Anisakis Species Research Articles

Occurrence of Anisakis pegreffii (Nematoda: Anisakidae) Larvae in Imported John Dory (Zeus faber) from Senegalese Coast Sold in Turkish Supermarkets.

West African goatfish Pseudupeneus prayensis, bluespotted seabream Pagrus caeruleostictus and John Dory Zeus faber are commercially marketed as fresh and frequently imported from Senegalese coast (FAO area 34.3.12) in Turkish supermarkets. The aim of the current study was to collect data of occurrence and molecular identification of Anisakis species in imported P. prayensis, P. caeruleostictus and Z. faber caught in the Senegalese coast and to support epidemiological report for a risk evaluation of Anisakis species in Turkish supermarkets. Forty imported fish from each species at a total of 120 samples were investigated for the presence of Anisakis larvae. Based on ITS region of RFLP analysis Anisakis larvae were identified and randomly selected five larvae were also sequenced for further confirmation for cox2 gene. No Anisakis larvae were isolated from P. prayensis, P. caeruleostictus whereas Anisakis larvae were only found in Z. faber. A total of 156 Anisakis larvae were collected from Z. faber. All larvae were molecularly identified as Anisakis pegreffii. The prevalence (%), intensity and abundance of Anisakis infection in Z. faber were detected to be 82.5%, 8.3 and 6.8, respectively. This is the first assessment of the occurrence of A. pegreffii in imported Z. faber from the Senegalese coast in Turkish supermarkets. Moreover, consuming imported P. prayensis and P. caeruleostictus present low to non-existent risk for anisakiasis in Turkish consumers. Furthermore, the presence of A. pegreffii larvae in imported Z. faber from the Senegal waters could have public health implications in Turkish consumers.

Differential proteolytic activity in Anisakis simplex s.s. and Anisakis pegreffii, two sibling species from the complex Anisakis simplex s.l., major etiological agents of anisakiasis

Proteolytic activity was studied in two sibling species of Anisakis (Nematoda: Anisakidae), A. simplex s.s. and A. pegreffii, throughout their in vitro development from third larval stage (L3) from the host fish (L3-0h) to fourth larval stage (L4) obtained in culture. Proteases have a significant role in the lifecycle of the parasite and in the pathogen–host relationship. Proteolytic activity peaks were detected at pH 6.0 and 8.5. Protease activity was detected in all the developmental stages of the two species studied at both pH values. These pH values were used for assaying with specific inhibitors which permitted the determination of metalloprotease activity, and, to a lesser extent, that of serine and cysteine protease. Aspartic protease activity was only detected at pH 6.0. At this pH, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p < 0.001), the majority of activity being due to metalloproteases and aspartic proteases, which could be related to nutrition, especially the latter, as occurs in invertebrates. At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p < 0.01). At this pH, the majority of activity was due to metalloproteases in all developmental phases of both species, although, in L3-0h, the activity of these proteases was significantly higher (p < 0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former. Serine proteases have frequently been implicated in the invasive capacity and pathogenicity of some parasites. This may be related to the significantly higher activity (p ≤ 0.05) of serine protease in all the larval stages of A. simplex studied at pH 6.0. Thus, there are interspecific differences in proteases that have been related to pathogenesis in nematodes. These differences could thus be contributing to the previously reported differences in pathogenicity between these two Anisakis species.

On the occurrence and molecular identification of Contracaecum larvae (Nematoda: Anisakidae) in Mugil cephalus from Turkish waters.

Anisakis and Contracaecum species are fish borne zoonotic nematodes. In our previous studies, other larval anisakid and raphidascarid nematodes, Anisakis and Hysterothylacium species, were genetically identified in marine fish from Turkish waters. However, there is no information on molecular identification of larval Contracaecum species in marine fish from Turkey. Therefore, the aim of this study was only to investigate the presence and molecular identification of Contracaecum species in commonly commercialized marine fish from Turkish waters. A total of 475 marine fish, which belong to 21 different species, were sampled from the Aegean (FAO 37.3.1), Mediterranean (FAO 37.3.2), and Black Sea (FAO 37.4.2). The prevalence of Contracaecum L3 larvae in the Aegean Sea was identified as 10% in Mugil cephalus. All Contracaecum L3 larvae were molecularly characterized with RFLP targeting the ITS region and rrnS gene. Moreover, all larvae were analyzed by sequencing of ITS region, rrnS and cox2 gene. All Contracaecum larvae were identified as C. overstreeti based on the cox2 sequence analysis. This is the first report of C. overstreeti larvae in M. cephalus as paratenic and intermediate hosts. Furthermore, the analysis reveals novel information on ITS region. Additionally, the rrnS gene of C. overstreeti was also achieved and deposited in Genbank for the first time. The PCR-RFLP patterns of the ITS region and rrnS gene from C. overstreeti were presented in the present study. Consequently, the presence of C. overstreeti larvae in M. cephalus from the Aegean Sea may alsopotentially capable of inducing allergic sensitization in humans.

Establishment and validation of ARMS (amplification-refractory mutation system) for identification of Anisakis species collected from Korean waters

The identification of Anisakis species in Korean waters was performed using an amplification-refractory mutation system (ARMS). ARMS is typically used to detect single nucleotide polymorphisms or allele types in the same species. However, the application of ARMS for species identification has not been reported. We designed a tetra-primer binding the internal transcribed spacer (ITS) region of 76 samples of Anisakis species and obtained reproducible results. ARMS revealed 380 bp and 130 bp ITS fragments in A. pegreffii, 380 bp and 280 bp fragments in A. simplex, a 130 bp fragments in A. typica and 380 bp, 280 bp and 130 bp fragments in an A. simplex – A. pegreffii hybrid. ARMS using a tetra-primer could be a more rapid, efficient, and reliable tool for monitoring Anisakis species more rapidly than restriction fragment length polymorphism.

Identification and prevalence of Anisakis pegreffii and A. pegreffii × A. Simplex (s.s.) hybrid genotype larvae in Atlantic horse Mackerel (Trachurus trachurus) from some North African Mediterranean coasts

Atlantic horse mackerel is one of the most popular fish for North African consumers. Mackerel constitutes a common host for the Anisakis species of infective larvae (L3). A. pegreffii and A. pegreffii × A. simplex (s.s.) hybrid genotype were identified using PCR-RFLP and entire ITS-DNA sequencing protocol. This research investigated the prevalence and mean intensity of A. pegreffii and its hybrid form in randomly collected mackerel samples throughout spring to summer seasons from the Libyan western coast. Briefly, 55 out of 240 (22.9%) fish samples were confirmed to be infected and the prevalence of A. pegreffii reached 22.08% with mean intensity of infection 29.13 ± 2.43 parasite/fish, while the prevalence of A. pegreffii × A. simplex (s.s.) hybrid genotype was 0.8% with mean intensity of infection 22 ± 0.85 parasite/fish. The prevalence of infection noticeably elevated during the summer season to reach 30.8%. Additionally, female fish samples were heavily infected as the mean intensity of infection was 34.75 ± 1.27 parasite/fish. Furthermore, results indicated that the highest intensity of infection was recorded in older and female mackerel. To sum up, the current study has provided substantial information about the impacts, diversity and epidemiology of A. pegreffii and its hybrid form in North African waters.

Molecular Epidemiology of Anisakis and Anisakiasis: An Ecological and Evolutionary Road Map.

This review addresses the biodiversity, biology, distribution, ecology, epidemiology, and consumer health significance of the so far known species of Anisakis, both in their natural hosts and in human accidental host populations, worldwide. These key aspects of the Anisakis species' biology are highlighted, since we consider them as main driving forces behind which most of the research in this field has been carried out over the past decade. From a public health perspective, the human disease caused by Anisakis species (anisakiasis) appears to be considerably underreported and underestimated in many countries or regions around the globe. Indeed, when considering the importance of marine fish species as part of the everyday diet in many coastal communities around the globe, there still exist significant knowledge gaps as to local epidemiological and ecological drivers of the transmission of Anisakis spp. to humans. We further identify some key knowledge gaps related to Anisakis species epidemiology in both natural and accidental hosts, to be filled in light of new 'omic' technologies yet to be fully developed. Moreover, we suggest that future Anisakis research takes a 'holistic' approach by integrating genetic, ecological, immunobiological, and environmental factors, thus allowing proper assessment of the epidemiology of Anisakis spp. in their natural hosts, in human populations, and in the marine ecosystem, in both space and time.

Integrative taxonomy of anisakid nematodes in stranded cetaceans from Brazilian waters: an update on parasite's hosts and geographical records.

Anisakidae are represented mainly by species of Anisakis Dujardin, 1845, Pseudoterranova Krabbe, 1878, and Contracaecum Railliet and Henry, 1913. Integrative taxonomy, based on morphological and genetic approaches, can provide a precise identification of species, increasing the knowledge of their systematics, biology, and evolution. In this study, integrative taxonomy was applied in order to identify, at generic and/or specific taxonomic level, 245 anisakids from 63 cetaceans of 12 different species, which stranded in the Brazilian coast. Parasites from nine hosts were preserved by freezing and, from 54 hosts, were fixed in 70% ethanol (n=43) or alcohol-formalin-acetic (n=11) for a period varying from 6 to 19years. The morphological analysis allowed the identification of 171 specimens at genus level: 120 as Anisakis sp. clade I, 47 as Anisakis sp. clade II, and four as Pseudoterranova sp. From those 15, specimens were identified at species level, seven as Anisakis typica, seven as Anisakis paggiae and one as Anisakis brevispiculata. Moreover, 74 specimens were identified as belonging to Anisakidae family. The genetic analysis based on cox2 gene allowed the identification/confirmation of Anisakis typica (n=33), Anisakis paggiae (n=1), and for the first time in the Brazilian coast, Anisakis brevispiculata (n=4) and Anisakis ziphidarum (n=1). Additionally, a probably new Anisakis species, genetically close to A. paggiae, is reported infecting a dwarf sperm whale. The results clearly indicated that the preservation of nematodes influenced significantly on the level of taxonomic definition, in both morphological and, mainly, genetic analyses. Pseudoterranova sp. was identified for the first time in the spinner dolphin, Stenella longirostris. The detection of nine A. paggiae specimens, in three Kogia breviceps animals, substantiates the austral occurrence of this species. The identification of A. brevispiculata in Kogia sima and K. breviceps confirms Kogiidae whales as their most common hosts. Anisakis typica was corroborated as a generalist species, the most widely distributed in the Brazilian coast, infecting a broad variety of Delphinidae and Kogiidae, with two new geographical records in Stenella frontalis and Lagenodelphis hosei. The study showed a new scenery of parasite diversity of marine anisakids, updating the Anisakis species distribution.

Anisakis species composition and infection characteristics in Atlantic mackerel, Scomber scombrus, from major European fishing grounds — reflecting changing fish host distribution and migration pattern

Atlantic mackerel (Scomber scombrus) ranks among the most valuable fish species in Europe. The NE Atlantic mackerel population is considered to comprise three main stocks (southern, western and North Sea), with variable proportions of these three intermixing at the northerly feeding grounds. The southern and western mackerel stocks have moved over the past 4–5 years further north- and westward. Consequently, large-scale mackerel fishing and processing have become thriving industries in Iceland and the Faroe Islands in just a few years. The mackerel population structure in the Mediterranean Sea is less well known but seems to comprise at least one, more or less isolated, spawning component. Although mackerel is an important food resource, systematic and concerted epidemiological surveys of Anisakis species in Atlantic mackerel from European fishing grounds have been lacking. As part of the EU FP7 PARASITE project (GA no. 312068), occurrence and specific identity of Anisakis spp. from 1801 mackerel from Northeast Atlantic and Mediterranean waters was investigated. In general, mackerel caught at the Atlantic fishing grounds exhibited markedly higher Anisakis spp. infection levels than fish from the Mediterranean localities. Mackerel caught off NW Spain and Portugal (ICES VIIIc, IXa) showed highest overall and muscular prevalence, reaching 87% and 52%, respectively, which differed significantly from all other Atlantic samples. Lowest overall Anisakis spp. prevalence and abundance was recorded in mackerel from Faroe Isles waters, while lowest muscular infection levels were found in the samples from the North Sea. Genetic nematode species identification showed that A. simplex (sensu stricto) is the dominating species in mackerel from the Atlantic areas, while A. pegreffii dominated in the samples from the Mediterranean Sea. The latter species showed generally low prevalence and intensity in the flesh, not exceeding 6% and one larva, respectively. While A. simplex (s. s.) and A. pegreffii seem to co-occur in mackerel from off NW Spain and Portugal, several A. pegreffii were also recorded in mackerel from the North- and Norwegian Seas. These findings imply that the actual mackerel started their feeding migration in waters south to the British Isles, which include parts of the sympatric area of the two sibling species. Thus, A. pegreffii could prove a useful supplementary marker to track migration routes of the different mackerel stock components in NE Atlantic waters.

Invasive anisakiasis by the parasite Anisakis pegreffii (Nematoda: Anisakidae): diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay

BackgroundAnisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen.MethodsParasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied.ResultsAccording to the mtDNA cox2 and the EF1 α − 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases.In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like.ConclusionThe Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis.

The Mediterranean European hake, Merluccius merluccius: Detecting drivers influencing the Anisakis spp. larvae distribution

The European hake Merluccius merluccius is one of the most commercially important and widely distributed fish species, occurring both in European and Mediterranean Sea fisheries. We analyzed the distribution and infection rates of different species of Anisakis in M. merluccius (N=1130 hakes), by site of infection in the fish host (viscera, dorsal and ventral fillets) from 13 different fishing grounds of the Mediterranean Sea (FAO area 37). The fillets were examined using the UV-Press method. A large number of Anisakis specimens (N=877) were identified by diagnostic allozymes, sequence analysis of the partial EF1 α-1 region of nDNA and mtDNA cox2 gene. Among these, 813 larvae corresponded to A. pegreffii, 62 to A. physeteris, 1 to A. simplex (s. s.), whereas one resulted as a F1 hybrid between A. pegreffii and A. simplex (s. s.). Remarkably high levels of infection with A. pegreffii were recorded in hakes from the Adriatic/Ionian Sea compared to the fish of similar length obtained from the western Mediterranean fishing grounds. A positive correlation between fish length and abundance of A. pegreffii was observed. Concerning the localization of A. pegreffii larvae in the fish, 28.3% were detected in the liver, 62.9% in the rest of the viscera, 6.6% in the ventral part of the flesh, whereas 2.1% in the dorsal flesh.

Pathogenic potential of Anisakis L3 after freezing in domestic freezers

Anisakis L3 were subjected to freezing in domestic freezers and the risk posed by those larvae that might possibly survive freezing was analysed by determining their agar penetration ability, survival in gastric juice, allergenic potential and oxygen consumption rate. Anisakis L3 gradually became non-viable as the temperature fell from −1 °C to −28 °C. The faster the freezing rate, the greater the decrease in survival, but viability was not found to be affected by the proportions of Anisakis species per batch (A. simplex s.s. 83–100%, A. pegreffii 0–17%, and their heterozygote genotypes at the ITS region of rDNA 0–10%). Surviving larvae after freezing presented a drastically reduced ability to penetrate into a layer of solid agar, as compared to controls, but still 5% of them penetrated into this medium. About 80% of surviving larvae died within 24 h under conditions simulating those of gastric fluid, whereas up to 85% of the controls were able to resist for 24 h. Allergen release during freezing and after freezing was higher than in controls, and this release was compatible with a contribution of both passive (by rupture of the cuticle) and active liberation of excretion/secretion allergens during and after freezing, suggesting that the pathogenic potential of these frozen and thawed larvae cannot be discarded. These results could be of use for more precise risk assessment and providing guidelines for consumers.

Validation of a commercial kit aimed to the detection of pathogenic anisakid nematodes in fish products

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005.Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova.Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005.

Comparison of anisakid infection levels between two species of Atlantic mackerel (&lt;em&gt;Scomber colias&lt;/em&gt; and &lt;em &gt;S. scombrus&lt;/em&gt;) off the Atlantic Portuguese coast

Anisakiasis is a problematic zoonotic infection associated with the consumption of raw or undercooked fish. Atlantic mackerel (Scomber colias) is of high commercial interest in Portugal and has been reported as a common host of Anisakisspp. In this study, the occurrence of anisakids is evaluated in S. colias and also Scomber scombrus, and the potential zoonotic risk associated with consumption of these two fishes is evaluated according to the recorded infection levels. These were found to be high for both fish species: a mean intensity and prevalence of 21.7 worms/fish and 85% for S. colias, and 16.4 worms/fish and 83.3% for S. scombrus, respectively. No correlation was detected between anisakid intensity and host total length, total weight, condition factor, and hepatosomatic and gonadosomatic indices for both fish species, but significantly higher intensity values were detected for more mature S. scombrus, i.e. fish recording a higher gonadosomatic index. Molecular tools allowed the identification of two species of Anisakis, A. simplex (s.s.) and A. pegreffii. They differed in their occurrence: in S. colias the prevalence of A. simplex (s.s.) was 18% and that of A. pegreffii was 82%, whereas in S. scombrus the prevalence of A. simplex (s.s.) was 73% and that of A. pegreffii was 27%. Occasionally, worms of Hysterothylacium aduncum were identified for both fish. The different infection levels of the two Anisakisspecies in both hosts off the Portuguese coast raise the hypothesis of a different life cycle at the level of the invertebrate intermediate host. S. colias lives in deeper waters than S. scombrus, and the differences found in infection levels suggest that A. pegreffii main first intermediate host also live in deeper waters, compared with A. simplex (s.s.) main first intermediate host. The higher infection levels of A. simplex (s.s.) (most infectious to humans) in S. scombrus suggest that its consumption when slightly cooked, as in grilled fish (so popular in Portugal), could be more problematic for the development of anisakiasis in humans than the consumption of S. colias and thus be of potential public health concern.

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.

Parasitic Helminth Fauna and Heavy Metals Analysis in Macrobrachium macrobranchion (Herklots, 1851) and Macrobrachium vollenhovenii (Herklots, 1857) From Lekki Lagoon, Lagos, Nigeria.

This study was carried out to investigate the uptake and bioaccumulation of heavy metals by intestinal helminths parasites in relation to the muscular tissues of the freshwater prawn (Macrobrachium species) obtained from Lekki Lagoon, Epe, Lagos, Nigeria. A total of 120 specimens composed of 82 Macrobrachium macrobrachion and 38 Macrobrachium species was Zn> Fe> Cu> Pb> Cr> Cd> Ni and Mn> Fe> Zn> Cr> Pb> Cu> Ni> Cd in the parasite.In the the non –infected specimens the trend shows; Mn > Fe> Cu> Zn> Cd> Cr, Ni> and Pb. Manganese (Mn) was found to have high concentration compared to other metal analysed .Two of the metals (Pb and Mn) were found in significantly higher concentrations vollenhovenii were examined for helminths parasites. Four species of helminths parasites were recovered; Procamallanus species, Cucullanus species, Anisakis species and Pomphorhynchus species. Out of 82 specimens of Macrobrachium macrobrachion examined, 16 (19.51%) were infected. These infected specimens had 16(19.51%) males and 0(0.00%) female while the non- infected individuals had 49 (59.76%) male and 17(20.73%) females. The Chi-square distribution was significant at 0.01 level (x2(2) =5.20, p< 0.01).Sixteen specimens (16) (42.11%) were infected out of the 38 Macrobrachium vollenhovenii examined. The infected Macrobrachium vollenhovenii had 16(42.11%) males and 0(0.00%) female and the non- infected individuals had 18(47.37%) males and 4(10.53%) females. The Chi-square distribution was significant at 0.01 level (x2(2) =5.20, p< 0.01). The condition factor in the study for Macrobrachium macrobrachion in female was 1.4±0.40 and in male 1.45±0.49, while the condition factor (K) of M. vollenhovenii in female was 1.18±0.20 and in male 1.63±0.61. The mean value for the condition factor of both in acanthocephalans compared with the host tissues. While five of the metals (Fe, Zn, Cd, Cu and Cr) were found in significant high concentration in the infected tissue than the parasites. Analysis of all the heavy metal sample recorded for the water and sediment media of Lekki lagoon, indicate Zn to be above the FME permissible limit. However, findings from this study indicate that the acanthocephalans interfere with the uptake and bioaccumulation of heavy metal in relation to the tissue of the freshwater prawn, and can be applied in biomonitoring procedure of environmental pollution.

Quantitative SYBR Green qPCR technique for the detection of the nematode parasite Anisakis in commercial fish-derived food

The extensive presence of anisakids in fish for human consumption has become a problem of food safety and quality. The aim of this study was to develop and assess the performance of a quantitative SYBR Green qPCR assay for the detection and quantification of Anisakis DNA in fish by-products. L3 nematode larvae of A. simplex (s.l.) (n=510), A. physeteris (n=3), Hysterothylacium sp. (n=10) and Pseudoterranova sp. (n=1), isolated from blue whiting, horse mackerel and monkfish, were used for the optimization of the molecular assay. In addition, molecularly typed larvae of A. simplex (s.s.) (n=10) and A. pegreffii (n=5) of the complex A. simplex (s.l.) were used for the specificity assay. Primers targeting the mitochondrial cytochrome c oxidase subunit II gene (COII) were selected. Analytical sensitivity and reproducibility were evaluated in a food matrix consisting of commercial fish-derived food spiked with larvae of A. simplex (s.l.). The assay proved to be specific for the three analyzed Anisakis species. A high reproducibility and sensitivity was detected, with a 95% limit of detection (LOD) of 0.30ng (95%CI 0.15–1.50) of A. simplex (s.l.) DNA per gram of food matrix and an operative LOD of 1.50ng after a PROBIT analysis. The assay was applied to study the presence of Anisakis in four types of processed commercial food, namely crab sticks, “gulas”, croquettes and burgers. Overall, 180 food samples from 15 commercial brands were studied, detecting Anisakis DNA in over half of them. The analyzed surimi-based products, “gulas” and crab sticks, showed the highest Anisakis burden (5.86±0.69 and 4.68±0.73ng of Anisakis DNA per gram of food, respectively). Our results indicate that the optimized SYBR Green qPCR technique is an accurate and sensitive method that may improve detection of Anisakis in fresh and processed products.

Anisakis - immunology of a foodborne parasitosis.

Anisakis species are marine nematodes which can cause zoonotic infection in humans if consumed in raw, pickled or undercooked fish and seafood. Infection with Anisakis is associated with abdominal pain, nausea and diarrhoea and can lead to massive infiltration of eosinophils and formation of granulomas in the gastrointestinal tract if the larvae are not removed. Re-infection leads to systemic allergic reactions such as urticarial or anaphylaxis in some individuals, making Anisakis an important source of hidden allergens in seafood. This review summarizes the immunopathology associated with Anisakis infection. Anisakiasis and gastroallergic reactions can be prevented by consuming only fish that has been frozen to -20°C to the core for at least 24hours before preparation. Sensitization to Anisakis proteins can also occur, primarily due to occupational exposure to infested fish, and can lead to dermatitis, rhinoconjunctivitis or asthma. In this case, exposure to fish should be avoided.

Environmental variables and definitive host distribution: a habitat suitability modelling for endohelminth parasites in the marine realm.

Marine nematodes of the genus Anisakis are common parasites of a wide range of aquatic organisms. Public interest is primarily based on their importance as zoonotic agents of the human Anisakiasis, a severe infection of the gastro-intestinal tract as result of consuming live larvae in insufficiently cooked fish dishes. The diverse nature of external impacts unequally influencing larval and adult stages of marine endohelminth parasites requires the consideration of both abiotic and biotic factors. Whereas abiotic factors are generally more relevant for early life stages and might also be linked to intermediate hosts, definitive hosts are indispensable for a parasite’s reproduction. In order to better understand the uneven occurrence of parasites in fish species, we here use the maximum entropy approach (Maxent) to model the habitat suitability for nine Anisakis species accounting for abiotic parameters as well as biotic data (definitive hosts). The modelled habitat suitability reflects the observed distribution quite well for all Anisakis species, however, in some cases, habitat suitability exceeded the known geographical distribution, suggesting a wider distribution than presently recorded. We suggest that integrative modelling combining abiotic and biotic parameters is a valid approach for habitat suitability assessments of Anisakis, and potentially other marine parasite species.

The Anisakis Transcriptome Provides a Resource for Fundamental and Applied Studies on Allergy-Causing Parasites.

BackgroundFood-borne nematodes of the genus Anisakis are responsible for a wide range of illnesses (= anisakiasis), from self-limiting gastrointestinal forms to severe systemic allergic reactions, which are often misdiagnosed and under-reported. In order to enhance and refine current diagnostic tools for anisakiasis, knowledge of the whole spectrum of parasite molecules transcribed and expressed by this parasite, including those acting as potential allergens, is necessary.Methodology/Principal FindingsIn this study, we employ high-throughput (Illumina) sequencing and bioinformatics to characterise the transcriptomes of two Anisakis species, A. simplex and A. pegreffii, and utilize this resource to compile lists of potential allergens from these parasites. A total of ~65,000,000 reads were generated from cDNA libraries for each species, and assembled into ~34,000 transcripts (= Unigenes); ~18,000 peptides were predicted from each cDNA library and classified based on homology searches, protein motifs and gene ontology and biological pathway mapping. Using comparative analyses with sequence data available in public databases, 36 (A. simplex) and 29 (A. pegreffii) putative allergens were identified, including sequences encoding ‘novel’ Anisakis allergenic proteins (i.e. cyclophilins and ABA-1 domain containing proteins).Conclusions/SignificanceThis study represents a first step towards providing the research community with a curated dataset to use as a molecular resource for future investigations of the biology of Anisakis, including molecules putatively acting as allergens, using functional genomics, proteomics and immunological tools. Ultimately, an improved knowledge of the biological functions of these molecules in the parasite, as well as of their immunogenic properties, will assist the development of comprehensive, reliable and robust diagnostic tools.

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.