Dirofilaria repens Testicular Infection in Child, Italy.

Testicular Dirofilaria repens infection was identified and confirmed by sequence analysis in a child in northeastern Italy. Because human dirofilariasis is emerging in southern and eastern Europe, this parasitic infection should be considered in the differential diagnosis of scrotal swelling in disease-endemic countries to avoid unnecessary interventions, such as orchiectomy.

A new species of Clinostomum Leidy, 1856 based on molecular and morphological analysis of metacercariae from African siluriform fishes.

In the Afrotropic region, the genus Clinostomum is represented by four accepted and four unnamed species distinguished using molecular data. Here, we describe one of the four unnamed species as Clinostomum ukolii n. sp. based on metacercariae from siluriform fishes (Synodontis batensoda, Schilbe intermedius) collected in Nigeria and South Africa. The new species is distinguished by molecular data (39 new sequences of partial cytochrome c oxidase I ≥ 6.7% divergent from those of other species) and morphological differences from named and unnamed species in the same region. Metacercariae of C. ukolii n. sp. can be distinguished based on size, tegumental spines, and various aspects of the genital complex, including its position, lobation of the anterior testis, and the disposition and shape of the cirrus pouch. Although descriptions of new species of digeneans are typically based on the morphology of adults, we argue that in cases where data are available from metacercariae from regionally known species, new species can be described based on metacercariae, particularly when supported by molecular data, as here. Moreover, sub-adult reproductive structures can be clearly visualized in metacercaria of Clinostomum. Considering metacercariae as potential types for new species could advance clinostome systematics more rapidly, because metacercariae are encountered much more often than adults in avian definitive hosts.

Molecular data show Clinostomoides Dollfus, 1950 is a junior synonym of Clinostomum Leidy, 1856, with redescription of metacercariae of Clinostomum brieni n. comb.

The genus Clinostomoides Dollfus, 1950 was erected to accommodate a single worm from Ardea goliath sampled in the Belgian Congo. The specimen was distinguished from other clinostomids by its large size and posterior genitalia. In the following years, metacercariae of Clinostomoides brieni, have been described in Clarias spp. in southern and western Africa. A few authors have referred to Clinostomum brieni, but all such usages appear to be lapsus calami, and the validity of Clinostomoides remains widely accepted. In this study our aim was: position C. brieni among the growing clinostomids molecular database, and redescribe the species with emphasis on characters that have emerged as important in recent work. We sequenced two nuclear (partial 18S and ITS) and one mitochondrial marker (partial cytochrome c oxidase I) and studied morphology in metacercariae from hosts and localities likely to harbour the type species (Clarias spp., Democratic Republic of the Congo, South Africa). Phylogenetic analysis shows C. brieni belongs within Clinostomum Leidy, 1856. We therefore transfer C. brieni to Clinostomum, amend the diagnosis for the genus Clinostomum and provide a critical analysis of other species in Clinostomoides, all of which we consider species inquirendae, as they rest on comparisons of different developmental stages.

A new species of Clinostomum Leidy, 1856 in East Asia based on genomic and morphological data.

Metacercariae of Clinostomum Leidy, 1856 are frequently encountered in freshwater fish. In 2015, a provisional species of Clinostomum in People's Republic of China (PRC) was distinguished from C. complanatum (Rudolphi, 1819) in Europe based on divergent cytochrome c oxidase I (CO1). However, in subsequent studies in East Asia, the same divergent CO1 genotype was identified as C. complanatum. These matching sequences suggest that either the provisional East Asian species was incorrectly distinguished from C. complanatum in 2015 or that C. complanatum in East Asia was misidentified in later studies. We tested these alternatives by sequencing the mitochondrial genome of C. complanatum in Italy, which was 5.7% divergent from a previously published sequence from Clinostomum in PRC, including differences in 80 of 3390 (2.4%) translated amino acids. Partial CO1 sequences of specimens from PRC and those from Italy, Romania, and Turkey also each formed reciprocally monophyletic clades. Partial CO1 from the East Asian clade varied by mean 3.6% (range 2.4-4.8%) from C. complanatum from Italy, Romania, and Turkey; mean intra-clade CO1 variation was 0.3% (range 0-1.9%). Metacercariae from Europe and East Asia display significant morphometric variation, and data from the literature suggest morphological differences in the genital complex of adults. Although sequences of nuclear rDNA did not differ between isolates from the west and East Asia, taken together, these results lead us to describe a new species of Clinostomum.

A morphological and molecular study of Clinostomid metacercariae from African fish with a redescription of Clinostomum tilapiae.

The genus Clinostomum Leidy, 1856 (Digenea: Clinostomidae) has been reported in all ecozones of the world and a clear separation between the species of the 'Old World' and 'New World' has been recognized based on molecular studies. Recent works on Afrotropical species include redescriptions of C. cutaneum and C. phalacrocoracis, while C. tilapiae has yet to be studied using modern taxonomic approaches. In the present research, morphological redescription of C. tilapiae metacercariae from a new host, Synodontis batensoda sampled at Anambra River Basin, Nigeria, together with molecular analysis of nuclear internal transcribed spacer rDNA and cytochrome c oxidase 1 mtDNA are reported. We also provide morphological and molecular data from four further putative species of Clinostomum (morphotypes 1-4) from different areas of Africa, as well as the first report of C. phalacrocoracis in South Africa.

First description of the adult stage of Clinostomum cutaneum Paperna, 1964 (Digenea: Clinostomidae) from grey herons Ardea cinerea L. and a redescription of the metacercaria from the Nile tilapia Oreochromis niloticus niloticus (L.) in Kenya.

The combined use of morphological and molecular studies allowed for the first time the recognition and description of the adult stage of Clinostomum cutaneum Paperna, 1964 from the grey heron Ardea cinerea L. in Kenya. A redescription of the metacercaria that infect Nile tilapia Oreochromis niloticus niloticus (L.) from the same aquatic environment is also presented. C. cutaneum differs from all other species of Clinostomum Leidy, 1856 in the shape of its uterus. Sequencing the rRNA confirmed the morphological similarity between adults from the grey heron and the metacercarial stage from tilapia, and a level of genetic similarity with the other previously sequenced Clinostomum spp. was observed. The need for a reorganisation of Clinostomum using both morphological and molecular methods is highlighted.

Multiplex PCR for simultaneous identification of the most common European Opisthorchiid and Heterophyid in fish or fish products.

Among others, the families Opisthorchiidae and Heterophyidae includes several genera causing fish-borne zoonoses and distributed also in European Countries and that are included in the ParaFishControl (Advanced Tools and Research Strategies for Parasite Control in European farmed fish) H2020 EU project. Due to the small size of the metacercariae, the infective stage for human, these parasites cannot be detected visually in fish and monitoring requires expert application of time-consuming techniques. The aim of this was to develop a rapid and affordable molecular method based on multiplex PCR for simultaneous identification of metacercariae of the most common European Opisthorchiid and Heterophyid in fish or fish products.

Gyrodactylus orecchiae sp. n. (Monogenea: Gyrodactylidae) from farmed populations of gilthead seabream (Sparus aurata) in the Adriatic Sea.

Gyrodactylus orecchiae sp. n. (Monogenea, Gyrodactylidae) is described from the skin, fins, eyes and gills of juvenile Sparus aurata L. (gilthead seabream) following two outbreaks of gyrodactylosis amongst stocks held in inshore floating cages on the Adriatic coast of Albania and Croatia. Fish were heavily infected (1000+ gyrodactylids/fish) with G. orecchiae which reportedly resulted in approximately 2-10% mortality amongst the infected stock. Morphologically, the haptoral hooks of G. orecchiae most closely resemble those of Gyrodactylus arcuatus Bychowsky, 1933 in the approximate shape of the ventral bar with its pronounced ventral bar processes and marginal hook sickles which possess a square line to the inner edge of the sickle blade and large rounded heels. The marginal hooks are also morphologically similar to those of Gyrodactylus quadratidigitus Longshaw, Pursglove et Shinn, 2003 and Gyrodactylus colemanensis Mizelle et Kritsky, 1967, but G. orecchiae can be readily discriminated from all three species by the characteristic infolding of the hamuli roots and the shape of the marginal hook sickle. Molecular sequencing of the ITS1, 5.8S, ITS2 regions (513+157+404 bp, respectively) of G. orecchiae and alignment with other gyrodactylids for which these same genomic regions have been determined, suggests that this is a new species. No similarities were found when the ITS1 region of G. orecchiae was compared with 84 species of Gyrodactylus available on GenBank.

Morphological and Molecular Characterization of Larval and Adult Stages of Eustrongylides excisus (Nematoda: Dioctophymatoidea) with Histopathological Observations.

The genus Eustrongylides includes nematodes known as the etiological agent of the "big red worm disease." The aim of this work was to identify Eustrongylides spp. larvae from fish and adults from great cormorant (Phalacrocorax carbo) sampled at Lake Trasimeno, Italy, by morphological and molecular analysis. Histopathological description of the lesions in birds was also provided. We described adults of Eustrongylides excisus for the first time in Italy, and we also linked larval stages 3 and 4 to adults. The use of molecular tools combined with the traditional taxonomy will help the identification of the species, including species inquirendae. Moreover, molecular analysis can also help to investigate the role of intermediate and paratenic hosts, to deepen the knowledge about geographical distribution of the different Eustrongylides spp. and to define the zoonotic potential of E. excisus, which has not yet been identified as causal agent of human cases.

Hysterothylacium fabri (Nematoda: Raphidascarididae) in Mullus surmuletus (Perciformes: Mullidae) and Uranoscopus scaber (Perciformes: Uranoscopidae) from the Mediterranean.

Raphidascarididae are among the most abundant and widespread parasitic nematodes in the marine environment. The life-cycle of most raphidascaridid species is poorly known and information about their distribution and host range is lacking in many geographical areas, as is the taxonomy of several species. A study of larval and adult stages of Hysterothylacium fabri (Rudolphi, 1819) Deardorff and Overstreet, 1980 (Nematoda: Raphidascarididae) infecting the striped goatfish Mullus surmuletus Linnaeus, 1758 (Mullidae) and the Mediterranean stargazer Uranoscopus scaber Linnaeus, 1759 (Uranoscopidae) from the Ionian Sea (central Mediterranean) has been carried out by combining light and scanning electron microscopy observations and molecular analyses through polymerase chain reaction-restriction fragment length polymorphism and sequencing of the ITS rDNA gene. Results indicate that U. scaber and M. surmuletus represent suitable definitive and intermediate/paratenic hosts of H. fabri, respectively, in the Mediterranean and highlight the importance of combining genetic and morphological data to study the taxonomy and epidemiology of parasites widely distributed in different fish species and aquatic ecosystems.

Development and validation of species-specific molecular diagnostic tool for Opisthorchis felineus (Digenea, Opisthorchiidae) metacercariae.

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method.

A Combined Morphometric and Molecular Approach to Identifying Metacercariae of Euclinostomum heterostomum (Digenea: Clinostomidae).

Metacercariae of species of Euclinostomum have been found encysted in kidney, liver, and muscles of several fish species, while adults occur in the mouth, pharynx, larynx, trachea, and upper esophagus of fish-eating birds. The aim of this work was to gather molecular and morphological data from the type species, Euclinostomum heterostomum, as a starting point for needed revisions of the genus. Metacercariae were collected from cichlids in Lake Kinneret, Israel, and all were identified as E. heterostomum based on morphology. This identification was further confirmed by principal components analyses that revealed no partitions in morphometric resemblance, indicating that the material represents a single species, and that the specimens were morphometrically similar to other descriptions of E. heterostomum. In phylogenetic analysis of concatenated partial internal transcribed spacer 1 and 2, 5.8S rDNA, and partial cytochrome c oxidase I barcode sequences, the isolates of Euclinostomum obtained in this study form a single, monophyletic group separate from recently published data from Euclinostomum sp. from Thailand and from Clinostomum species, which are also monophyletic. The morphological and molecular data reported in the present work can provide a useful point of reference for future studies.

Redescription of Clinostomum phalacrocoracis metacercariae (Digenea: Clinostomidae) in cichlids from Lake Kinneret, Israel.

Clinostomidae are digeneans characterized by a complex taxonomic history, continuously under revision based on both morphological and molecular analysis. Among the 14 species considered valid so far Clinostomum phalacrocoracis has been well described only at the adult stage, whereas the morphology of the metacercarial stage has been reported only once. During a parasitological survey carried out on 262 wild cichlids sampled from Lake Kinneret (Israel) metacercariae referable to C. phalacrocoracis were found in 18 fingerlings. In this study, we report this clinostomid species for the first time in wild fish from Israel describing the metacercarial stage of Clinostomum phalacrocoracis, coupling its morphological description with molecular analysis carried out on ITS rDNA and COI mtDNA sequences.

Morphological and molecular differentiation of Clinostomum complanatum and Clinostomum marginatum (Digenea: Clinostomidae) metacercariae and adults.

The separation of Clinostomum complanatum Rudolphi, 1814 and Clinostomum marginatum Rudolphi, 1819 has long been unclear. Recent data confirm the validity of the junior species, C. marginatum , by ∼ 1% differences in its 18S rDNA sequences. We collected adults and metacercariae of C. complanatum and C. marginatum and found reliable morphological differences in the genital complex at both developmental stages. In addition, we identified basic morphometrics (distance between suckers, body width) in metacercariae that may be useful for discriminating the species. The morphological differences were supported by the comparison of sequences of internal transcribed spacers of ribosomal DNA and of the mitochondrial gene cytochrome c oxidase I (COI) from 39 specimens. In 36 specimens, the average divergence between the species was 7.3% in ITS and 19.4% in COI sequences. Two specimens from North America and 1 from Europe had sequences that did not allow them to be clearly allied with either species.

The first report of Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes, Monogenea) on Italian cultured stocks of rainbow trout (Oncorhynchus mykiss Walbaum).

The monogenean Gyrodactylus salaris Malmberg, 1957 is considered one of the most important parasites of wild salmonids in the European Community due to the heavy ecological and economical damage it has inflicted on Atlantic salmon (Salmo salar) parr populations. Rainbow trout (Oncorhynchus mykiss) is susceptible to G. salaris and can act as a suitable carrier host and, consequently, its trade in EU territory is restricted in relation to the status of "recognized free" zones. Despite the economic importance of rainbow trout farming in Italy, information on the Italian gyrodactylid fauna is lacking and prior to this study, G. salaris had not been officially reported. During a routine health examination of farmed rainbow trout stock throughout Central and Northern Italy in 2004-2005, five fish farms were found to be infected with G. salaris alongside three other gyrodactylids. Morphological and molecular characterisation confirmed the presence of G. salaris, Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999 and Gyrodactylus derjavinoides Malmberg, Collins, Cunningham et Jalali, 2007, while Gyrodactylus truttae Gläser, 1974 was identified by morphological analysis only. The findings from this study extend the distribution of G. salaris within Europe and highlight the importance of the rainbow trout trade in its dissemination.