Detection of Acipenser European Iridovirus (AcIV-E) in Sturgeon Farms in Northern Italy between 2021-2023.

Sturgeon farming is rapidly expanding in Europe, where Italy ranks first in farmed caviar production. A major threat to sturgeon health in captivity is infection with Acipenser European Iridovirus (AcIV-E), a viral disease definitively identified in 2016. Here we present data on the occurrence of AcIV-E in 482 sturgeons (age ≤ 12 months, species of the genus Acipenser and the species Huso huso) collected from sturgeon farms in northern Italy between January 2021 and December 2023. The health status of each specimen was determined by necroscopy and virological assay. Virological analysis was performed on gill samples and real-time PCR specific to the MCP gene of the iridovirus viral capsid. Molecular analysis revealed positivity to the virus in 204 samples (42.68% of the total), while anatomopathological examination of nearly all fish with positive real-time PCR disclosed swollen abdomen, hepatic steatosis, splenomegaly, and increased gill volume. Two challenges to timely diagnosis are the absence of pathognomonic symptoms and the inability to isolate the virus on cell monolayers. Continuous and widespread health monitoring is therefore crucial for disease management and to effectively control spread of the virus.

The Gut Microbiota of Farmed and Wild Brook Trout (Salvelinus fontinalis): Evaluation of Feed-Related Differences Using 16S rRNA Gene Metabarcoding.

The gut microbiota has become a topic of increasing importance in various fields, including aquaculture. Several fish species have been the subject of investigations concerning the intestinal microbiota, which have compared different variables, including the intestinal portions, the environment, and diet. In this study, the microbiota of farmed and wild brook trout (Salvelinus fontinalis) were analyzed, in which the wall and content of the medial portion of the intestine were considered separately. A total of 66 fish (age class 2+) were sampled, of which 46 were wild and 20 were farmed brook trout, in two different years. Microbiota data were obtained using a 16S metabarcoding approach by analyzing the V3-V4 hypervariable regions of the corresponding 16S rRNA. The data showed that the core microbiota of these species consist of Proteobacteria (Alpha- and Gammaproteobacteria), Actinobacteria, Firmicutes (Bacilli and Clostridia), and, only for farmed animals, Fusobacteria. The latter taxon's presence is likely related to the fishmeal-based diet administered to farmed brook trout. Indeed, alpha and beta diversity analysis showed differences between wild and farmed fish. Finally, statistically significant differences in the microbiota composition were observed between the intestinal walls and contents of wild fish, while no differences were detected in reared animals. Our work represents the first study on the intestinal microbiota of brook trout with respect to both farmed and wild specimens. Future studies might focus on the comparison of our data with those pertaining to other fish species and on the study of other portions of the brook trout intestine.

Isolation and Genetic Characterization of Streptococcus iniae Virulence Factors in Adriatic Sturgeon (Acipenser naccarii).

The first case of infection of Streptococcus iniae in Adriatic sturgeon (Acipenser naccarii) was recently reported in a raceway system located in Northern Italy. A second episode of infection in sturgeons with absence of mortality and evident clinical signs, was registered in November 2020 in the same farm and is reported in this study. Histopathological changes observed in infected organs are described. The strains isolated in the two episodes were compared using molecular analysis based on PCR, phylogeny and virulence factors analysis. Not all the major virulence factors were detected for the two strains; in particular the strains 78697, isolated in November, lacks cpsD, compared to the strains 64844, isolated in September. Moreover, genetic variations were reported for lctO and pmg genes. These findings let us hypothesize a different virulence of the strains in accordance with clinical findings related to the sturgeons.

Surveys on Exposure to Reptile-Associated Salmonellosis (RAS) in the Piedmont Region-Italy.

Reptile-associated salmonellosis (RAS), Salmonella infection in humans, is acquired through contact with reptiles. Reptiles have become popular pet animals, and RAS is likely to be an underestimated but growing problem. No epidemiological data about RAS are routinely collected in Italy. In order to estimate the occurrence of RAS in the Italian human population and to investigate the exposure, two epidemiological studies on patients with sporadic salmonellosis were carried out in the Piedmont region, along with an evaluation of human exposure in public places displaying reptiles and with a survey on people awareness. RAS appeared make up 7% of sporadic salmonellosis in the first study and 3% in the second, more extensive study. A prevalence of 11.7% and 5.7%, respectively, were calculated for the age range of 0-21 years. It was observed that in public places displaying reptiles, it was possible to easily come into contact with the animals and their environment. Some knowledge about RAS emerged from the interviews with the general population, but preventive measures are not completely applied by reptile owners. In conclusion, RAS in Italy is present and constitutes a proportion of the human salmonellosis cases in line with the percentages reported in other countries. Exposure to reptiles should always be considered as a risk factor, and people should be more informed about RAS and the related preventive measures.

Teleost fish: a new spontaneous model for the study of Lambl's excrescences.

Lambl's excrescences (LE) are fibrous strands typically occurring at coaptation lines of cardiac valves. Although they have been widely reported in human medicine, information on LE occurrence in veterinary medicine is still scarce. The present study aimed to investigate the presence of LE in different freshwater fish and in swordfish. A total of 185 hearts were collected from different fish species, and samples of different cardiac areas (sinus venosus, atrial wall with sinoatrial valves, ventricular wall with atrioventricular valves and bulb with bulboventricular valves) were submitted to histopathological evaluation. LE were detected in 6 of 103 freshwater fish (5.8%) and 19 of 82 swordfish (23.2%). LE developed in atrioventricular, sinoatrial and bulboventricular valves. All affected valves also showed endocardiosis. Based on the results of the present work, teleost fish, specifically swordfish, could be proposed as a novel spontaneous model for the study of LE pathogenesis.

Relationship between the prevalence of Dibothriocephalus latus (Cestoda: Diphyllobothriidea) and the load of Escherichia coli: New findings in a neglected fish-borne parasitic zoonosis.

The sub-Alpine lakes of Switzerland, Italy and France have long been reported as an endemic area of diphyllobothriosis, a parasitic zoonosis caused by Dibothriocephalus latus. With this study, we explored the hypothesis for a relationship between the prevalence of D. latus in Perca fluviatilis and the Escherichia coli load in lake water. To do this, we identified eleven sampling sites in three areas (north, centre and south) of Lake Iseo (north Italy) to determine E. coli load and the prevalence of D. latus in P. fluviatilis. Prevalence and 95% confidence interval (CI) of D. latus infestation ranged from 0% (95% CI: 0.71-0.0) in Sarnico (southern area) to 20% (95% CI: 33.0-11.2) in Pisogne (northern area). There were significant differences in prevalence between the sites (χ2  = 31.12; p-value = .0006) and in E. coli load (Kruskal-Wallis test; p-value = .0005). There was decreasing gradient of E. coli load and prevalence of D. latus infestation from north to south. A significant positive correlation (r = .881; p-value = .003) was found between E. coli load and prevalence of D. latus. Also, linear regression showed a significant relationship between E. coli load and prevalence of infestation (R2  = .775). Our findings offer an explanation for the link between E. coli load in water and D. latus prevalence. The potential factors in this link are the efficiency of the local wastewater treatment plant, the bathymetric profile of the lake and the life cycle of D. latus, which is mainly affected by light and water temperature.

Investigation of Potential Reservoirs of Non-Tuberculous Mycobacteria in a European Sea Bass (Dicentrarchus labrax) Farm.

Fish mycobacteriosis is a widespread global problem caused by species of non-tuberculous mycobacteria (NTM). Mycobacterium marinum is one of the species most often involved in disease episodes of aquarium and farmed fish. Since there is currently no available effective therapy or vaccine, a prompt search for routes of entry is key to limiting the damage induced by the disease. Here we report a case of mycobacteriosis follow up in a European sea bass (Dicentrarchus labrax) farm located in Northern Italy, in which environmental samples and newly added fish batches were analyzed. Samples from fish present on the farm, sediment, and periphyton all resulted positive for M. marinum, whereas the new fish batches and the water samples resulted negative. The environmental resistance of NTM (alcohol-acid resistance, biofilm formation) and the lack of prophylactic and therapeutic strategies make these diseases difficult to manage. Prompt identification of biotic and abiotic reservoirs, combined with good zootechnical hygiene practices, are the most effective measures to control fish mycobacteriosis in intensive farms.

Unusual Localization of Pennella Sp. in Swordfish (Xiphias gladius) Hearts.

The genus Pennella comprises hematophagous parasites of marine aquatic species, including cephalopods, marine mammals, and pelagic fish. Nine species have been officially included in the genus Pennella plus another six species inquirendae. They are most often found in the host's musculature, without penetrating internal organs. For the present study, 83 hearts from swordfish (Xiphias gladius) caught in the Mediterranean Sea were sampled and immediately fixed in formalin for histopathological analysis. In total, 10 (12.05%) hearts were found to be parasitized by copepods of the genus Pennella. Macroscopically, there was mild-to-severe fibrinous pericarditis with atrial wall thickening and multiple parasitic nodules. Histologically, the parasitic nodules were surrounded by an inflammatory-necrotizing reaction. Parasitic infestation by Pennella spp. is common in pelagic fish and in swordfish, in particular. Here, however, we report atypical cardiac localization. A future area of focus is the evaluation of cardiac Pennella spp. infestation by histopathology and genetic identification of the parasites.

Draft Genome Sequence of Rhodococcus qingshengii Strain PN_19, Isolated from a Moribund Individual of Pinna nobilis in Sardinia, Italy.

During an epidemiological survey that aimed to discover the causes for the mass mortality of Pinna nobilis, a strain of Rhodococcus was found in a moribund individual. Here, we report its 7,037,134-bp draft genome sequence, which, after the annotation and genome survey, was identified as belonging to Rhodococcus qingshengii PN_19.

Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members.

The Mycobacterium fortuitum group (MFG) consists of about 15 species of fast-growing nontuberculous mycobacteria (NTM). These globally distributed microorganisms can cause diseases in humans and animals, especially fish. The increase in the number of species belonging to MFG and the diagnostic techniques panel do not allow to clarify their real clinical significance. In this study, biomolecular techniques were adopted for species determination of 130 isolates derived from fish initially identified through biochemical tests as NTM belonging to MFG. Specifically, gene sequencing and phylogenetic analysis were used based on a fragment of the gene encoding the 65 KDa heat shock protein (hsp65). The analyzes made it possible to confirm that all the isolates belong to MFG, allowing to identify the strains at species level. Phylogenetic analysis substantially confirmed what was obtained by gene sequencing, except for six strains; this is probably due to the sequences present in NCBI database. Although the methodology used cannot represent a univocal identification system, this study has allowed us to evaluate its effectiveness as regards the species of MFG. Future studies will be necessary to apply these methods with other gene fragments and to clarify the real pathogenic significance of the individual species of this group of microorganisms.

The unusual isolation of carnobacteria in eyes of healthy salmonids in high-mountain lakes.

Carnobacteria are common bacteria in cold and temperate environments; they are also reported during fish mortality events. In a previous study, carnobacteria were isolated from the eyes of healthy wild salmonids from a high-mountain lake. To better understand these findings, salmonids were captured from three high-mountain lakes (Lower and Upper Balma Lake, Rouen Lake; northwest Italy) during August 2019 and subjected to bacteriological and histological examination. Although all were healthy, 8.7% (Lower Balma Lake), 24% (Upper Balma Lake), and 32.6% (Rouen Lake) were positive for carnobacteria colonization of the eyes. A Trojan-horse effect was hypothesized to explain carnobacteria isolation in the eye. This immune-escaping macrophage-mediated mechanism has been identified in other Gram-positive bacteria. Biochemical, molecular, and phylogenetic analysis were carried out on isolated bacteria (Carnobacterium maltaromaticum and C. divergens). Based on previous references for carnobacteria isolated from fish, C. maltaromaticum strains were tested for the pisA precursor gene of the bacteriocin piscicolin 126. Carnobacterium maltaromaticum strains were found to display genotypic heterogeneity and a low percentage of pisA positive amplification. Features of geomorphology, geographic isolation, and microbiota common to the three lakes are thought to be possibly related to our findings. Moreover, terrestrial insects collected from the lake shoreline and the stomach contents were screened for the presence of carnobacteria. The salmonids in these high-mountain environments feed mainly on terrestrial insects, which are considered possible vectors for carnobacteria that might catabolize the exoskeleton chitin. All insects tested negative for carnobacteria, but as a small number of samples were analyzed, their role as possible vectors of infection cannot be excluded. Further studies are needed to corroborate our research hypothesis.

Reply to Pavlik et al. Clinical Relevance and Environmental Prevalence of Mycobacterium fortuitum Group Members. Comment on "Mugetti et al. Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members. Microorganisms 2021, 9, 797".

We appreciate the valuable comment of Pavlik et al. [...].

Multiple Non-Species-Specific Pathogens Possibly Triggered the Mass Mortality in Pinna nobilis.

The fan mussel, Pinna nobilis, represents the largest bivalve endemic to the Mediterranean Sea. Since 2016, dramatic mass mortality of this species has been observed in several areas. The first surveys suggested that Haplosporidium pinnae (currently considered species-specific) was the main etiological agent, but recent studies have indicated that a multifactorial disease may be responsible for this phenomenon. In this study, we performed molecular diagnostic analyses on P. nobilis, P. rudis, and bivalve heterologous host species from the island of Sardinia to shed further light on the pathogens involved in the mass mortality. The results support the occurrence of a multifactorial disease and that Mycobacterium spp. and H. pinnae are not necessarily associated with the illness. Indeed, our analyses revealed that H. pinnae is not species-specific for P. nobilis, as it was present in other bivalves at least three years before the mass mortality began, and species of Mycobacterium were also found in healthy individuals of P. nobilis and P. rudis. We also detected the species Rhodococcus erythropolis, representing the first report in fan mussels of a bacterium other than Mycobacterium spp. and Vibrio spp. These results depict a complicated scenario, further demonstrating how the P. nobilis mass mortality event is far from being fully understood.

Bioremediation with freshwater bivalves: A sustainable approach to reducing the environmental impact of inland trout farms.

Integrated multi trophic aquaculture (IMTA) is well developed in seawater, while the only suitable organisms for bioremediation in freshwater are bivalves. The aim of this research has been to investigate a novel system, based on freshwater bivalves integrated with rainbow trout, for the bioremediation of inland aquaculture systems. The Unionid species selected for the experiments was Sinanodonta woodiana. Five mesocosm experiments were conducted to evaluate the efficiency of mussels in filtering the total bacteria and Aeromonas hydrophila, as well as their clearance efficiency on trout farm wastewater. The temperatures, dissolved oxygen, nitrates, and phosphates were monitored and were all within the physiological tolerance range of the species in all the experiments. The feasibility of bioremediation with S. woodiana in trout farming has thus been demonstrated, and among the tested rearing densities (3.75; 7.5; 15; 30 and 60 kg m-3) that of 7.5 kg m-3 was found to be optimal. The net reduction of the total bacterial concentration was as much as 72%, while that of A. hydrophila reached a level of 95-98%. No relevant effects of the mussels on particulate suspended material or sedimented material was observed, regardless of the temperature. The efficiency of freshwater bivalves in reducing the bacterial load, in particular toward A. hydrophila, indicates a bioremediation system with the possibility of interesting applications on inland fish farms, and as a biotechnological tool against the diffusion of antibiotic resistance in aquaculture.

Mycobacterium pseudoshottsii in Mediterranean Fish Farms: New Trouble for European Aquaculture?

Mycobacterium pseudoshottsii, a slow-growing mycobacterium closely related to M. marinum, has been isolated only in wild fish in the United States and in Japanese fish farms to date. Here, we report cases of mortality in three farmed fish species (Dicentrarchus labrax, Sparus aurata, and Sciaenops ocellatus) caused by M. pseudoshottsii in Italy. Samples underwent necropsy, histology, and culture with pathogen identification based on PCR and sequencing of housekeeping genes (16S rRNA, hsp65, rpoB). Multifocal to coalescing granulomatous and necrotizing inflammation with acid-fast bacilli were observed in the parenchymatous organs, from which M. pseudoshottsii was isolated and identified. Phylogenetic analysis confirmed the results of gene sequencing and allowed subdivision of the isolates into three distinct groups. M. pseudoshottsii poses a potential threat for Mediterranean aquaculture. Its origin in the area under study needs to be clarified, as well as the threat to the farmed fish species.

Occurrence and Spatial Distribution of Dibothriocephalus Latus (Cestoda: Diphyllobothriidea) in Lake Iseo (Northern Italy): An Update.

Dibothriocephalus latus (Linnaeus, 1758) (Cestoda: Diphyllobothriidea; syn. Diphyllobothrium latum), is a fish-borne zoonotic parasite responsible for diphyllobothriasis in humans. Although D. latus has long been studied, many aspects of its epidemiology and distribution remain unknown. The aim of this study was to investigate the prevalence, mean intensity of infestation, and mean abundance of plerocercoid larvae of D. latus in European perch (Perca fluviatilis) and its spatial distribution in three commercial fishing areas in Lake Iseo (Northern Italy). A total of 598 specimens of P. fluviatilis were caught in 2019. The total prevalence of D. latus was 6.5%. However, there were significant differences between areas (10.2% North; 7.3% Center; 1.5% South) (Chi-square test, p = 0.0018). The mean intensity of infestation ranged from 1 larva in southern area to 1.2 larvae in both the central and northern (Pisogne) areas. In addition, the mean abundance ranged from 0.02 in the southern area to 0.26 in the northern area (Pisogne). The total number of larvae (anterior dorsal-AD = 21; anterior ventral-AV = 1; posterior dorsal-PD = 15; posterior ventral-PV = 5) differed significantly between the four anatomical quadrants (Kruskal-Wallis test; p = 0.0001). The prevalence of D. latus plerocercoid larvae in European perch from Lake Iseo has long been investigated, but without an appropriate sampling design. With the present study, a broader analysis in spatial distribution has been added to the existing literature, revealing new information about D. latus distribution and occurrence in Lake Iseo, with new data that will be useful for health authorities and future studies.

First Occurrence of Eustrongylides spp. (Nematoda: Dioctophymatidae) in a Subalpine Lake in Northwest Italy: New Data on Distribution and Host Range.

The genus Eustrongylides includes nematodes that infect fish species and fish-eating birds inhabiting freshwater ecosystems. Nematodes belonging to the genus Eustrongylides are potentially pathogenic for humans; infection occurs after the consumption of raw or undercooked fish. In the two-year period 2019-2020, a total of 292 fish belonging to eight species were examined for the occurrence of Eustrongylides spp. from Lake San Michele, a small subalpine lake in northwest Italy. The prevalence of infestation was 18.3% in Lepomis gibbosus, 16.7% in Micropterus salmoides, and 10% in Perca fluviatilis. The other five fish species (Ameiurus melas, Ictalurus punctatus, Squalius cephalus, Carassius carassius, and Scardinius erythrophthalmus) were all negative for parasite presence. There were no significant differences in prevalence between the three fish species (Fisher's exact test; p = 0.744). The mean intensity of infestation ranged from 1 (M. salmoides and P. fluviatilis) to 1.15 (L. gibbosus), and the mean abundance ranged from 0.1 (P. fluviatilis) to 0.28 (L. gibbosus). There were significant differences in the infestation site between the four muscle quadrants (anterior ventral, anterior dorsal, posterior ventral, and posterior dorsal) and the visceral cavity (Kruskal-Wallis test; p = 0.0008). The study findings advance our knowledge about the distribution and host range of this parasite in Italy.

The Old and the New on Viral Diseases in Sturgeon.

Although sturgeon production by aquaculture has increased worldwide, a major factor limiting its expansion are infectious diseases, although few data about viral diseases are available however. This review provides a rapid overview of viral agents detected and described to date. Following a general introduction on viral diseases are four sections arranged by virus classification: sturgeon nucleocytoplasmic large DNA viruses, herpesviruses, white sturgeon adenovirus 1, and other viruses. Molecular diagnosis is currently the best tool to detect viral diseases, since cell culture isolation is not yet applicable for the detection of most sturgeon viruses.

First Report of Clinostomum complanatum (Trematoda: Digenea) in European Perch (Perca fluviatilis) from an Italian Subalpine Lake: A Risk for Public Health?

Clinostomum complanatum, a digenean trematode of the Clinostomidae family, is a fish-borne zoonotic parasite responsible for Halzoun syndrome in humans and is transmitted through the consumption of raw or undercooked freshwater fish. Of the total of 112 specimens of European perch (Perca fluviatilis) sampled from a subalpine lake (Lake Endine) in North Italy in 2019, 21 (18.75%) tested positive for encysted metacercariae in the fillet. This study reports the first isolation of C. complanatum in P. fluviatilis and highlights the possible zoonotic risk for consumers, since P. fluviatilis is a food fish used in the traditional local cuisine.