Molecular and morphological studies on Contracaecum rudolphii A and C. rudolphii B in great cormorants (Phalacrocorax carbo sinensis) from Italy and Israel.

The distribution of parasites is shaped by a variety of factors, among which are the migratory movements of their hosts. Israel has a unique position to migratory routes of several bird species leaving Europe to winter in Africa, however, detailed studies on the parasite fauna of birds from this area are scarce. Our study investigates occurrence and distribution of sibling species among Contracaecum rudolphii complex in Phalacrocorax carbo sinensis from Italy and Israel, to acquire further information on the geographical range of these species to gain deeper knowledge on the ecology of these parasites and their bird host. A total of 2383 Contracaecum were collected from the gastric mucosa of 28 great cormorants (18 from Israel and 10 from Italy). A subsample was processed for morphological analyses in light and scanning electron microscopy (SEM), and for molecular analyses through amplification and sequencing of the ITS rDNA and the cox2 mtDNA, and through PCR-RFLP. All the 683 Contracaecum subjected to molecular identification belonged to C. rudolphii s.l., (300 C. rudolphii A and 383 C. rudolphii B). SEM micrographs provided, for the first time, details of taxonomic structures in male specimens from both sibling species, and the first SEM characterization of C. rudolphii B. This work presents the first data on the occurrence of sibling species of C. rudolphii in Israel and provides additional information on the distribution of C. rudolphii A and B in Italy, confirming the high prevalence and intensity of infection observed in Ph. carbo sinensis from other Italian areas.

Advancing understanding of the taxonomy and diversity of the genus Contracaecum in the great white pelican (Pelecanus onocrotalus).

Despite the wide distribution and health importance of anisakids of the genus Contracaecum, epidemiological data on their occurrence in definitive bird hosts are scarce, particularly from certain parts of the world that represent important wintering sites or migration stopovers for different bird species. In the present study, Contracaecum spp. infecting six great white pelicans (Pelecanus onocrotalus) in Israel were identified using light and scanning electron microscopy and phylogenetic analyses of nuclear internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase II (cox2). A PCR-RFLP method was also developed and applied to screen large numbers of Contracaecum parasites. Most (415/455) worms recovered were C. micropapillatum, followed by C. gibsoni (31/455), C. quadripapillatum (8/455), and C. multipapillatum E (1/455). Contracaecum micropapillatum from Israel and C. bancrofti from Australia are distinguishable by cox2 but less well resolved with ITS sequences, and could not be distinguished morphologically. Worms with cox2 matching C. gibsoni had ITS matching specimens identified as C. multipapillatum A. To the authors' knowledge, this represents the first of such studies in Israel and provides useful data on the ecology and distribution of different Contracaecum species of health and economic interest.

Histological assessment of systemic granulomatosis progression in meagre Argyrosomus regius during cage ongrowing phase.

Meagre Argyrosomus regius is a potential candidate for Mediterranean aquaculture diversification, although several health issues still represent important bottlenecks for its sustainable production, including systemic granulomatosis (SG). To evaluate the SG progression in meagre during a 10 mo period of cage ongrowing, a histopathological investigation was carried out on 108 meagre fed 3 different diets (commercial pellets, hydrated commercial pellets and defrosted sardines). Histological sections of the gills and visceral organs were examined and lesions referable to SG scored from 1-3 according to the severity of the granulomatosis. The kidney and liver were the most affected organs, showing the highest percentage of positivity for granulomas and severity of lesions along the whole observation period. Using a statistical mixed model (GLMM) followed by odds ratio analysis, an effect of diet and temperature was found: the severity of liver and digestive tract SG scores decreased in the Cage 3 group (defrosted sardines) and with increasing temperature (p < 0.05, negative estimates, odds ratio <1). These observations, in accordance with the literature, suggest that SG in meagre could be related to nutritional-metabolic factors with the possible influence of environmental factors such as temperature.

Negligible risk of zoonotic anisakid nematodes in farmed fish from European mariculture, 2016 to 2018.

BackgroundThe increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment.AimTo evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon.MethodsFrom 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae.ResultsNo zoonotic parasites were found in any of the fish investigated.ConclusionsThe risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation.

In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains.

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment-friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon™ S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10-fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.

Mortality outbreak by perch rhabdovirus in European perch (Perca fluviatilis) farmed in Italy: Clinical presentation and phylogenetic analysis.

This work reports a mortality outbreak, occurred in 2015 and affecting juveniles of European perch (Perca fluviatilis L.) farmed in Italy. Perch rhabdovirus (PRV) was detected by viral isolation and biomolecular investigations. Phylogenetic analysis clustered our isolate into genogroup B, which also includes PRV isolates from Perca fluviatilis identified in France (2004-2009); diagnostic investigations also revealed opportunistic bacteria (Aeromonas hydrophila) and parasites (Chilodonella piscicola). Since, occasionally, PRV has been reported in the natural environment, which is often a source of eggs and broodstock for farms, it could be possible that both similar France and Italian isolate were imported from a same place elsewhere and have a common origin. Improving biosecurity measures (batch control) and disinfection of egg strings with an iodine-based solution helps prevent apparent vertical transmission of PRV.

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.

Assessment of Detoxifying Markers for Florfenicol in Rainbow Trout Liver.

Florfenicol (FF) is employed in fish farms to contest or prevent bacterial infections. However, this pharmaceutical may produce reactive oxygen species that may cause biochemical changes in antibiotic-treated fish. The aim of this study was to evaluate the effect of FF on Rainbow Trout Oncorhynchus mykiss treated for 10 d with 7.5 and 15 mg/kg FF followed by a withdrawal period of 5 d. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyoxalase I and glyoxalase II, total glutathione, lactic dehydrogenase, and alkaline phosphatase were investigated in the livers of treated and untreated fish. A general impairment of antioxidant enzymes and metabolic indicators was measured in FF-treated Rainbow Trout. Onset of oxidative damage may have occurred during the antibiotic treatment as a consequence of the effect of FF toxicity at mainly the highest dose. Nevertheless, the rise in levels of total glutathione and glutathione S-transferase even after the withdrawal period may shield the antibiotic-mediated oxidative processes. Received December 22, 2015; accepted May 26, 2016.

FIRST ISOLATION OF TENACIBACULUM MARITIMUM IN A CAPTIVE SAND TIGER SHARK (CARCHARIAS TAURUS).

This report describes a case of the first isolation of Tenacibaculum maritimum from a captive-bred adult female sand tiger shark (Carcharias taurus) housed at the Cattolica Aquarium (Italy). The animal showed, between the second dorsal fin and the precaudal pit, skin lesions characterized by the presence of abundant whitish necrotic tissue. Through routine bacteriological examination, a bacterium was isolated from a skin lesion and subsequently identified as T. maritimum by phenotypic characters and species-specific polymerase chain reaction. The antimicrobial sensitivity of the isolated strain was evaluated for 11 antimicrobial agents by disk diffusion method. Antibiotic therapy was conducted with enrofloxacin at 10 mg kg(-1) i.m. on alternate days for 10 days. One month after the end of treatment skin lesions showed complete resolution and the shark recovered completely. The case presented here represents the first report of infection by T. maritimum in a sand tiger shark and highlights the potential pathogenic role of this microorganism in elasmobranchs kept in an aquarium.

Disseminated mycobacteriosis in a stranded loggerhead sea turtle (Caretta caretta).

A loggerhead sea turtle (Caretta caretta) was found stranded alive along the Adriatic coast close to Ancona, Italy, displaying obtundation, tachypnea, and increased respiratory effort. It died a few hours after admission, and a postmortem examination was immediately performed. Miliary yellowish nodules were evident in the liver, and a lower number in the heart, stomach, and gut wall. Hundreds of whitish nodules were scattered in the lungs, with the majority of the pulmonary parenchyma being replaced by the lesions. Histologically, all nodular lesions consisted of a small central area of necrosis with acid-fast bacilli surrounded by epithelioid cells, macrophages, and lymphocytes. Giant cells were found in the spleen and the liver. Kidneys, lungs, liver, spleen, brain, and skin lesions were inoculated aseptically onto general isolation media and selective isolation media for mycobacteria. The isolate showed a restriction pattern identical to Mycobacterium chelonae by polymerase chain reaction-restriction fragment length polymorphism. To the best of the authors' knowledge, this is the first description of a disseminated infection caused by a potentially pathogenic mycobacteria in a stranded, free-ranging loggerhead sea turtle. Veterinary staff and biologists who handle sea turtles with suspected mycobacterial disease should protect themselves appropriately.

Morphological and molecular data on acuariid nematodes in European great cormorants (Phalacrocorax carbo sinensis) and pygmy cormorants (Microcarbo pygmaeus).

The family Acuariidae is a speciose group of parasitic nematodes, infecting mostly birds as definitive hosts. This study focused on the characterization of two species of acuariids, collected in two different species of piscivorous birds, the European great cormorant Phalacrocorax carbo sinensis from Italy, and the pygmy cormorant Microcarbo pygmaeus from Israel. Parasites were analyzed using light and scanning electron microscopy and by amplification and sequencing of the 28S rDNA. The results of morphological and molecular analyses showed that Ph. carbo sinensis was infected by the acuariid Syncuaria squamata (12 females) and Cosmocephalus obvelatus (1 female), whereas M. pygmaeus was infected by C. obvelatus (2 males, 12 females). The present results provide new data on the distribution of acuariid parasites of piscivorous birds, the first report of Acuariidae in Israel, and the first molecular data on S. squamata and C. obvelatus, which will be useful in future epidemiological and phylogenetic studies of these widely distributed, but less molecularly studied parasites.

Sphaerospora sensu stricto: taxonomy, diversity and evolution of a unique lineage of myxosporeans (Myxozoa).

Myxosporeans (Myxozoa) are eukaryotic parasites, primarily of fish, whose classification is in a state of flux as taxonomists attempt to synthesize the traditional morphology-based system with emerging DNA sequence-based phylogenies. The genus Sphaerospora Thélohan, 1892, which includes pathogenic species that cause significant impacts on fisheries and aquaculture, is one of the most polyphyletic taxa and exemplifies the current challenges facing myxozoan taxonomists. The type species, S. elegans, clusters within the Sphaerospora sensu stricto clade, members of which share similar tissue tropism and long insertions in their variable rRNA gene regions. However, other morphologically similar sphaerosporids lie in different branches of myxozoan phylogenetic trees. Herein, we significantly extend taxonomic sampling of sphaerosporids with SSU+LSU rDNA and EF-2 sequence data for 12 taxa including three representatives of the morphologically similar genus Polysporoplasma Sitjà-Bobadilla et Álvarez-Pellitero, 1995. These taxa were sampled from different vertebrate host groups, biogeographic realms and environments. Our phylogenetic analyses and statistical tests of single and concatenated datasets revealed Sphaerospora s. s. as a strongly supported monophyletic lineage, that clustered sister to the whole myxosporean clade (freshwater+marine lineages). Generally, Sphaerospora s. s. rDNA sequences (up to 3.7 kb) are the longest of all myxozoans and indeed metazoans. The sphaerosporid clade has two lineages, which have specific morphological, biological and sequence traits. Lineage A taxa (marine Sphaerospora spp.) have a single binucleate sporoplasm and shorter AT-rich rDNA inserts. Lineage B taxa (freshwater/brackish Sphaerospora spp.+marine/brackish Polysporoplasma spp.) have 2-12 uninucleate sporoplasms and longer GC-rich rDNA inserts. Lineage B has four subclades that correlate with host group and habitat; all Polysporoplasma species, including the type species, cluster together in one of these subclades. We thus suppress the genus Polysporoplasma and the family Polysporoplasmidae and emend the generic diagnosis of the genus Sphaerospora. The combination of morphological, biological and DNA sequence data applied in this study helped to elucidate an important part of the taxonomic puzzle within the phylum Myxozoa.

Parasitic infection in the scyphozoan Rhizostoma pulmo (Macri, 1778).

Very little information is reported for parasites of cnidarians, therefore, the present work aimed to investigate parasitic infections in one of the most widespread jellyfish in the Mediterranean Sea, Rhizostoma pulmo. The goals were to determine prevalence and intensity of parasites in R. pulmo, identify the species involved through morphological and molecular analysis, test whether infection parameters differ in different body parts and in relation to jellyfish size. 58 individuals were collected, 100% of them infected with digenean metacercariae. Intensity varied between 18.7 ± 6.7 per individual in 0-2 cm diameter jellyfish up to 505 ± 50.6 in 14 cm ones. Morphological and molecular analyses suggest that the metacercariae belonged to the family Lepocreadiidae and could be possibly assigned to the genus Clavogalea. Prevalence values of 100% suggest that R. pulmo is an important intermediate host in the life cycle of lepocreadiids in the region. Our findings also support the hypothesis that R. pulmo is an important part in the diet of teleost fish, which are reported as definitive hosts of lepocreadiids, since trophic transmission is necessary for these parasites to complete their life cycles. Parasitological data may therefore be useful to investigate fish-jellyfish predation, integrating traditional methods such as gut contents analysis.

Low prevalence of Contracaecum third-stage larvae parasitizing Sea of Galilee fisheries: 1-year survey after 57 years of no information.

Freshwater and marine ecosystems are a suitable habitat for parasitic nematodes of the genus Contracaecum (family: Anisakidae) to complete their complex life cycle. Several fish species of the Sea of Galilee (Lake Kinneret) were reported in 1964 as second intermediate/paratenic hosts of Contracaecum spp. larvae. The lack of taxonomically relevant morphological features of these larvae hindered their proper identification. Here we report the results of a 1-year survey conducted in 2021, 57 years after the first (and only) such survey. We analyzed 352 specimens from 10 fish species (native and non-native) of the Sea of Galilee (Israel) ichthyofauna. We compared our results with those of the first parasitological survey conducted by Paperna in 1964; the overall prevalence of nematodes referable to Contracaecum larvae was 16.8% and 0.85% in 1964 and in 2021, respectively. Different from the first survey that identified Contracaecum larvae morphologically, we used both morphological and molecular tools. Two wild native cyprinids-Jordan himri (Carasobarbus canis) and Jordan barbel (Luciobarbus longiceps)-were infected (a single specimen each) with Contracaecum quadripapillatum larvae in their abdominal cavity. A single specimen of blue tilapia (Oreochromis aureus) was infected with two larvae of Contracaecum multipapillatum E, localized in the pericardial cavity. The findings of our study, which is part of a large project focused on Contracaecum spp. infecting both piscivorous birds and fish collected in Israel, advance our knowledge about the distribution and host range of this potentially zoonotic parasite in fishery products of the Sea of Galilee.

The first new species of European Ascocotyle Looss, 1899 (Digenea: Heterophyidae) described in more than half a century.

Ascocotyle (Phagicola) trentinii n. sp. is described based on adults from experimentally infected ducklings (Anas platyrhynchos domesticus) fed with metacercariae from the visceral serosa of the Mediterranean banded killifish, Aphanius fasciatus (Cyprinodontiformes: Aphaniidae), from coastal lagoons in northeastern Italy (Emilia-Romagna Region). The new species is placed into the subgenus Phagicola because of the presence of a single row of circumoral spines, vitelline follicles being confined between the ventral sucker and testes, and uterine loops not reaching anterior to the ventral sucker. Ascocotyle (P.) trentinii n. sp. differs from other members of the subgenus Phagicola, as well as other species of Ascocotyle, by the number (27-33) of circumoral spines which are 13.5-17 µm long and 3.5-5 µm wide, and by the morphology of a gonotyl which is composed of about 8 large refractile pockets. The occurrence of metacercariae in A. fasciatus indicates that the life cycle of the new species is completed in brackish water lagoons. It is the fourth species of Ascocotyle described in Europe and may be endemic to the Mediterranean region because its second (fish) intermediate host is endemic to this region.

Morphological description and molecular characterization of Contracaecum larvae (Nematoda: Anisakidae) parasitizing market-size hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and red drum (Sciaenops ocellatus) farmed in Israel.

Nematodes belonging to the genus Contracaecum (family: Anisakidae) are heteroxenous parasites with a complex life cycle. Contracaecum larvae infecting farmed fish and fishery products are economically important causing market rejection in massive infection and may have zoonotic potential. In Israel, Contracaecum larvae have been described morphologically in several fish species; however, none of these descriptions were supported by molecular tools. In 2019-2020, hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and red drum (Sciaenops ocellatus), farmed in polyculture were found to be heavily infected with nematodes referable to Contracaecum larvae. Prevalence of infection in hybrid tilapia and red drum was 53.8% and 40.9%, respectively. A combined (morphological and molecular) approach revealed that both infected fish species were parasitized by the same species of Contracaecum, although larvae in hybrid tilapia were localized in the pericardial cavity whereas in red drum, they were observed in the abdominal cavity. Genetic analysis of internal transcribed spacer rDNA and cox2 mtDNA showed high similarity to unidentified Contracaecum larvae detected in several fish species in Ethiopia, Egypt and Kenya. In this study, molecular and morphological analyses place the possible new species in the C. multipapillatum complex and was provisionally named C. multipapillatum E. Further analyses combining morphological and molecular approaches are required on adult specimens collected from piscivorous birds living in the same area to support the identification of a potentially new species.

Occurrence of Neobenedenia girellae (Monogenea: Capsalidae) in Gilthead Seabream Sparus aurata (Actinopterygii: Sparidae) Cultured in Portugal.

Monogenean capsalids of the genus Neobenedenia are widespread parasites of wild and farmed marine fish, and represent a potential threat to mariculture due to their pathogenicity and ability to cause mortality in fish maintained in controlled conditions. The identification of Neobenedenia species and, consequently, the definition of their host specificity is often problematic due to their highly conserved morphology; therefore, in order to establish their specific identity, microscopic observation should be complemented with molecular analysis. The present work aims at characterizing Neobenedenia specimens infecting the skin of cage reared gilthead seabream Sparus aurata from Portugal. Parasite samples obtained from caged fish were processed for morphological analysis, through observation in light and scanning electron microscopy, and for molecular analysis, through amplification and sequencing of 28S rDNA and cytB, aimed at identifying them to the species level. Our results showed that the collected parasites belonged to the species Neobenedenia girellae; the susceptibility of S. aurata towards this pathogenic capsalid monogenean highlighted in the present work represents an important risk in the farming of this valuable fish species.