Anisakis Type Research Articles

Quality assessment of Zeus faber (Peter's fish) ovaries regularly commercialized for human consumption.

In the last few years, the consumption of fish eggs has increased rapidly, finding widespread use also in mass catering. This increase has involved also those of the Peter’s fish (Zeus faber). Females of this species, by their reproductive characteristics, have highly developed gonads in different periods of the year, making the raw material easy to find. The aim of the present study was to perform a quality assessment of Zeus faber ovaries regularly commercialized for human consumption. A total number of 34 samples, divided in fresh (11) and frozen (23), were processed for microbiological characterization, parasitological and histological evaluations. Fresh and frozen samples have significant (P<0.01) differences in total bacterial charge, with values of 4.75±0.5 Log CFU/g and 3.65±0.7 Log CFU/g respectively. The mean value of Enterobacteriaceae was 2.58±0.7 Log CFU/g in fresh products, while 52.17% (12) of frozen samples reported loads of <1 Log CFU/g. No Salmonella spp. and Listeria monocytogenes were found. Aeromonas spp. was detected in two frozen sample (with loads of 2.2 and <1 Log CFU/g) and in 5 fresh ovaries with value ranged from 1.70 to 3.48 Log CFU/g. Vibrio spp. was found in 4 (36.36%) and 3 (13.04%) of fresh and frozen samples respectively, with loads always <1 Log CFU/g. All 31 Vibrio strains isolated, were identified as Vibrio alginolyticus, and 61.29% (19) of them was positive for the ToxRS factor and 6.45% (2) for ToxR. The 47.06% (16) of total samples showed infestations by larvae of Anisakis Type 1 in the serous and inside the ovary. In this last case, histologically it was found to be free larvae. This study attested satisfactory hygiene conditions for Zeus faber ovaries currently marked for human consumption. The presence of potentially pathogenic strains of V. alginolyticus and Aeromonas spp., but above all the frequent infestation by Anisakis larvae, represent a potentially hazard for the consumer.

Keberadaan Anisakis typica (Anisakidae) dari Ikan Tongkol dan Ikan Layang dari perairan Sulawesi Barat

Anisakis (L3-stage) is a parasitic nematode commonly found in marine fish or squid serve as intermediate or paratenic host. The purpose of this research was to identify Anisakis larvae using PCR-RFLP and sequencing methods. A total of 30 individuals of each species: bullet tuna (Auxis rochei) and Indian scad(Decapterus russeli) were examined for presence of Anisakis spp. Fish samples caught by fishermen around Mamuju waters were purchased at a fish market. The fish was necropsied and the organs: liver, intestine wall, heart and muscle were removed and put on separated petri dishes and examined for parasites under a stereo microscope. Anisakis found was cleaned and fixed in 70% alcohol. Initial identification was based on ventriculus shape and presence of mucron to distinguish Anisakis type I and Anisakis type II. All Anisakis found belong to Anisakis type I. Ten Anisakis type I were isolated, cleaned and stored in microfuge tubes containing 70% alcohol. The parasite gDNA was extracted using Wizard genomic DNA extraction and purification kit (Promega).Species of Anisakis was determined by PCR-RFLP at ITS1-5.8S-ITS2 region using two restriction enzymes: Taq I and Hinf I and PCR-sequencing in mtDNA cox2 region. Analysis by PCR-RFLP showed that all Anisakis type Iexamined was in the same pattern as Anisakis typica. Sequencing in mtDNAcox-2 region and the phylogenetic analysis showed that all samples were in the same cluster as A. typica. Based on PCR-RFLP and sequencinganalysis, all Anisakis found in this study belong to A. typica.

Anisakis sp. and Hysterothylacium sp. larvae in anchovies (Engraulis encrasicolus) and chub mackerel (Scomber colias) in the Mediterranean Sea: Molecular identification and risk factors

Larval ascaridoids in fish destined to human consumption represent an important public health issue, besides to be an economical problem. Indeed, marine ascaridoids are the etiological agents of the fish-borne zoonosis anisakidosis. Due to an increase of new cases reported worldwide, a continuous monitoring of infection in fish is mandatory. The study was aimed to evaluate the risk of infection by larval ascaridoids in fishes from Mediterranean Sea. Two species of fishes among those representing a major potential threat for human health were selected. Epidemiological and molecular study was carried out. At Milan Fish Market, Italy, 179 anchovies (Engraulis encrasicolus) and 84 chub mackerels (Scomber colias) caught in different fishing areas in the Mediterranean Sea were sampled and inspected for the presence of larvae. For each fish, larvae were counted and morphologically identified. Predictors of infections were investigated through general linear models. A subsample of 100 larvae was molecular characterized with PCR–RFLP targeting the nuclear ribosomal internal transcribed spacer (ITS) region. Moreover, 26 Hysterothylacium spp. larvae were analyzed by sequencing of both nuclear ITS and mitochondrial ribosomal rrnS regions.Overall, 1080 anisakids larvae were collected from 103 anchovies (P = 57.5%) and 53 chub mackerels (P = 63.09%). Larvae were morphologically identified as Anisakis Type I larvae (P = 6.14% in anchovies and P = 55.95% in chub mackerels) and as Hysterothylacium spp. (P = 54.18% in anchovies and P = 13.09% in chub mackerels). Fishing area and fish weight resulted predictors of both Anisakis Type I land Hysterothylacium spp. infections in anchovies; in chub mackerels, only fishing areas resulted to be associated to both infections. Molecular analysis on ITS region identified Anisakis pegreffii, heterozygote genotype between A. pegreffii and A. simplex sensu stricto, and Hysterothylacium aduncum. Sequences analysis on Hysterothylacium specimens revealed a great homogeneity in rrnS marker, with eight variable nucleotides and an average evolutionary divergence over all sequence of 0.3%.

Parasites of the Lessepsian invasive fish Lagocephalus sceleratus (Gmelin 1789) in the eastern Mediterranean Sea

ABSTRACTLagocephalus sceleratus, is a Lessepsian invasive fish species introduced into the Mediterranean Sea through the Suez Canal. This study investigated it as host of parasites. Out of 41 individuals caught from northern and southern locations of the eastern Aegean Sea, nine were found to host parasites. Gnathia sp. praniza larva, an ectoparasite, was associated with severe damage of the primary and secondary structures of the gills and it was found in one specimen (prevalence 2.4%). Regarding endoparasites, Anisakis type I third-stage larvae were found in two specimens (prevalence 4.9%). In addition, adult nematodes of Hysterothylacium aduncum were found in another six specimens (prevalence 14.6%). Lagocephalus sceleratus is a new host for some indigenous parasites, with low host specificity, in the Mediterranean Sea, providing an additional niche for the success and increase of local populations of these parasites and, to our knowledge, this is the first record of L. sceleratus parasites in this area. Whether these parasites can control the invasion success of L. sceleratus needs to be further investigated. However, the low mean intensity of infection found in this study does not support such control.

Survey for the presence of ascaridoid larvae in the cinnamon flounder Pseudorhombus cinnamoneus (Temminck & Schlegel) (Pleuronectiformes: Paralichthyidae)

The cinnamon flounder Pseudorhombus cinnamoneus is a frequently consumed marine fish in China. However, the occurrence of ascaridoid larvae in P. cinnamoneus remains unclear. In the present study, a total of 85 P. cinnamoneus caught from the Yellow Sea (off Shidao, 36°52′57″N, 122°26′42″E) in 2011, which is located between mainland China and the Korean Peninsula, was investigated for ascaridoid larval infection. Four ascaridoid larval types, including Anisakis type I of Berland (1961), Hysterothylacium type of Smith (1983), Hysterothylacium type HL of Guo et al. (2014) and Raphidascaris type of Zhao et al. (2016), were detected in this important food fish. These larval types were identified as Anisakis pegreffii, Hysterothylacium aduncum, H. sinense and Raphidascaris lophii, respectively, using PCR-based restriction fragment length polymorphism (PCR–RFLP) analysis and sequencing of the ITS region of nuclear ribosomal DNA (rDNA). The third-stage larvae of H. sinense are reported from Chinese waters for the first time. The prevalence of H. sinense was 100% and represents the predominant species of the ascaridoid larvae found in P. cinnamoneus. The prevalences of A. pegreffii and H. aduncum were 44.7% and 81.2%, respectively. Phylogenetic analyses based on ITS sequences were performed to elucidate the genetic relationships of these ascaridoid nematodes. The present study increases the knowledge and distribution of ascaridoid larvae in this area of Yellow Sea. The high prevalence of ascaridoid larvae in P. cinnamoneus shows that an assessment needs to be undertaken to assess the risk these parasites may pose to public health.

Occurrence of ascaridoid nematodes in selected edible fish from the Persian Gulf and description of Hysterothylacium larval type XV and Hysterothylacium persicum n. sp. (Nematoda: Raphidascarididae)

Despite several reports on the presence of the potentially zoonotic nematodes among edible fishes in the Persian Gulf, there is still no study on the specific identification of these parasites or their genetic characterisation. In the present study, a total of 600 fish belonging to five popular species of fish in the region, including Otolithes ruber, Psettodes erumei, Saurida tumbil, Scomberomorus commerson and Sphyraena jello were examined for infection with nematode parasites. Detailed microscopy of nematodes found in the present study followed by characterisation of the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) showed that they belong to five distinct taxa that could be potentially zoonotic. Anisakis type I was found in four species of fish, had identical ITS sequences as Anisakis typica previously reported in Australian waters and was different from those reported in the Nearctic. Hysterothylacium type VI in the present study was morphologically similar to those previously described from Australasian waters and ITS sequences were identical among Australian specimens and those found in the present study. Another Hysterothylacium larval type was also found in the present study which had identical ITS sequences and similar morphology to those previously reported and identified as H. amoyense in China Sea. Since no ITS sequence data from a well identified adult H. amoyense with an identifiable museum voucher number is yet available and due to some other issues discussed in the article we suggest assignment of this larval type from the China Sea and the Persian Gulf to H. amoyense is doubtful until future studies on a well identified male specimen of H. amoyense or other species reveals the specific identity of this larval type. We propose to refer to this larval type as Hysterothylacium larval type XV. In the present study we also describe a new species, Hysterothylacium persicum and discuss how to differentiate it from closely related species. We also found some adult females with distinct morphology and ITS sequence but due to lack of male specimens they have been referred as Hysterothylacium sp. in this paper. They had the same ITS sequence data as Hysterothylacium larval type VI. This study shows the presence of a relatively broad diversity of potentially zoonotic nematodes in edible fish of the Persian Gulf. Therefore educational campaigns for public and local health practitioners are suggested to protect consumers from becoming infected with these parasites.

Anisakis Dujardin, 1845 infection (Nematoda: Anisakidae) in Pygmy Sperm Whale Kogia breviceps Blainville, 1838 from west Pacific region off the coast of Philippine archipelago.

Cetaceans are definitive hosts of anisakid nematodes known to cause human anisakidosis. Despite the reported strandings of different cetaceans in the Philippines, studies on anisakids from these definitive hosts are limited. Here, the morphologically and molecularly identified anisakid species, specifically those of the genus Anisakis Dujardin, 1845 in stranded Pygmy Sperm Whale Kogia breviceps Blainville, 1838 in the west Pacific region off Philippine waters are presented. Morphological data using SEM and LM revealed multi-infections with different Anisakis species belonging to Anisakis type I and type II groups. Molecularly, PCR-RFLP on the ITS rDNA and sequence data analyses of both ITS rDNA and mtDNA cox2 regions identified those from Anisakis type I group as A. typica (Diesing, 1860), whereas those from type II group as A. brevispiculata Dollfus, 1968, and A. paggiae Mattiucci et al. (Syst Parasitol 61:157-171, 2005). This is the first record of Anisakis infection from this host stranded in the west Pacific region off the coast of Philippine waters and new geographical record for A. paggiae.

High prevalence of Anisakidae larvae in marketed frozen fillets of pink salmons (Oncorhynchus gorbuscha)

The wild pink salmon (Oncorhynchus gorbuscha) has a great economic importance having a meat quality higher than that of breeding salmons; however, the consumption of wild pink salmons is a public health concern due to the high risk of infection with the third-stage nematode larvae (L3) of the family Anisakidae. Only L3 of Anisakis and Pseudoterranova genera are the causative agents of anisakidosis and allergies, which can be even fatal. The aims of the present work were to investigate the infection prevalence and species identity of Anisakidae larvae in fillets of North Pacific wild pink salmons. A total of 244 samples of frozen fillets, collected between 2009 and 2013, were examined by the artificial digestion method. L3 were detected in 177 (72.5%) fillets. Out of 708 recovered L3, 705 were identified as Anisakis type I and 3 as Pseudoterranova sp. by morphological examination; of these, one L3 from each infected fish, was identified as Anisakis simplex sensu stricto (#174) or as Pseudoterranova decipiens (#3) by PCR-RFLP. Despite the risk of acquiring human anisakidosis is efficiently prevented by fish fillet freezing, our findings highlight how the high prevalence of Anisakidae larvae in frozen fillets of pink salmon, may be the source of allergic reactions in sensitized persons. The increased interest of consumers in the pink salmon may pose considerable food safety problems that merits to be systematically investigated.

Potential risk analysis of human anisakiasis through the consumption of mackerel, Scomber scombrus, sold at Spanish supermarkets

Herein, the potential risk of human anisakiasis through the consumption of mackerel, Scomber scombrus, sold at nationwide Spanish supermarket chains is assessed, by means of the identification of anisakid and other nematode species found, and the analysis of intrinsic and extrinsic factors that may influence the prevalence and abundance of nematode worms, as well as their presence in the viscera or in the flesh. 231 fresh mackerel specimens caught between 2008 and 2014 (140 from the Atlantic and 91 from the Mediterranean), captured in autumn and spring and acquired at nationwide Spanish supermarket chains, were examined dissecting their viscera and submitting their flesh to artificial digestion. Helminthological identification was based on the morphological criteria of L3. Moreover, 62 larvae of different origins and seasons were molecularly characterized by RFLP-PCR. The target region, ITS1-5.8S rDNA-ITS2, was amplified and the restriction enzyme HinfI was used for digestion. The potential influence of intrinsic (fish length and weight) and extrinsic (origin and season of capture, and days after catch) factors was statistically analysed. Total Anisakis type I prevalence was 58.4% (55.4% in viscera; 26.0% in the flesh) and 4.8% Hysterothylacium spp. 45 Anisakis simplex (s.s.), 12 hybrid genotype (A. simplex s.s./Anisakis pegreffi) and only one larva of A. pegreffi were identified in the 58 L3 molecularly characterized. The four Hysterothylacium larvae were identified as belonging to the species H. aduncum. There were statistically significant differences in the presence of Anisakis type I from the Atlantic and the Mediterranean (χ2 = 47.341; p < 0.0001), in viscera as opposed to the flesh (χ2 = 42.630; p < 0.0001), also in the longest specimens (χ2 = 73.170; p < 0.0001) and in the highest weight (χ2 = 27.469; p < 0.0001). A significant correlation (χ2 = 22.458; p = 0.004) was found between the presence of larvae in the flesh and the number of days passed between the catch and the analysis. Hence, apart from the recommendation of freezing fish at −20 °C for at least 24 h in case the fish is consumed raw or poorly cooked, the aforementioned factors should be considered by the consumer when purchasing this fish species to avoid any potential anisakiasis risk.

Anisakiasis: Report of 15 Gastric Cases Caused by Anisakis Type I Larvae and a Brief Review of Korean Anisakiasis Cases.

The present study was performed to report 15 anisakiasis cases in Korea and to review the Korean cases reported in the literature. Total 32 Anisakis type I larvae were detected in the stomach of 15 patients by the endoscopy. Single worm was detected from 12 cases, and even 9 larvae were found from 2 cases. Epigastric pain was most commonly manifested in almost all cases, and hemoptysis and hematemesis were seen in 1 case each. Symptom manifestations began at 10-12 hr after eating fish in 73.3% cases. Endoscopy was performed 1-2 days after the symptom onset in most cases. The common conger, Conger myriaster, was the probable infection source in 7 cases. In the review of Korean anisakiasis cases, thus far, total 645 cases have been reported in 64 articles. Anisakis type I larva was the most frequently detected (81.3%). The favorable infection site of larvae was the stomach (82.4%). The common conger was the most probable source of human infections (38.6%). Among the total 404 cases which revealed the age and sex of patients, 185 (45.8%) were males, and the remaining 219 (54.2%) were female patients. The age prevalence was the highest in forties (34.7%). The seasonal prevalence was highest in winter (38.8%). By the present study, 15 cases of gastric anisakiasis are added as Korean cases, and some epidemiological characteristics of Korean anisakiasis were clarified.

Natural products: Perspectives in the pharmacological treatment of gastrointestinal anisakiasis.

To evaluate the activity of different natural products against L3 larvae of Anisakis type I. Information on investigations into the activity of natural products against the L3 larvae of Anisakis simplex was collected from scientific journals, books, theses and reports via a library and electronic search (using Pubmed, Scopus, Medline, Web of Science and ScienceDirect). The search terms included: natural products, medicinal plants, essential oils, terpenic derivatives, Anisakis, antinematodal activity. In the literature reviewed numerous papers were found concerning the invitro and invivo activity against Anisakis type I of various natural products (plant extracts, essential oils and their major components). Analysis of the results showed that invitro tests the Melaleuca alternifolia essential oil and the ar-turmerone isolated from Curcuma longa displayed the greatest activity. Invivo, the most active compound was perillaldehyde, the main component of Perilla frutescens essential oil. This study shows that some natural products exhibited promising antianisakis properties.

Anisakis pegreffii Larvae in Sea Eels (Astroconger myriaster) from the South Sea, Republic of Korea.

Anisakis simplex sensu stricto (s.s.), Anisakis pegreffii, Anisakis berlandi (=A. simplex sp. C), and Anisakis typica are the 4 major species of Anisakis type I larvae. In the Republic of Korea (Korea), A. pegreffii, A. berlandi, and A. typica larvae in fish hosts has seldom been documented. In this study, molecular analysis was performed on Anisakis larvae from the sea eels (Astroconger myriaster), the major source of human anisakiasis in Korea, collected from Tongyeong City, a southern coastal area of Korea. All 20 sea eels examined were infected with Anisakis type I larvae (160 larvae; 8 per fish). Their species were analyzed using PCR-RFLP patterns and nucleotide sequences of internal transcribed spacers (ITS1, 5.8 subunit gene, and ITS2) and mitochondrial cytochrome c oxidase 2 (cox2). Most (86.8%; 112/129) of the Anisakis type I larvae were A. pegreffii, and 7.8% (10/129) were A. typica. The remaining 5.4% (7/129) was not identified. Thus, A. pegreffii is the major species of anisakid larvae in sea eels of the southern coast of Korea.

Characterisation of Ascaridoid larvae from marine fish off New Caledonia, with description of new Hysterothylacium larval types XIII and XIV

Here we report occurrence of six different morphotypes of ascaridoid type larvae from 28 species of fish collected from New Caledonian waters. The larvae were morphologically identified as Anisakis type I, Hysterothylacium type VI and new larval types XIII and XIV, Raphidascaris larval type and Terranova larval type II. Representatives of each morphotype were subjected to the amplification of the second internal transcribed spacers (ITS-2) of ribosomal DNA (rDNA) and those sequences were compared with ITS-2 sequences of other ascaridoid nematodes previously deposited in GenBank. ITS-2 sequences of Anisakis larval type I were identical to those of A. typica. ITS-2 sequences of Hysterothylacium larval type VI in the present study were identical to those previously found in Eastern Australian waters. No match was found for ITS-2 sequences of Hysterothylacium larval types XIII and XIV; therefore, the specific identities of these larval types remain unclear. ITS-2 sequences of Raphidascaris larval type were identical to those of R. trichiuri, which have previously been reported in Taiwanese waters. Terranova larval type II in the present study had identical ITS-2 sequences with Terranova larval types reported from Australian waters, however, the specific identity is unknown. This taxonomic work is essential if further research on these zoonotic parasites is to be effective. This includes investigations into such aspects as life cycle studies, impacts on human health and risk assessment for their transmission to humans.

Occurrence and prevalence of fish-borne Anisakis larvae in the spotted mackerel Scomber australasicus from Taiwanese waters.

Anisakid nematodes have been found in a variety of marine fishes throughout the world and they are known to cause anisakiasis in human hosts. The present study investigated the prevalence of potentially zoonotic anisakid larvae in spotted mackerel caught from Taiwanese waters where fish represents an important food sources. Anisakis third-stage larvae (L3, n=502) were isolated from 250 spotted mackerel Scomber australasicus. Anisakis L3 larvae were divided morphologically into two types, Anisakis type I larvae had a longer ventriculus and mucron while type II larvae had a shorter ventriculus and no mucron. Anisakis species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer (ITS) regions of ribosomal DNA and direct sequencing. A simple molecular taxonomic key, utilizing RFLP by two restriction enzymes HinfI and HhaI, enabled the differentiation of the genus Anisakis. The prevalence, mean intensity and mean abundance of Anisakis nematodes recorded for the total specimens were 72.8%, 2.8 (1-15) and 2.0 (0-15), respectively. Anisakis pegreffii was determined to be the dominant species (prevalence=57.2%) and important agent of human anisakiasis. A recombinant genotype (Anisakis simplex sensu stricto × A. pegreffii) was identified as the subdominant species (25.3%) followed by Anisakis typica (10%), Anisakis physeteris (4.0%), Anisakis paggiae (3.0%) and Anisakis brevispiculata (0.5%). The topology of the maximum likelihood and neighbor-joining trees show two well supported clades: one includes the species of A. pegreffii and the other includes A. paggiae, A. physeteris and A. brevispiculata, while A. typica has basal position to all other Anisakis spp. analyzed. This study advances our knowledge of the prevalence of different Anisakis spp. in the spotted mackerel from Taiwanese waters, which is helpful for monitoring the fish populations throughout a diverse array of aquatic ecosystems. More importantly, we provide the concise characterization of multiple Anisakis spp. by PCR-RFLP, which could also be applicable for the rapid diagnosis of human anisakiasis.

Molecular analysis of Anisakis type I larvae in marine fish from three different sea areas in Korea.

Anisakiasis, a human infection of Anisakis L3 larvae, is one of the common foodborne parasitic diseases in Korea. Studies on the identification of anisakid larvae have been performed in the country, but most of them have been focused on morphological identification of the larvae. In this study, we analyzed the molecular characteristics of 174 Anisakis type I larvae collected from 10 species of fish caught in 3 different sea areas in Korea. PCR-RFLP and sequence analyses of rDNA ITS and mtDNA cox1 revealed that the larvae showed interesting distribution patterns depending on fish species and geographical locations. Anisakis pegreffii was predominant in fish from the Yellow Sea and the South Sea. Meanwhile, both A. pegreffii and A. simplex sensu stricto (A. simplex s.str.) larvae were identified in fish from the East Sea, depending on fish species infected. These results suggested that A. pegreffii was primarily distributed in a diverse species of fish in 3 sea areas around Korea, but A. simplex s.str. was dominantly identified in Oncorhynchus spp. in the East Sea.

Parasites of the deep-sea fish Mora moro ( ) from the NW Mediterranean Sea and relationship with fish diet and enzymatic biomarkers

Specimens of Mora moro were collected in two seasons and three localities of the Balearic Sea (NW Mediterranean Sea) and parasitological, dietary (to prey-species level), enzymatic and histological data were obtained, alongside with environmental information (T, S, O2). The relationships among fish parasite load, condition indices, diet, enzymatic activity of muscular acetylcholinesterase (AChE) and lactate dehydrogenase (LDH), intensity of splenic melano-macrophage centres (MMC) and hepatic granulomas were tested. M. moro showed a rich and abundant parasite fauna, and was a new host record for 17 out of the 18 different endoparasite taxa found. Significant differences were detected among locality-season groups, in turn related to different environmental variables, for Anisakidae gen. sp., Anisakis Type II and Tetraphyllidea fam. gen. sp.; thus, they are proposed as potentially useful as biological tags for geographical discrimination of M. moro in the NW Mediterranean Sea. Detailed relationships were found between parasite taxa and prey ingested (e.g. Anisakidae gen. sp. related with meso-bathypelagic crustaceans; Anisakis Type I with benthopelagic squids). Most parasites were linked to samples with highest levels of near-bottom O2, which is consistent with direct relationships found between near-bottom O2 and zooplankton biomass in the Balearic Basin. Total parasite abundance and the abundance of Tetraphyllidea fam. gen. sp. showed a significant relationship with the activity of AChE and the abundance of Anisakis Type II with LDH. AChE was associated with hepatosomatic index (HSI) and condition factor (K); LDH with gonadosomatic index (GSI), K and fish total length (TL). LDH activity showed differences among sampling groups. Splenic MMC and hepatic granulomas were not associated with fish parasite load. A positive relationship was found between MMC area and fish TL and LDH activity.

Occurrence of Anisakis simplex sensu stricto in imported Atlantic mackerel (Scomber scombrus) represents a risk for Turkish consumers

Anisakid larvae are a prevalent food-borne pathogen that has been found in numerous fish species destined for human consumption. The accidental consumption of infected raw or poorly cooked fish may cause gastroenteric diseases and allergies in humans. In spite of the fact that thorough cooking or freezing kills Anisakis worms, this method does not destroy their allergenic capacity. The presence of A. simplex (s.s.) in seafood products may present a health risk for consumers. In Turkey, Atlantic mackerels are marketed as frozen and mainly imported from Norway. The aim of this study was to identify the Anisakis species found in deep-frozen whole Atlantic mackerel (Scomber scombrus) destined for human consumption in fish markets that imported fish from Norway to Turkey. All Anisakis larvae isolated from imported Atlantic mackerel were identified via morphology as third larvae of Anisakis Type I. The ITS region (ITS-1, 5.8S subunit, ITS-2) was amplified and digested with the restriction enzymes Hinf I and Hha I. Larvae of the genus Anisakis were identified via PCR-RFLP as belonging to Anisakis simplex (s.s.), and this was confirmed by sequencing the cox2 gene. The overall prevalence of Anisakis larvae was 25% (95% confidence limits: 13–41%), and the mean intensity was 19.1 (bootstrap 95% confidence limits: 15.3–25.5). Recognized zoonotic A. simplex (s.s.) larvae found in imported Atlantic mackerel could represent a risk. Those who consume them could acquire parasitic allergies. The results will have an important impact on public health risk assessment in that they suggest reviewing critical control points at the Hazard Analysis Critical Control Point (HACCP) programmer to reduce the risk of anisakid-induced allergies among consumers. Consequently, the present study provides the first data regarding the occurrence of A. simplex (s.s.) larvae in imported Atlantic mackerel in Turkish markets.

Occurrence and Molecular Identification of Anisakis Dujardin, 1845 from Marine Fish in Southern Makassar Strait, Indonesia

Anisakis spp. (Nematoda: Anisakidae) parasitize a wide range of marine animals, mammals serving as the definitive host and different fish species as intermediate or paratenic hosts. In this study, 18 fish species were investigated for Anisakis infection. Katsuwonus pelamis, Euthynnus affinis, Caranx sp., and Auxis thazard were infected with high prevalence of Anisakis type I, while Cephalopholis cyanostigma and Rastrelliger kanagurta revealed low prevalence. The mean intensity of Anisakis larvae in K. pelamis and A. thazard was 49.7 and 5.6, respectively. A total of 73 Anisakis type I larvae collected from K. pelamis and A. thazard were all identified as Anisakis typica by PCR-RFLP analysis. Five specimens of Anisakis from K. pelamis and 15 specimens from A. thazard were sequenced using ITS1-5.8S-ITS2 region and 6 specimens from A. thazard and 4 specimens from K. pelamis were sequenced in mtDNA cox2 region. Alignments of the samples in the ITS region showed 2 patterns of nucleotides. The first pattern (genotype) of Anisakis from A. thazard had 100% similarity with adult A. typica from dolphins from USA, whereas the second genotype from A. thazard and K. pelamis had 4 base pairs different in ITS1 region with adult A. typica from USA. In the mtDNA cox2 regions, Anisakis type I specimens from A. thazard and K. pelamis showed similarity range from 94% to 99% with A. typica AB517571/DQ116427. The difference of 4 bp nucleotides in ITS1 regions and divergence into 2 subgroups in mtDNA cox2 indicating the existence of A. typica sibling species in the Makassar Strait.

Molecular identification of Anisakis species (Nematoda: Anisakidae) from marine fishes collected in Turkish waters

Anisakid nematodes are important etiological agents for zoonotic human anisakiasis (or anisakidosis). These parasites in the Turkish waters still remain unexplored. This study aims the molecular identification of Anisakis species in Turkey's coast from Black, Aegean and Mediterranean Sea and specifically to screen for zoonotic species in commonly commercialized a total of 1145 fish belonging to 31 different species using both polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) and sequencing of the ribosomal internal transcribed spacer (ITS) regions and the mitochondrial cytochrome C oxidase subunit II (cox2) gene. A total of 776 Anisakis type I larvae were isolated in 56/1145 (4.8%) fish of 7 species from Turkish waters. The combining all of our results, e.g., morphology, PCR-RFLP, ITS region, and the cox2 gene, conclusively supported the identification of 3 Anisakis spp. taken from marine fish hosts, namely Anisakis pegreffii, Anisakis typica and Anisakis simplex sensu stricto (s.str.)/A. pegreffii hybrid genotype. No Anisakis larvae were isolated from the Black Sea whereas A. pegreffii, A. typica and A. simplex s.str./A. pegreffii hybrid genotype was found in the Aegean Sea and A. pegreffii was only isolated from the Mediterranean Sea. This study represents the first identification of A. typica and A. simplex s.str./A. pegreffii hybrid genotypes from Turkish waters. Moreover, in the present study first record of the presence of A. pegreffii is also reported from Turkish coasts of Aegean and Mediterranean Sea. No zoonotic Anisakis species were found in commonly commercialized 1025 fish belonging to 16 different species from the Black Sea, thus Turkish populations who consume captured fish from the Black Sea may have a less risk of human anisakiasis or allergies. However, the prevalence of larvae were 47.1% and 46% and recognized zoonotic A. pegreffii were identified from the Aegean and Mediterranean Sea coast, suggesting a high threat of anisakiasis or allergies for Turkish populations who consume fish originating in these regions.

A histological study of eosinophilic granuloma in mice following infestation with Anisakis larvae

Anisakidosis is a parasitic anthropozoonosis caused by the nematode larvae of the Anisakidae family. Anisakid nematodes belonging to the Anisakis pegreffii are highly prevalent in several fish species living in the Mediterranean sea. The larvae can accidentally infect humans following the ingestion of infected raw or undercooked sea fish. A migrating larva causes a clinical disease when it invades the stomach or intestinal wall and peritoneum, mimicking an eosinophilic gastroenteritis or an ulcer. In this preliminary experiment, the histopathology of the newly-formed parasitic granulomas in mice infested with third stage Anisakis pegreffii larvae, was studied and described. The larvae were morphologically identified as Anisakis type I by the presence of a boring tooth and a mucron, without ventriculus and caecum. The larval DNA was extracted and amplified by polymerase chain reaction (PCR). After PCR, the samples were processed to undergo restriction fragment length polymorphism analysis (RFLP). This was done to scan the restriction profiles for genetic identification. PCR products were purified and sequenced. The sequences were analysed to detect the relationship between nucleotides and perform a phylogenetic analysis. The paraffin-embedded granuloma samples showed worms having a diameter of 0.55 mm x 0.37 mm, polymyarian muscle cells and a circular intestine. The histological profiles showed a primary lesion at the site of anisakid penetration marked by oedema and neutrophil and eosinophil infiltration. The presence of histiocytes or epithelioid histiocytes, lymphocytes, monocytes, and plasma cells was also possible. Fibrinous exudation, hemorrhage, or vascular damage were detected within the first week of the acute intestine infection with a massive eosinophilic infiltration. Two weeks after the infestation, the infiltrating host cells formed a granuloma in the tissue surrounding the penetrated worm mainly consisting of eosinophils, a large number of fibroblasts and a varying number of admixed multinucleated giant cells. In order to explain the origin of the eosinophilic granulomas, a study into the produced substance attracting eosinophils and other host white blood cells to the area will carry out.