can be observed in oesophageal and intestinal damage.

Table 2.

In vitro study of the effect of the EOs against the L3 of Anisakis type I.

Total L3		Container				Muscle
		Total	NS	S	X	Total	NS	S	X
Spanish lavender	57	41 (71.9%)	32 (78.1%)	7 (17.1%)	2 (4.9%)	16 (28.1%)	16 (100%)	–	–
Cumin	237	196 (82.7%)	28 (14.3%)	123 (62.8%)	45 (23.0%)	41 (17.3%)	5 (12.2%)	33 (80.5%)	3 (7.3%)
Lavender	126	107 (84.9%)	71 (66.4%)	36 (33.7%)	–	19 (15.1%)	15 (79.0%)	4 (21.1%)	–
Marjoram	89	78 (87.6%)	78 (100%)	–	–	11 (12.4%)	10 (100%)	–	–
Oregano	102	62 (60.8%)	–	9 (14.5%)	53 (85.5%)	40 (39.2%)	15 (37.5%)	23 (57.5%)	2 (5%)
Rosmary	49	34 (69.4%)	33 (100%)	–	–	15 (30.6%)	15 (100%)	–	–
Thyme	88	69 (78.4%)	21 (30.4%)	38 (55.1%)	10 (14.5%)	19 (21.6%)	10 (52.6%)	9 (47.4%)	–

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NS: L3 alive with no stimulus; S: L3 alive with stimulus; X=L3 dead.

Discussion

Human anisakidosis presents a risk to public health that must be reduced through basic prevention. Steps to control the disease include various measures and checks to detect and kill the larvae in fish, thus preventing infection of the consumer. The main recommendations are freezing the fish or exposing it to temperatures over 60°C. However, essential oils have long been recognized as effective plant-based antimicrobials and have been used to preserve foods for centuries, thus they could be good alternatives to the abovementioned methods to protect against anisakidosis. Indeed, all seven essential oils tested in this study did somehow appear to stimulate a high percentage of the Anisakis L3 in the fish musculature to abandon it and migrate into the container (Table 2): the values were ≥70% for the lavender, Spanish lavender, marjoram, cumin, thyme and rosemary EOs, and slightly lower for the oregano one (60.8%). These data are of particular interest given that the muscle is the part of the fish that humans consume, and thus the reduced presence of parasites in this part would reduce the risk of contracting this disease. Furthermore, during this process a certain number of larvae in the fish died or lost spontaneous mobility on coming into contact with the essential oils (Table 2): the two most effective EOs in this sense were cumin and oregano, for which only 13.9% and 14.7% of the larvae, respectively, exhibited spontaneous movement. The favourable results obtained for these two EOs against Anisakis larvae are in keeping with those described by Valero et al. (2006), although this study was conducted under different conditions. The GC–MS analysis of cumin EO revealed the presence of its major components: cuminaldehyde (34.1%), 2-caren-10-al (20.8%), p-cymene (12.3%) and 3-caren-10-al (11.8%); carvacrol was detected in a high proportion (88.4%) in the oregano oil, while a low proportion of p-cymene (3.3%) and aromadendrene (2.4%) was found (Table 1). These compounds have been identified in both EOs, displaying inhibitory activity against certain pathogenic agents (Martinez-Velazquez et al. 2011; Dussault et al. 2014; Kedia et al. 2014; Fournomiti et al. 2015). The chemical profile of essential oil of a particular plant species shows different chemotypic variations because of ecological and geographical conditions, age of plant and time of harvesting (Prakash et al. 2010). Such chemotypic variations would definitely affect the biological activity of the essential oil (Kedia et al. 2014). Thyme EO exhibited less anti-anisakis activity: it only achieved the death of 11.4% of larvae but a high percentage of larvae (53.4%) needed stimulus to move. For thyme EO at the same concentration (5%), Giarratana et al. (2014) indicated 100% mortality after 14h of treatment; this difference in larvicidal activity could be due to the test conditions and the chemical composition of the essential oil used by these authors [thymol (50%), linalool (7%), carvacrol (3%), α-pinene and β-pinene (6%)] in comparison with the thyme EO employed by us [thymol (76.3%), p-cymene (17.7%), carvacrol (2.4%) and linalool (1.4%)] (Table 1). The phenolic molecules thymol and carvacrol have been observed as the more active constituents (di Pasqua et al. 2005), being principally responsible for the antimicrobial activity, for example, against E. coli, K. oxytoca and K. pneumoniae (Fournomiti et al. 2015). The rest of the EOs did not display biocidal activity against the L3s.

With regard to the in vivo trials, the action of oregano, cumin and Spanish lavender EOs on the L3s in the fish muscle resulted in the larvae not causing any lesions in the animals’ digestive tract. The antimicrobial activity of essential oils, which are markedly lipophilic compounds, is related to their chemical composition, in which there is a predominance of terpenic derivatives (mono- and sesquiterpenes). Although its mechanism of action has not yet been fully explained, it is likely that in most cases it involves disruptions of the membrane, increasing the permeability of the bacterial cytoplasmic membranes, and of the mitochondrial membranes in eukaryotic organisms (Cowan 1999; Burt 2004). A decrease in the hydrophobicity of certain terpenes by the addition of methyl groups reduces their antimicrobial activity (Mendoza et al. 1997; Usano 2012). Furthermore, the main component of oregano EO, carvacrol, is known to interact with the cell membrane of some organisms, where it dissolves in the phospholipid bilayer and aligns between the fatty acid chains. The distortion of the physical structure causes expansion of the membrane, thus bringing about an increase in its permeability (García-García & Palou-García 2008). Moreover, carvacrol and 1,8-cineole, alone or in combination, severely affected the viability of the bacteria and caused dramatic changes in the cell membrane permeability, leading to cell death, as observed by confocal laser microscopy (de Sousa et al. 2015).

In the case of cumin EO, the activity of its main component, cuminaldehyde, against various pathogenic agents including Anisakis L3 larvae has been described, although the exact way in which it acts is still unknown (Hierro et al. 2004; Yeom et al. 2012).

With regard to the action of Spanish lavender EO, we should point out that although it did not show larvicidal action in the in vitro studies, in the in vivo trials no lesions were observed in the infected animals treated with this EO. The chemical composition identified in this oil was: fenchone (48.7%), camphor (40.0%) and 1,8-cineole (5.2%), so the EO analyzed could be considered a camphor-fenchone chemotype, according to Granger et al. (1973). Others authors detected these molecules in the Spanish lavender EO as its most representative components (Zrira & Benjilal 2003; Dadalioglu & Evrendilek 2004), although differences in the percentages have been found: fenchone, 14.9–75.5%; camphor, 2.5–56.2%; and 1,8-cineole, 0.2–8% (Ristocelli et al. 1998). Considering the high amount of fenchone detected in our oil, the difference in activity between the in vitro and in vivo trials for this EO may be related to a possible conversion, facilitated by the gastric pH, of fenchone into its alcohol derivative, fenchol, a compound which has been shown to be effective against G+and G−bacteria, as well as against various fungi (Kotan et al. 2007; Guleria et al. 2013).

It should also be highlighted that in the case of the larvae treated with marjoram EO, although one larva was found in the body cavity of the animal, there were no visible signs of lesions in the digestive tract. This leads us to speculate that this EO, whose main component is 1,8-cineole (68.1%), may have favoured the reabsorption of the haemorrhage. This could be due to the anti-inflammatory activity of 1,8-cineole which, in agreement with the results obtained by Abu-Darwish et al. (2013) would exert an anti-inflammatory effect through a mechanism involving the inhibition of LPS-induced NO production. This molecule has also been shown to be the majority constituent in the chromatogram of this EO, followed by terpenoids (Waller et al. 2016).

Conclusions

The essential oils tested in this work provoked the migration of the Anisakis larvae out of the fish, therefore they might well be added to the marinated fish plates. This fact, together with the biocidal action of some of them, particularly Spanish lavender, cumin and oregano EOs, are promising active ingredients that could be used in the development of effective products for the control of anisakidosis. Further studies on safety and palatability of the marinating with essential oils should be carried out.

References