Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.     

Ani s 1, the major allergen of Anisakis simplex: purification by affinity chromatography and functional expression in Escherichia coli

Third stage larvae of the nematode Anisakis simplex often infect marine fish and invertebrates. When the larvae are ingested orally via seafood, they can cause IgE-mediated allergic reactions as well as anisakiasis. Of the known A. simplex allergens, Ani s 1 (Kunitz/bovine pancreatic trypsin inhibitor family protein) has been demonstrated to be a major allergen, being expected to be a useful tool for diagnosis of A. simplex allergy. For a diagnostic purpose, sufficient amounts of either natural Ani s 1 (nAni s 1) or recombinant Ani s 1 (rAni s 1) with an IgE-binding capacity should be stably supplied whenever needed. In this study, therefore, we first developed a simple and rapid purification method for Ani s 1 that is based on affinity chromatography using anti-Ani s 1 antibodies as ligands. The method was shown to produce nAni s 1 with a higher yield than the previously reported methods. Then, an attempt was made to express rAni s 1 in Escherichia coli as a His-tagged protein. rAni s 1 obtained as an inclusion body was solubilized in a solvent containing denaturing and reducing reagents and purified by nickel-chelate chromatography. Refolding of rAni s 1 was accomplished by dialysis in the presence of arginine, followed by that in the absence of arginine. Fluorescence ELISA and inhibition ELISA data revealed that rAni s 1 is IgE reactive enough to be used as a diagnostic tool.     

Identification of novel three allergens from Anisakis simplex by chemiluminescent immunoscreening of an expression cDNA library

Anisakis simplex is a representative nematode parasitizing marine organisms, such as fish and squids, and causes not only anisakiasis but also IgE-mediated allergy. Although 10 kinds of proteins have so far been identified as A. simplex allergens, many unknown allergens are considered to still exist. In this study, a chemiluminescent immunoscreening method with higher sensitivity than the conventional method was developed and used to isolate IgE-positive clones from an expression cDNA library of A. simplex. As a result, three kinds of proteins, Ani s 11 (307 amino acid residues), Ani s 11-like protein (160 residues) and Ani s 12 (295 residues), together with three known allergens (Ani s 5, 6 and 9), were found to be IgE reactive. Furthermore, ELISA data showed that both recombinant Ani s 11 and 12 expressed in Escherichia coli are recognized by about half of Anisakis-allergic patients. Ani s 11 and Ani s 11-like protein are characterized by having six and five types of short repetitive sequences (5-16 amino acid residues), respectively. Both proteins share as high as 78% sequence identity with each other and also about 45% identity with Ani s 10, which includes two types of short repetitive sequences. On the other hand, Ani s 12 is also structurally unique in that it has five tandem repeats of a CX(13-25)CX(9)CX(7,8)CX(6) sequence, similar to Ani s 7 having 19 repeats of a CX(17-25)CX(9-22)CX(8)CX(6) sequence. The repetitive structures are assumed to be involved in the IgE-binding of the three new allergens.     

Ani s 10, a new Anisakis simplex allergen: cloning and heterologous expression

Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy.     

Molecular characterization of Anisakis larvae from fish caught off Sardinia

Anisakis spp. larvae are parasitic, and potentially zoonotic, nematodes transmitted by marine fish and cephalopods, which are the main intermediate hosts of the third larval stage. The accidental consumption of infected raw or poorly cooked fish may cause gastroenteric diseases and allergies in humans. The aim of the present study was to use polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to define the occurrence, species variability, and host preferences of Anisakis spp. larvae in fish caught off the coast of Sardinia. Necropsy was used on 285 samples; 552 Anisakis spp. L3 larvae were isolated from 87 fish that tested positive for this nematode. Anisakis pegreffii was most frequently encountered (90.6%), with a primary preference for Scomber scombrus, Zeus faber, and Trachurus mediterraneus. In contrast, the prevalence of Anisakis physeteris was only 1.3%. A hybrid genotype of Anisakis simplex sensu stricto and Anisakis pegreffii was also observed, which confirms the results of previous studies carried out in the western Mediterranean. Interestingly, no Anisakis simplex s.s. larvae were recovered. These results indicate that the diversity of Anisakis species is low in Sardinia waters, probably because of its geographic position.     

Molecular characterization of larval anisakid nematodes from marine fishes of Madeira by a PCR-based approach, with evidence for a new species

One-hundred and fifteen anisakid larvae from 3 different fish hosts, Aphanopus carbo, Scomber japonicus, and Trachurus picturatus, caught in Madeiran waters, were identified by PCR-RFLP. Three distinct species were identified in A. carbo, namely Anisakis simplex sensu srricto, Anisakis pegreffii, and Anisakis ziphidarum; 5 in S. japonicus, i.e., A. simplex s.s., A. pegreffii, Anisakis physeteris, Anisakis typica, and A. ziphidarum; and 3 in T. picturatus, i.e., A. simplex s.s., A. pegreffii, and A. typica. Anisakis simplex s.s. was the most frequent species in both A. carbo and S. japonicus (54% and 23.5%, respectively). Anisakis pegreffii and A. physeteris occurred with a frequency of 20.6% in S. japonicus, whereas in T. picturatus the most frequent species was A. typica (41.9%), followed by A. simplex s.s. (32.3%). Furthermore, A. carbo and S. japonicus were infected by an apparently undescribed taxon, provisionally named Anisakis sp. A. Based on estimations of the genetic distance, this new taxon seems to be more similar to A. ziphidarum (0.0335) than to other species of the genus.     

Distribution of Anisakis species larvae from fishes of the Japanese waters

Human anisakiasis is caused by the consumption of raw, marinated or undercooked fish and squid infected with nematodes of the genus Anisakis Dujardin, 1845. In view of food safety, this study was carried out to examine the distribution of Anisakis species in marine fishes within Japanese waters. Seven fish species from six localities were collected and examined for Anisakis infection. Morphological and molecular (ITS region and mtDNA cox2 gene) characterization revealed the presence of two, among the three sibling species of Anisakis simplex, viz. A. simplex sensu stricto (s.s.) and A. pegreffii. Distribution data were collated with the results from the previous researches to better understand Anisakis distribution in Japanese waters. Distributions of Anisakis species were found to be locality-specific rather than host-specific, particularly between the two major species, A. simplex s.s. and A. pegreffii. Anisakis simplex s.s. is mainly found in fishes from northern Japan to Pacific sides, whereas A. pegreffii is in fishes from the Sea of Japan to East China Sea sides.     

Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder

The third-stage larvae of Anisakis simplex sensu lato (s.l.) are found in many marine fishes. To ensure food safety, it is important to determine whether these larvae are present in the body muscle of commercial fish species. However, there is little information regarding the tissue specificity of Anisakis and two of its sibling species, A. simplex sensu stricto (s.s.) and Anisakis pegreffii, that are common in marine fish in Japanese waters. We orally challenged rainbow trout (Oncorhynchus mykiss (Walbaum)), and olive flounder (Paralichthys olivaceus (Temminck and Schlegel)) with L3 larvae of these two sibling species and monitored infection for 5weeks. In rainbow trout, A. simplex s.s., but not A. pegreffii larvae, migrated into the body muscle. A small number of freely moving A. pegreffii larvae were recovered within the body cavity. In olive flounder, A. simplex s.s. larvae were found in both the body cavity and body muscle. A. pegreffii larvae were found only in the body cavity and primarily encapsulated in lumps. Our results indicate that there are differences in the sites of infection and host specificity between the two sibling species of A. simplex s.l.     

Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47

We present a new application of the noncompetitive phage anti-immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile noncompetitive sandwich-type format using immunocomplex binding phage-borne peptides to detect the brominated flame retardant, brominated diphenyl ether 47 (BDE 47). Three phage-displayed 9-mer disulfide-constrained peptides that recognize the BDE 47-polyclonal antibody immunocomplex were isolated. The resulting PHAIAs showed variable sensitivities, and the most sensitive peptide had a dose-response curve with an SC₅₀ (concentration of analyte producing 50% saturation of the signal) of 0.7 ng/ml BDE 47 and a linear range of 0.3-2 ng/ml, which was nearly identical to the best heterologous competitive format (IC₅₀ of 1.8 ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive assay. The validation of the PHAIA with extracts of house furniture foam as well as human and calf sera spiked with BDE 47 showed overall recovery of 80-113%. The PHAIA was adapted to a dipstick format (limit of detection of 3.0 ng/ml), and a blind test with six random extracts of local house furniture foams showed that the results of the PHAIA and dipstick assay were consistent, giving the same positive and negative detection.     

Development and characterization of a competitive polyclonal antibody enzyme-immunoassay for salmon insulin-like growth factor-II

This paper describes the development and validation of a competitive, polyclonal antibody enzyme-immunoassay (EIA) for the measurement of salmon and trout insulin-like growth factor-II (IGF-II). A polyclonal antiserum was raised against a synthetic peptide epitope, corresponding to amino acid residues 1-9 of the N-terminus of mature Atlantic salmon (Salmo salar) IGF-II. The antiserum was purified by hydrophobic charge induction chromatography (HCIC). The partially purified immunoglobulins were used in an enzyme-immunoassay system (EIA) resulting in a highly specific assay for salmon IGF-II with cross-reactivity of less than 0.01% for recombinant salmon IGF-I and recombinant salmon growth hormone (GH), and 5.57% for salmon insulin (sIns). The recombinant salmon IGF-II (rsIGF-II) standard curve limit of detection was 1.37 ng/ml with an EC(50) of 44.97+/-0.82 ng/ml. Intra- and interassay coefficients of variation were determined at 7.47% (n=15) and 7.42% (n=15), respectively. Added rsIGF-II was adequately recovered from acid-treated Atlantic salmon and rainbow trout (Oncorhynchus mykiss) plasma samples. Parallel dose-response inhibition curves were demonstrated for the plasma of both fish species tested. Circulating IGF-II levels of 22.26+/-2.66 and 18.24+/-1.43 ng/ml were determined for acid-treated plasma of normal adult Atlantic salmon and rainbow trout, respectively. This EIA should prove to be useful in the study of factors which influence circulating plasma levels of IGF-II in these fish species.     

An immunoassay method for quantitative detection of proteins using single antibodies

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01 ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose-response was 3.3 to 0.01 ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.     

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner’s fiber

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner’s fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.     

Distribution of Anisakis larvae, identified by genetic markers, and their use for stock characterization of demersal and pelagic fish from European waters: an update

In the present paper, recent results obtained on the use of different distributions observed in larval species of Anisakis, genetically identified by means of allozyme markers, for stock characterization of demersal (Merluccius merluccius), small (Trachurus trachurus) and large pelagic (Xiphias gladius) finfish species in European waters, are reviewed and discussed. Several species of Anisakis were identified in the three fish hosts: A. simplex (s.s.), A. physeteris, A. typica, A. ziphidarum, A. pegreffii, A. brevispiculata and A. paggiae. Canonical discriminant analysis performed on all the samples of the three fish species collected in areas comprising their geographical range, according to the different species of Anisakis identified, showed distinct fish populations in European waters. In all the three fish hosts, the pattern of distribution of Anisakis larvae allowed discrimination of Mediterranean stocks from Atlantic stocks. In the case of swordfish, the possible existence of a southern Atlantic stock separated from a northern one is also suggested. Congruence and discordance with the population genetic data inferred from allozyme markers on the same samples of the three fish species are also discussed.     

Population structure of three species of Anisakis nematodes recovered from Pacific sardines (Sardinops sagax) distributed throughout the California Current system

Members of the Anisakidae are known to infect over 200 pelagic fish species and have been frequently used as biological tags to identify fish populations. Despite information on the global distribution of Anisakis species, there is little information on the genetic diversity and population structure of this genus, which could be useful in assessing the stock structure of their fish hosts. From 2005 through 2008, 148 larval anisakids were recovered from Pacific sardine (Sardinops sagax) in the California Current upwelling zone and were genetically sequenced. Sardines were captured off Vancouver Island, British Columbia in the north to San Diego, California in the south. Three species, Anisakis pegreffii, Anisakis simplex 'C', and Anisakis simplex s.s., were identified with the use of sequences from the internal transcribed spacers (ITS1 and ITS2) and the 5.8s subunit of the nuclear ribosomal DNA. The degree of nematode population structure was assessed with the use of the cytochrome c oxidase 2 (cox2) mitochondrial DNA gene. All 3 Anisakis species were distributed throughout the study region from 32°N to 50°N latitude. There was no association between sardine length and either nematode infection intensity or Anisakis species recovered. Larval Anisakis species and mitochondrial haplotype distributions from both parsimony networks and analyses of molecular variance revealed a panmictic distribution of these parasites, which infect sardines throughout the California Current ecosystem. Panmictic distribution of the larval Anisakis spp. populations may be a result of the presumed migratory pathways of the intermediate host (the Pacific sardine), moving into the northern portion of the California Current in summer and returning to the southern portion to overwinter and spawn in spring. However, the wider geographic range of paratenic (large piscine predators), and final hosts (cetaceans) can also explain the observed distribution pattern. As a result, the recovery of 3 Anisakis species and a panmictic distribution of their haplotypes could not be used to confirm or deny the presence of population subdivision of Pacific sardines in the California Current system.     

In vivo larvicidal activity of monoterpenic derivatives from aromatic plants against L3 larvae of Anisakis simplex s.l.

In view of the lack of an effective pharmacological treatment against human anisakiosis, a disease produced by L(3) larvae of the genus Anisakis present in raw fish, we studied the in vivo larvicidal effect of certain monoterpenic derivatives against L(3) of A. simplex s.l. The aldehydic monoterpene citral and the alcoholic citronellol, when they are administered together to the larvae of the nematode at the concentration of 46.90 mg/0.5 ml in olive oil, achieve 85.90% and 67.53% dead L(3), respectively, and also stop rats suffering gastrointestinal hemorrhages produced by the larvae.     

Seroprevalence of antibodies against Anisakis simplex larvae among health-examined residents in three hospitals of southern parts of Korea

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.     

Effects of ivermectin and albendazole against Anisakis simplex in vitro and in guinea pigs

The activity of ivermectin and albendazole against larval Anisakis simplex was tested in vitro and in experimentally infected guinea pigs. Before drug exposure the medium for half of the larvae was adjusted to pH 2.0 with 1 N HCl, whereas the other half was held at pH 7.0. To these solutions, ivermectin was added to full concentrations of 1, 2, 5, 10, 50, 100, or 200 microg/ml, and for albendazole, 300, 400, and 500 microg/ml. Animals from group I were given 0.1 ml of 1% (3.3 mg/kg) ivermectin, whereas guinea pigs from group II were each given 5-7 mg (16.6-23.3 mg/kg) of albendazole orally. The efficacy of both drugs against L, A. simplex was high in vitro and in vivo against the larvae in different organs of guinea pigs.     

The nematode Anisakis simplex in American shad (Alosa sapidissima) in two Oregon rivers

This paper represents the first report of the nematode Anisakis simplex in the American shad (Alosa sapidissima) in its introduced range in the American Pacific Northwest. All the adult shad sampled from spawning populations in the Willamette (n = 9) and Umpqua (n = 12) rivers were infected with A. simplex with intensities ranging from 6 to 89 worms per fish. This preliminary investigation contrasts sharply with previous studies in the native range of American shad and confirms that this fish may be an important intermediate host for A. simplex in the Pacific Northwest. It is suggested that this new parasite-host relationship has led to an ecological expansion into rivers and Anisakis may present an emerging health risk for wildlife and some human consumers.     

Molecular identification of the etiological agent of the human anisakiasis in Japan

Anisakis simplex complex presently comprises three sibling species, A. simplex sensu stricto, A. pegreffii and A. simplex C. A. simplex is a common parasite in fishes and cephalopods and capable of causing anisakiasis in humans. Therefore, identification of sibling species of A. simplex was important for human health. In this study, one hundred Anisakis type I larvae isolated from eighty five patients with anisakiasis in Hokkaido and Kyushu in Japan were analyzed by adapting the new molecular method that can identify the sibling species of A. simplex complex. Based on the restriction fragment length polymorphism (RFLP) pattern of ITS regions including 5.8 subunit rRNA gene, we identified two sibling species, A. simplex s. str. and A. pegreffii. However, the infection rate of A. simplex s. str. was significantly higher than that of A. pegreffii. Eighty four (98.8%) out of the eighty five patients were infected with A. simplex s. str. On the contrary, one patients (1.2%) in Kyushu infected with A. pegreffii. This study provided basic information about human infection with A. simplex complex. Furthermore, we suggested that A. simplex s. str. is the most important etiological agent in Japan.     

Morphological and molecular characterization of Anisakis larvae (Nematoda: Anisakidae) in Beryx splendens from Japanese waters

The third-stage (L3) larvae of Anisakis, which are the etiological agents of human anisakiasis, have been categorized morphologically into Anisakis Type I larvae and Anisakis Type II larvae. Genetic analysis has allowed easy identification of these larvae: Anisakis Type I larvae include the species Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis simplex C, Anisakis typica, Anisakis ziphidarum, and Anisakis nascettii, whereas Anisakis Type II larvae include the species Anisakis physeteris, Anisakis brevispiculata, and Anisakis paggiae. Since human consumption of raw fish and squid is common in Japan, we investigated Anisakis L3 larvae in 44 specimens of Beryx splendens from Japanese waters. A total of 730 Anisakis L3 larvae collected from B. splendens were divided morphologically into 4 types: Type I, Type II, and 2 other types that were similar to Anisakis Type III and Type IV described by Shiraki (1974). Anisakis Type II, Type III, and Type IV larvae all had a short ventriculus, but their tails were morphologically different. In addition, data from genetic analysis indicated that Anisakis Type II, Type III, and Type IV larvae could be identified as A. physeteris, A. brevispiculata, and A. paggiae, respectively. Therefore, A. physeteris, A. brevispiculata, and A. paggiae can be readily differentiated not only by genetic analysis but also by morphological characteristics of L3 larvae.