Parasitic Effects on the Congenital Transmission of Trypanosoma cruzi in Mother-Newborn Pairs.

Maternal parasitemia and placental parasite load were examined in mother-newborn pairs to determine their effect on the congenital transmission of Trypanosoma cruzi. Parasitemia was qualitatively assessed in mothers and newborns by the microhematocrit test; parasite load was determined in the placental tissues of transmitting and non-transmitting mothers by the detection of T. cruzi DNA and by histology. Compared to transmitter mothers, the frequency and prevalence of parasitemia were found to be increased in non-transmitter mothers; however, the frequency and prevalence of parasite load were higher among the transmitter mothers than among their non-transmitter counterparts. Additionally, serum levels of interferon (IFN)-γ were measured by an enzyme-linked immunosorbent assay (ELISA) in peripheral, placental, and cord blood samples. Median values of IFN-γ were significantly increased in the cord blood of uninfected newborns. The median IFN-γ values of transmitter and non-transmitter mothers were not significantly different; however, non-transmitter mothers had the highest total IFN-γ production among the group of mothers. Collectively, the results of this study suggest that the anti-T. cruzi immune response occurring in the placenta and cord is under the influence of the cytokines from the mother's blood and results in the control of parasitemia in uninfected newborns.

Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

Optimization of a fast and sensitive method based on matrix solid-phase dispersion-LC-ms/ms for simultaneous determination of phthalates and bisphenols in mussel samples.

Bisphenols and phthalates are wide classes of endocrine disrupting chemicals (EDCs) extensively used as additives in plastic products. In this study, a fast and reliable analytical method based on matrix solid-phase dispersion (MSPD) coupled with LC-MS/MS was developed and optimized for simultaneous determination of 8 bisphenols and 7 phthalates in raw mussel extract. The LC-MS/MS method was tested for linearity (R2), inter- and intra-day repeatability, limit of detection and quantification, both for matrix-free and matrix-matched solutions. The MSPD method was optimized in terms of ratio between sample and sorbent, and the type and quantity of the eluents in order to maximize the recoveries and to minimize matrix effects. The obtained recoveries (values between 75% and 113%), limits of detection (values between 0.048 and 0.36 µg kg-1), limits of quantification (values between 0.16 and 1.28 µg kg-1), repeatability (RSD% between 1.30% and 8.41%) and linearity (R2 > 0.998) were satisfactory and suitable for the determination of target micropollutants in food samples. In addition, the low solvent consumption and fast execution make this method ideal for routinely determinations of bisphenols and phthalates in mussels.

An update and ecological perspective on certain sentinel helminth endoparasites within the Mediterranean Sea.

The Mediterranean Sea is recognized as a marine biodiversity hotspot. This enclosed basin is facing several anthropogenic-driven threats, such as seawater warming, pollution, overfishing, bycatch, intense maritime transport and invasion by alien species. The present review focuses on the diversity and ecology of specific marine trophically transmitted helminth endoparasites (TTHs) of the Mediterranean ecosystems, aiming to elucidate their potential effectiveness as 'sentinels' of anthropogenic disturbances in the marine environment. The chosen TTHs comprise cestodes and nematodes sharing complex life cycles, involving organisms from coastal and marine mid/upper-trophic levels as definitive hosts. Anthropogenic disturbances directly impacting the free-living stages of the parasites and their host population demographies can significantly alter the distribution, infection levels and intraspecific genetic variability of these TTHs. Estimating these parameters in TTHs can provide valuable information to assess the stability of marine trophic food webs. Changes in the distribution of particular TTHs species can also serve as indicators of sea temperature variations in the Mediterranean Sea, as well as the bioaccumulation of pollutants. The contribution of the chosen TTHs to monitor anthropogenic-driven changes in the Mediterranean Sea, using their measurable attributes at both spatial and temporal scales, is proposed.

De novo transcriptome assembly of an Antarctic nematode for the study of thermal adaptation in marine parasites.

Understanding the genomic underpinnings of thermal adaptation is a hot topic in eco-evolutionary studies of parasites. Marine heteroxenous parasites have complex life cycles encompassing a free-living larval stage, an ectothermic intermediate host and a homeothermic definitive host, thus representing compelling systems for the study of thermal adaptation. The Antarctic anisakid Contracaecum osculatum sp. D is a marine parasite able to survive and thrive both at very cold and warm temperatures within the environment and its hosts. Here, a de novo transcriptome of C. osculatum sp. D was generated for the first time, by performing RNA-Seq experiments on a set of individuals exposed to temperatures experienced by the nematode during its life cycle. The analysis generated 425,954,724 reads, which were assembled and then annotated. The high-quality assembly was validated, achieving over 88% mapping against the transcriptome. The transcriptome of this parasite will represent a valuable genomic resource for future studies aimed at disentangling the genomic architecture of thermal tolerance and metabolic pathways related to temperature stress.

Black spot diseases in seven commercial fish species from the English Channel and the North Sea: infestation levels, identification and population genetics of Cryptocotyle spp.

Fish are often speckled with "black spots" caused by metacercarial trematode infection, inducing a host response. Cryptocotyle spp. (Opisthorchiidae) are among the parasites responsible for this phenomenon. So far, the impact on human health is still unknown. In addition, few publications dealing with black spot recovery, identification, distribution and diversity among commercially important fish are available. Moreover, "black spots" have been observed by fishermen on marine fish, revealing an appreciable but unquantified presence in consumed fish. An epidemiological survey of 1,586 fish from seven commercial species (herring, sprat, whiting, pout, dab, flounder, and plaice) was conducted in the Eastern English Channel and the North Sea in January 2019 and 2020. Encysted metacercariae were found in 325 out of 1,586 fish, with a total prevalence of 20.5%. Intensity of infection varied from 1 to 1,104 parasites. The recorded encysted metacercariae were identified either by microscopic examination or with molecular tools. Partial sequences of the mtDNA cox1 gene and of the rDNA ITS region were obtained. Two species of Cryptocotyle, Cryptocotyle lingua (Creplin, 1825) and Cryptocotyle concava (Creplin, 1825) were found. Metacercariae belonging to other trematode families were also identified. Molecular phylogenetic analysis and haplotype network construction were performed to confirm the identification and to study the potential presence of different populations of Cryptocotyle spp. This survey enabled us to describe the distribution of two species of Cryptocotyle in the English Channel and North Sea ecosystems. The observed differences in infestation levels between fish species and geographical areas will contribute to better understanding of the ecology of these parasites.

Title: Maladies des points noirs chez sept espèces commerciales de poissons de la Manche et de la mer du Nord : niveaux d'infestation, identification et génétique des populations de Cryptocotyle spp. Abstract: Les poissons sont souvent parsemés de « points noirs ¼ causés par une infection par des métacercaires de trématodes induisant une réponse de l'hôte. Les Cryptocotyle spp. (Opisthorchiidae) font partie des parasites responsables de ce phénomène. Jusqu'à présent, leur impact sur la santé humaine est inconnu. De plus, il existe peu de publications traitant de la récupération, l'identification, la distribution et la diversité des « points noirs ¼ parmi les poisons d'importance commerciale. Par ailleurs, des observations de « points noirs ¼ sur les poissons marins ont été constatées par les pêcheurs révélant une présence assez importante mais non quantifiée dans les poissons consommés. Une enquête épidémiologique portant sur 1 586 poissons de sept espèces commerciales (hareng, sprat, merlan, tacaud, limande, flet et plie) a été menée en Manche orientale et en Mer du Nord, en janvier 2019 et 2020. Des métacercaires enkystées ont été trouvées chez 325 poissons parmi 1 586, avec une prévalence totale de 20,5 %. L'intensité de l'infection variait de 1 à 1 104 parasites. Les métacercaires enkystées répertoriées ont été identifiées soit par examen microscopique, soit avec des outils moléculaires. Des séquences partielles du gène cox1 de l'ADNmt et de la région ITS de l'ADNr ont été obtenues. Deux espèces de Cryptocotyle, Cryptocotyle lingua (Creplin, 1825) et Cryptocotyle concava (Creplin, 1825) ont été trouvées. Des métacercaires appartenant à d'autres familles de trématodes ont également été identifiées. Une analyse phylogénétique moléculaire et la construction d'un réseau d'haplotypes ont été effectuées pour confirmer l'identification et étudier la présence potentielle de différentes populations de Cryptocotyle spp. Cette étude a permis de décrire la distribution de deux espèces de Cryptocotyle dans les écosystèmes de la Manche et de la Mer du Nord. Les différences observées dans les niveaux d'infestation entre les espèces de poissons et les zones géographiques contribueront à une meilleure compréhension de l'écologie de ces parasites.

Basophil Activation Test in the Diagnosis of Anisakis Allergy: An Observational Study from an Area of High Seafood Consumption in Italy.

The rising popularity of undercooked or raw seafood containing larvae of the Anisakis parasite has led to issues of public health concern due to allergic manifestations. We conducted an observational study on the use of an innovative Anisakis allergy diagnostic algorithm in a convenience sample of 53 allergic outpatients recruited in Western Sicily, between April 2021 and March 2022. We included individuals with an anamnesis suggestive of IgE sensitization to Anisakis reporting clinical manifestation in the last month due to allergic reactions after eating fresh fish, or in subjects at high exposure risk with sea products while abstaining from fish ingestion, excluding those with documented fish sensitization. Outpatients were tested via Skin Prick Test, IgE-specific dosage and Basophil Activation Test (BAT). Twenty-six outpatients were diagnosed with Anisakis, while 27 with Chronic Urticaria (CU). We found a seven-fold excess risk for Anisakis (p4) positivity in the Anisakis allergic outpatients, as compared to the CU ones. BAT showed the best diagnostic accuracy (92.45%) and specificity (100%), while specific IgE to Ascaris (p1) documented the best sensitivity (92.31%) but a very low specificity (37.04%). In conclusion, our findings may represent a potentially useful contribution to the future development of updated clinical guidelines.

Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae).

Introduction: Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized. Methods: Genetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis. Results and discussion: EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.

Genetic diversity of Contracaecum rudolphii sp. A (Nematoda: Anisakidae) parasitizing the European Shag Phalacrocorax aristotelis desmarestii from the Spanish Mediterranean coast.

Sibling species of the Contracaecum rudolphii (s.l.) complex are habitual endoparasites of cormorants of the Phalacrocoracidae family, worldwide. In Europe, the two species, C. rudolphii sp. A and C. rudolphii sp. B, have been identified. However, information regarding the occurrence and distribution of these anisakids in cormorants from Spain is scarce. In the present study, 20 specimens of the European Shag, Ph. aristotelis desmarestii, from the western Mediterranean Spanish marine coast were parasitologically analyzed for the presence of nematodes. All hosts were found parasitized with Contracaecum specimens (n = 1,517). A representative subsample was genetically identified as C. rudolphii sp. A by sequence analysis of the mtDNA cox2 gene and the ITS1 and ITS2 regions of the rDNA. This represents the first report of C. rudolphii sp. A from the Spanish Mediterranean waters. Population genetic analysis was performed including other C. rudolphii sp. A specimens from the west Sardinian and the Tyrrhenian Sea. At the intraspecific level, a significant genetic differentiation (Fst ≈ 0.08, p < 0.00001) between the metapopulation from the Spanish Mediterranean coast and that from the Sardinian waters was observed; whereas, no differentiation was found between metapopulations of the parasite from the Spanish and the Tyrrhenian Italian coast. The findings highly support the hypothesis of the adaptation of the life cycle of C. rudolphii sp. A in brackish and marine ecosystems. Furthermore, the results on the population genetics of C. rudolphii sp. A suggest the possible role of the migration routes of wintering populations of cormorants in the Mediterranean Sea in influencing the parasite genetic structure.

Ascaridoid parasites in European sardine throughout the annual cycle: Variability in parasitic load according to host stock features.

In recent years, a drop in the condition of the European sardine has been observed. Although several causes have been attributed to this issue, as overfishing and climate change, little is known about the link between ascaridoid nematode parasitisation and fish status. In this study, sardines were obtained from four fishing grounds along the Mediterranean (Alboran, Northern Spain, Northern Adriatic, and Aegean), and one location in the Atlantic Ocean (Southern Portugal). After analysing individual fish body condition (by direct tissue fat content measurements and condition indices), and reproductive status (by a detailed gonadal examination) throughout the entire annual cycle, ascaridoids were recognised by combining naked eye and UV-press method along flesh, viscera, and gonads. Afterwards, sequence analysis of the rDNA internal transcribed spacers region (ITS) and the mtDNA cox2 gene were used to identify and characterise the different species of ascaridoids from the fish host in the localities throughout the seasons. The main species found along different areas was Hysterothylacium aduncum, present in the Northern Adriatic (prevalence of 7.6%, mean intensity 1.700), the Atlantic (7.5%, 3.889), and the Northern Spain (3.9%, 1.600). Moreover, few individuals of Anisakis simplex (s.s.) and A. pegreffii were observed in the Atlantic (1.7% and 0.8%, respectively), and the latter species was also found in the Adriatic stock (0.8%). All ascaridoid specimens were found in viscera. Obtained results seem to indicate that in stocks with medium sizes, small variations in length are related to parasite intensity. This study highlights the importance of seasonal parasitological analyses at stock level and, especially, in capital breeders, as relationships between condition and reproduction parameters and parasitism are conditioned by seasonality.

Anisakid nematodes in Trichiurus lepturus and Saurida undosquamis (Teleostea) from the South-West Indian Ocean: Genetic evidence for the existence of sister species within Anisakis typica (s.l.), and food-safety considerations.

Nematode parasites of the genus Anisakis (Nematoda, Anisakidae) are considered among the most important biological hazards in seafood products worldwide. In temperate and tropical waters, the most common species appears to be Anisakis typica, generally found around the viscera and sporadically in the flesh of various fish host species. This study investigated the infection sites and genetic diversity of A. typica infecting commercial fishes from the South-West Indian Ocean. Largehead hairtail (N = 20) and brushtooth lizardfish (N = 72) fished off Tanzania were inspected for anisakid nematodes by UV-press. A subsample of 168 nematodes were identified by sequence analyses of the cox2 mtDNA gene and ITS region of rDNA. The species A. typica (s.l.) (N = 166), Pseudoterranova ceticola (N = 1) and Anisakis paggiae (N = 1) were molecularly identified. Phylogenetic analysis of A. typica (s.l.) sequences based on both genes, indicated the existence of two distinct phylogenetic lineages forming two well-supported clades. The first clade comprised 12 A. typica specimens including individuals from its type locality (central Atlantic Ocean). The second clade comprising 154 specimens, clustered with reference sequences retrieved from GenBank including one apparently undescribed taxon, i.e., Anisakis sp. 1, and A. typica var. indonesiensis. The two reciprocally monophyletic clades are closely related and correspond to two distinct sister species within A. typica (s.l.), presently indicated as A. typica sp. A and A. typica sp. B. Two and four fixed alternative nucleotide substitutions (SNPs), i.e., diagnostic positions, between the two taxa, respectively, were found at the mtDNA cox2 and the ITS region of rDNA. The genetic data, as well as their occurrence in sympatry, strengthens the hypothesis that the actual specimens represent two distinct gene pools. The occurrence of both A. typica sp. A and A. typica sp. B in the musculature of freshly examined T. lepturus and S. undosquamis, suggests that both species can migrate intra-vitam into the flesh. Although the zoonotic potential of A. typica s.l. is still unclear, the presence of these parasites in the musculature, edible part of the fish, raises health concerns for consumers.

Distribution and genetic diversity of Anisakis spp. in cetaceans from the Northeast Atlantic Ocean and the Mediterranean Sea.

Parasite biodiversity in cetaceans represents a neglected component of the marine ecosystem. This study aimed to investigate the distribution and genetic diversity of anisakid nematodes of the genus Anisakis sampled in cetaceans from the Northeast Atlantic Ocean and the Mediterranean Sea. A total of 478 adults and pre-adults of Anisakis spp. was identified by a multilocus genetic approach (mtDNA cox2, EF1 α - 1 nDNA and nas 10 nDNA gene loci) from 11 cetacean species. A clear pattern of host preference was observed for Anisakis spp. at cetacean family level: A. simplex (s.s.) and A. pegreffii infected mainly delphinids; A. physeteris and A. brevispiculata were present only in physeterids, and A. ziphidarum occurred in ziphiids. The role of cetacean host populations from different waters in shaping the population genetic structure of A. simplex (s.s.), A. pegreffii and A. physeteris was investigated for the first time. Significant genetic sub-structuring was found in A. simplex (s.s.) populations of the Norwegian Sea and the North Sea compared to those of the Iberian Atlantic, as well as in A. pegreffii populations of the Adriatic and the Tyrrhenian Seas compared to those of the Iberian Atlantic waters. Substantial genetic homogeneity was detected in the Mediterranean Sea population of A. physeteris. This study highlights a strong preference by some Anisakis spp. for certain cetacean species or families. Information about anisakid biodiversity in their cetacean definitive hosts, which are apex predators of marine ecosystems, acquires particular importance for conservation measures in the context of global climate change phenomena.

Integrative Taxonomy Reveals Molicola uncinatus and Gymnorhynchus gigas (Cestoda: Trypanorhyncha) Coinfection in the Atlantic Pomfret Brama brama From the Mediterranean Sea, With Notes on the Phylogenetic Position of G. gigas Within the Family Gymnorhynchidae.

The cestode family Gymnorhynchidae Dollfus, 1935 (Trypanorhyncha) comprises three genera and six valid species that are typically intestinal parasites of large pelagic sharks. Members of Gymnorhynchidae show a wide geographic distribution and represent a global sanitary concern because as larvae they infect the edible flesh of several commercially important fishes with some species having allergenic potential. Larval Gymnorhynchidae collected from the muscles of the Atlantic pomfret Brama brama from various localities in the Mediterranean Sea were identified and characterized by combining traditional morphology, scanning electronic microscopy, and molecular analyses using newly generated nuclear 18S and 28S rRNA sequences. Overall, 98 larvae were collected from 20 (100%) Atlantic pomfrets (intensity of infection: 4.9; range: 1-12). High-quality sequences were obtained for 54 larvae. Of these, 11 and 43 larvae were identified as Molicola uncinatus and Gymnorhynchus gigas, respectively. The phylogenetic analysis showed the existence of three main clades within Gymnorhynchidae. The first included species of G. gigas and M. uncinatus from the Mediterranean and Atlantic; the second and third major clades included an unidentified species of Molicola from the Indian Ocean and specimens of Gymnorhynchus isuri from the Mediterranean and Atlantic, respectively. Finally, Chimaerarhynchus rougetae was the basal and most diverging taxon. The phylogenetic analysis suggested that G. gigas is more closely related to the members of Molicola. We demonstrated the coinfection of M. uncinatus and G. gigas from all localities studied and extended the intermediate hosts and geographical range of M. uncinatus by including the Atlantic pomfret and the Tyrrhenian and Ionian Seas, respectively. The present results supported the previous proposal that G. gigas and Molicola species should be included in the same genus. Our study demonstrated the usefulness of the integrative taxonomy for the unequivocal recognition of larval trypanorhynch species, resolving the current difficulties in the taxonomy, and elucidating the poorly known ecological and biological aspects of members of Gymnorhynchidae.

Anisakis spp, DNA detection in paraffin-embedded tissue biopsies recovered from patients with gastritis using real-time PCR in Bushehr, Persian Gulf, Iran.

Anisakiasis is a zoonotic fish-born parasitic disease caused by anisakid nematodes. Paraffin-embedded blocks containing biopsy samples taken from patients suffering gastritis with unknown causes were investigated by real-time PCR, in the Bushehr region, Iran; where human anisakiasis has not been reported, so far. A total of 50 paraffin-embedded blocks were randomly selected from 250 archived blocks of the patients with gastritis. A SYBER green-based real-time PCR targeting the ITS1 region was developed for the identification of Anisakis genus. An 86 bp partial fragment of the Anisakis spp. ITS1 gene was amplified successfully. A total of 3 out of 50 samples (6 %) had positive amplification in the samples and their pathology reports showed a significant finding of moderate chronic gastritis with or without ulcers. In conclusion, the developed qPCR could be used for detecting Anisakis spp. larval DNA in human biopsy blocks. This study showed the hidden human cases of anisakiasis in the Bushehr for the first time.

De novo transcriptome assembly and annotation of the third stage larvae of the zoonotic parasite Anisakis pegreffii.

OBJECTIVES: Anisakis pegreffii is a zoonotic parasite requiring marine organisms to complete its life-history. Human infection (anisakiasis) occurs when the third stage larvae (L3) are accidentally ingested with raw or undercooked infected fish or squids. A new de novo transcriptome of A. pegreffii was here generated aiming to provide a robust bulk of data to be used for a comprehensive "ready-to-use" resource for detecting functional studies on genes and gene products of A. pegreffii involved in the molecular mechanisms of parasite-host interaction. DATA DESCRIPTION: A RNA-seq library of A. pegreffii L3 was here newly generated by using Illumina TruSeq platform. It was combined with other five RNA-seq datasets previously gathered from L3 of the same species stored in SRA of NCBI. The final dataset was analyzed by launching three assembler programs and two validation tools. The use of a robust pipeline produced a high-confidence protein-coding transcriptome of A. pegreffii. These data represent a more robust and complete transcriptome of this species with respect to the actually existing resources. This is of importance for understanding the involved adaptive and immunomodulatory genes implicated in the "cross talk" between the parasite and its hosts, including the accidental one (humans).

Ascaridoid nematodes infecting commercially important marine fish and squid species from Bangladesh waters in the Bay of Bengal.

Parasitic ascaridoid nematodes occur in a wide range of marine organisms across the globe. Some species of the anisakid family (Ascaridoidea: Anisakidae) can cause gastrointestinal disease in humans (i. e. anisakidosis). Despite their importance as potentially hazardous parasites, the occurrence and infection characteristics of ascaridoids are still poorly known from many host species and geographical areas. This study investigated the diversity and infection levels of ascaridoid parasites in various commercial fish and squid host species off Bangladesh. Fish and squid specimens were visually inspected for nematodes using the UV-press method. Nematodes were assigned to genus level based on morphology and identified by sequence analyses of the entire ITS region and partial 28S rDNA and mtDNA cox2 genes. Third-stage larvae (L3) of Anisakis typica occurred at low prevalence (P = 10% and 8%, respectively) in the viscera of Selar crumenophthalmus and Trichiurus lepturus, while Hysterothylacium amoyense occurred in the viscera of Sardinella fimbriata (P = 1%) and the viscera and muscle of Harpadon nehereus (P = 32%) and T. lepturus (P = 76%). Lappetascaris sp. Type A L3 occurred in the mantle of the squid Uroteuthis duvaucelii (P = 11%). Anisakis and Lappetascaris species, and H. amoyense were firstly identified in the Bay of Bengal. The potentially zoonotic A. typica was only found in fish viscera. Hysterothylacium amoyense and Lappetascaris sp., both generally regarded as non-zoonotic, occurred at low prevalence in the muscle or mantle of fish or squid, respectively. Since consumption of raw or lightly processed seafood seems to be rare in Bangladesh, the risk of acquiring anisakidosis from consuming fishery products from off Bangladesh appears to be low. Due to its reddish appearance, the visual presence of H. amoyense larvae in fish flesh may represent a food quality issue.

Molecular Subtyping of Blastocystis sp. Isolated from Farmed Animals in Southern Italy.

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.

Molecular and morphological characterization of Bolbosoma balaenae (Acanthocephala: Polymorphidae), a neglected intestinal parasite of the fin whale Balaenoptera physalus.

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.

Investigating the genetic structure of the parasites Anisakis pegreffii and A. berlandi (Nematoda: Anisakidae) in a sympatric area of the southern Pacific Ocean waters using a multilocus genotyping approach: first evidence of their interspecific hybridization.

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.

Insights into the role of deep-sea squids of the genus Histioteuthis (Histioteuthidae) in the life cycle of ascaridoid parasites in the Central Mediterranean Sea waters.

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.