Presence of Anisakid Larvae in the European Anchovy, <I

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Journal of Food Protection, Vol. 76, No. 9, 2013, Pages 1643–1648 doi:10.4315/0362-028X.JFP-13-092 Copyright G, International Association for Food Protection

Research Note Presence of Anisakid Larvae in the European Anchovy, Engraulis encrasicolus, Fished Off the Tyrrhenian Coast of Central Italy

CLAUDIO DE LIBERATO,1* TERESA BOSSU` ,1 PAOLA SCARAMOZZINO,1 GIUSEPPE NICOLINI,2 PIETRO CEDDIA,3 SALVATORE MALLOZZI,4 SERENA CAVALLERO,5 AND STEFANO D’AMELIO5

1 2 Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Rome, Italy; Azienda Sanitaria Locale ASL RM/F, Via Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/9/1643/1686929/0362-028x_jfp-13-092.pdf by guest on 27 September 2021 Terme di Traiano 39/A, 00053 Civitavecchia (Rm), Italy; 3Azienda Sanitaria Locale ASL RM/H, Viale Severiano 5, 00042 Anzio (Rm), Italy; 4Azienda Sanitaria Locale ASL Latina, Comprensorio Formia/Gaeta, Via degli Eroi, 04026 Minturno (Lt), Italy; and 5Department of Public Health and Infectious Diseases, Parasitology Section, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy

MS 13-092: Received 7 March 2013/Accepted 18 April 2013

ABSTRACT A survey was carried out to determine the prevalence of anisakid nematode larvae in European anchovy (Engraulis encrasicolus) fished off the Tyrrhenian coast of central Italy. From February through July 2012, 1,490 specimens of E. encrasicolus caught in three different fishing areas (off Civitavecchia, Anzio, and Gaeta in the northern, central, and southern Lazio region of Italy, respectively) were tested for the presence of anisakid larvae, both by visual microscopic inspection and enzymatic digestion. In each of the three fishing areas, each of two sampling times produced 250 fish (with the exception of one sampling time in Gaeta that produced 240 fish). Larvae of the family Anisakidae were detected with an overall estimated prevalence of 2.3%, and each positive fish harbored a single larva. No anisakid larvae were detected in fish caught off Gaeta. Fish with larvae were significantly longer (standard length) than fish without larvae. Twenty-six larvae (74.3%) were detected by visual inspection of the viscera, eight larvae (22.8%) were detected by visual inspection of the fillets, and one larva (2.8%) was detected after digestion of pooled fillets. Molecular analysis to fully characterize the 35 detected larvae revealed 15 specimens of Anisakis pegreffii, 10 specimens of Hysterothylacium aduncum, and one hybrid genotype of A. pegreffii | Anisakis simplex. For nine specimens, no visible product was obtained after PCR amplification. The overall prevalence for A. pegreffii and H. aduncum was 1.0 and 0.7%, respectively. A comparison between fishes harboring A. pegreffii larvae and those harboring H. aduncum revealed that those with A. pegreffii were significantly heavier. The prevalence of anisakid larvae found in the present study is lower then that reported previously in E. encrasicolus collected in the Mediterranean Sea.

Nematodes of the family Anisakidae are parasites of Human anisakidosis was first described in The Nether- marine mammals and fish-eating birds and are distributed lands in 1960 (36) and occurs more frequently in those worldwide, both in temperate and cold waters. Second-stage countries where consumption of raw or undercooked fish is larvae hatching from eggs must be consumed by an a traditional practice (e.g., Japan, countries bordering the intermediate host, usually a crustacean. Fish or squid Mediterranean Sea, and countries in northern Europe). The (paratenic hosts) become infected after eating an infected incidence of this parasitosis is growing worldwide because crustacean. The life cycle is completed after the paratenic of increasing use of raw fish and the growing populations of host is ingested by a definitive host. Humans can acquire the definitive hosts recorded in many areas of the world since infection by eating raw or undercooked infected marine fish the introduction of regulations aimed at the protection of or squids (1). Humans are dead-end hosts for these parasites, marine mammals (4, 9). Increasing apparent incidence of because anisakid larvae cannot transform to adults in anisakidosis also may be due to improved endoscopic humans and usually are excreted without causing any diagnostic techniques and an increased awareness of this clinical disease. However, occasionally the intake of these parasitosis. At present, 20,000 cases of human anisakidosis parasites can cause gastrointestinal disorders and/or allergic have been described (90% originating from Japan) (20), and reactions (5). From a zoonotic point of view, the relevant 2,000 new cases are reported annually. In Italy, 30 human genera of the family Anisakidae are Anisakis and Pseudo- cases of anisakidosis have been described, mainly in the terranova, in particular the complex of species Anisakis Puglia region (southern Italy). Several new cases (not simplex and Pseudoterranova decipiens, although larvae of described in the literature) are also reported annually by the genera Contracaecum and Hysterothylacium have been sanitary authorities in many regions of central and southern rarely associated with disease in humans (20, 38). Italy. Given the high prevalence of anisakid larvae in fish species of high commercial value, some of which are * Author for correspondence. Tel: z390679099336; Fax: z3906790993 commonly consumed raw in traditional dishes (3), the real 31; E-mail: [email protected]. incidence of this parasitosis in humans is likely to be 1644 DE LIBERATO ET AL. J. Food Prot., Vol. 76, No. 9 underestimated, both because of missed diagnosis and the (41u059N, 13u399E). Fishing areas were chosen based on coverage high frequency of self-limiting infections. of the entire coast of the Lazio Region and the presence of a fishing Many fish species can harbor anisakid larvae (2, 11, fleet dedicated to anchovy fishing. 31). In the Mediterranean Sea, anisakid nematodes have Between February and July 2012, 1,490 E. encrasicolus were been found in several economically relevant fish species, tested for the presence of anisakid larvae. In each of the fishing areas, two sampling sessions were used, each one producing 250 with prevalence exceeding 50% in Lepidopus caudatus, fish (240 fish at the second sample session off Gaeta). Anchovies Scomber scombrus, and Dicentrarchus labrax and 30% in caught by professional fishermen were randomly selected by Local Zeus faber and Lophius piscatorius (4, 7, 10, 24). Anisakis Sanitary Authority veterinarians after the fishing boats landed and and Hysterothylacium larvae have been recorded in 43.8% immediately transferred to the Istituto Zooprofilattico Sperimentale of all fish marketed in two towns of the Adriatic coast of delle Regioni Lazio e Toscana. Fish were stored at 4uC during central Italy (17). transport and storage until they were examined for parasites. The European anchovy (Engraulis encrasicolus)isa Fish inspection. After standard length and weight measure-

common paratenic host for Anisakidae in the Mediterranean Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/9/1643/1686929/0362-028x_jfp-13-092.pdf by guest on 27 September 2021 and a significant source of infection for humans. In Italy and ments were obtained, fish were tested individually for the presence Spain, consumption of marinated anchovies (Italian ac- of nematode larvae of the family Anisakidae. Viscera and fillets of ciughe marinate and Spanish boquerones en vinagre)is each fish were placed in two different petri dishes and examined separately. Tissue were examined for anisakid larvae by visual considered the main risk factor for human anisakidosis (6, inspection under a stereomicroscope. After visual inspection, fillets . Many researchers have reported the presence of 22, 23, 29) were pooled up to 100 g of material. Pooled fillets were then Anisakis spp. and Hysterothylacium aduncum third-stage digested following the procedure of Angelucci et al. (4) and filtered larvae in E. encrasicolus in the Mediterranean Sea, with through 1- and 0.5-mm-pore-size sieves. Material collected on the prevalence values of 26.6 to 77.0% (3, 4, 17, 27, 28). H. sieves was then observed under the stereomicroscope. aduncum is usually more commonly reported than Anisakis Collected larvae were individually preserved in 70% ethanol sp. in anchovy. Rello et al. (28) studied four different and transferred to the Parasitology Section of the Sapienza fishing areas of the western Mediterranean Sea. H. aduncum University of Rome for molecular identification. was more prevalent than Anisakis sp. in all four areas, with prevalences of 68.9 and 3.9% for H. aduncum and Anisakis Molecular identification. Nematodes collected from fish sp., respectively, in the Gulf of Lion. These authors stated samples were identified to species using a molecular approach that prevalence of infected anchovies originating from based on PCR–random fragment length polymorphism analysis of the nuclear ribosomal internal transcribed spacer (ITS) region. different fishing areas could differ significantly, with higher Genomic DNA was isolated using the Wizard Genomic DNA prevalences estimated in the northwestern Mediterranean purification kit (Promega, Madison, WI), according to the (Gulf of Lion and Ligurian Sea). Other studies carried out manufacturer’s protocol. on different fish species have highlighted differences in The entire ITS (ITS1, 5.8S rRNA gene, and ITS2) was anisakid prevalence in relation to the fishing area, probably amplified by PCR using 5.0 ml of template DNA (20 to 40 ng), linked to lower or higher abundances of the definitive hosts 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2 (Bioline, Taunton, of these parasites (25, 26). MA), 40 mM nucleotide mix (Promega), 50 pM/ml concentrations Based on number of landed tons (54,388 tons [4.9 | of the forward primer NC5 (59-GTAGGTGAACCTGCGGAAG- 107 kg] in 2009), the anchovy is the most relevant species GATCAT-39) and reverse primer NC2 (59-TTAGTTTCTT- for the Italian fishery, representing 23% of the national CCTCCGCT-39) (39), and 1.0 U of BIOTAQ DNA polymerase fishery production. Lazio Region (central Italy, where (Bioline) in a final volume of 50 ml. The PCR was performed in a marinated anchovies are a traditional food) contributes GeneAmp PCR system 2400 (Applied Biosystems, Foster City, CA) under the following conditions: 10 min at 95 C (initial 7.9% of Italian anchovy fishery, and although most of the u denaturation), 30 cycles of 30 s at 95uC (denaturation), 40 s at product is consumed locally, anchovies originating from this 52uC (annealing), and 75 s at 72uC (extension), and a final area are also exported to other European Union countries elongation step of 7 min at 72uC. A negative control (without such as Spain, Greece, and Germany. genomic DNA) was included for each set of amplification Because data regarding anisakids in E. encrasicolus reactions. Aliquots (5 ml) of individual PCR products were fished off the Tyrrhenian coast of the Lazio Region are separated by electrophoresis on agarose gels (1%), stained with inadequate, the present study was conducted to better define ethidium bromide (0.4 mg/ml), and visualized with UV transillu- the risk of exposure to larvae of the family Anisakidae mination. Gel images were captured electronically and analyzed through consumption of E. encrasicolus. An additional using the program Multi-Analyst (v. 1.1, Bio-Rad, Hercules, CA). reason for conducting this study was to document the well- ITS-positive amplicons were subsequently digested with the known differences in anisakid prevalence in fish from restriction endonuclease HinfI because the resulting restriction different fishing areas, which makes it difficult to generalize patterns have been useful for the identification of anisakid species findings even to nearby areas. (13). Sampling and statistical analysis. The aim of sampling was MATERIALS AND METHODS to estimate the prevalence of anisakid larvae in anchovy, which is Study area. Three fishing areas off the coast of Lazio Region, known to harbor this parasite. Sample size was determined central Italy, were selected to cover a linear distance of about according to the following criteria: 20% expected prevalence 200 km (Fig. 1): Civitavecchia in the north (41u599N, 11u419E), (28), 5% precision, and 95% confidence level. Test sensitivity and Anzio in the center (41u209N, 12u289E), and Gaeta in the south specificity were both assumed to be 100%. An analysis of variance J. Food Prot., Vol. 76, No. 9 ANISAKID LARVAE IN E. ENCRASICOLUS FROM CENTRAL ITALY 1645

FIGURE 1. Study area, Lazio Region of central Italy, indicating the landing har- bors of each fishing area. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/9/1643/1686929/0362-028x_jfp-13-092.pdf by guest on 27 September 2021

was used to test differences in size and weight of fishes from the were found in fillets via visual inspection, and only 1 (2.8%) three fishing areas. Association between variables was tested using was detected after digestion of pooled fillets. 2 the x test, and the significance of the differences between positive Both in Civitavecchia and Anzio, estimated prevalences and negative fish in size and weight was tested using a t test (95% were higher during the first sampling time of the season. In confidence interval). The cutoff for significance was set at P ~ Civitavecchia, the prevalence was 6.0% in February and 0.05. Confidence intervals in Table 1 were calculated using Open- 3.2% in May; in Anzio, the prevalence was 3.6% in April epi software (14). and 1.2% in June (P ~ 0.025). Molecular analysis produced 26 ITS-positive ampli- RESULTS cons, and the restriction patterns allowed identification of Average lengths and weights of examined anchovies the nematodes to species. Fifteen larvae produced three are shown in Table 1. Fish from Anzio were not bands of about 370, 300, and 250 bp, which were significantly longer or heavier than those from Gaeta, and representative of A. pegreffii, 10 larvae produced two bands those from Civitavecchia were in the middle (P . 0.05 for of about 700 and 350 bp, representative of H. aduncum, and all comparisons). 1 larva produced four bands (620, 370, 300, and 250 bp), Thirty-five larvae of nematodes of the family Anisaki- indicative of a heterozygote genotype of A. pegreffii | A. dae were detected, with an overall prevalence of 2.3% simplex s.s. Nine larvae could not be identified to species (Table 1). All infected fish harbored only a single larva. because DNA amplification was not successful; therefore, Infected anchovies were significantly longer (10.7 ¡ they were recorded only as Anisakidae. A. pegreffii and H. 1.1 cm) than uninfected fish (9.9 ¡ 1.1 cm) (P , 0.05). aduncum were present in both fishing areas (Table 1), with No anisakid larvae were found in fish caught in the Gaeta A. pegreffii more prevalent. H. aduncum was more abundant fishing area, but 4.6 and 2.4% of fish from Civitavecchia in Civitavecchia than in Anzio; in Anzio A. pegreffii was and Anzio respectively, were infected (Table 1). Twenty-six more prevalent than H. aduncum (Table 1). Fish harboring (74.3%) of the 35 larvae were found in viscera, 8 (22.8%) A. pegreffii larvae were significantly heavier than those

TABLE 1. Fishing area, sample size, standard length and weight of the Engraulis encrasicolus tested for anisakid larvae, and the prevalence of these larvaea

Mean (95% CI) prevalence (% of fish examined) of:

No. of fish Mean (SD) fish Mean (SD) fish Unidentified Anisakis Hysterothylacium Fishing area examined length (cm) wt (g) All anisakids anisakids pegreffii aduncum

Civitavecchia 500 10.2 (0.7) 8.6 (3.2) 4.6 (2.9–6.8) 1.6 (0.7–3.1) 1.4 (0.6–2.9) 1.6 (0.7–3.1) Anzio 500 10.5 (1.4) 11.3 (3.5) 2.4 (1.2–4.1) 0.2 (0.0–1.1) 1.6 (0.7–3.1) 0.4 (0.0–1.4) Gaeta 490 9.2 (0.7) 7.2 (1.4) 0 0 0 0 Total 1,490 9.9 (1.1) 9.0 (3.3) 2.3 (1.6–3.2) 0.6 (0.3–1.1) 1.0 (0.6–1.6) 0.7 (0.3–1.2) a SD, standard deviation; CI, confidence interval. 1646 DE LIBERATO ET AL. J. Food Prot., Vol. 76, No. 9 infected by H. aduncum (12.3 ¡ 5.1 and 8.3 ¡ 3.0 g, prevalence estimated in the present study could be due to respectively; P , 0.05). Although 33.3% (5 of 15) of A. both the relative small fish size (Table 1) (18) and to the pegreffii larvae were detected in fillets, only one H. paucity of definitive hosts (cetaceans) in the waters off aduncum larva (10%) was found in fillets. The hybrid central Italy (26). genotype A. pegreffii | A. simplex s.s. also was found in Infection intensity (one larva per infected host) was fillets. lower than values reported by previous studies, where intensities ranged from 1.0 to 17.0 larvae per infected host DISCUSSION (4, 28). The intensity estimated in the present study In terms of number of tested fish, this survey is the apparently fits with the overall finding of general low largest carried out to detect and estimate the prevalence of abundance of Anisakidae in the considered host species and anisakid larvae in E. encrasicolus from the Tyrrhenian Sea. fishing areas. Moreover, infection intensity is also reported The overall prevalence in the present study is lower than to be related to fish size (27). Many authors have reported migration of larvae from those previously published for Mediterranean anchovy, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/9/1643/1686929/0362-028x_jfp-13-092.pdf by guest on 27 September 2021 viscera to musculature after fish death (30, 37), which also which were 7.14 to 77.0%. In 2004, Fioravanti et al. (17) occurs in the genus Engraulis (21), with a consequent reported 53.1% prevalence of Anisakis sp. and Hysterothy- increased risk of infection in the edible part of the fish. lacium sp. larvae in 414 anchovies fished off the coasts of However, larval migration from viscera to fillets is Marche Region (Adriatic Sea) in 2001 and 2002. A 3-year controversial (15) and has not been confirmed in some study (2003 through 2005) on fish marketed along the studies. In the present study, the majority of larvae were Adriatic coast of central Italy revealed an overall 26.6% detected in viscera 18 to 24 h after fish death, theoretically prevalence of Anisakidae in 2,636 examined anchovies, enough time for larvae to migrate to muscles. Similar with values ranging from 9.8 to 56.5% depending on the findings were reported by Rello et al. (28) and Mladineo et fishing area (16). More recently, Rello et al. (28) reported a al. (27). Thus, the risk for human infection linked to larval 31.9% overall prevalence of anisakid larvae in anchovies in migration to edible parts of the fish probably would be four different fishing areas of the western Mediterranean lower for fish placed on ice (rather than refrigerated) Sea, with prevalence estimates close to our findings in the immediately after they were caught, as usually occurs for Alboran Sea fishing area, where Anisakis sp. and H. fish sent to retail markets. aduncum had prevalences of 1.4 and 2.8 , respectively. % Anisakis sp. and H. aduncum are the more frequently Angelucci et al. (4) reported 77.0% anisakid prevalence in reported anisakid taxa from Mediterranean fish and E. anchovies fished off the Sardinian coast. The only encrasicolus in particular (4, 8, 10, 17, 28). In almost all exceptions to these findings are the absence of anisakid studies, H. aduncum has been reported to be more larvae recorded in E. encrasicolus fished off Sicily (160 commonly found than Anisakis sp. Fioravanti et al. (17) examined specimens) (12) and the Catalan coast of reported that 98.6% of infected anchovies caught in the Terragona (153 examined specimens) (18). Those negative Adriatic Sea harbored Hysterothylacium larvae, and this findings are comparable to ours in the southernmost fishing type of larvae was the most prevalent in all fishing areas of area (Gaeta) of the Lazio Region. According to Guite´rrez- Adriatic Sea that have been examined. The overall Galindo et al. (18), lack of anisakids in anchovies could be prevalences of Anisakis sp. and H. aduncum in the western associated with fish size; anchovies would start feeding on Mediterranean anchovy were 9.35 and 24.5%, respectively intermediate Anisakidae hosts only after they have reached a (28), with H. aduncum dominant in all four fishing areas certain size. However, given the low prevalence recorded in examined. The same situation was reported by Angelucci our study, sample size of both these studies (12, 18) could et al. (4) from waters off Sardinia, with a 34.6% prevalence be too small to detect the presence of anisakid larvae in of Anisakis sp. and a 67.3% prevalence of Hysterothylacium anchovies in the fishing areas surveyed. sp. The present findings differ from those of these other A positive correlation between anisakid prevalence and reports in that A. pegreffii was more common than H. fish length and weight has been recorded in many fish aduncum, even though prevalence values for these two species (2, 19, 32–35). Regarding E. encrasicolus, Anasta- species were comparable and low. Hybrid specimens of A. sio et al. (3) found a 7.1% prevalence in anchovies , 9.5 cm pegreffii | A. simplex s.s. have been previously reported in long and a 42.8% prevalence in fish exceeding this length, anchovies caught in the Tyrrhenian Sea (8). in a fishing area close to those considered in the present As reported by Rello et al. (28), we found a higher study (gulf of Naples, Central Italy). The same finding was percentage of A. pegreffii larvae than H. aduncum larvae in reported by Rello et al. (28) and Mladineo et al. (27) in their the fish fillets, which represents a greater risk to consumers studies carried out in Western Mediterranean and Adriatic because A. pegreffii is more frequently reported as a cause Seas respectively. The relationship between anisakid of human anisakidosis. prevalence and fish size is probably due to an age/dependent In the present study, visual inspection was highly accumulation effect of parasites in fish (7); this association effective for detection of larvae; only a single additional could explain the absence of these parasites in Gaeta fishing larva was found after muscle digestion. However, visual area, where examined anchovies were smaller (albeit non inspection was carried out with a stereomicroscope, and the significantly) and therefore younger than those fished off relatively small size of the fish allowed thorough evaluation Civitavecchia and Anzio. In conclusion, the overall low of the transparent whole fillets. Similar results should not be J. 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