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Molecular identification of Anisakis spp. from fishes collected in the Tyrrhenian Sea (NW Mediterranean)
Veterinary Parasitology 187 (2012) 563–566
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Molecular identification of Anisakis spp. from fishes collected in the Tyrrhenian Sea (NW Mediterranean) S. Cavallero a , A. Ligas b , F. Bruschi c , S. D’Amelio a,∗ a b c
Department of Public Health and Infectious Diseases, Section of Parasitology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy Centro Interuniversitario di Biologia Marina ed Ecologia Applicata “G. Bacci”, Livorno, Italy Department of Experimental Pathology, M.B.I.E., Università di Pisa, Pisa, Italy
a r t i c l e
i n f o
Article history: Received 11 November 2011 Received in revised form 18 January 2012 Accepted 23 January 2012 Keywords: Anisakis Tyrrhenian Sea ITS PCR-RFLP Anisakidosis
a b s t r a c t The accurate identification of anisakid nematodes at any life cycle stage is important both to deepen the knowledge on their taxonomy, ecology, epidemiology and for diagnosis and control, as larval stages cause a clinical disease in humans known as anisakidosis. With the aim to investigate the presence of anisakid larvae, specimens of horse mackerel, Trachurus trachurus (Linnaeus, 1758), silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), European anchovy, Engraulis encrasicolus (Linnaeus, 1758) and opah fish, Lampris guttatus (Brunnich, 1788), were collected by trawling at depths ranging from 50 to 400 m. A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region was used to identify Anisakis spp. larvae recovered in fish samples. Restriction profiles showed three banding patterns, corresponding to Anisakis pegreffii, Anisakis physeteris and to heterozygote pattern between A. pegreffii and Anisakis simplex s.s. Specimens showing the heterozygote restriction pattern were also analyzed by sequencing of the entire ITS region, to confirm the heterozygote status. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nematodes of the genus Anisakis are parasites of fishes and cephalopods at their larval stage and of marine mammals at their adult stage. Accidental ingestion of these larvae can cause a gastrointestinal infection known as anisakidosis, as well as a series of mild to severe allergic reactions. Their occurrence in fishery products can cause both public health and economic problems, therefore it is currently raising considerable concern by the veterinary and health services in charge of the fishery products control. Human infections usually involve Anisakis simplex (s.l.) and Pseudoterranova decipiens (s.l.) (Yu et al., 2001; Audicana et al., 2002; Suzuki et al., 2010; Arizono et al., 2011), both existing as a complex of cryptic species, morphologically similar but with different genetic and
∗ Corresponding author. Tel.: +39 0649914671. E-mail address: [email protected] (S. D’Amelio). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2012.01.033
ecologic traits. Other nematodes, such as those of the genera Contracaecum and Hysterothylacium spp., can rarely cause anisakidosis and food allergies (Ishikura et al., 1993; Yagi et al., 1996; Valero et al., 2003). Therefore the accurate identification of anisakid nematodes at any life cycle stage is crucial to deepen the knowledge on their ecology and epidemiology, for diagnosis and control, and to screen the safety of fish products. In the last two decades the use of molecular and genetic approaches have revised the systematics of anisakids providing rapid and unambiguous identification methods (Mattiucci et al., 1997; D’Amelio et al., 2000; Mattiucci and Nascetti, 2008). A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region with informative endonucleases (D’Amelio et al., 2000) was used to identify Anisakis spp. larvae recovered in fish samples. RFLP analysis allowed the identification of all the nine species so far described and to recognize the heterozygote genotype between A. simplex sensu stricto and Anisakis pegreffii (Abollo et al., 2003; Martín-Sánchez et al.,
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2005; Umehara et al., 2006; Farjallah et al., 2008; Abattouy et al., 2011; Chaligiannis et al., 2011; Meloni et al., 2011). The aim of the present study was to investigate the presence and to identify at species level anisakid larvae in four commonly commercialized fish species: the horse mackerel, Trachurus trachurus (Linnaeus, 1758), the silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), the opah fish, Lampris guttatus (Brunnich, 1788) and the European anchovy Engraulis encrasicolus (Linnaeus, 1758). The presence of anisakid nematodes was extensively previously reported in the horse mackerel (Mattiucci et al., 1986, 2007; Adroher et al., 1996; Manfredi et al., 2000; Abollo et al., 2001; Costa et al., 2003; Farjallah et al., 2008; Kijewska et al., 2009; Costa et al., 2011; Meloni et al., 2011), in the scabbardfish (Orecchia et al., 1986; Ioli et al., 1998; Klimpel et al., 2006) and in the anchovy (de la Torre Molina et al., 2000; Mattiucci and Nascetti, 2007; Rello et al., 2009), while the opah fish has not been so far checked for anisakids. 2. Materials and methods Anisakid nematodes at larval stage were collected from four fish species: 46 horse mackerels, two silver scabbardfish, one opah fish and 61 European anchovies. Fishes were collected during 2009 (from April to the end of June) by trawling at depths ranging from 50 to 400 m in the Tyrrhenian Sea, northwestern Mediterranean (43◦ 00 –42◦ 00 N; 10◦ 00 –11◦ 30 E), during the sampling activities carried out in the framework of the Data Collection Framework (DCF, EC Reg. no. 199/2008). The abdominal cavity was checked for the presence of nematodes. A total of 131 larvae morphologically referable to the genus Anisakis were collected and stored in ethanol 70% for molecular characterization. DNA was isolated from single larvae using the Wizard® Genomic DNA purification kit (Promega), according to the manufacturer’s protocol. The nuclear ribosomal ITS region (plus intervening 5.8S rRNA gene) was amplified by PCR using 5.0 l of template DNA (20–40 ng), 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2 (Bioline), 40 mM of a nucleotide mix (Promega), 50 pM/l each of the forward primer NC5 (5 -GTAGGTGAACCTGCGGAAGGATCAT-3 ) and the reverse primer NC2 (5 -TTAGTTTCTTCCTCCGCT-3 ) (Zhu et al., 2000) and 1.0 U of BIOTAQ DNA Polymerase (Bioline) in a final volume of 50 l. PCR were performed in a GeneAmp PCR System 2400 (Applied Biosystems) using the following conditions: 10 min at 95 ◦ C (initial denaturation), 30 cycles of 30 s at 95 ◦ C (denaturation), 40 s at 52 ◦ C (annealing) and 75 s at 72 ◦ C (extension), and a final elongation step of 7 min at 72 ◦ C. A negative control (without genomic DNA) was included in each set of amplification reactions. Aliquots (5 l) of individual PCR products were separated by electrophoresis using agarose gels (1%), stained with ethidium bromide (0.4 g/ml) and detected using ultraviolet transillumination. Positive ITS amplicons were digested with the restriction endonucleases HinfI and TaqI because they are useful for the identification of anisakid species (D’Amelio et al., 2000). Digests were resolved by electrophoresis in 2% agarose gels, stained with ethidium bromide and detected with transillumination and the sizes of fragments
Fig. 1. RFLP profiles obtained by digestion of ITS region with the restriction enzymes HinfI showing Anisakis pegreffii pattern (three bands) and heterozygote pattern (H) between A. pegreffii and A. simplex s.s. (four bands). L: 100 bp ladder.
determined by comparison with a 100 bp DNA ladder marker (Promega). Specimens that showed a heterozygote restriction pattern between A. simplex s.s. and A. pegreffii with HinfI digestion were also analyzed by sequencing of the entire ITS region, to confirm the presence of both heterozygote positions (Abollo et al., 2003): PCR products were purified by SureClean (Bioline), following the manufacturer’s instructions and then sequenced by MWG EurofinsDNA. The resulting electropherograms were analyzed manually using Chromas Lite (www.technelysium.com.au) to check the presence of both nucleotides (C and T) in the correct position. 3. Results and discussion Seventy-six anisakid larvae were found infecting the body cavity of the 46 specimens of T. trachurus; 12 larvae were observed in L. caudatus, 15 in L. guttatus and 28 larvae were found in 61 specimens of E. encrasicolus. Seventy three larvae from T. trachurus, 12 from L. caudatus and 23 from E. encrasicolus showed the restriction profiles corresponding to A. pegreffii with HinfI digestion: three fragments of about 370, 300 and 250 bp (Fig. 1); 3 individuals collected from T. trachurus and 5 from E. encrasicolus showed the heterozygote pattern between A. pegreffii/A. simplex s.s. (Fig. 1); all the 15 larvae from L. guttatus showed the restriction patterns of Anisakis physeteris: three fragments of about 380, 280 and 250 bp using HinfI (Fig. 2a). The identification of A. physeteris larvae was confirmed using the TaqI endonucleases, which provided the
Fig. 2. RFLP profiles obtained by digestion of ITS region with the restriction enzymes HinfI (a) and TaqI (b) showing Anisakis physeteris pattern. L: 100 bp ladder.
S. Cavallero et al. / Veterinary Parasitology 187 (2012) 563–566
banding pattern described by D’Amelio et al. (2000) for this species (Fig. 2b). The eight nematode specimens showing the heterozygote digestion pattern at HinfI were also analyzed by sequencing of the entire ITS region, to confirm the presence of the two heterozygote position of the polymorphisms. Five individuals showed both heterozygote positions (C/T), confirming their putative hybrid identity; two specimens showed a C in the first variable position and a C/T in the second position and one specimen showed a C in both position, the specific polymorphism of A. pegreffii. In this last case the heterozygote pattern is probably due to an incomplete digestion of the amplified DNA. The data obtained in the present study further confirm the widespread occurrence of A. pegreffii in fish off the marine Italian coasts, as the most frequent anisakid species. The epidemiological survey of zoonotic anisakids species in fish usually intended for food use (as anchovy or horse mackerel) is of particular interest. In fact several previously reported cases of human anisakidosis were supposed to be caused by infected typical Italian preparations based on marinated anchovies (Moschella et al., 2004; Fumarola et al., 2009; Mattiucci et al., 2011). This raw dish is highly popular also in other Mediterranean countries and it is supposed to be the cause of most cases of anisakidosis in Spain (Rello et al., 2009). Ioli et al. (1998) described a human case of anisakidosis in Sicily caused by an infected fish, L. caudatus. This species is known to harbor a large number of Anisakis larvae, with high values of prevalence and mean intensity (Mattiucci and Nascetti, 2006). The finding that the opah fish was infected only by A. physeteris is of particular interest, as this host species was not checked before for anisakids. This record may be related to opah feeding habits, mainly based on mesopelagic fishes and invertebrates, especially squids (Palmer, 1986). However, other Anisakis species were previously reported in mesopelagic and deep-water fishes, such as A. paggiae, A. nascettii and A. pegreffii (Pontes et al., 2005; Kijewska et al., 2009; Klimpel et al., 2011), and additional samplings of this host may reveal a more diverse anisakid fauna. Even if the opah is occasionally taken as a by-catch of tuna fisheries, it is considered a very valuable fish and it can be used for fresh food (sashimi). Infection in humans with A. physeteris has been reported only in Japan (Asato et al., 1991) and in Spain (Clavel et al., 1993), stressing the need to improve the knowledge about the presence of different anisakids species with zoonotic potential in all possible paratenic hosts. Moreover, in the present study new records of the presence of the heterozygote genotype at rDNA marker are reported. This kind of putative hybrid pattern was previously identified in larval nematodes collected off the Iberian, Tunisian and Japanese waters (Abollo et al., 2003; Umehara et al., 2006; Farjallah et al., 2008) and more recently in Sardinian waters (Meloni et al., 2011), but their identity and evolutive meaning still remain unsolved. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that
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could inappropriately influence or bias the content of the paper. References Abattouy, N., Valero, A., Benajiba, M.H., Lozano, J., Martín-Sánchez, J., 2011. Anisakis simplex s.l. parasitization in mackerel (Scomber japonicus) caught in the North of Morocco – prevalence and analysis of risk factors. Int. J. Food Microbiol. 150, 136–139. Abollo, E., Gestal, C., Pascual, S., 2001. Anisakis infestation in marine fish and cephalopods from Galician waters: an update perspective. Parasitol. Res. 87, 492–499. Abollo, E., Paggi, L., Pascual, S., D’Amelio, S., 2003. Occurrence of recombinant genotypes of Anisakis simplex s.s. and Anisakis pegreffii (Nematoda: Anisakidae) in an area of sympatry. Infect. Genet. Evol. 3, 175–181. Adroher, F.J., Valero, A., Ruiz-Valero, J., Iglesias, L., 1996. Larval anisakids (Nematoda: Ascaridoidea) in horse mackerel (Trachurus trachurus) from the fish market in Granada (Spain). Parasitol. Res. 82, 253–256. Arizono, N., Miura, T., Yamada, M., Tegoshi, T., Onishi, K., 2011. Human infection with Pseudoterranova azarasi roundworm. Emerg. Infect. Dis. 17, 555–556. Asato, R., Wakuda, M., Sueyoshi, T., 1991. A case of human infection with Anisakis physeteris larvae in Okinawa, Japan. Jpn. J. Parasitol. 40, 181–183. Audicana, M.T., Ansotegui, I.J., de Corres, L.F., Kennedy, M.W., 2002. Anisakis simplex: dangerous—dead and alive? Trends Parasitol. 18, 20–25. Chaligiannis, I., Lalle, M., Pozio, E., Sotiraki, S., 2011. Anisakidae infection in fish of the Aegean Sea. Vet. Parasitol., doi:10.1016/j.vetpar.2011.09.007. Clavel, A., Delgado, B., Sánchez Acedo, C., Carbonell, E., Castillo, J., Ramírez, J., 1993. A live Anisakis physeteris larva found in the abdominal cavity of a woman in Zaragoza, Spain. J. Parasitol. 42, 445–448. Costa, G., Pontes, T., Mattiucci, S., D’Amelio, S., 2003. The occurrence and infection dynamics of Anisakis larvae in the black-scabbard fish, Aphanopus carbo, chub mackerel, Scomber japonicus, and oceanic horse mackerel, Trachurus picturatus from Madeira, Portugal. J. Helminthol. 77, 163–166. Costa, G., Melo-Moreira, E., Pinheiro de Carvalho, M.A., 2011. Helminth parasites of the oceanic horse mackerel Trachurus picturatus Bowdich 1825 (Pisces: Carangidae) from Madeira Island, Atlantic Ocean, Portugal. J. Helminthol., doi:10.1017/S0022149X11000502. D’Amelio, S., Mathiopoulos, K.D., Santos, C.P., Pugachev, O.N., Webb, S.C., Picanc¸o, M., Paggi, L., 2000. Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: Ascaridoidea) defined by polymerase chain reaction-based restriction fragment length polymorphism. Int. J. Parasitol. 30, 223–226. de la Torre Molina, R., Pérez Aparicio, J., Hernández Bienes, M., Jurado Pérez, R., Martínez Ruso, A., Morales Franco, E., 2000. Anisakiasis in fresh fish sold in the north of Córdoba. Rev. Esp. Salud Publica 74, 517–526. Farjallah, S., Slimane, B.B., Busi, M., Paggi, L., Amor, N., Blel, H., Said, K., D’Amelio, S., 2008. Occurrence and molecular identification of Anisakis spp. from the North African coasts of Mediterranean Sea. Parasitol. Res. 102, 371–379. Fumarola, L., Monno, R., Ierardi, E., Rizzo, G., Giannelli, G., Lalle, M., Pozio, E., 2009. Anisakis pegreffi etiological agent of gastric infections in two Italian women. Foodborne Pathog. Dis. 6, 1157–1159. Ioli, A., Leonaldi, R., Gangemi, C., Lo Giudice, L., Bottari, M., Petithory, J.C., 1998. Apropos of 1 case of anisakiasis contracted in Sicily. Bull. Soc. Pathol. Exot. 91, 232–234. Ishikura, H., Kikuchi, K., Nagasawa, K., Ooiwa, T., Takamiya, H., Sato, N., Sugane, K., 1993. Anisakidae and anisakidosis. In: Sun, T. (Ed.), Progress in Clinical Parasitology, vol. III. Springer-Verlag, New York, NY, pp. 43–102. Kijewska, A., Dzido, J., Shukhgalter, O., Rokicki, J., 2009. Anisakid parasites of fishes caught on the African shelf. J. Parasitol. 95, 639–645. Klimpel, S., Kuhn, T., Busch, M.W., Karl, H., Palm, H.W., 2011. Deep-water life cycle of Anisakis paggiae (Nematoda: Anisakidae) in the Irminger Sea indicates kogiid whale distribution in north Atlantic waters. Polar Biol. 34, 899–906. Klimpel, S., Rückert, S., Piatkowski, U., Palm, H.W., Hanel, R., 2006. Diet and metazoan parasites of silver scabbard fish Lepidopus caudatus from the Great Meteor Seamount (North Atlantic). Mar. Ecol. Prog. Ser. 315, 249–257.
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Manfredi, M.T., Crosa, G., Galli, P., Ganduglia, S., 2000. Distribution of Anisakis simplex in fish caught in the Ligurian Sea. Parasitol. Res. 86, 551–553. Martín-Sánchez, J., Artacho-Reinoso, M.E., Díaz-Gavilán, M., Valero-López, A., 2005. Structure of Anisakis simplex s.l. populations in a region sympatric for A. pegreffii and A. simplex s.s. Absence of reproductive isolation between both species. Mol. Biochem. Parasitol. 141, 155–162. Mattiucci, S., Nascetti, G., 2006. Molecular systematics, phylogeny and ecology of anisakid nematodes of the genus Anisakis Dujardin, 1845: an update Anisakis Dujardin. Parasite 13, 99–113. Mattiucci, S., Nascetti, G., 2007. Genetic diversity and infection levels of anisakid nematodes parasitic in fish and marine mammals from Boreal and Austral hemispheres. Vet. Parasitol. 148, 43–57. Mattiucci, S., Nascetti, G., 2008. Advances and trends in the molecular systematics of anisakid nematodes, with implications for their evolutionary ecology and host-parasite co-evolutionary processes. Adv. Parasitol. 66, 47–148. Mattiucci, S., Nascetti, G., Cianchi, R., Paggi, L., Arduino, P., Margolis, L., Brattey, J., Webb, S., D’Amelio, S., Orecchia, P., Bullini, L., 1997. Genetic and ecological data on the Anisakis simplex complex with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae). J. Parasitol. 83, 401–416. Mattiucci, S., Abaunza, P., Damiano, S., Garcia, A., Santos, M.N., Nascetti, G., 2007. Distribution of Anisakis larvae, identified by genetic markers, and their use for stock characterization of demersal and pelagic fish from European waters: an update. J. Helminthol. 81, 117–127. Mattiucci, S., Paoletti, M., Borrini, F., Palumbo, M., Palmieri, R.M., Gomes, V., Casati, A., Nascetti, G., 2011. First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy. BMC Infect. Dis. 31, 11–82. Mattiucci, S., Smith, J.W., Paggi, L., Orecchia, P., Nascetti, G., Bullini, L., 1986. Geographic distribution and hosts of Anisakis simplex and Anisakis simplex B (Ascaridida: Anisakidae). Ann. Ist. Super. Sanità 22, 353–355. Meloni, M., Angelucci, G., Merella, P., Siddi, R., Deiana, C., Orrù, G., Salati, F., 2011. Molecular characterization of Anisakis larvae from fish caught off Sardinia. J. Parasitol. 97, 908–914.
Moschella, C.M., Mattiucci, S., Mingazzini, P., De Angelis, G., Assenza, M., Lombardo, F., Monaco, S., Paggi, L., Mondini, C., 2004. Intestinal anisakiasis in Italy: case report. J. Helminthol. 78, 271–273. Orecchia, P., Paggi, L., Mattiucci, S., Smith, J.W., Nascetti, G., Bullini, L., 1986. Electrophoretic identification of larvae and adults of Anisakis (Ascaridida: Anisakidae). J. Helminthol. 60, 331–339. Palmer, G., 1986. Lamprididae. In: Whitehead, P.J.P., Bauchot, M.-L., Hureau, J.-C., Nielsen, J., Tortonese, E. (Eds.), Fishes of the NorthEastern Atlantic and the Mediterranean, vol. 2. UNESCO, Paris, pp. 725–726. Pontes, T., D’Amelio, S., Costa, G., Paggi, L., 2005. Molecular characterization of larval anisakid nematodes from marine fishies of Madeira by a PCR-based approach, with evidence for a new species. J. Parasitol. 91, 1430–1434. Rello, F.J., Adroher, F.J., Benítez, R., Valero, A., 2009. The fishing area as a possible indicator of the infection by anisakids in anchovies (Engraulis encrasicolus) from southwestern Europe. Int. J. Food Microbiol. 129, 277–281. Suzuki, J., Murata, R., Hosaka, M., Araki, J., 2010. Risk factors for human Anisakis infection and association between the geographic origins of Scomber japonicus and anisakid nematodes. Int. J. Food Microbiol. 31, 88–93. Umehara, A., Kawakami, Y., Matsui, T., Araki, J., Uchida, A., 2006. Molecular identification of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) from fish and cetacean in Japanese waters. Parasitol. Int. 55, 267–271. Valero, A., Terrados, S., Díaz, V., Reguera, V., Lozano, J., 2003. Determination of IgE in the serum of patients with allergic reactions to four species of fish-parasite anisakids. J. Investig. Allergol. Clin. Immunol. 13, 94–99. Yagi, K., Nagasawa, K., Ishikura, H., Nakagawa, A., Sato, N., Kikuchi, K., Ishikura, H., 1996. Female worm Hysterothylacium aduncum excreted from human: a case report. Jpn J. Parasitol. 45, 12–23. Yu, J.R., Seo, M., Kim, Y.W., Oh, M.H., Sohn, W.M., 2001. A human case of gastric infection by Pseudoterranova decipiens larva. Kor. J. Parasitol. 39, 193–196. Zhu, X., D’Amelio, S., Paggi, L., Gasser, R.B., 2000. Assessing sequence variation in the internal transcribed spacers of ribosomal DNA within and among members of the Contracaecum osculatum complex (Nematoda: Ascaridoidea: Anisakidae). Parasitol. Res. 86, 677–683.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Molecular identification of Anisakis spp. from fishes collected in the Tyrrhenian Sea (NW Mediterranean) S. Cavallero a , A. Ligas b , F. Bruschi c , S. D’Amelio a,∗ a b c
Department of Public Health and Infectious Diseases, Section of Parasitology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy Centro Interuniversitario di Biologia Marina ed Ecologia Applicata “G. Bacci”, Livorno, Italy Department of Experimental Pathology, M.B.I.E., Università di Pisa, Pisa, Italy
a r t i c l e
i n f o
Article history: Received 11 November 2011 Received in revised form 18 January 2012 Accepted 23 January 2012 Keywords: Anisakis Tyrrhenian Sea ITS PCR-RFLP Anisakidosis
a b s t r a c t The accurate identification of anisakid nematodes at any life cycle stage is important both to deepen the knowledge on their taxonomy, ecology, epidemiology and for diagnosis and control, as larval stages cause a clinical disease in humans known as anisakidosis. With the aim to investigate the presence of anisakid larvae, specimens of horse mackerel, Trachurus trachurus (Linnaeus, 1758), silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), European anchovy, Engraulis encrasicolus (Linnaeus, 1758) and opah fish, Lampris guttatus (Brunnich, 1788), were collected by trawling at depths ranging from 50 to 400 m. A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region was used to identify Anisakis spp. larvae recovered in fish samples. Restriction profiles showed three banding patterns, corresponding to Anisakis pegreffii, Anisakis physeteris and to heterozygote pattern between A. pegreffii and Anisakis simplex s.s. Specimens showing the heterozygote restriction pattern were also analyzed by sequencing of the entire ITS region, to confirm the heterozygote status. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nematodes of the genus Anisakis are parasites of fishes and cephalopods at their larval stage and of marine mammals at their adult stage. Accidental ingestion of these larvae can cause a gastrointestinal infection known as anisakidosis, as well as a series of mild to severe allergic reactions. Their occurrence in fishery products can cause both public health and economic problems, therefore it is currently raising considerable concern by the veterinary and health services in charge of the fishery products control. Human infections usually involve Anisakis simplex (s.l.) and Pseudoterranova decipiens (s.l.) (Yu et al., 2001; Audicana et al., 2002; Suzuki et al., 2010; Arizono et al., 2011), both existing as a complex of cryptic species, morphologically similar but with different genetic and
∗ Corresponding author. Tel.: +39 0649914671. E-mail address: [email protected] (S. D’Amelio). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2012.01.033
ecologic traits. Other nematodes, such as those of the genera Contracaecum and Hysterothylacium spp., can rarely cause anisakidosis and food allergies (Ishikura et al., 1993; Yagi et al., 1996; Valero et al., 2003). Therefore the accurate identification of anisakid nematodes at any life cycle stage is crucial to deepen the knowledge on their ecology and epidemiology, for diagnosis and control, and to screen the safety of fish products. In the last two decades the use of molecular and genetic approaches have revised the systematics of anisakids providing rapid and unambiguous identification methods (Mattiucci et al., 1997; D’Amelio et al., 2000; Mattiucci and Nascetti, 2008). A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region with informative endonucleases (D’Amelio et al., 2000) was used to identify Anisakis spp. larvae recovered in fish samples. RFLP analysis allowed the identification of all the nine species so far described and to recognize the heterozygote genotype between A. simplex sensu stricto and Anisakis pegreffii (Abollo et al., 2003; Martín-Sánchez et al.,
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2005; Umehara et al., 2006; Farjallah et al., 2008; Abattouy et al., 2011; Chaligiannis et al., 2011; Meloni et al., 2011). The aim of the present study was to investigate the presence and to identify at species level anisakid larvae in four commonly commercialized fish species: the horse mackerel, Trachurus trachurus (Linnaeus, 1758), the silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), the opah fish, Lampris guttatus (Brunnich, 1788) and the European anchovy Engraulis encrasicolus (Linnaeus, 1758). The presence of anisakid nematodes was extensively previously reported in the horse mackerel (Mattiucci et al., 1986, 2007; Adroher et al., 1996; Manfredi et al., 2000; Abollo et al., 2001; Costa et al., 2003; Farjallah et al., 2008; Kijewska et al., 2009; Costa et al., 2011; Meloni et al., 2011), in the scabbardfish (Orecchia et al., 1986; Ioli et al., 1998; Klimpel et al., 2006) and in the anchovy (de la Torre Molina et al., 2000; Mattiucci and Nascetti, 2007; Rello et al., 2009), while the opah fish has not been so far checked for anisakids. 2. Materials and methods Anisakid nematodes at larval stage were collected from four fish species: 46 horse mackerels, two silver scabbardfish, one opah fish and 61 European anchovies. Fishes were collected during 2009 (from April to the end of June) by trawling at depths ranging from 50 to 400 m in the Tyrrhenian Sea, northwestern Mediterranean (43◦ 00 –42◦ 00 N; 10◦ 00 –11◦ 30 E), during the sampling activities carried out in the framework of the Data Collection Framework (DCF, EC Reg. no. 199/2008). The abdominal cavity was checked for the presence of nematodes. A total of 131 larvae morphologically referable to the genus Anisakis were collected and stored in ethanol 70% for molecular characterization. DNA was isolated from single larvae using the Wizard® Genomic DNA purification kit (Promega), according to the manufacturer’s protocol. The nuclear ribosomal ITS region (plus intervening 5.8S rRNA gene) was amplified by PCR using 5.0 l of template DNA (20–40 ng), 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2 (Bioline), 40 mM of a nucleotide mix (Promega), 50 pM/l each of the forward primer NC5 (5 -GTAGGTGAACCTGCGGAAGGATCAT-3 ) and the reverse primer NC2 (5 -TTAGTTTCTTCCTCCGCT-3 ) (Zhu et al., 2000) and 1.0 U of BIOTAQ DNA Polymerase (Bioline) in a final volume of 50 l. PCR were performed in a GeneAmp PCR System 2400 (Applied Biosystems) using the following conditions: 10 min at 95 ◦ C (initial denaturation), 30 cycles of 30 s at 95 ◦ C (denaturation), 40 s at 52 ◦ C (annealing) and 75 s at 72 ◦ C (extension), and a final elongation step of 7 min at 72 ◦ C. A negative control (without genomic DNA) was included in each set of amplification reactions. Aliquots (5 l) of individual PCR products were separated by electrophoresis using agarose gels (1%), stained with ethidium bromide (0.4 g/ml) and detected using ultraviolet transillumination. Positive ITS amplicons were digested with the restriction endonucleases HinfI and TaqI because they are useful for the identification of anisakid species (D’Amelio et al., 2000). Digests were resolved by electrophoresis in 2% agarose gels, stained with ethidium bromide and detected with transillumination and the sizes of fragments
Fig. 1. RFLP profiles obtained by digestion of ITS region with the restriction enzymes HinfI showing Anisakis pegreffii pattern (three bands) and heterozygote pattern (H) between A. pegreffii and A. simplex s.s. (four bands). L: 100 bp ladder.
determined by comparison with a 100 bp DNA ladder marker (Promega). Specimens that showed a heterozygote restriction pattern between A. simplex s.s. and A. pegreffii with HinfI digestion were also analyzed by sequencing of the entire ITS region, to confirm the presence of both heterozygote positions (Abollo et al., 2003): PCR products were purified by SureClean (Bioline), following the manufacturer’s instructions and then sequenced by MWG EurofinsDNA. The resulting electropherograms were analyzed manually using Chromas Lite (www.technelysium.com.au) to check the presence of both nucleotides (C and T) in the correct position. 3. Results and discussion Seventy-six anisakid larvae were found infecting the body cavity of the 46 specimens of T. trachurus; 12 larvae were observed in L. caudatus, 15 in L. guttatus and 28 larvae were found in 61 specimens of E. encrasicolus. Seventy three larvae from T. trachurus, 12 from L. caudatus and 23 from E. encrasicolus showed the restriction profiles corresponding to A. pegreffii with HinfI digestion: three fragments of about 370, 300 and 250 bp (Fig. 1); 3 individuals collected from T. trachurus and 5 from E. encrasicolus showed the heterozygote pattern between A. pegreffii/A. simplex s.s. (Fig. 1); all the 15 larvae from L. guttatus showed the restriction patterns of Anisakis physeteris: three fragments of about 380, 280 and 250 bp using HinfI (Fig. 2a). The identification of A. physeteris larvae was confirmed using the TaqI endonucleases, which provided the
Fig. 2. RFLP profiles obtained by digestion of ITS region with the restriction enzymes HinfI (a) and TaqI (b) showing Anisakis physeteris pattern. L: 100 bp ladder.
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banding pattern described by D’Amelio et al. (2000) for this species (Fig. 2b). The eight nematode specimens showing the heterozygote digestion pattern at HinfI were also analyzed by sequencing of the entire ITS region, to confirm the presence of the two heterozygote position of the polymorphisms. Five individuals showed both heterozygote positions (C/T), confirming their putative hybrid identity; two specimens showed a C in the first variable position and a C/T in the second position and one specimen showed a C in both position, the specific polymorphism of A. pegreffii. In this last case the heterozygote pattern is probably due to an incomplete digestion of the amplified DNA. The data obtained in the present study further confirm the widespread occurrence of A. pegreffii in fish off the marine Italian coasts, as the most frequent anisakid species. The epidemiological survey of zoonotic anisakids species in fish usually intended for food use (as anchovy or horse mackerel) is of particular interest. In fact several previously reported cases of human anisakidosis were supposed to be caused by infected typical Italian preparations based on marinated anchovies (Moschella et al., 2004; Fumarola et al., 2009; Mattiucci et al., 2011). This raw dish is highly popular also in other Mediterranean countries and it is supposed to be the cause of most cases of anisakidosis in Spain (Rello et al., 2009). Ioli et al. (1998) described a human case of anisakidosis in Sicily caused by an infected fish, L. caudatus. This species is known to harbor a large number of Anisakis larvae, with high values of prevalence and mean intensity (Mattiucci and Nascetti, 2006). The finding that the opah fish was infected only by A. physeteris is of particular interest, as this host species was not checked before for anisakids. This record may be related to opah feeding habits, mainly based on mesopelagic fishes and invertebrates, especially squids (Palmer, 1986). However, other Anisakis species were previously reported in mesopelagic and deep-water fishes, such as A. paggiae, A. nascettii and A. pegreffii (Pontes et al., 2005; Kijewska et al., 2009; Klimpel et al., 2011), and additional samplings of this host may reveal a more diverse anisakid fauna. Even if the opah is occasionally taken as a by-catch of tuna fisheries, it is considered a very valuable fish and it can be used for fresh food (sashimi). Infection in humans with A. physeteris has been reported only in Japan (Asato et al., 1991) and in Spain (Clavel et al., 1993), stressing the need to improve the knowledge about the presence of different anisakids species with zoonotic potential in all possible paratenic hosts. Moreover, in the present study new records of the presence of the heterozygote genotype at rDNA marker are reported. This kind of putative hybrid pattern was previously identified in larval nematodes collected off the Iberian, Tunisian and Japanese waters (Abollo et al., 2003; Umehara et al., 2006; Farjallah et al., 2008) and more recently in Sardinian waters (Meloni et al., 2011), but their identity and evolutive meaning still remain unsolved. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that
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