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Ascaridoid nematodes comprise a wide range of heteroxenous parasites infecting top fish predators and marine mammals as definitive hosts, with crustaceans, squids, and fishes acting as intermediate/paratenic hosts. Limited data exist on the species and role of several intermediate and paratenic hosts in the life cycle of these parasites. In the aim of adding knowledge on the role of squid species in their life cycle, we have here investigated the larval ascaridoid nematodes collected from the deep-sea umbrella squid Histioteuthis bonnelli and the reverse jewel squid Histioteuthis reversa captured in the Central Mediterranean Sea (Tyrrhenian Sea). Morphological study and sequence analysis of the internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) and the mitochondrial cytochrome c oxidase subunit 2 (mtDNA cox2) gene locus revealed the occurrence of Anisakis physeteris and of an unidentified species of the genus Lappetascaris. Sequence analysis revealed that specimens of Lappetascaris from both squid species matched at 100% sequences previously deposited in GenBank from larval ascaridoids collected in octopuses of the genus Eledone of the Mediterranean Sea. The Bayesian inference tree topology obtained from the analysis of the fragments amplified showed that Lappetascaris specimens were included in a major clade comprising Hysterothylacium species collected in fishes of the families Xiphiidae and Istiophoridae. As regards the site of infection in the squid host species, A. physeteris larvae predominated (60.7%) in the gonads, while those of Lappetascaris (76.3%) were found infecting the mantle musculature. The overall high values of parasitic load suggest both squid species as transmitting hosts of third stage larvae of Lappetascaris to top predator fishes, as well as the umbrella squid as an intermediate/paratenic host in the life cycle of A. physeteris in the Mediterranean Sea.

Blastocystis is a common intestinal protist distributed worldwide, infecting humans and a wide range of domestic and wild animals. It exhibits an extensive genetic diversity and, so far, 25 distinct small subunit ribosomal RNA (SSU rRNA) lineages termed subtypes (STs)) have been characterized, among them, 12 have thus far been reported in humans. The aims of the present study were to detect and genetically characterize Blastocystis sp. in synantropic animals to improve our current knowledge on the distribution and zoonotic transmission of Blastocystis STs in Italy. Samples were collected from N = 193 farmed animals and submitted to DNA extraction and PCR amplification of the SSU rRNA. Blastocystis was detected in 60 samples (31.08%) and successfully subtyped. Phylogenetic analysis evidenced that the isolates from fallow deer, goats, and pigs (N = 9) clustered within the ST5, those from pheasants (N = 2) in the ST6, those from chickens (N = 8) in the ST7, those from sheep (N = 6) in the ST10, and those from water buffaloes (N = 9) in the ST14 clade. The comparison between the present isolates from animals and those previously detected in humans in Italy suggested the animal-to-human spillover for ST6 and ST7. The present study represents the widest Blastocystis survey performed thus far in farmed animals in Italy. Further epidemiological studies using molecular approaches are required to determine the occurrence and distribution of Blastocystis STs in other potential animal reservoirs in Italy and to define the pathways of zoonotic transmission.

Trypanorhynch tapeworms are marine heteroxenous parasites, involving sharks and rays as definitive hosts, invertebrates (i.e. crustaceans) as first intermediate hosts, and various teleost fishes as intermediate/paratenic ones. The massive presence of Trypanorhyncha larval forms in the flesh or body cavity of the fish hosts reduces their market value, making them unappealing to consumers, or causing discards of fish products. The identification of merocercoids (N = 100) of Molicola uncinatus and plerocercoids (N = 46) of Hepatoxylon trichiuri parasitizing the silver scabbardfish, Lepidopus caudatus, from the Central Mediterranean Sea (off Malta coast), was obtained, for the first time, by using sequences analysis of the complete small subunit (ssrDNA) and the partial large subunit (lsrDNA) ribosomal RNA gene loci. The Bayesian Inference from the concatenated sequences obtained at the two gene loci showed that the individuals of M. uncinatus and H. trichiuri clustered, respectively, in two well distinct and supported phylogenetic lineages, corresponding to the two cestode species. High prevalence (P) and mean abundance (MA) values were observed in the infection with merocercoids of M. uncinatus (P = 100%, A = 28.81), with the ventral musculature of the silver scabbardfish as the most parasitized site. Plerocercoids of H. trichiuri were found to infect only the mesenteries, attached to various fish tissues, with a mean abundance rate of 1.28. Even if harmless to humans, a heavy parasitic load with larval stages of Trypanorhyncha, particularly in the edible part of the silver scabbardfish (as here reported for merocercoids of M. uncinatus), can compromise the appearance of the fish-food, making it repugnant and unappealing for the final consumer. It has been also discussed that when managing the risk related to parasites affecting the fish quality and safety of fisheries products, the fishing ground where the life cycle of those parasites takes place should be also taken into account, as it would include the ecological drivers of the infection with those parasites. This wider ecological approach would be of help to the Food Business Operators (FBOs), gathering knowledge useful to manage fisheries in a wide “One-Health” perspective, aware of sustainability, quality and consumer safety aspects of sea-food production.

The southern Pacific Ocean, off the New Zealand coast, has been reported as one sympatric area of the two parasite species Anisakis pegreffii and A. berlandi. Here, a multilocus genotyping approach, based on a panel of eleven DNA microsatellite (SSR) loci plus the sequences analysis of the nuclear nas10 nDNA and the mitochondrial mtDNA cox2 gene loci, was applied to a total of N = 344 adults and larvae of Anisakis spp. from cetacean and fish species, respectively. Out of the newly scored SSR loci, Anisl 15 and Anisl 2 showed fixed alternative alleles between A. pegreffii and A. berlandi resulting as 100% diagnostic loci. Out of SSRs Anisl 00314 and Anisl 7 previously disclosed, two additional loci, i.e., Anisl 4 and Anisl 22, were found to be sex-linked. The Bayesian genotypes clustering approach (STRUCTURE) allowed identification, with a 100% of probability value, N = 208 specimens to the “pure parental” A. pegreffii, N = 133 to the “pure parental” A. berlandi, while one adult and two larval stages showed mixed ancestry between the two groups having, in all cases, a Q-value = 0.50. NEWHYBRIDS analysis assigned (100% of probability) those specimens to their F1 hybrid category. This represents the first evidence of contemporary hybridization between the two parasite species in a sympatric area. The pairwise FST values estimated at intraspecific and interspecific level, inferred from both SSR loci and mitochondrial mtDNA cox2 sequences, have also demonstrated the existence of two distinct panmictic units in this study area, corresponding respectively to A. pegreffii and A. berlandi. The results obtained support the useful application of a multilocus approach in the identification of sibling species and their hybrid categories in sympatric areas. The possible use of sex-linked SSR loci of the two species of the A. simplex (s. l.), for sex determination of their larval stages, is also suggested.

A survey on Anisakis simplex (sensu stricto (s.s.)) from blue whiting, Micromesistius poutassou, in the north-eastern Atlantic Ocean revealed the occurrence of high infection levels of third larval stages in visceral organs and flesh. Larvae were genetically identified with a multilocus approach as A. simplex (s.s.). Histochemical, immunohistochemical and ultrastructural observations were conducted on 30 M. poutassou specimens. Gonads, pyloric caeca and flesh harboured encapsulated larvae of A. simplex (s.s.) but no intense host reaction was encountered around the parasite in the above organs. In the liver, the most infected organ, the larvae co-occurred with the coccidian Goussia sp. Within the granuloma around the A. simplex (s.s.) larvae, two concentric layers were recognized, an inner mostly comprising electron-dense epithelioid cells and an outer layer made of less electron-dense epithelioid cells. Macrophages and macrophage aggregates (MAs) were abundant out of the granulomas, scattered in parenchyma, and inside the MAs, the presence of engulfed Goussia sp. was frequent. In liver tissue co-infected with Goussia sp. and A. simplex (s.s.), hepatocytes showed cytoplasmic rarefaction and acute cell swelling. Results suggest that the host-induced encapsulation of A. simplex (s.s.) larvae is a strategic compromise to minimize collateral tissue damage around the larval infection sites, to facilitate the survival of both parasite and host.

Post-mortem examination of a fin whale Balaenoptera physalus stranded in the Mediterranean Sea led to the finding of Bolbosoma balaenae for the first time in this basin. In this work, we describe new structural characteristics of this parasite using light microscopy and scanning electron microscopy approaches. Moreover, the molecular and phylogenetic data as inferred from both ribosomal RNA 18S-28S and the mitochondrial DNA cytochrome oxidase c subunit 1 (cox1) for adult specimens of B. balaenae are also reported for the first time. Details of the surface topography such as proboscis's hooks, trunked trunk spines of the prebulbar foretrunk, ultrastructure of proboscis's hooks and micropores of the tegument are shown. The 18S + 28S rRNA Bayesian tree (BI) as inferred from the phylogenetic analysis showed poorly resolved relationships among the species of Bolbosoma. In contrast, the combined 18S + 28S + mtDNA cox1 BI tree topology showed that the present sequences clustered with the species of Bolbosoma in a well-supported clade. The comparison of cox1 and 18S sequences revealed that the present specimens are conspecific with the cystacanths of B. balaenae previously collected in the euphausiid Nyctiphanes couchii from the North Eastern Atlantic Ocean. This study provided taxonomic, molecular and phylogenetic data that allow for a better characterization of this poor known parasite.

Adult Anisakis Dujardin, 1845 were found in two specimens of killer whale Orcinus orca and one specimen of franciscana Pontoporia blainvillei stranded from off the coast of Buenos Aires Province, Argentina. Genetic identification of the nematodes (N = 144) was performed by sequence analysis of the mitochondrial (mtDNA cox2) and the nuclear (nas 10 nDNA) gene loci. Anisakis pegreffii and Anisakis berlandi were detected in the two individuals of O. orca, while Anisakis typica and A. pegreffii were identified in P. blainvillei. Morphological and morphometric analysis also carried out on adult specimens of A. pegreffii and A. berlandi has allowed to underlining the usefulness of genetic/molecular markers in their recognition. This represents the first record of A. pegreffii in O. orca and P. blainvillei and of A. berlandi in O. orca. This is also the first sympatric and syntopic occurrence, as adults, of A. pegreffii and A. berlandi from the Austral Region of the Atlantic Ocean waters. These results provide insights into the knowledge of the host ranges and geographical distribution of these parasites in the basin waters of the region. Pontoporia blainvillei showed low abundance values of infection with Anisakis spp., which is the general pattern for coastal dolphins in the area, whereas O. orca harboured higher abundance of Anisakis spp. than those previously recorded among cetacean species in the Argentine Sea. Differences in the Anisakis spp. distribution and their parasitic loads, observed among the three host specimens, are discussed in relation to the oceanographic parameters, as well as to the host ecology. The usefulness of genetic/molecular markers in the recognition of adults of the sibling species A. pegreffii and A. berlandi with considerable overlapping in morphometric and morphological characters was underlined. The distribution of Anisakis species from Southwestern Atlantic waters is discussed in relation to their value as indicators for studies on the zoogeography of their hosts at a regional-scale level.

The study aims to perform, for the first time, the molecular identification of anisakid larvae in commercial fish from the Southeastern Pacific Ocean off the Peru coast, and to provide data on their infection level by fishing ground, fish host, and site of infection. Fish specimens (N = 348) from the northern and the central coast of Peru were examined for parasites. The fish fillets were examined by the UV-press method. Anisakis spp. larvae (N = 305) were identified by mtDNA cox2 sequences analysis and by the ARMS-PCR of the locus nas10 nDNA. Two hundred and eighty-eight Anisakis Type I larvae corresponded to Anisakis pegreffii, whereas 17 Anisakis Type II larvae clustered in a phylogenetic lineage distinct from Anisakis physeteris deposited in GenBank, and corresponding to a phylogenetic lineage indicated as Anisakis sp. 2, previously detected in fish from both Pacific and Atlantic waters. Anisakis pegreffii was found to infect both the flesh and viscera, while Anisakis sp. 2 occurred only in the viscera. The average parasitic burden with A. pegreffii in the examined fish species from the two fishing grounds was significantly higher than that observed with Anisakis sp. 2. The results obtained contribute to improve the knowledge on the distribution and occurrence of Anisakis species in Southeastern Pacific waters and their implications in seafood safety for the local human populations.

Studies describing the parasite fauna of sunfish species from the Mediterranean Sea are to date limited, despite information gained through parasitological examination may reveal unknown ecological and biological aspects of both hosts and parasites. Moreover, recent molecular studies on sunfish taxonomy revealed the presence of two species belonging to the genus Mola in the Mediterranean basin, namely M. mola and M. alexandrini. These two fish taxa have long been synonymized or confused among them, which implies that the majority of the studies carried out so far reported the parasites infecting both species under a single host species, generally referred to as M. mola. We hereby investigated the parasite fauna of a 43 cm long M. mola specimen from the Mediterranean Sea, whose identification was confirmed by molecular tool, and provided the first evidence of the occurrence of the nematode Anisakis simplex (s.s.) and of the cestode Gymnorhynchus isuri in Mola species anywhere. The use of helminth species as biological tags for the sunfish is also discussed.

Few reports exist upon the occurrence and localization of zoonotic anisakid nematodes in T. sagittatus, especially in the mantle of the squid. The occurrence and site of infection of larval anisakids in 98 T. sagittatus caught West off St. Kilda, NE Atlantic Ocean, were investigated. Squids were examined for anisakids using the UV-Press method. In total, 689 nematodes were detected in the viscera and mantle. According to morphology, all the larvae (L3) were assigned to genus Anisakis. Diagnostic allozymes and mtDNA cox2 sequence analysis permitted to genetically identify all larvae as Anisakis simplex (s.s.) (N = 100). Overall prevalence (P = 81%) and mean intensity (mI = 8.6) of infection with A. simplex are provided. Most of the larvae present in the mantle cavity were embedded in the stomach wall or attached in the outer layer of the stomach and caecum (49%). Over a third of squids (37%) hosted A. simplex (s.s.) larvae in the mantle. A novel schematized representation of larvae distribution in the mantle is provided, showing where they were mostly located. According to the results obtained, the risk of anisakiasis associated with consumption of raw or undercooked T. sagittatus should be considered.

The third-stage larvae of the parasitic nematode genus Anisakis tend to encapsulate in different tissues including the musculature of fish. Host tissue penetration and degradation involve both mechanic processes and the production of proteins encoded by an array of genes. Investigating larval gene profiles during the fish infection has relevance in understanding biological traits in the parasite&rsquo, s adaptive ability to cope with the fish hosts&rsquo, defense responses. The present study aimed to investigate the gene expression levels of some proteins in L3 of A. simplex (s.s.) infecting different tissues of blue whiting Micromesistius poutassou, a common fish host of the parasite in the NE Atlantic. The following genes encoding for Anisakis spp. proteins were studied: Kunitz-type trypsin inhibitor (TI), hemoglobin (hb), glycoprotein (GP), trehalase (treh), zinc metallopeptidase 13 (nas 13), ubiquitin-protein ligase (hyd) and sideroflexin 2 (sfxn 2). Significant differences in gene transcripts (by quantitative real-time PCR, qPCR) were observed in larvae located in various tissues of the fish host, with respect to the control. ANOVA analysis showed that relative gene expression levels of the seven target genes in the larvae are linked to the infection site in the fish host. Genes encoding some of the target proteins seem to be involved in the host tissue migration and survival of the parasite in the hostile target tissues of the fish host.

13 pages, 6 figures, 4 tables, Atlantic cod (Gadus morhua) is among the most important commercial fish species on the world market. Its infection by ascaridoid nematodes has long been known, Pseudoterranova even being named cod worm. In the present study, 755 individuals were sampled in the Barents, Baltic and North Seas during 2012–2014. Prevalences for Anisakis in whole fish and in fillets in the different fishing areas varied from 16 to 100% and from 12 to 90% respectively. Abundance was also greatly influenced by the sampling area. Generalized additive model results indicate higher numbers of Anisakis in the North Sea, even after the larger body size was accounted for. Numbers and prevalence of Anisakis were positively related to fish length or weight. The prevalence of parasites in whole fish and in fillets was also influenced by the season, with the spring displaying a peak for the prevalence in whole fish and, at the same time, a drop for the prevalence in fillets. Whereas 46% of cod had Anisakis larvae in their fillets, the majority (39%) had parasites mainly in the ventral part of the fillet and only 12% had parasites in their dorsal part. This observation is of importance for the processing of the fish. Indeed, the trimming of the ventral part of the cod fillet would allow the almost total elimination of ascaridoids except for cod from the Baltic Sea where there was no difference between the dorsal and the ventral part. The presence of other ascaridoid genera was also noticeable in some areas. For Pseudoterranova, the highest prevalence (45%) in whole fish was observed in the Northern North Sea, whereas the other areas had prevalences between 3 and 16%. Contracaecum was present in every commercial size cod sampled in the Baltic Sea with an intensity of up to 96 worms but no Contracaecum was isolated from the Central North Sea. Non-zoonotic Hysterothylacium was absent from the Baltic Sea but with a prevalence of 83% in the Barents and the Northern North Sea. A subsample of worms was identified with genetic-molecular tools and assigned to the species A. simplex (s.s.), A. pegreffii, P. decipiens (s.s.), P. krabbei, C. osculatum and H. aduncum. In addition to high prevalence and abundance values, the cod sampled in this study presented a diversity of ascaridoid nematodes with a majority of fish displaying a co-infection. Out of 295 whole infected fish, 269 were co-infected by at least 2 genera., The present survey was financially supported by the European Union's 7th Framework Program for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant agreement (GA) no. 312068.

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.

This review provides an inventory of the biodiversity of the anisakid species identified so far from fish and marine mammals of the NE Atlantic Ocean. The paper reviews and discusses various taxonomical and epidemiological aspects related to biodiversity assessment, with emphasis on: (1) taxa recognized as ‘biological species’ based on molecular/genetic markers; (2) current molecular/genetic approaches to identify the species at different developmental stages; (3) ecological data related to the actual geographical distribution and definitive host preferences of the species; (4) their distribution in various, commercially important fish species in northern European waters; (5) their possible occurrence in farmed fish; and, finally, (6) an update of their zoonotic potential as causative agents of anisakidosis in humans.

Sulcascaris sulcata (Anisakidae), a pathogenic nematode of sea turtles, may cause ulcerous gastritis with different degrees of severity. Previous studies demonstrated a high prevalence of infection in the Mediterranean loggerhead turtle (Caretta caretta), although no data on the potential intermediate hosts of this nematode has been published thus far from the Mediterranean basin. Here, using molecular analyses, we demonstrated that the cross sections of nematode larvae observed histologically in Mediterranean mussels (Mytilus galloprovincialis) collected from a farm along the Tyrrhenian coast of southern Italy belong to S. sulcata. The BLAST analysis of sequences at the ITS2 region of rDNA and mtDNA cox2 gene loci here obtained from samples of two Mediterranean mussels containing nematode larvae showed 100% homology with those at the same gene loci from the adults of S. sulcata collected from the Mediterranean Sea and deposited in GenBank. To our knowledge, this study is the first to present data on a potential intermediate host of S. sulcata in the Mediterranean basin and to report a nematode parasite from the Mediterranean mussel.

Background Loiasis is a vector-borne parasitic infection endemic across many areas of Central and West Africa. Its treatment is tricky due to the risk of serious neurologic adverse events occurring after the administration of microfilaricidal drugs, like diethylcarbamazine or ivermectin, in subjects with high pre-treatment microfilarial load. Albendazole is currently recommended to slowly reduce microfilaremia before curative regimen is prescribed. Case presentation We report the case of a 25-year-old man from Guinea-Conakry who was incidentally diagnosed with highly microfilaremic Loa loa infection. A three weeks regimen of albendazole was prescribed. Minor neurologic side effects occurred after two weeks of administration, while serious encephalopathy developed one week later. Clinical and electroencephalographic features of the patient resembled those of an immune-mediated encephalitis. After exclusion of other causes of encephalopathy, treatment-related Loa loa encephalopathy induced by albendazole was suspected. Corticosteroid treatment was administered and the patient recovered. Discussion Our case confirms that Loa loa treatment-related encephalopathy may occur even during albendazole treatment. The clinical and electroencephalographic similarities between Loa loa albendazole-related encephalopathy and immune-mediated encephalitis suggest the possibility of an underlying inflammation-based pathogenesis. Although corticosteroid administration is not recommended in Loa loa ivermectin-induced encephalopathy, in this case of Loa loa albendazole-induced encephalopathy it may have played a therapeutic role.

Contracaecum rudolphii (s. l.) is a complex of sibling species of anisakid nematodes having the fish-eating birds belonging to the Family Phalacrocoracidae as final hosts. The great cormorant Phalacrocorax carbo sinensis is parasitized by C. rudolphii A and C. rudolphii B. Adults and L4 specimens of C. rudolphii (s. l.) (N = 3282) were collected in cormorants from brackish and freshwater ecosystems of Central Italy. Third-stage larvae of Contracaecum (N = 882) were obtained from the fish species Dicentrarchus labrax, Anguilla anguilla, Aphanius fasciatus, Atherina boyeri, Leuciscus cephalus, Barbus barbus, and Carassius carassius captured in the same geographical areas of cormorants’ standings. Contracaecum rudolphii A and C. rudolphii B were identified by a multilocus genetic approach: allozymes, sequences analysis of the mtDNA cox2, and ITS region of rDNA gene loci. Differential distribution of the two parasite species was observed in different aquatic environments. Contracaecum rudolphii B outnumbered C. rudolphii A in wintering cormorants from freshwater ecosystems; the opposite trend was found in cormorants from brackish water. Analogously, C. rudolphii A larvae were more prevalent in brackish water fish, while C. rudolphii B larvae were found infecting only freshwater fish. The findings seem to confirm that C. rudolphii A and C. rudolphii B would have a life-cycle adapted to brackish and freshwater environments, respectively. A differential feeding behavior of wintering cormorants, the ecology of the infected fish species, and abiotic factors related to early stages of the parasites are supposed to maintain the distinctiveness of the two parasite species’ life cycles in the two different aquatic ecosystems.

An epidemiological survey on Blastocystis was carried out enrolling a total of 2524 subjects referred to the Umberto I Academic Hospital in Rome, for the routine parasitological exams, during 2017–2018. The studied population included a sample of immunocompromised individuals (N = 130) followed at the same hospital. DNA sequencing of the small subunit rRNA gene (SSU rDNA) locus was performed on samples positive to the coproparasitological analysis to molecular characterize the Blastocystis-subtypes. Microscopical analysis detected Blastocystis in 192/2524 (7.6%) of the enrolled subjects. It was the organism most frequently identified in the analysed faecal samples diagnosed in single infection (5.6%) or in co-infection with other enteric protozoa (2%). Furthermore, it was found mainly in immunocompromised patients (22.3%) compared to immunocompetent ones (6.8%). As expected, ST3 was the most occurring subtype identified in 40% of the subjects, followed by ST1 (29%), ST2 (16%), ST4 (12%), and ST7 (3%). Next-generation sequencing (NGS) of the 16S rDNA was performed on a sub-sample of Blastocystis-ST3-carriers, homogenous by age and gender, as well as on Blastocystis-free subjects, to profile and compare their gut bacterial composition. A higher bacterial diversity was found in ST3-Blastocystis-carriers, which exhibited a high abundance of Prevotella, Methanobrevibacter and Ruminococcus while, a high percentage of Bacteroides was found in Blastocystis-free subjects. This study evidenced the presence of Blastocystis in 7.6% of faecal samples in Italy and a high circulation of the protist among immunocompromised patients (22.3%). Molecular characterization of positive samples evidenced the occurrence of five different subtypes, including zoonotic ST such as the ST7, highlighting the risk of transmission from animals. Study of the gut microbiota composition confirms previous evidences according to which, the colonisation by Blastocystis would be linked with an eubiotic gut characterized by potentially beneficial species such as Prevotella and Ruminococcus, rather than with a dysbiotic state, with a high abundance of Enterobacteriaceae, and corroborated the role of the protist as “an old friend” of the human gut., Highlights • Microscopical analysis detected Blastocystis in 7.6% subjects in Italy. • Molecular methods allowed the identification of 5 STs (ST1, ST2, ST3, ST4, ST7). • ST3 resulted the most frequent subtype. • NGS of the 16S rDNA gene was performed in Blastocystis-ST3-carriers. • Blastocystis-ST3 resulted correlated to an eubiotic gut characterized by potentially beneficial species.

The genus Anisakis represents one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognized in the Anisakis simplex (s. l.) complex: A. pegreffii, A. simplex (s. s.) and A. berlandi. They are widely distributed in populations of their intermediate/paratenic hosts (fish and squids) and definitive hosts (cetaceans). A novel nuclear gene locus, metallopeptidase 10 (nas 10) (451 bp), was sequenced and validated on a total of 219 specimens of the three species of Anisakis, collected in fish and cetacean hosts from allopatric areas included in their ranges of distribution. The specimens of Anisakis were first identified by allozymes and sequence analysis of the mtDNA cox2 and EF1α-1 nDNA. The novel nuclear marker has shown fixed alternative nucleotide positions in the three species, i.e. diagnostic at 100%, permitting the species determination of a large number of specimens analyzed in the present study. In addition, primers to be used for amplification-refractory mutation system (ARMS) PCR of the same gene locus were designed at these nucleotide positions. Thus, direct genotyping determination, by double ARMS, was developed and validated on 219 specimens belonging to the three species. Complete concordance was observed between the tetra-primer ARMS-PCR assays and direct sequencing results obtained for the nas 10 gene locus. The novel nuclear diagnostic marker will be useful in future studies on a multi-locus genotyping approach and also to study possible hybridization and/or introgression events occurring between the three species in sympatric areas.Un nouveau marqueur nucléaire et développement d'un test ARMS-PCR ciblant le locus de la métallopeptidase 10 (nas 10) pour identifier les espèces du complexe Anisakis simplex (s. l.) (Nematoda, Anisakidae).Le genre Anisakis représente l’un des groupes de nématodes ascaridoïdes les plus répandus dans l’écosystème marin. Trois taxons étroitement apparentés sont reconnus dans le complexe Anisakis simplex (s. l.) : A. pegreffii, A. simplex (s. s.) et A. berlandi. Ils sont largement répartis dans les populations de leurs hôtes intermédiaires/paraténiques (poissons et calmars) et définitifs (cétacés). Un nouveau locus de gène nucléaire, la métallopeptidase 10 (nas 10) (451 pb), a été séquencé et validé sur un total de 219 spécimens des trois espèces d’Anisakis, collectés chez des hôtes poissons et cétacés de zones allopatriques incluses dans leur aire de répartition. Les échantillons d’Anisakis ont d’abord été identifiés par des allozymes et une analyse des séquences de l’ADNmt cox2 et de l’ADNn EF1α-1. Le nouveau marqueur nucléaire a montré des positions de nucléotides alternatives fixes dans les trois espèces, c’est-à-dire qu’il a permis un diagnostic à 100%, permettant la détermination de l’espèce d’un grand nombre d’échantillons analysés dans la présente étude. De plus, des amorces à utiliser pour la PCR par système de mutation réfractaire à l’amplification (ARMS) du même locus génique ont été conçues à ces positions nucléotidiques. Ainsi, la détermination directe du génotypage, par double ARMS, a été développée et validée sur 219 spécimens appartenant aux trois espèces. Une concordance complète a été observée entre les dosages ARMS PCR tétra-amorces et les résultats de séquençage direct obtenus pour le locus du gène nas 10. Le nouveau marqueur de diagnostic nucléaire sera utile dans les travaux futurs d’une approche de génotypage multi-locus et également pour étudier les éventuels événements d’hybridation et/ou d’introgression se produisant entre les trois espèces dans des zones sympatriques.

Background Blastocystis is one of the most common intestinal protozoa in human faecal samples with uncertain impact on public health. Studies on the prevalence of Blastocystis in HIV-positive patients are limited and dated. Methods A cross-sectional study was carried out involving 156 HIV-positive patients to evaluate the prevalence of Blastocystis-subtypes by molecular amplification and sequencing the small subunit rRNA gene (SSU rDNA), to identify the risk factors for its transmission, to examine the relationship between the presence of the protist and gastrointestinal disorders. Furthermore, the evaluation of the faecal calprotectin by immunoassay from a sample of subjects was performed to evaluate the gut inflammation in Blastocystis-carriers. Results Blastocystis-subtypes ST1, ST2, ST3, ST4 were identified in 39 HIV-positive patients (25%). No correlation was found between the presence of the protist and virological or epidemiological risk factors. Blastocystis was more frequently detected in homosexual subjects (p = 0.037) infected by other enteric protozoa (p = 0.0001) and with flatulence (p = 0.024). No significant differences in calprotectin level was found between Blastocystis-carriers and free ones. Conclusions Blastocystis is quite common in HIV-positive patients on ART showing in examined patients 25% prevalence. Homosexual behaviour may represent a risk factor for its transmission, while CD4 count and viremia didn’t correlate with the presence of the protist. The pathogenetic role of Blastocystis remains unclear and no gut inflammation status was detected in Blastocystis-carriers. The only symptom associated with Blastocystis was the flatulence, evidencing a link between the presence of the protist and the composition and stability of gut microbiota.

Eight microsatellite loci, recently developed in the species Anisakis pegreffii, were successfully amplified in Anisakis berlandi, sibling species of the A. simplex (s. l.) complex. They were validated on adult specimens (n = 46) of the parasite species, collected from two individuals of the definitive host, the long-finned pilot whale Globicephala melas from New Zealand waters. Among the eight loci scored, one, Anisl 07132, had null alleles in A. berlandi and was thus excluded from the subsequent genetic analysis. Two loci, Anisl 00314 and Anisl 10535, were monomorphic. In addition, as also previously detected in the other species of the A. simplex (s. l.) complex, the Anisl 7 locus was seen to be sex-linked, showing hemizygosity in male specimens. Differential allele frequency distributions of A. berlandi, with respect to those previously observed in A. pegreffii and A. simplex (s. s.), were found at some microsatellite loci. The Anisl 7 locus provided 100% diagnosis between A. berlandi and A. pegreffii, while others resulted in 99% diagnosis between A. berlandi and the other two species. Simple sequence repeat (SSR) loci also allowed us to estimate the genetic differentiation of A. berlandi from A. pegreffii (FUtilité des loci microsatellites pour la caractérisation génétique interspécifique d’Anisakis berlandi (Nematoda, Anisakidae).Huit loci microsatellites, récemment développés chez l’espèce Anisakis pegreffii, ont été amplifiés avec succès chez Anisakis berlandi, espèce sœur du complexe A. simplex (s. l.). Ils ont été validés sur des spécimens adultes (n = 46) de l’espèce, récoltés chez deux individus de l’hôte définitif, le globicéphale commun Globicephala melas, des eaux néo-zélandaises. Parmi les huit loci notés, l’un, Anisl 07132, avait des allèles nuls chez A. berlandi et a donc été exclu de l’analyse génétique ultérieure. Deux loci, Anisl 00314 et Anisl 10535, étaient monomorphes. De plus, comme cela a également été détecté précédemment dans les autres espèces du complexe A. simplex (s. l.), le locus Anisl 7 était lié au sexe, montrant une hémizygosité chez les spécimens mâles. Chez A. berlandi, des distributions de fréquences d’allèles, différentielles par rapport à celles précédemment observées chez A. pegreffii et A. simplex (s. s.), ont été trouvées pour certains loci microsatellites. Le locus Anisl 7 a fourni un diagnostic à 100 % entre A. berlandi et A. pegreffii, tandis que d’autres ont abouti à un diagnostic à 99 % entre A. berlandi et les deux autres espèces. Les loci des SSR ont également permis d’estimer la différenciation génétique d’A. berlandi par rapport à A. pegreffii (F

Anisakis pegreffii, a recognised etiological agent of human anisakiasis, is a parasite of homeothermic hosts at the adult stage and of ectothermic hosts at the third larval stage. Among distinct factors, temperature appears to be crucial in affecting parasite hatching, moulting and to modulate parasite-host interaction. In the present study, we investigated the gene transcripts of proteins having an antigenic role among excretory secretory products (ESPs) (i.e., a Kunitz-type trypsin inhibitor, A.peg-1; a glycoprotein, A.peg-7; and the myoglobin, A.peg-13) after 24 h, in A. pegreffii larvae maintained in vitro, under controlled temperature conditions. Temperatures were 37 °C and 20 °C, resembling respectively homeothermic and ectothermic hosts conditions, and 7 °C, the cold stress condition post mortem of the fish host. Primers of genes coding for these ESPs to be used in quantitative real-time PCR were newly designed, and qRT-PCR conditions developed. Expression profiles of the genes A.peg-1 and A.peg-13 were significantly up-regulated at 20 °C and 37 °C, with respect to the control (larvae kept at 2 °C for 24 h). Conversely, transcript profiles of A.peg-7 did not significantly change among the chosen temperature conditions. In accordance with the observed transcript profiles, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of the three target ESPs at 37 °C, while only A.peg-13 was observed at 7 °C. The results suggest that temperature conditions do regulate the gene expression profiles of A.peg-1 and A.peg-13 in A. pegreffii larvae. However, regulation of the glycoprotein A.peg-7 is likely to be related to other factors such as the host’s immune response.

This study reports the presence of two distinct MHC class II β genes in the Antarctic icefish Chionodraco hamatus, belonging to the classical (ChhaDAB) and nonclassical (ChhaDBB) evolutionary lineages. By the application of targeted sequencing approach, a remarkable molecular diversity in the exon 2 sequence of the highly expressed gene ChhaDAB has been observed, resulting in an estimate of 92 different variants translated in 87 different peptides from 54 analysed icefish individuals. A highly conservative estimate, based on a 95% sequence identity threshold clustering, translate this variability in 41 different peptide clusters belonging to four different clades and showing the signature of different kinds of selection. In stark contrast, the poorly expressed ChhaDBB gene displayed a very low level of molecular diversity within exon 2, in agreement with expectations for a nonclassical MHC class II β gene.

Capelin (Mallotus villosus) is among the most abundant fish species in the Barents Sea, and represents a critical food source for many predators in the area including Atlantic cod and harp seal. In Norway, the fish is of economic importance since whole capelin and roe are valuable export products. Despite its economic and ecological importance, the parasites of Barents Sea capelin are poorly known. However, the presence of parasites in the edible parts may adversely affect product quality and consumer safety. During the main annual catching seasons of 2009-2012, we investigated the diversity and infection dynamics of ascaridoid nematodes in capelin (n = 620) from the southern Barents Sea. Three anisakid species were identified by genetic or molecular methods; Anisakis simplex (s.s.), Contracaecum osculatum sp. B, and Hysterothylacium aduncum, with C. osculatum sp. B as the most prevalent and abundant species. The present findings suggest that the ascaridoid species composition in capelin reflects its trophic position in the Barents Sea ecosystem. There appears to be a link between infection level of the nematode species and the preferred prey organisms of the different developmental phases of capelin. Thus, the higher abundance of C. osculatum sp. B compared to A. simplex (s.s.) and H. aduncum may be related to more extensive feeding on calanoid copepods over a wider ontogenetic size range including adolescence, while the main intermediate hosts of the latter nematode species, i.e. euphausiids and amphipods, appear to be the preferred prey of larger capelin.

We describe a rare case of Dirofilaria repens infection presenting as peripheral lung nodules and mimicking a metastatic focus from a previously diagnosed cutaneous melanoma. To avoid invasive investigations before arriving at the correct diagnosis, dirofilariasis should be included as a part of the diagnostic process in subjects with lung nodules who live in (or have traveled to) endemic regions.

Myxozoans of the genus Kudoa (Myxosporea, Multivalvulida) infect marine and estuarine fish species worldwide. Some Kudoa species are of concern to the seafood industry since they may generate macroscopic cysts in the fish host's musculature, or cause post mortem myoliquefaction, commonly known as 'soft flesh'. One of the economically most important species is K. thyrsites, a myoliquefactive myxosporean parasite that occurs in many wild and cultured marine fish species worldwide. Here we identified a K. thyrsites isolate as the causative agent of myoliquefaction in silver scabbardfish Lepidopus caudatus from the Alboran Sea (western Mediterranean Sea). For comparative and validation purposes, the morphological and molecular characteristics of the isolate were compared with fresh spores of a K. thyrsites isolate infecting Atlantic mackerel Scomber scombrus from the Norwegian Sea. Myxospores of both isolates shared a stellate appearance and contained 4 unequal pyriform polar capsules (1 large, 1 small and 2 intermediate). These morphological traits were consistent with all other previously described K. thyrsites isolates. Moreover, the small subunit rDNA sequences of the Mediterranean and Norwegian Sea isolates revealed 100% similarity, and matched 100% with K. thyrsites isolates previously recorded in myoliquefactive Atlantic mackerel from the North Sea and off southern England. The findings suggest that K. thyrsites is the primary cause of myoliquefaction in silver scabbardfish from the Alboran Sea. This report represents the first morphological and molecular characterization of K. thyrsites in the Mediterranean Sea. A set of new allometric characters is proposed as additional descriptors for more accurate and specific description of kudoid myxospores.

The species ofAnisakisconstitute one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognised in theA. simplex(s. l.) complex, i.e.A. pegreffii,A. simplex(s. s.) andA. berlandi.They are distributed in populations of their intermediate/paratenic (fish and squids) and definitive (cetaceans) hosts. A panel of seven microsatellite loci (Anisl 05784,Anisl 08059,Anisl 00875,Anisl 07132, Anisl 00314, Anisl 10535andAnisl 00185), were developed and validated on a total ofN= 943 specimens ofA. pegreffiiandA. simplex(s. s.), collected in fish and cetacean hosts from allopatric areas within the range of distribution of these parasite species. In addition, the locusAnisl 7, previously detected in thoseAnisakisspp., was investigated. The parasites were first identified by sequence analysis of the EF1α-1 nDNA. The panel of the microsatellites loci here developed have allowed to: (i) detect diagnostic microsatellite loci between the two species; (ii) identify specimens of the two speciesA. pegreffii, A. simplex(s. s.) in a multi-marker nuclear genotyping approach; (iii) discover two sex-linked loci in bothAnisakisspecies and (iv) estimate levels of genetic differentiation at both the inter- and intra-specific level.

The Argentinean short-finned squid (Illex argentinus) is an oceanic, neritic species widely distributed off the east coast of South America, representing the most abundant commercially exploited squid species in these waters. Despite the great commercial importance of Argentinean short-finned squid as a food resource, and as frozen product exported to Europe, the presence of zoonotic anisakid nematodes, especially in the mantle of the squid, is poorly known. The occurrence and site of infection of larval ascaridoid nematodes in 70 I. argentinus caught off the Falkland Islands were investigated. Squids were examined using the UV-Press method. In total, 30 nematodes were detected in the viscera and mantle. According to morphology, 27 were third-stage larvae (L3) belonging to genus Anisakis, while three were L3 assigned to Hysterothylacium. Anisakis pegreffii (n = 27) were identified by sequence analysis of the mtDNA cox2 and the partial EF1 α-1 region of nDNA genes; Hysterothylacium aduncum (N = 3) were identified by sequence analysis of the ITS rDNA region. These findings represent the first molecular identification of A. pegreffii and H. aduncum in I. argentinus. Both prevalence (P = 15.7%) and abundance (A = 0.39) of infection with A. pegreffii were low, and even lower values of infection were recorded for H. aduncum (P = 2.1%, A = 0.04). Only 3 out of 70 (4.3%) squids hosted A. pegreffii larvae in the mantle. Larvae infecting viscera were coiled and mainly attached to outer surface of visceral organs. Mantle-infecting larvae were situated in the posterior half. Thus, these results suggest that – although low - the risk of acquiring anisakiasis from consumption of raw, marinated and/or undercooked short-finned squid products still exists.

Limited data exist on the occurrence of the dwarf sperm whale Kogia sima in the Mediterranean Sea and its parasite fauna. Here, the occurrence of the anisakid species Anisakis physeteris and A. pegreffii in the stomach chambers of an adult female dwarf sperm whale, stranded in southern Italy, is reported. In addition, the occurrence of Phyllobothrium delphini larvae infecting the blubber of the caudal peduncle region was recorded. A. physeteris and A. pegreffii represent the 2 parasite species of the genus, mostly distributed in the Mediterranean Sea in fish and squids. The finding of A. pegreffii and A. physeteris in the dwarf sperm whale represents a new record in this host species for the Mediterranean Sea. The study of gastrointestinal content also revealed a massive presence of cephalopod beaks identified as belonging to pelagic squids including the umbrella squid Histioteuthis bonnellii, the reverse jewel squid H. reversa, the long-armed squid Chiroteuthis veranii, and the comb-finned squid Ctenopteryx sicula. The feeding habits of the dwarf sperm whale, as well as the occurrence of these squid residuals in the cetacean host, suggest that these squid species play a major role in maintaining the life cycle of anisakid parasite species and P. delphini.

Anisakid nematodes belonging to the species Anisakis pegreffii are distributed in a wide variety of fishes from the Mediterranean Sea and they are known to cause the human zoonosis anisakiasis. The present study investigated, for the first time, the response of A. pegreffii larvae (identified to species level by allozymes and mtDNA cox2 sequence analysis) to the storage temperature of European anchovies, Engraulis encrasicolus . The larval motility of A. pegreffii was studied in 1300 fish specimens, captured from a highly infested area (FAO 37.2.1, 43°8′N, 14°16′E), maintained under different temperatures (2 °C, 5 °C, 7 °C), and examined at different time intervals (immediately after fishing, 24 h, 48 h and 72 h). Parasitological analysis was carried out with the UV-press method. The results showed that the increase of infection values with A. pegreffii in the fillets of anchovies was statistically positively related to the increase of the temperature (at 5 °C and 7 °C) and time of storage (after 24 h, 48 h, and 72 h). Accordingly, a significant statistical correlation between the increasing of the worm burden in the fillets and a decreasing of A. pegreffii in the viscera was observed. In contrast, those fish constantly maintained at 2 °C showed no statistically significant variation in infection either in the viscera or the fillets, after 24, 48 and 72 h. In the same batches of anchovies, larvae of the non-zoonotic nematode parasite Hysterothylacium aduncum (identified to the species level by ITS rDNA sequences analysis) were found, but they were never observed infecting the musculature of the anchovies. Our results suggest that temperature plays an important role in the post-mortem motility of A. pegreffii larvae in anchovies. In addition, the presence of A. pegreffii in the fillets inspected immediately after their capture indicates that intra-vitam migration may also occur. As a consequence, the importance of the adoption of rules to prevent human anisakiasis, as the deep freezing to −20 °C for 24 h, was underlined.

Mass strandings of sperm whales (Physeter macrocephalus) are rare in the Mediterranean Sea. Nevertheless, in 2014 a pod of 7 specimens stranded alive along the Italian coast of the Central Adriatic Sea: 3 individuals died on the beach after a few hours due to internal damages induced by prolonged recumbency; the remaining 4 whales were refloated after great efforts. All the dead animals were genetically related females; one was pregnant. All the animals were infected by dolphin morbillivirus (DMV) and the pregnant whale was also affected by a severe nephropathy due to a large kidney stone. Other analyses ruled out other possible relevant factors related to weather conditions or human activities. The results of multidisciplinary post-mortem analyses revealed that the 7 sperm whales entered the Adriatic Sea encountering adverse weather conditions and then kept heading northward following the pregnant but sick leader of the pod, thereby reaching the stranding site. DMV infection most likely played a crucial role in impairing the health condition and orientation abilities of the whales. They did not steer back towards deeper waters, but eventually stranded along the Central Adriatic Sea coastline, a real trap for sperm whales.

Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.

10 pages, 4 figures, 2 tables, 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., The present survey was financially supported by the European Union's 7th Framework Program for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant agreement (GA) no. 312068

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.

18 pages, 10 figures, 6 tables.-- Arne Levsen ... et al., Harvesting and exploiting limited fisheries resources in a sustainable manner also implies achieving maximum added value from the raw material. However, the presence of parasites in the products may adversely affect consumer perception and/or pose a direct health hazard. As a major stepping-stone of the PARASITE project, an epidemiological survey was carried out to provide the basis for analysis and prediction of consumer exposure risk due to the presence of anisakid nematodes in fish from European wild-catch fisheries. The project consisted of nine separate workpackages (WP) where the exposure risk assessment survey was organized within WP2. The surveillance task also provided the data or samples needed for data management and sample storage (WP3, Biobank), molecular and genetic parasite species identification (WP4), and statistical modelling and inference (WP8). In total 17,760 fish belonging to 16 teleost species were examined for anisakids, with special emphasis on economically and ecologically important species such as Atlantic mackerel, herring, European hake, Atlantic cod and anchovy. The target fish species were sampled at four major European fishing areas including the Barents Sea, North Sea, Baltic Sea, Grand Sole Bank, waters off NW Spain and Portugal, central and western parts of the Mediterranean Sea, and the Adriatic Sea. Thus, the survey represents the largest and most comprehensive epidemiological data compilation of anisakids ever generated in terms of geographical range as well as number of fish host species and sample size. An important requirement of the survey was the use of commonly accepted nematode detection methods, i.e. the UV-press method or artificial digestion, to quantify infection level and spatial distribution of anisakid larvae in the target fish species. The basic layout, set-up and principles of the method, along with a discussion of possible source of errors are described. Additionally, the molecular and genetic markers which were used to identify and characterize different species and populations of anisakids from the targeted fish host species and geographical areas, are reviewed as well. Some basic parasite infection characteristics of each fish host species, and any relationships with the presumably most important infection predictors, i.e. fish host body size and fishing locality, are presented and discussed. Emphasis is put on anisakid occurrence in the flesh of the fish. Based on the findings, a graphical exposure risk profile is introduced, including fish species or products thereof, which due to common processing or preparation practices, are at highest risk to act as source of anisakiasis in Europe, The present work was financially supported by the European Union’s 7th Framework Programme for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant Agreement (GA) no. 312068

Human dendritic cells (DCs) show remarkable phenotypic changes when matured in the presence of helminth-derived products. These modifications frequently elicited a polarization towards Th2 cells and regulatory T cells thus contributing to immunological tolerance against these pathogens. In this study, the interaction between DCs and larvae of the zoonotic anisakid nematode Anisakis pegreffii was investigated. A. pegreffii larvae were collected from fish hosts, and monocyte-derived DCs were cocultured in the presence of the live larvae (L) or its crude extracts (CE). In both experimental conditions, A. pegreffii impacted DC viability, hampered DC maturation by reducing the expression of molecules involved in antigen presentation and migration (ie HLA-DR, CD86, CD83 and CCR7), increased the phagosomal radical oxygen species (ROS) levels and modulated the phosphorylation of ERK1,2 pathway. These biological changes were accompanied by the impairment of DCs to activate a T-cell-mediated IFNγ. Interestingly, live larvae appeared to differently modulate DC secretion of cytokines and chemokines as compared to CE. These results demonstrate, for the first time, the immunomodulatory role of A. pegreffii on DCs biology and functions. In addition, they suggest a dynamic contribution of DCs to the induction and maintenance of the inflammatory response against A. pegreffii.

11 pages, 5 figures, 3 tables, Haddock and whiting are two species of the family Gadidae that are exploited by fisheries mainly in European waters, the former being one of the most important seafood resources in Scotland (UK). The present study aimed to quantify prevalence, abundance and intensity of infection of the zoonotic parasite Anisakis spp. and other ascaridoid nematodes in these fish as part of a study on the risk to consumers. Fish were sourced from research trawling surveys during 2013–2015, aiming to cover a range of size-classes and different times of year. Samples were obtained from the North Sea, west coast of Scotland and the Barents Sea. Fish were stored frozen prior to separation of viscera and fillets and the use of pressing and UV visualisation to locate, identify and count Anisakis spp. and other ascaridoids in fillets (divided into 8 parts), liver and other viscera. A subsample of Anisakis spp. collected from haddock were identified genetically by means of two diagnostic markers (mtDNA cox2 and EF1 α-1 nuclear DNA), and all were identified as A. simplex (s. s.). Both fish species showed moderate to high infection levels with Anisakis spp. (likely A. simplex (s. s.)) in the visceral organs and cavity, and rather low infection in the musculature. Those A. simplex (s. s.) larvae present in the musculature were mostly found in the anterior ventral area. In both fish species, the infection rate increased with fish length and in haddock a negative relationship was observed between Anisakis spp. numbers in muscle and body condition. From our sampling of Anisakis spp. in fish muscle, in Scotland, whiting presents a greater human health risk than haddock in Scotland. The risk arising from consumption of haddock from the Barents Sea (with 72% prevalence of Anisakis) is markedly higher, although in all cases the risk of infection in humans can be minimised if fish are frozen or adequately cooked prior to consumption. We present previously unpublished information on reported cases of anisakiasis in the United Kingdom during 2000–2013, which included 4 cases from Scotland among 22 overall. While these findings suggest that anisakiasis is relatively rare in the UK, underreporting is likely and further investigation of the incidence of anisakiasis is needed, The present work was supported by the European Union's 7th Framework Programme for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant Agreement (GA) no. 312068. GJP acknowledges additional support from the University of Aveiro and Caixa Geral de Depósitos (Portugal)

9 pages, 3 figures, 5 tables.-- Simonetta Mattiucci ... et al., The Atlantic herring is a schooling, pelagic species that inhabits both sides of the North Atlantic Ocean. Herring stock identification is usually based on several approaches, including fish meristic characters, population genetic analysis and the use of parasite species composition. A total of 654 Anisakis spp. larvae collected from herring of four fishing grounds in the Norwegian Sea, Baltic Sea, North Sea, and the English Channel off the French coast, was identified to species level using diagnostic allozymes and sequence analysis of EF1 α−1 nDNA and the mtDNA cox2 genes. Population genetic differentiation of Anisakis simplex (s. s.) among the different fishing areas was estimated, at the intraspecific level, on the basis of mtDNA cox2 sequences analysis. Spatial comparison based on molecular variance analysis and Fst values was performed for the collected specimens (among regions). Haplotype network construction showed relevant differences in haplotype frequencies between samples of A. simplex (s. s.) from the different geographical areas. Results indicate a genetic sub-structuring of A. simplex (s. s.) obtained from herring in different areas, with the population from the Norwegian Sea being the most differentiated one, and with North Sea and Baltic Sea populations being most similar. The population genetic structure of A. simplex (s. s.) was in accordance with the herring population genetic structure throughout the host’s geographical range in the NE Atlantic. Results suggest that mtDNA cox2 is a suitable genetic marker for A. simplex (s. s.) population genetic structure analysis and a valuable tool to elucidate the herring stock structure in the NE Atlantic Ocean, The research was carried out with funding received from the European Union’s Seventh Framework Programme for research, technological development and demonstration, under the grant agreement no. 312068 “PARASITE”

European pilchards are traditionally eaten marinated or salted in the Mediterranean countries often without thermal processing or gutting due to small size. Since ingestion of live third stage Anisakis larvae represents a causing agent in the onset of anisakiasis, the aim of our study was to assess prevalence and intensity of Anisakis infection in European pilchards originating from different Mediterranean regions in a three-year sampling period (2013–2015). A total of 1564 specimens of European pilchard collected from two geographically distinct sampling regions (western Mediterranean and Adriatic Sea) were examined using the UV-Press method, which utilises the fluorescence of frozen anisakids in flattened and subsequently frozen fillets and viscera. A subsample of 67 isolated larvae was identified as A. pegreffii by diagnostic allozyme markers and sequence analyses of the mtDNA cox2 locus. The overall prevalence in pilchards was 12.2% (range 0–44.9% for different sampling points) and mean intensity 1.8. More importantly, we have observed an overall larval prevalence of 1.5% in fillets. The highest prevalence (44.9%) was recorded in pilchards caught in western parts of the Mediterranean. As fish host size was a significant predictor of parasite abundance, it should be highlighted that these pilchards were also the largest (mean total length 173.2 mm) ; on average > 2 cm larger than the rest of the samples. Other isolated nematode species included Hysterothylacium sp. in viscera, showing almost a double of A. pegreffii prevalence, 20.1%. In summary, our study demonstrates that the presence of A. pegreffii in European pilchards from the Mediterranean Sea is highly influenced by both geographic and host size variation. This implies that, before future risk management measures are developed, these variables should be assessed in order to minimize public health concerns.

A retrospective analysis on human anisakiasis in Italy since its first description in 1996 was performed by conducting a literature search. Inclusion criteria based on the presence of a larva and on parasite identification were applied. Epidemiological data and clinical features were analysed. Particular attention was paid to the source of infection. In total, 73 cases were included in the analysis, while 34 were excluded. Cases were reported from eight Italian regions, most frequently Abruzzo, Apulia and Latium. The parasite was detected by endoscopy (51.4%) or laparotomy (48.6%). The site of infection was intestinal (42.5%), gastric (43.8%), oesophageal (1.4%) or ectopic (12.3%). Most of the parasites (71.0%) were identified as Anisakis sp. or A. simplex (s.l.). However, when molecular methods were used (21 cases), A. pegreffii was always identified. In most of the patients (65.7%), the source of infection was raw or undercooked anchovies, followed by “anchovies or sardines” (15.1%), generic “raw seafood” (15.1%), and sardines (1.4%). In only 2 cases (2.7%), the source was not available. This is the first systematic analysis of Italian cases of anisakiasis. The main conclusions derived from the results are: i) attention should be given to the history, in particular when raw marinated anchovies, proven to be the main source of human anisakiasis in Italy, are consumed; ii) in order to assess correct epidemiological data, a confirmed and specific etiological identification should always be sought.

Myctophids (lantern fish) and cephalopods play a key role in trophic webs from the continental slope and oceanic waters linking the zooplankton to top predators. Many cetaceans feed on both lantern fish and cephalopods, and such prey would thus be expected to bridge the trophic gap in the life-cycles of helminths infecting cetaceans. However, information on the life-cycles of most of these helminths is extremely scanty. We examined the parasite fauna of myctophids and cephalopods in two areas from the western Mediterranean where at least 21 helminth taxa from cetaceans have been reported and both cetacean diversity and abundance is high. A total of 1012 individuals of 8 lantern fish species, namely, Ceratoscopelus maderensis, Lampanyctus crocodilus, Notoscopelus elongatus, Benthosema glaciale, Myctophum punctatum, Lobianchia dofleini, Diaphus holti and Hygophum benoiti, and 792 individuals of 2 cephalopod species, Alloteuthis media and Sepietta oweniana, were collected from the Gulf of Valencia and Alboran Sea (Spanish Mediterranean) during 2010–2012 and examined for larval helminths. All these species have been reported as prey for at least some cetacean species in the area. Only five helminth taxa were found. The nematodes Anisakis pegreffii and Anisakis physeteris were detected in N. elongatus and C. maderensis (overall prevalence for Anisakis: 8.1% and 0.5%, respectively). Their prevalence in N. elongatus was significantly higher than that from the other three myctophid species with n>50 individuals. A single individual of Hysterothylacium sp. was found in N. elongatus (prevalence: 0.5%) and Raphidascarididae gen. spp. in N. elongatus and L. crocodilus (prevalence: 20.3% and 0.7%, respectively). Juvenile didymozoid digeneans (Torticaecum type) were detected in N. elongatus and L. crocodilus (prevalence: 18.5% and 4.3%, respectively). Two unidentified cestode plerocercoids were collected from N. elongatus. Our study suggests, for the first time, that myctophids could play a role as paratenic hosts in the oceanic life-cycle of A. pegreffii and A. physeteris in the western Mediterranean. None of the other larvae identified at least to family level infect cetaceans, but some of them can be transmitted to large predatory fish. The extreme scarcity of such cetacean parasites in this, and previous parasitological surveys of mesopelagic fish and cephalopods is at odds with the key role of these preys in the diet of oceanic cetaceans.

Anisakiasis 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. Parasite 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. According 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. The 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.

We used morphological and genetic identification to document metazoan parasites of cetaceans and pinnipeds from 28 carcasses salvaged in California between 1974 and 2002, including whales, dolphins, porpoises, sea lions, and seals. Nematodes found included Anisakis simplex (s.l.), A. simplex (s.s.), Pseudoterranova decipiens (s.l.), Pseudoterranova sp., Contracaecum ogmorhini (s.l.), Contracaecum sp., Stenurus cf. minor, Pharurus cf. dalli, and Otostrongylus circumlitus. Cestodes found included Phyllobothrium delphini and Tetrabothrius sp. The acanthocephalan Bolbosoma capitatum, and the arthropod Halarachne miroungae, also were documented. Tetrabothrius sp. in the pygmy beaked whale (Mesoplodon peruvianus) and P. delphini in the Eastern North Pacific long-beaked common dolphin (Delphinus delphis bairdii) constitute two new host records.

17 páginas, 7 tablas, 4 figuras.-- This work is licensed under a Creative Commons Attribution 4.0 International License, Anisakiasis is an emerging zoonosis caused by the fish parasitic nematode Anisakis. Spain appears to have the highest reported incidence in Europe and marinated anchovies are recognised as the main food vehicle. Using data on fishery landings, fish infection rates and consumption habits of the Spanish population from questionnaires, we developed a quantitative risk assessment (QRA) model for the anchovy value chain. Spaniards were estimated to consume on average 0.66 Anisakis per untreated (non-frozen) raw or marinated anchovy meal. A dose-response relationship was generated and the probability of anisakiasis was calculated to be 9.56 × 10−5 per meal, and the number of annual anisakiasis cases requiring medical attention was predicted between 7,700 and 8,320. Monte Carlo simulations estimated post-mortem migration of Anisakis from viscera to flesh increases the disease burden by >1000% whilst an education campaign to freeze anchovy before consumption may reduce cases by 80%. However, most of the questionnaire respondents who ate untreated meals knew how to prevent Anisakis infection. The QRA suggests that previously reported figures of 500 anisakiasis per year in Europe is a considerable underestimate. The QRA tool can be used by policy makers and informs industry, health professionals and consumers about this underdiagnosed zoonosis, M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial support of the contract from the EU Project PARASITE (grant number 312068).

Inventorying of anisakid nematode biodiversity is the discovering, surveying, quantifying and mapping of species, populations and their genetic diversity and variability. This aim, however, is compromised if discrimination of anisakid taxa relies solely on morphological features. Therefore, the accurate detection and delimitation of cryptic anisakid species requires molecular-based assessments. This, in turn, permits elucidation of patterns and process in their evolution and ecology, including biogeography, host-parasite association and co-evolution. In addition, a true picture of anisakids and their genetic diversity facilitates understanding of their temporal and spatial distribution also related to their hosts demographic changes and marine ecosystem food webs. This review represents an inventorying of the biodiversity, at species and gene level, of those anisakid species so far discovered belonging to the genera Anisakis, Pseudoterranova and Contracaecum, from the Austral Region, including: (1) taxa recognized as “biological species” based on the application of different molecular genetic markers; (2) current molecular/genetic approaches to identify them at any life-history stage; (3) ecological data relating to the geographical distribution, definitive host-association and host-preferences; (4) estimates of genetic variability values inferred from nuclear and mitochondrial genes, as a possible indicator of the integrity of marine food webs; (5) data so far known concerning the possible zoonotic role of those anisakid species to humans. Fil: Mattiucci, Simonetta. Università degli studi di Roma "La Sapienza"; Italia Fil: Paoletti, Michela. Universita Di Tuscia; Italia Fil: Cipriani, Paolo. Università degli studi di Roma "La Sapienza"; Italia. Universita Di Tuscia; Italia Fil: Webb, Stephen C.. Cawthron Institute; Nueva Zelanda Fil: Timi, Juan Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina Fil: Nascetti, Giuseppe. Universita Di Tuscia; Italia

Summary IgE sensitization to Anisakis pegreffii in Italian subjects suffering from gastro-allergic anisakiasis (GAA) (N= 5), or showing chronic urticaria (CU+) after fish consumption (N= 100), was investigated. A control group (N= 5) was also included. IgE response was analyzed by immunoblotting (WB) assay, using both excretory/secretory products (ESPs) and crude extract (CE) of A. pegreffii larvae. The results were compared with those achieved by the conventional immunological method for Anisakis allergy (i.e. immunoCAP). Among the 110 subjects, 28 showed IgE positivity with both WB and iCAP methods; 13 proved IgE reactivity, in WB assay, to EPS antigens of A. pegreffii, here provisionally indicated as Ani s 1-like, Ani s 7-like, Ani s 13-like; only 15 sera have shown IgE-WB reaction to Ani s 7-like and Ani s 13-like. iCAP and WB exhibited a high concordance value (k=1.00) when iCAP value was 50.0 (positive result). In the sera samples recorded as positive to Anisakis allergy, Ani s 1-like was responsible for 46.4% of the sensitivity, while Ani s 7-like and Ani s 13-like for 100%. They could be considered as major antigens in the diagnosis of allergic anisakiasis caused by A. pegreffii. This article is protected by copyright. All rights reserved.

A new taxon, Rhabdias esculentarum n. sp., is described based on DNA sequence analysis at multiple loci (i.e. mtDNA cox-1, 12S rRNA, ITS-1 and partial ITS-2 regions of the nuclear rDNA) and morphometric analysis carried out on specimens collected from the green frogs of the Rana esculenta species complex in Italy (i.e. R. lessonae Camerano and R. esculenta Linnaeus, identified genetically by diagnostic allozyme loci). Rhabdias esculentarum n. sp. was differentiated genetically, at both mitochondrial and nuclear levels, from Rh. bufonis (Schrank, 1788) (sensu Hartwich, 1972) and Rh. sphaerocephala Goodey, 1924 recovered from the toad Bufo bufo Linnaeus collected sympatrically with the specimens of Rana lessonae and R. esculenta examined in the present study. Moreover, the new taxon proved to be different from the other species of Rhabdias from anurans, which had previously been sequenced using the same genes and deposited in GeneBank. Phylogenetic analyses (MP and ML) inferred from mitochondrial (mtDNA cox-1 and 12S ribosomal RNA) and nuclear (ITS-1 and ITS-2 of the rDNA regions) sequences datasets were congruent in depicting Rh. esculentarum n. sp. as forming a highly supported clade distinct from the sympatric species Rh. bufonis, as well as from Rh. sphaerocephala, characterised on the basis of the same loci. Morphometric analysis and the differential diagnosis of genetically characterised specimens of the new species have revealed differences in several features in comparison with the type-species, Rh. bufonis. Material of the latter species included voucher specimens from Germany deposited by Hartwich (1972) and other specimens collected from B. bufo in Italy. Among the diagnostic characters, the particular cup-shaped buccal capsule characterising Rh. esculentarum is clearly different from the tear-shaped buccal capsule observed in material of R. bufonis obtained from Berlin Museum and collected in the same geographical area as the green frogs under study. Rh. esculentarum was also found to differ in some measurements and allometric characters from Rh. bufonis (sensu Moravec et al., 1997). The data so far collected appear to indicate a host-preference of Rh. esculentarum for Rana lessonae and R. esculenta, which belong to the R. esculenta hybridogenetic species complex in Italy.

Garcia, A., Mattiucci, S., Damiano, S., Santos, M. N., and Nascetti, G. 2011. Metazoan parasites of swordfish, Xiphias gladius (Pisces: Xiphiidae) from the Atlantic Ocean: implications for host stock identification. – ICES Journal of Marine Science, 68: 175–182. The metazoan parasite fauna composition of the swordfish Xiphias gladius is reported from 157 fish caught in four areas of the Atlantic Ocean: northwest (NW), central north (CN), eastern tropical (ET), and central south (CS). Samples were collected in 2003, 2006, and 2007. In all, 18 parasite taxa were identified by morphological and genetic methods. These data, together with earlier analyses from the tropical equatorial (TEQ) Atlantic revealed the association of some parasite taxa with distinct areas based on their presence/absence and infection levels. Hysterothylacium corrugatum (s.l.) and Rhadinorhynchus pristis were mostly associated with swordfish caught in the NW, and Anisakis paggiae and H. incurvum with fish caught in the southern areas (ET, TEQ, and CS). Anisakis simplex (s.s.) could be used as a marker for all northern samples. Parasitological data on Atlantic swordfish seem to support the existence of at least two discrete stocks in the Atlantic, supporting swordfish genetic stock structure.

Two new species of Contracaecum Railliet & Henry, 1912, previously referred to as C. multipapillatum sp. A and C. multipapillatum sp. B by Nascetti et al. (1990) from the Dalmatian pelican Pelecanus crispus (L.) in the Ambracian Gulf off Greece, are described as C. gibsoni n. sp. and C. overstreeti n. sp., respectively. Morphological analysis and the differential diagnosis of genetically recognised male specimens of C. gibsoni and C. overstreeti with respect to C. multipapillatum (von Drasche, 1882) (sensu lato) from Egretta alba (L.) in northern Colombia and other morphologically related Contracaecum spp. enabled the detection of differences between the two species in a number of characters, including spicule length and the shape of its tip, and the arrangement of the proximal and distal papillae on the male tail. Accordingly, formal descriptions are presented for C. gibsoni n. sp. and C. overstreeti n. sp. from P. crispus. The genetic characterisation of the two taxa is based on 20 allozyme loci and sequence analyses (519 bp) of the mtDNA cox2 gene. Reproductive isolation was demonstrated between these two taxa, which sympatrically infect the same definitive host, and fixed allele differences between the two species were found at some (Aat-2, PepC-1, PepC-2 and Pgm-1) of the 20 allozyme loci analysed. Their genetic divergence, estimated at the allozyme level, was D Nei = 0.31. The genetic relationships of C. gibsoni and C. overstreeti with respect to C. multipapillatum (s. l.) collected from E. alba in Colombia, as well as with other congeners from fish-eating birds which had previously been genetically characterised using the same genetic markers, i.e. C. rudolphii A and C. rudolphii B of Bullini et al. (1986), C. septentrionale Kreis, 1955, C. microcephalum (Rudolphi, 1809), C. bioccai Mattiucci et al., 2008, C. pelagicum Johnston & Mawson, 1942 and C. micropapillatum (Stossich, 1890), were inferred from mtDNA cox2 sequence analysis. The MP, NJ and BI trees obtained were congruent in depicting C. gibsoni and C. overstreeti as closely related species but quite distinct from each other and forming a subclade with specimens of C. multipapilllatum (s. l.) from E. alba (L.) in Colombia. This subclade was also found to be distinct from the remainder of the taxa considered.