Anisakiasis, or more broadly anisakidosis (infection with anisakid nematode larvae; Anisakidae and Raphidascarididae), is a typical example of fishborne parasitic zoonoses in localities where marine fish or cephalopods are consumed raw or improperly cooked [1]. The etiological agents include species of Anisakis (A. pegreffii, A. simplex s.s., and A. simplex), Pseudoterranova (P. decipiens s.s., P. azarasi, and P. cattani), Contracaecum (C. osculatum), and Hysterothylacium (H. aduncum) [2,3]. However, 4 species, A. pegreffii, A. simplex, P. decipiens, and P. azarasi, are most frequently involved in highly endemic countries, especially Korea and Japan [3,4]. Natural definitive hosts include dolphins, porpoises, whales, seals, and rarely, large marine fish [2]. Animal or human (accidental host) infection is caused by third-stage larvae found in the viscera or muscles of various fish and cephalopod (mollusks) species [1].

The concept of this zoonotic disease (initially called ‘herring worm disease’ or ‘codworm disease’) was f irst introduced in 1960 by van Thiel et al. [5], who reported 11 patients complaining of acute abdominal syndrome in the Netherlands, followed by Ashby et al. [6] in England in 1964, who reported 2 such patients and reviewed 89 cases possibly due to this disease. However, the diagnosis of the causative worms at this time was erroneously Eustoma rotundatum [5,6]. In 1965, Asami et al. [7] and Yokogawa and Yoshimura [8] reported similar human infections in Japan and assigned the causative worms to Anisakis-like larvae. Since then, human anisakidosis has been reported in various locations worldwide, including Japan, Korea, the Netherlands, Spain, France, Italy, Germany, England, and the USA [1,2]. To date, at least 50,000 clinical cases have been reported in these countries [1,2]. However, this is a significant underestimation of the actual situation. For example, in Japan, an average annual incidence of 19,737 anisakidosis cases was estimated from 2018 to 2019 [4], and in Spain, an annual incidence of 7,700-8,320 anisakidosis cases was estimated [9].

The most common site of anisakidosis in humans is the stomach, followed by the intestines (small and large intestines), and rarely the oral cavity, pharynx, tonsils, and esophagus [1,2]. Extra-gastrointestinal infections may also occur in rare instances, in the peritoneum, liver, lymph nodes, omentum, mesentery, pancreas, ovaries, and lungs [2]. In gastrointestinal anisakiasis, acute abdomen, nausea, vomiting, and mucosal bleeding are the most common clinical manifestations, although some cases reveal no special clinical problems, and the larvae can be incidentally found during surgery or endoscopy [2]. Diagnosis is mainly based on the recovery of anisakid larvae from the affected lesions through gastrointestinal endoscopy or surgery. In some cases, the diagnosis is made based on sectional morphologies of the larvae in histopathological specimens [2]. Serology to detect serum antibodies, for example, enzyme-linked immunosorbent assay (ELISA), and radiography, are other diagnostic methods used to diagnose acute and/or chronic anisakidosis [2]. No anthelmintic drugs have been proven to be effective in anisakidosis patients.

In Korea, human anisakidosis is common because Koreans generally prefer to eat the raw or undercooked f lesh of marine fish or cephalopods (cuttlefish, squid, and octopus). Since the first case was reported in 1971 [10], over a hundred articles have been published on clinical anisakidosis cases until 2022. The status was briefly reviewed by Sohn et al. [11] in 2015, in which 64 articles were analyzed concerning the number of cases, locality of patient occurrence, gender characteristics, source of infection (fish or cephalopods), morphological types of larvae, and predilection sites. However, since then, there has been no further review of the anisakidosis status in Korea.

Besides clinical case reports, there have been reports on the detection of anisakid larvae from marine fish and cephalopods, the occurrence of animal anisakidosis, and immunologic studies on experimental anisakidosis in animals. Thus, the present study focused on reviewing the Korean literature on human and animal anisakidosis (occurrence of clinical cases, clinicopathologic characteristics, immunity, diagnosis, and natural and experimental animal infections), as well as the status of anisakid larval infections in fish and cephalopods, larval morphology, molecular analyses, and resistance of anisakid larvae to physicochemical agents.

We searched and analyzed Korean literature on Anisakis, Pseudoterranova, anisakiasis, and anisakidosis from sources, including Google Scholar, PubMed Central, and KoreaMed. International literature to refer was also obtained from the same data sources.

In Korea, human anisakidosis was first reported in 1971 [10] in a patient living in Seoul complaining of a foreign body sensation and dull pain in the palatine tonsil. It was evident that these symptoms were due to infection with a larva of Anisakis sp. penetrating the palatine tonsil, which was extracted using forceps. The second case was reported in 1980 by Cho et al. [12] in a patient living in Seoul with an acute abdomen due to duodenal ulcer perforation. An anisakid larva section was incidentally found in the ileum of this patient.

By the end of 2022, at least 108 articles (including several abstracts) had been published on human anisakidosis cases in Korea (Tables 1-4) [3,10-116]. The total number of cases reported was 851, including 131 cases during 1971-1990, 225 cases during 1991-2000, 229 cases during 2001-2010, and 268 cases during 2011-2022, which demonstrated a chronologically increasing trend (Fig. 1). Seropositive cases were excluded if anisakid larvae were not parasitologically confirmed [117-120]. In addition, an increasing number of anisakiasis cases were reported from 2011 to 2018 in Korea based on big data analysis of Health Insurance Review Assessment claims [121]. According to this article, the annual number of anisakiasis cases registered in the Korean Health Insurance Database (2011-2018) was 333-818, including 409 cases in 2011, 333 cases in 2012, 359 cases in 2013, 409 cases in 2014, 450 cases in 2015, 491 cases in 2016, 685 cases in 2017, and 818 cases in 2018, totaling 3,954 cases during the 8 years (avg. 494 cases/year) [121]. Therefore, at least 800 cases of human anisakidosis occur annually in the Korean population. However, most of these cases were not officially reported in scientific publications (it is also uncertain whether these cases were parasitologically proven) and thus were not included in the clinical case analyses of this review.

The geographic locations where anisakidosis cases were reported in Korea were variable, including Jeju (229 cases; 26.9%), Seoul (182; 21.4%), Jinju (159; 18.7%), Busan (93; 10.9%), Incheon (42; 4.9%), Gwangju (21; 2.5%), Daegu (21; 2.5%), Pohang (20; 2.4%) and other cities (Fig. 2). A strong trend was observed for cases to be more commonly diagnosed in coastal areas than in inland areas. In addition, the frequency of reports seemed to depend on the number of medical facilities available in each area.

The possible sources of infection (fish or cephalopod dishes) were mentioned in most articles (Table 5). Of the 851 anisakidosis patients, approximately a quarter (25.4%) stated that they had eaten undefined or mixed f ish dishes (over 2 kinds of fish). The next most common source of infection was sea eel (19.9%), followed by squid (5.2%), yellow corvina (4.2%), Japanese flounder or flatfish (3.4%), codfish (2.6%), yellowtail (1.6%), and rockfish (1.4%) (Table 5). Anchovy, seabream, skate, and shad were the next most common dishes consumed by patients with anisakidosis in Korea (Table 5). It is of special note that approximately 1/5 of the cases involved the consumption of raw or undercooked sea eels. Considering that mixed fish dishes frequently include sea eel, the number of cases associated with sea eel consumption would increase to over 1/3 of all cases. A significant concern related to this is that anisakid larvae are commonly found in the muscles of sea eel, whereas larvae are generally found in the viscera and abdominal cavity of most other types of marine fish [122-126]. Therefore, if the sea eel muscle is consumed raw, it could be possible to be infected with anisakid larvae that are frequently found alive in the muscles of this fish.

Among the 851 cases analyzed in this review, the most frequently involved organs were the stomach (698 cases; 82.0%) followed by the small and large intestines (88 and 40 cases, respectively, 128 cases in total; 15.0%), esophagus (8 cases; 0.9%), tonsils (2 cases; 0.2%), oral cavity/oral mucosa (2 cases; 0.2%), mesocolic lymph nodes (1 case; 0.1%), greater omentum (1 case; 0.1%), peritoneum (1 case; 0.1%), and unknown location (15 cases; 1.8%) (Fig. 3). The involvement of multiple organs, more than 2 of the stomach, ileum, and colon, has been reported in 5 cases [73,80,89,98,103].

Recurrent anaphylaxis due to A. simplex larva infection was first reported by Audicana et al. [135] in Spain. Now it has been well established that acute allergic symptoms, such as urticaria, angioedema, or anaphylaxis, accompanied by epigastric pain, nausea, and vomiting, called gastroallergic anisakiasis, are elicited by live (not dead or heat-inactivated) third-stage larvae of A. simplex and A. pegreff ii [136]. This hypersensitivity reaction is often accompanied by an acute IgE-mediated generalized reaction with cutaneous (urticaria to angioedema) and/or respiratory (asthma and rhinitis) symptoms [136]. IgE bound to mast cells or basophils recognizes specific allergens, and degranulation of these cells leads to local and/or systemic allergic reactions [136]. IgE-producing parasite-derived antigens have been isolated and characterized, including Ani s 1 through Ani s 14, which are somatic antigens, excretory-secretory products (ESP), or of unknown origin [137]. Major allergens, as defined by the recognition of > 50% of sensitized individuals, were 5 kinds, including Anis 1 (ESP; similar to thermostable serine protease inhibitors), Ani s 2 (somatic antigen; paramyosin), Ani s 7 (ESP; function unknown), Ani s 12 (origin and function unknown), and Ani s 13 (ESP; hemoglobin) [136,137]. Ani s 1 and Ani s 7 have high allergenic potential and no cross-reactivity with other allergens, whereas Ani s 2 is a pan-allergen with cross-reactivity with other foods or inhalant allergens [137].

In Korea, Cho and coworkers [138-142] performed immunological and serological studies on anisakiasis in experimental animals and humans. Kim et al. [138] found that the ESP of A. simplex third-stage larvae (L3) contained important allergens (12 low-molecular-weight bands ranging from 10 to 186 kDa by immunoblotting) necessary to induce Anisakis allergy in rats. In human anisakiasis, the binding patterns of L3-specific antibodies varied depending on the different L3-ESP preparations, and low-molecular-weight proteins appeared to be strong and specific antigens [139]. Choi et al. [140] reported high serum titers of IgG4 and IgE antibodies in 4 of 9 Anisakis allergy patients in Korea. Cho et al. [141] demonstrated that allergic responses induced by L3 oral infection were predominantly caused by reinfection rather than primary infection, accompanied by elevated IgE and IgM levels. The role of specific IgG, especially IgG4, has also been suggested in immune responses to intraperitoneally injected L3, as IgG4 binds to epitopes recognized by specific IgE [143]. In human anisakidosis, the importance of specific IgA has been suggested; however, its role has not been demonstrated in experimental mice and rats [139,142].

Choi et al. [143] reported that A. pegreffii larval extract induced airway inflammation and asthma 4 weeks after the challenge, enhancing the expression of Th2-type immune responses (IL-4, IL-5, and IL-13) in the lungs of mice. Conversely, Park et al. [144] cloned a macrophage migration inhibitory factor (MIF)-like protein from L3 of A. simplex. This MIF-like protein was shown to reduce Th2-related cytokines in the bronchoalveolar lavage fluid and allergen-specific IgG2a in the sera of mice, which is probably associated with host immune modulation. Treg cells were recruited to the spleen and lungs of these MIF-treated mice [144]. Park et al. [145] identified a 24 kDa protein from the ESP of A. simplex larvae as a possible allergen, homologous to the 22U protein of filarial nematodes and distinct from the Ani s 1 antigen, although having the same molecular weight. Park et al. [146] performed repeated intranasal applications of recombinant 22U of A. simplex 6 times and found that this antigen induces airway allergic inflammation accompanied by enhanced lung Th2 and Th17 responses. Kim et al. [147] isolated and characterized an α-methylacetyl CoA racemase originating from the ventriculus of A. simplex larvae, which may have a function in the growth and development of the larvae. Jeon et al. [148] compared the hydrolase activities (esterases, peptidases, proteases, and glycosidases) of ESP and the somatic proteins of A. simplex and A. pegreff ii larvae collected from salmon in Yangyang (Namdae Stream) and mackerels in the southern sea of Korea, respectively. Cho et al. [149] studied the allergenicity of Ani s 1 (major antigen) and Ani s 9 (minor antigen) by repeated intranasal inoculation into mice and observed airway inflammation in mice with significantly elevated lung Th2 and Th17 cytokine responses. An interesting study by Cha and Ock [150] demonstrated that antigenic proteins from A. simplex larvae could change cytokine profiles and prevent drug-induced Crohn’s disease in experimental mice.

A relationship between chronic anisakid nematode infection and stomach or colon cancers has been suggested [151-154]. Some clinical cases have shown the co-localization of anisakidosis and cancer in the gastrointestinal tract [155,156]. Garcia-Perez et al. [151] provided data showing an epidemiological link between previous Anisakis infections and gastrointestinal cancer. In addition, products from Anisakis were shown to cause inflammation and DNA damage in the host [152], and the complete extract of Anisakis worms could induce changes in epithelial cells in vitro and in vivo (rats) [153]. However, in Korea, only 1 case of the co-existence of ascending colon anisakiasis and sigmoid colon cancer has been reported [86]. It is noteworthy that whereas carcinogenic stimuli should continue for a long time to undergo a neoplastic transformation of the mucosal tissues of the stomach or colon, most anisakidosis cases are detected in the acute stage, and the disease does not continue for a long time. Notably, in experimental anisakiasis in rabbits, the pathological lesions almost completely resolved in the submucosa of the stomach within 5 months after infection, and the worms were calcified, became tiny, and encircled by fibrous tissues infiltrated by inflammatory cells [157]. However, attention should be paid to patients who are repeatedly infected with anisakid nematodes because in such cases mucosal changes in the stomach and/or colon may persist for a considerably long time.

Studies of experimental anisakiasis in laboratory animals (regarding susceptibility, habitat, and pathology/ histopathology) have been reported in at least 3 articles in Korea. Kwon and Chyu [158] observed the distribution of anisakid larvae in 2 experimental rabbits after oral inoculation with 30 L3 from day 2 to 14 post-infection (PI). On days 2-3 PI, a total of 42 larvae (live) were recovered in the stomach (33 larvae), omentum (8), and mesentery (1), and on days 7-14 PI, only 7 larvae (dead) were detected in the abscess cavity (6) and fundus submucosa (1) of the stomach wall [158]. Choi and Kim [159] infected rats and rabbits with Anisakis L3 and chronologically observed the distribution of the larvae within 1 day (rats) and during days 1-14 PI (rabbits); in rats, 1 larva penetrated the stomach wall within 1 hour PI, and a total of 4 larvae were found in the stomach wall by 8 hours PI, while some larvae were found in the omentum, intestinal wall, abdominal cavity, and liver within 24 hours PI. In rabbits, 22 larvae penetrated the stomach wall within 1 day, and some larvae were found in the omentum, intestinal wall, abdominal cavity, and mesentery during days 3-14 PI [159]. Hong and Lee [157] observed larval locations and gross and histopathologic findings in the stomach, small intestine, colon, and mesentery from day 13 to day 150 PI; on day 13 PI, larvae were found in the stomach (15 of 60 larvae inoculated), omentum (1), intestine (2), mesentery (5), and abdominal wall (2), and on day 90 PI, larvae were found in the stomach (12 of 60 larvae inoculated), omentum (3), intestine (2), mesentery (1), and abdominal wall (1). Only 1 larva was detected on day 150 PI in the stomach wall of a rabbit [157]. The involved tissues showed severe histopathological changes, including granuloma formation, inflammatory cell infiltrations, fibrosis, and calcification until day 90 PI, and only a few sections of tiny, calcified larvae were detected in the submucosal layer of the stomach on day 150 PI [157]. Jeon and Kim [160] performed in vitro (agar block plates) and in vivo (rats) experiments on the pathogenic potential of 2 sibling species, A. simplex and A. pegreffii, and found that A. pegreff ii had higher penetration ability (in vitro) and longer survival time in rats (in vivo) than A. simplex.

A finless porpoise was stranded on the coast of Jeju Island with adhesive bowel disease (ABD), which is life-threatening, and nematode parasites, including Anisakis sp. larvae and Crassicauda sp., were found at autopsy. The primary cause of ABD is presumed to be a reaction related to these parasitic infections [167]. The first detection of A. simplex s.s. adult nematodes (n = 2) containing eggs was reported by Kim et al. [168] in the gastrointestinal tract of a common minke whale (Balaenoptera acutorostrata Lacépède, 1804) that died in the sea between Namhae and Bangeojin, Korea. The diagnosis was molecularly confirmed using the gene sequences of the mitochondrial cytochrome c oxidase 2 (cox2) [168].

The first detection of anisakid larvae in marine fish in Korea was reported by Chun et al. [169] in 1968 (Table 7). A total of 313 fish specimens (17 species) caught from the western (Yellow Sea) and southern seas were examined, and 312 (99.7%) were found to be infected with anisakid larvae (1-334 larvae per fish) [169]. The next study was reported by Lim [170]; a total of 1,940 fish specimens (15 species) were purchased from 5 localities (caught from western and southern seas) in Korea, and the average number of anisakid larvae per f ish ranged from 0.2 (damselfish; Chromis notata) to 156.0 (yellow corvina; P. manchurica). The third study was a morphological analysis of anisakid larvae collected from the yellow corvina caught in the western sea; a total of 1,026 identifiable anisakid larvae were collected from 30 fish, and Anisakis type I was the most common (859 larvae; 80.4%) followed by Contracaecum type Dʹ (77; 7.2%), Contracaecum type C′ (55; 5.1%), Contracaecum type D (18; 1.7%), Contracaecum type A (13; 1.2%), Contracaecum type V (3; 0.28%), and Raphidascaris sp. (1; 0.09%) [123].

Thereafter, at least 19 studies reported anisakid larval infections in fish and cephalopods in the vicinity of Korean seawater [171-189]. Eight studies that examined more than 7 species of fish or cephalopods are presented in Table 7 [125,169,170,171,177,178,182,183]. Among the 99 species of fish/cephalopods examined, 82 were infected with anisakid larvae (mostly Anisakis spp.) (Table 7). The major (positive in over 3 studies) infected fish/cephalopods were 15 species, including sea eel (C. myriaster), chub mackerel (Scomber japonicus), hairtail (Trichiurus lepturus), anchovy (Engraulis japonicus), silver pomfret (Pampus argenteus), yellow corvina (P. manchurica), yellowtail (Seriola quinqueradiata), jack mackerel (Trachurus japonicus), white croaker (Argyrosomus argentatus), herring (Clupea palassii), red seabream (Pagrus major), Japanese flounder (P. olivaceus), flathead (Platycephalus indicus), rockfish (Sebastes inermis), and Japanese common squid (T. pacificus) (Table 7). The reason why chub mackerels are not ranked among the 12 most common fish consumed by human patients in Korea (Table 5) seems to be that mackerels are less frequently consumed raw by the Korean people.

In addition, chum salmon (Oncorhynchus keta) purchased from the eastern coastal areas (Taep’o Port in Sokcho City and Namdae Stream in Yangyang, Gangwon-do) were examined in 3 studies; the infection rate was 100%, with the mean number of larvae per fish ranging from 28.9 to 108.0 [172,179,181]. Masu salmon (Oncorhynchus masou masou) purchased from the eastern part (Joomunjin) of Gangwon-do were also examined, and the recovered larvae were molecularly identified as A. simplex s.s. L3 by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis [180]. Sea eels (C. myriaster) were studied 5 more times in Korea; Chai et al. [124] examined 26 sea eels purchased from the Noryangjin Fish Market in Seoul, and among them, 15 (57.5%) were positive for anisakid larvae (90.1 larvae/fish), including Anisakis type I (564/1,351 larvae; 41.7%) followed by Contracaecum spp. (787/1,351; 58.3%). Song and Hwang [126] examined 382 sea eels purchased from Busan, of which 259 (67.8%) were infected with 1,768 anisakid larvae (Anisakis spp. and Contracaecum spp.). Cho et al. [183] examined 20 sea eels caught in Tongyeong City, Gyeongsangnam-do, and recovered 129 Anisakis type I larvae; these larvae were molecularly analyzed using PCR-RFLP and sequencing of the ITS region and cox2 genes, and 112 (86.8%) were confirmed to be A. pegreff ii, 10 (7.8%) were Anisakis typica, and 7 (5.4%) were unidentified [183]. Lee et al. [186] examined sea eels (C. myriaster), chub mackerels (S. japonicus), and hairtails (T. lepturus) from the seawater of Korea and performed molecular analyses (PCR-RFLP) on the larvae; 136 (97.1%) of 140 larvae were A. pegreffii, and the remaining 4 (2.9%) were A. typica. Neves [188] also examined sea eels (C. myriaster), chub mackerels (S. japonicus), and yellow croakers (Larimichthys polyactis) caught from the western, southern, and eastern coasts of Korea to detect anisakid larvae. Bak et al. [184] caught chub mackerels from the western, southern, and eastern coasts of Korea and examined them for anisakid larvae; 231 (55.4%) of the 417 mackerels were positive, with a total number of larvae of 1,628 (7.0 larvae/fish). Molecular studies (PCR-RFLP of ITS and sequencing of cox2) of these larvae revealed that most of them were A. pegreff ii (94.3%), and small proportions were A. simplex s.s. (0.4%), a hybrid of A. pegreffii and A. simplex (2.5%), or Hysterothylacium sp. (2.8%) [184].

Jeong and Song [176] examined yellow corvina (P. manchurica) (n = 551) purchased from seawater around Jeju Island and recovered 4,386 larvae (8.5 larvae/fish) comprising 3,428 Anisakis type I larvae and 958 Contracaecum spp. larvae. Anchovies were examined in 2 more studies [174,189] in addition to the 4 studies in Table 7 [170,171,177,182]; Song et al. [174] purchased a total of 2,180 anchovies (E. japonica) from the eastern and southern seas and 171 (7.8%) were found to be infected with anisakid larvae (Anisakis type I or II, Anisakis sp., and Contracaecum type B or type C). Chang et al. [189] purchased anchovies from the southern sea and found anisakid larvae in 39 (19.5%) of 200 specimens (51 larvae in total; 1.3/ fish); molecular analyses using ITS gene sequences revealed that 28 larvae were A. pegreffii, 12 were Hysterothylacium sinense, and 11 were H. aduncum. The large-head hairtail (T. japonicus) (n = 9) purchased from Jeju Island was examined for anisakid larvae (1,259; 139.9/fish) using PCR-RFLP of ITS and cox2 sequencing; A. pegreff ii larvae (1,243/1,259; 98.7%) were most commonly detected, followed by a hybrid of A. pegreffii and A. simplex s.s. (6/1,259; 0.5%), Hysterothylacium sp. larvae (2/1,259; 0.2%), and unknown species (8/1,259; 0.5%) [187]. Regarding a squid species (T. pacificus), 1 more study was available [175] in addition to 3 studies shown in Table 7 [125,177,182]; 272 squids purchased from Busan (southern sea) were examined, and Anisakis type I, type II, Contracaecum types A and D larvae were recovered.

There may be questions about the possibility of anisakid larval infection in cultured marine fish. As fish farms are usually located near coastal areas, remote from the sea where whales and other sea mammals thrive, and within the farm there seems to be no infected first intermediate host (shrimp-like crustaceans) with anisakid larvae, cultured fish can be regarded to be safe from anisakid larval infection.

Various morphological types of anisakid larvae have been reported since the reports by Yamaguti [190], Berland [191], and Koyama et al. [192]. Anisakis type I (A. pegreffii, A. simplex, and A. typica) and type II larvae (A. simplex) were named by Berland [191]; Contracaecum types I through V by Yamaguti [190]; and Terranova types A (P. decipiens), B (P. decipiens), Contracaecum types A through D (C. osculatum), and Raphidascaris sp. by Koyama et al. [192]. In Korea, Chai et al. [123,124] found variants of Contracaecum types A, C, and D larvae in sea eel and yellow corvina, which were named Contracaecum types A′, C′, and D′, respectively.

Studies on anisakid larvae in fish from Korea started in 1966 but were officially reported in 1968 by Chun et al. [169]. Anisakis type I larvae (as Anisakis sp.) were detected in marine fish from the western and southern seas. Since then, several articles have been published regarding the morphology of anisakid larvae found in Korea. Rim [193] described the morphology of Anisakis type I larvae obtained from marine f ish and swine in Gwangju, South Korea. Kim et al. [171,172] detected Anisakis type I; Terranova type B; Contracaecum types A, B, and D; and Raphidascaris sp. larvae in marine fish purchased from fish markets in Seoul and Sokcho (Gangwon-do). Woo and Kim [125] detected Anisakis types I and II, Terranova types A and B, Contracaecum types A, B, C, and D, and Raphidascaris sp. larvae in marine fish purchased from Jeju Island. Sohn and Lee [194] and Sohn [195] observed the sectional morphologies of Anisakis type I, Contracaecum type A, and type D’ larvae by light and transmission electron microscopies. Chai et al. [196] studied the morphologies (light microscopic, sectional, and scanning electron microscopic) of P. decipiens larvae detected from the codfish (Notothenia neglecta) caught in the Antarctic Ocean near the South Pole.

Hong and Lee [157] studied the changing patterns of IgG in experimental rabbit anisakiasis for 150 days PI. IgG levels started to increase from day 3 PI, peaked on day 30 PI, maintained a plateau during days 30-90 PI, and then decreased to almost control levels on day 150 PI [157]. The serological diagnosis of human anisakidosis patients in Korea was first reported by Lee et al. [197], who used ELISA on 12 parasiteconfirmed anisakidosis cases and observed significantly elevated optical density values in 11 of the 12 patients (0.229-1.547 in 12 patients vs 0.103-0.353 in 5 controls). They also performed immunoblotting on sera from humans and experimental rabbits and found that the 46 kDa L3 antigen was highly sensitive and specific for Anisakis infection [197]. Quan et al. [198] compared different types of antigens used in ELISA for anisakiasis in rabbits and found that ESP was the most suitable antigen. Yang et al. [199] performed ELISA and immunoblotting to observe the changing patterns of serum IgM and IgG in experimental rabbits and detected at least 41 reactive bands; IgM reacted to 7 bands and IgG reacted to 8 bands. Choi et al. [200] observed the antibody responses of experimental rabbits infected with A. simplex L3 using ELISA and immunoblotting; serum IgG began to increase at days 7-9 PI, reached their maximum levels by days 1215 PI, and then decreased, while serum IgM began to increase from day 5 to 7 PI, reached a peak at day 12 PI, and then decreased. Significantly elevated levels of total and L3-specific IgE were observed in 10 gastroallergic patients [92]. However, serum IgM levels have not been studied in human anisakiasis patients in Korea and require further investigation.

Rim et al. [201] performed a seroepidemiological study using ELISA for 6 major parasitic diseases in 6,074 individuals selected from several localities in Korea. The number of anisakiasis-seropositive cases was 495 (8.1%) and varied by locality; it was higher in the southern areas of Jeollanam-do (16.9%) and some parts of Gyeongsangbuk-do (10.6%) than in other localities. Kim et al. [202] detected 33 seropositive cases by ELISA against A. simplex L3 ESP antigen in 498 health-examined people in 3 hospitals from southern parts of Korea; the allergen of 130 kDa could be a candidate for the serodiagnosis of anisakiasis. Chung and Lee [134] reported that 14 out of 17 patients with suspected gastroallergic anisakiasis on Jeju Island were strongly positive for Anisakis-specific IgE.

In Korea, molecular studies of anisakid larvae were initiated by Kim et al. [203] in 2006 who analyzed the complete mitochondrial genome of A. simplex L3 from sea eel (C. myriaster). Thereafter, at least 23 studies were performed on the molecular genetic characteristics of anisakid larvae from Korean seawater [147,178,180,181,184-189,203-215]. Yu et al. [204] analyzed the cDNA library of A. simplex L3 and detected 493 expressed sequence tags (ESTs), including 21 ESTs that matched previously reported A. simplex genes or proteins in addition to many other EST clones related to cell functions and worm growth and development.

Subsequently, the PCR-RFLP technique was applied to detect anisakid larvae in several fish species caught in Korean seawater [205-207]. Kang et al. [205] molecularly analyzed anisakid larvae from marine fish and squids caught in Korean seawater using PCR-RFLP of the ITS gene and found that they were larvae of A. pegreffii. Lee et al. [206] also molecularly confirmed that most larvae from Korean sea-fish were A. pegreffii (47 of 60 larvae), and small proportions were A. typica (10 larvae), A. simplex s.s. (1 larva), and a hybrid of A. pegreffii and A. simplex (2 larvae). Choi et al. [178] performed molecular studies (18S ribosomal DNA; 18S rDNA) on 10 Anisakis type I larvae recovered from marine fish in Korean seawater, and the larvae demonstrated high identity with A. pegreff ii. Park et al. [208] molecularly analyzed 45 anisakid larvae collected from rockfish (S. schlegeli) from the western sea and identified 3 species, which included A. simplex (17 larvae), A. pegreffii (4 larvae), and Hysterothylacium sp. (24 larvae). Kim et al. [209] molecularly analyzed 26 anisakid larvae from fish in the southern sea and found that A. pegreffii was the most common (17 larvae) followed by H. aduncum (2 larvae), Hysterothylacium fabri (1 larva), Raphidascaris lophii (1 larva), and hybrid forms (5 larvae). However, Jeon et al. [180] and Setyobudi et al. [181] reported that the anisakid larvae from masou salmon (O. masou masou) and chum salmon (O. keta), respectively, from the eastern sea analyzed by PCR-RFLP of the ITS region and cox2 gene were A. simplex s.s. Subsequently, this discrepancy was well explained by Setyobudi et al. [210] that A. pegreff ii larvae were distributed mainly in the southern (90.7% of all anisakid larvae detected) and western seas (98.9% of all larvae detected) but less in the eastern sea of Korea based on molecular analyses of larvae collected from common squids (T. pacificus). A similar but interesting finding was observed by Sohn et al. [211]; all 48 larvae from 3 species of fish from the Yellow Sea (western sea) were A. pegreffii, whereas 79 of 80 anisakid larvae from 7 species of fish from the southern sea were A. pegreff ii, and only 1 larva was A. simplex s.s. In f ish from the eastern sea, the species of anisakid larvae were different depending on the species of fish; the larvae detected in Japanese flounder (P. olivaceus) and rockfish (S. schlegeli) were all A. pegreffii, whereas those found in 2 species of salmon (O. masou masou and O. keta) were determined to be A. simplex s.s. [211]. Salmon fish are considered to have been infected by anisakid larvae when they migrate from the east coast of Korea through Hokkaido (Japan), the northern North Pacific Ocean, and the Bering Sea. Similar features were observed in the distribution of A. pegreff ii and A. simplex s.s. larvae in chub mackerel (S. japonicus) caught from the eastern, southern, and western seas [184], and it seemed that A. pegreffii is the dominant species in Korean chub mackerels which are mainly caught from the Tsushima Current stock, whereas Japanese chub mackerels are 2 stocks and harbor 2 kinds of larvae; A. pegreffii in the Tsushima Current stock and A. simplex s.s. in the Pacific Current stock.

The predominance of A. pegreff ii larvae was repeatedly observed in different fish species collected from the western and southern seawater of Korea (Fig. 4); sea eel (C. myriaster) [185,186,188], Pacific cod (Gadus macrocephalus) [212], chub mackerel (S. japonicus) [186,188], hairtail (T. lepturus) [186], large-head hairtail (Trichiurus japonicus) [187], yellow croaker (L. polyactis) [188], and anchovy (E. japonica) [189]. Nurhidayat et al. [213] examined walleye pollock (G. chalcogrammus) caught from the East Sea of Korea and found that 73.5% of the anisakids were A. simplex; only 1.4% were A. pegreffii (Fig. 4). Walleye pollock inhabits cold and deep-water columns and is abundant in the northern North Pacific Ocean (Pacific Current) and the Bering Sea [213]. In addition, the predominance of A. simplex larvae in chum salmon caught from the East Sea (Namdae Stream, Yangyang) of Korea was confirmed by Kim et al. [214], who performed comparative transcriptome analyses of the larvae. Kim et al. [215] developed a new genetic technique to discriminate the species of anisakid larvae collected from Korean seawater and the amplification-refractory mutation system (ARMS) was found to be effective in discriminating A. pegreffii, A. simplex, and their hybrids.

In human infections in Korea, specific molecular diagnosis of anisakid larvae has been performed in 3 studies [3,107,113]. Lim et al. [107] performed molecular analyses of 26 larvae extracted from human infections using ITS region sequences, and the larvae from 15 (25 larvae) of 16 cases were confirmed to be A. pegreffii, while the larva from only 1 case (1 larva) was A. simplex s.s. Song et al. [113] performed molecular analyses of anisakid larvae from 20 human cases (health check-up patients) and confirmed that all of them were infected with A. pegreffii. Molecular studies of Pseudoterranova sp. larvae were performed in only 1 study by Song et al. [3]; 12 larvae collected from 12 human cases in Korea were analyzed using sequences of the mitochondrial cox1 and nd1 genes, all of which were confirmed to be P. decipiens s.s. larvae.

In Korea, it has been shown that anisakid larvae are highly resistant to various physicochemical stimuli, including temperature (high and low), chemical substance (salinity, acidity, alcohol, and others), irradiation, ultrasound, hydrostatic pressure, and anthelmintic drugs [170,216-224]. Lee and Chyu [216] reported that anisakid larvae survived 1, 4, 6, 3, and > 30 hours at -20℃, -15℃, -10℃, -5℃, and 0℃, respectively, and were killed when stored at 30% saline for 17 hours, 20% saline for 2 days, and 10% saline for 5 days; however, some (17%-19%) larvae lived > 10 days at 0.85% or 5% saline. In addition, anisakid larvae survived < 1 hour at 20% acetic acid, < 24 hours at 10% acetic acid, < 10 days at 5% acetic acid, < 2 days at 40% ethanol, < 3 days at 20% ethanol, and < 6 days at 10% ethanol. The larvae were highly resistant to variable concentrations of soy sauce, Japanese soy sauce, mustard, and Japanese mustard (wasabi) surviving 1 day or longer [216]. Lim [170] reported similar results. Chai et al. [217] observed high radioresistance of anisakid larvae; they demonstrated active movements even after irradiation with a surprisingly high dose of 10,000 Gy, although their infectivity to experimental rats and rabbits decreased considerably. This radioresistance was suggested to be due to the role of superoxide dismutase [218]. Jeon and Jee [219] studied the inhibitory effects of ivermectin, doramectin, and ethanol in vitro and found that ivermectin and ethanol had some effect on the migration and movement of anisakid larvae, whereas doramectin had no effect. However, the inhibitory effects of ivermectin were lower in vivo in experimental rats [220]. Herbal extracts (Meliae ezadarach, Dryopteris crassirhizoma, and Quisqualis indica var. villosa) have been tested for their inhibitory effects in vitro against anisakid larvae [221]; however, the in vivo effects need to be determined. Oh et al. [222] studied the effects of freezing, salting, and combined treatment with chlorine and ultrasound on anisakid larvae inoculated in salt-fermented squid and pollock tripe; all larvae were inactivated after 48 hours at -20℃ and 24 hours at -40℃. The viability of the larvae was 81.7% and 26.7% in 5% and 10% saline solutions, respectively, after 7 days of storage, and all larvae were inactivated when submerged in 15% saline for 7 days or in 20% saline for 6 days of storage [222]. The viability of the larvae (isolated from the f ish) was significantly reduced after combined treatment with low concentrations (< 2,000 ppm) of chlorine and ultrasound for 5-30 min; however, this combined treatment exerted no effects on the viability of the larvae located within the viscera of heavily infected sea eels [222]. Lee et al. [223] examined the effect of high hydrostatic pressure (HHP) on the viability of anisakid larvae in the flesh of sea eels and found that HHP at 200 MPa for 5 min could be used as a potential measure for the inactivation of anisakid larvae in f ish without changes in color and sensory quality of the fish. Nam et al. [224] observed the larvicidal effects of various food condiments, including soybean sauce, Japanese mustard, vinegar, red pepper paste, saline solution, ethanol, and soju (Korean liquor); all these exhibited significant effects after several hours, but L3 is exposed to these condiments only for seconds before ingestion, so these conditions cannot be used to prevent anisakid larval infections.