Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) for six taxa of anisakids, namely, Anisakis simplex (s.s.), Anisakis typica, Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium sp, and Contracaccum osculatum C, specific primers were designed in the ITS-1 and/or ITS-2 for each of the six anisakid taxa. These specific primers were used to develop polymerase chain reaction (PCR) tools for the identification of these anisakid taxa of sea fish by amplifying partial ITS-1 and/or ITS-2 of rDNA from anisakid nematodes. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the DNA fragments amplified. The minimum amounts of DNA detectable using the PCR assays were 0.5–1 ng. These PCR tools were then applied to ascertain the specific identity of 143 anisakid larval samples collected from fish in China, Canada, Thailand, and Indonesia, and these anisakid samples were identified to represent one of the six anisakid taxa. These PCR assays based on ITS sequences should provide useful molecular tools for the accurate identification and molecular epidemiological investigations of anisakid infections in humans and fish.
Anisakid nematodes are common parasites of fish, mammals, fish-eating birds, and reptiles with a worldwide distribution, causing diseases in animals and significant economic losses (Hartwich 1974; Rohlwing et al. 1998; Abollo et al. 2001; Costa et al. 2003; Doupe et al. 2003; Zhou et al. 2008). More importantly, human infection with the third-stage larvae of anisakids of some genera, such as Anisakis, Contracaecum, and Pseudoterranova, causes significant clinical diseases (i.e., gastric or intestinal anisakiasis) in many countries, even in some countries where less fish are consumed (Adams et al. 1997; Maggi et al. 2000; McCarthy and Moore 2000; Couture et al. 2003; Chai et al. 2005; Pellegrini et al. 2005).
The success of control for any parasitic disease is dependent on the accurate identification of the parasites causing the diseases. The accurate identification of anisakid nematodes at any life cycle stage is central to the diagnosis of anisakid infections in humans and animals and is therefore an important step for disease surveillance and control. The taxonomy within this group of nematodes based on morphological characters was determined mainly to the level of genera, but identification of species by microscopic examinations is difficult and sometimes impossible due to their small size and poor morphological distinctions of these nematodes, in particular their larvae (Olson et al. 1983; Fagerholm 1988; Dick et al. 1991; Szostakowska et al. 2002). In clinical situations, most of nematodes are isolated from frozen fish or patient’s organs and tissues; therefore, such identification is sometimes difficult or impossible due to damage of nematodes during isolation or when only small portions of worms are available for identification (Dick et al. 1991). Therefore, it is necessary to develop a reliable molecular tool to allow identification of anisakid nematodes even in the case of their damage, as well as retrospectively in a material frozen or preserved in alcohol.
Polymerase chain reaction (PCR)-based approaches, using appropriate genetic markers, provide useful alternatives for the accurate identification of parasites and the diagnosis of the diseases they cause (Masiga et al. 2000; Prichard and Tait 2001; Gasser 2006; Li et al. 2007; Lin et al. 2007). In particular, using internal transcribed spacers (ITS-1 and/or ITS-2) of nuclear ribosomal DNA (rDNA) as genetic markers, various previous studies have demonstrated the usefulness of PCR-linked restriction fragment length polymorphism and single strand conformation polymorphism for the accurate identification of anisakid nematodes (Zhu et al. 1998, 2001, 2007a, b; D’Amelio et al. 2000; Szostakowska et al. 2002; Zhang et al. 2007). A previous study has demonstrated the potential of specific PCR assays for the identification of some anisakids (Umehara et al. 2008). The objectives of the present study were to establish, using genetic markers in the ITS-1 and/or ITS-2, specific PCR tools for the specific delineation of six taxa of anisakid nematodes of fish with zoonotic significance commonly found in Poland and China (Zhu et al. 1998, 2007a), namely A. typica, A. simplex (s.s.), A. pegreffii, Hysterothylacium sp., H. aduncum, and C. osculatum C, and to apply these tools to examine the specific identity of anisakid samples collected from fish in China, Canada, Thailand, and Indonesia.
Materials and methods
Larval anisakid nematodes (n = 143) were collected from different fish species from Poland, Thailand, Canada, Indonesia, and China (Table 1). Individual anisakid larvae were repeatedly washed in physiological saline (pH 7.3) and identified to species or generic level based on the host and tissue from which they were derived, the geographical origin of the host/parasite, and the morphology of the parasites (Hartwich 1974; Olson et al. 1983; Nascetti et al. 1993; Mattiucci et al. 1997).
Extraction of genomic DNA and quality evaluation
Genomic DNA was isolated from individual anisakid nematodes by sodium dodecyl-sulfate/proteinase K treatment, column-purified (WizardTM DNA Clean-Up, Promega), and eluted into 40 μl of H2O according to the manufacturer’s recommendations. DNA was also isolated from the musculature of fish using the same method.
In order to evaluate the quality of the anisakid DNA samples, PCR was used to amplify the entire ITS fragment (including ITS-1, 5.8S, ITS-2, and primer flanking sequences of ~90 bp) with primers NC5 (forward: 5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and NC2 (reverse: 5′-TTAGTTTCTTTTCCTCCGCT-3′; Zhu et al. 2007a). PCR reactions (in a volume of 25 μl) were performed in 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 3 mM MgCl2, 250 μM each of dNTP, 50 pmol of each primer, and 2 U Taq polymerase (Takara) in a thermocycler (Biometra) under the following conditions: 94°C for 5 min (pre-denaturation), followed by 35 cycles of 94°C, 30 s (denaturation), 55°C, 30 s (annealing), 72°C, 1 min (extension), and a final extension of 72°C for 5 min. One microliter of genomic DNA was added to each PCR reaction. Samples with fish DNA or without genomic DNA were included in each PCR run as ‘negative’ controls. An aliquot (4 μl) of each PCR product was examined on a 1% w/v agarose gel, stained with ethidium bromide, and photographed using a gel documentation system (UVItec). One amplicon representing each of the taxa was sequenced to confirm their identity.
Design of species-specific primers and optimization of specific PCR assays
Based on the comparison of the ITS-1 and ITS-2 sequences of A. typica, A. simplex (s.s.), A. pegreffii, Hysterothylacium sp, H. aduncum, and C. osculatum C (Zhu et al. 1998, 2007a, also see GenBank™ accession numbers AJ225062-AJ225070, AJ937669-AJ937674, AM706345, AY826724, AM706344, AY826720, AB196671, and AB277823), specific forward and reverse primers were specifically designed for each of the six common anisakid taxa, and the sequences of these specific primers are listed in Table 2. Because A. pegreffii and A. simplex (s.s.) have identical ITS-2 sequences, primer set CQ3F/CQ3R (Table 2) was designed in the ITS-2 to amplify partial ITS-2 from both species, allowing the differentiation of these two closely related taxa from other examined taxa. In order to further differentiate between A. pegreffii and A. simplex (s.s.), the forward primer CQ6F (see Table 2) was specifically designed for A. pegreffii and used together with the conserved reverse primer NC2 to amplify partial ITS-1, complete 5.8S, and ITS-2 rDNA. The PCR conditions were optimized for specificity by varying the annealing temperatures and magnesium concentrations in the buffer. The six primer sets were also evaluated separately for their specificity using some other members of the family Anisakidae, namely Contracaecum rudolphii B, Pseudoterranova decipiens (s.s.), Contracaecum ogmorhini, and Contracaecum eudyptulae, as well as DNA from fish and human as controls in addition to the six taxa. The specificity of one representative amplicon produced using each specific primer set was verified by directly sequencing them (Zhu et al. 2007b). The smallest amount of target DNA detectable by each of the specific primer sets was estimated by serial titration of genomic DNA from one specimen for each of A. typica, A. simplex (s.s.), A. pegreffii, Hysterothylacium sp., H. aduncum, and C. osculatum C.
Results and discussion
The entire ITS fragment including ITS-1, 5.8S, ITS-2, and primer flanking sequences amplified from each individual specimen varied in size from 1,000 to 1,100 bp, corresponding to the expected fragment lengths on agarose gels (not shown), demonstrating the presence of amplifiable DNA in and absence of components inhibitory to the PCR from each sample used in the present study. Sequencing of one amplicon representing each of the six anisakid taxa confirmed their identity.
The specific PCR protocols were optimized by varying the annealing temperatures and titrating the magnesium concentrations. The optimal magnesium concentration was 3 mM and the optimal annealing temperature was 55°C for each of the six primer sets. Therefore, the conditions of specific PCR protocols were the same as for primer set NC5/NC2. Under the optimized cycling conditions, primer set CQ1F/CQ1R amplified a product of 255 bp solely from A. typica, primer set CQ2F/CQ2R amplified a product of 464 bp solely from Hysterothylacium sp., primer set CQ3F/CQ3R amplified a product of 580 bp from A. simplex (s.s.) and A. pegreffii, primer set CQ4F/CQ4R amplified a product of 641 bp solely from Hysterothylacium aduncum, primer set CQ5F/CQ5R amplified a product of 434 bp exclusively from C. osculatum C, and primer set CQ6F/NC2 amplified a product of 692 bp exclusively from A. pegreffii. No product was amplified from DNA samples of heterogeneous anisakids, fish, human, or no-DNA controls. The representative results using primer set CQ5F/CQ5R (specific for C. osculatum C) are shown in Fig. 1. One representative amplicon produced using each specific primer set was sequenced with corresponding primers and proved to be partial ITS of the appropriate taxon, hence demonstrating the specificity of the primers, cycling conditions, and the PCR assays. The smallest amounts of DNA detectable for each specific PCR assay were 0.55 ng for A. typica, 1 ng for Hysterothylacium sp., 0.53 ng for A. simplex (s.s.), 0.5 ng for Hysterothylacium aduncum, 0.69 ng for C. osculatum C, and 0.65 ng for A. pegreffii. The representative results using primer set CQ5F/CQ5R (specific for C. osculatum C) are shown in Fig. 2. These findings showed that these PCR assays were specific and sensitive, allowing the detection and delineation of all of the six common anisakids found in fish in Poland and China (Zhu et al. 1998, 2007a).
The specific PCR assays were then applied to determine the specific identity of 143 anisakid samples from sea fish in Thailand, Canada, Indonesia, and China (cf. Table 1), and representative amplification results using specific primer set for C. osculatum C (primers CQ5F/CQ5R) are shown in Fig. 3. Sequencing of representative PCR products verified the identification achieved by PCR amplification. For 9 anisakid samples from China and Canada, specific PCR products were amplified by using the primer pair specific to Hysterothylacium sp., a non-characterized member of the genus Hysterothylacium (cf. Zhu et al. 2007a), and it will be characterized in further studies.
Infection with anisakid larvae has been recorded in approximately 200 fish species worldwide (Abollo et al. 2001). In China, more than 150 fish species have been found to be infected with anisakid larvae, with several fish species having a prevalence of 100% (Tang et al. 2001; Ruan and Zhang 2007). The anisakid nematodes of fish are of considerable public health significance worldwide (Adams et al. 1997; Maggi et al. 2000; McCarthy and Moore 2000; Couture et al. 2003; Pellegrini et al. 2005), and human infection with larval anisakids has been considered an emerging fish-borne zoonotic disease in a number of countries in recent years due to the habit of eating raw or undercooked fish and the pursuit of eating exotic and delicate foods such as sushi (in which raw fish is the main component) and cisheng (sliced raw fish; Costa et al. 2003; Couture et al. 2003; Zhou et al. 2008). However, there remain difficulties in the accurate diagnosis of anisakiasis in different host species, mainly as a consequence of limitations in identifying larval stages using morphological characters (Olson et al. 1983; Fagerholm 1988; Dick et al. 1991) or when only small portions of worms are available for identification (Dick et al. 1991).
Given the difficulty in the accurate identification of anisakid nematodes, particularly their larvae, using morphological approach, the established specific PCR assays using genetic markers in the ITS sequences should provide useful tools for the unequivocal identification and differentiation of anisakids and have implications for studying the ecology, epidemiology, and population genetics of these anisakid nematodes, as well as for the diagnosis of their infections in various hosts.
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Project support was provided by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (Garnt No. IRT0723) to XQZ. The authors are grateful to Dr. M. Podolska of Sea Fisheries Institute, Poland for providing some anisakid samples from Poland. The experiments comply with the current laws of the countries in which the experiments were performed.
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Chen, Q., Yu, H.Q., Lun, Z.R. et al. Specific PCR assays for the identification of common anisakid nematodes with zoonotic potential. Parasitol Res 104, 79–84 (2008). https://doi.org/10.1007/s00436-008-1161-7
- Polymerase Chain Reaction Assay
- Specific Polymerase Chain Reaction
- Anisakid Nematode
- Specific Polymerase Chain Reaction Product