Red mullet (Mullus barbatus) is a commercially relevant fish species, yet epidemiological data on anisakid nematode infestation in M. barbatus are scarce. To fill this gap, we report the occurrence of Anisakis larvae in red mullet in the Ligurian Sea (western Mediterranean). This survey was performed between 2018 and 2020 on fresh specimens of M. barbatus (n = 838) from two commercial fishing areas (Imperia, n = 190; Savona, n = 648) in the Ligurian Sea. Larvae morphologically identified as Anisakis spp. (n = 544) were characterized using PCR-RFLP as Anisakis pegreffii. The overall prevalence of A. pegreffii was 24.46%; the prevalence at each sampling site was 6.32% for Imperia and 29.78% for Savona. Furthermore, 3300 larvae of Hysterothylacium spp. were detected in the visceral organs of fish coinfected with A. pegreffii, showing that coinfection with two parasitic species is not rare. This study provides a timely update on the prevalence of ascaridoid nematodes in red mullet of the Ligurian Sea, an important commercial fishing area in the Mediterranean.

The Atlantic horse mackerel (Trachurus trachurus) is a commercially important fish species. Most parasitological studies on the anisakid nematodes occurrence in T. trachurus to date have involved fish from the Atlantic Ocean, while few studies are available for the Mediterranean Sea. Since T. trachurus is used raw and cooked in Ligurian cuisine (northwest Italy), assessment of possible consumer health risks (i.e., anisakiasis and allergic issues) is warranted. With this study we investigated the occurrence of zoonotic anisakid nematode larvae in the Atlantic horse mackerel. Between 2017 and 2019 a parasitological survey was carried out on 481 T. trachurus from four commercial fishing areas (Sanremo, n = 45; Imperia, n = 30; Savona, n = 300; Arenzano, n = 106) in the Ligurian Sea (western Mediterranean Sea). All isolated larvae morphologically assigned to the genus Anisakis (n = 937) were identified as A. pegreffii by PCR-RFLP. The total prevalence of A. pegreffii was 60.9% (95% confidence interval [CI] 56.5-65.3); the prevalence by area was: Savona (89.3%; 95% CI 85.8-92.8) > Imperia (20%; 95% CI 4.8-35.2) > Arenzano (16%; 95% CI 8.9-23.1) > Sanremo (4.4%; 95% CI 0-10.7). Anisakis pegreffii larvae were found in visceral organs (99.15%) and muscle tissue (0.85%). In addition, 411 larvae morphologically referable to the genus Hysterothylacium sp. were also isolated but only in the viscera. This parasitological survey provides new data on the occurrence and the level of anisakid nematode infection in the Atlantic horse mackerel.

Tributyltin-based (TBT) antifouling paints, widely used for the treatment of flooded surfaces, have been banned in 2008 for their high environmental persistence and bioaccumulation in aquatic organisms. Although it is still present in aquatic ecosystems, oxidative stress driven by TBT has been still poorly investigated in fish. The aim of the study was to examine the time-course stress responses in liver of rainbow trout that received a single intraperitoneal injection of tributyltin chloride (TBTC) or tributyltin ethoxide (TBTE), both at a dose of 0.05 and 0.5 mg/kg. Levels of metallothioneins, total glutathione, malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase were evaluated at 3 and 6 days post-injection. Tin load was measured in the muscle of the same fish. Differences were observed in the time-course accumulation of tin with a clear dose-response relationship. Although individual oxidative stress biomarkers varied, the biomarker profile indicated different stress mechanisms caused by both TBTC and TBTE. The weak induction of metal-trapping metallothioneins and the changes of oxidative stress biomarkers suggested a stress-pressure in both TBT-treated trout, advising for an ecotoxicological risk for freshwater ecosystems.

Biofouling is a costly burden for the shipping industry. Metal-based antifouling paints are widely used to protect submerged surfaces, but the release of metals from coatings and the recoating of hulls can leach large amounts of copper and zinc into aquatic environments, posing a risk for aquatic ecosystems and biodiversity. With this study, we studied the time-course metal accumulation and oxidative stress in the digestive gland and the gills of Hyriopsis bialatus, an Asian freshwater mussel, exposed to sublethal concentrations of cuprous chloride (50 and 5 µg/L) and zinc sulfate (1000 and 100 µg/L). Time-dependent accumulation was observed after exposure to copper, but zinc uptake was negligible. Integrated biomarker response (IBRv2) and statistical analysis of individual biomarker levels showed a greater biomarker response in the digestive gland and the gills after exposure to the higher concentration of CuCl and ZnSO4. Both compounds elicited a biochemical response, especially in the digestive gland. Glutathione peroxidase activity was increased after exposure to both metals at both concentrations, suggesting a powerful defense against lipid peroxidation. The biological impact of zinc was less than that of copper, suggesting mitigated ecological pressure.

The introduction of fish for stoking purpose is a common operation to manage fisheries. This may cause ecological and sanitary risks to native aquatic biodiversity especially in fishless Alpine lakes. In these ecosystems, fish have been introduced by anglers for ages. Europe imposes strictly controls on aquaculture facilities that breed fish for stoking purpose only for viral disease but not for bacterial or parasitic ones. Moreover, the role of alien fish as carriers of pathogens is poorly studied. For these reasons, we performed two fish sampling campaigns in two Italian Alpine lakes (Dimon Lake and Balma Lake) to have qualitative information about alien fish populations and to perform a bacteriological survey on captured fish. In Dimon Lake we captured individuals of Cottus gobio and of Phoxinus phoxinus, while only individuals of Salvelinus fontinalis were sampled in Balma Lake. We isolated Aeromonas sobria and Plesiomonas shigelloides in Dimon Lake, which are bacteria widespread in aquatic environment. In Balma Lake, one fish was positive for Yersinia ruckeri, a primary pathogen present in aquaculture facilities that reared salmonids. Our study show how alien species may introduce bacteria that could be more virulent for native aquatic fauna or even pathogenic bacteria.

Lactococcosis is one of the main bacterial diseases affecting rainbow trout (Oncorhynchus mykiss), with significant economic and sanitary repercussion. Vaccination and antibiotic treatments are commonly used to prevent and control the infection outbreaks; however, these strategies have some drawbacks including limited coverage, handling costs, induction of antibiotic resistance and chemical residues in the environment. Selective breeding programs represent a promising complementary approach for increasing fish disease resistance in commercial farms and some immunological parameters may be tentatively used as indirect indicators for this purpose. The present study investigated for the first time some innate and adaptive immune responses in two groups of rainbow trout derived from selected lines (susceptible and resistant) showing a different “in field” phenotypical resistance to Yersinia ruckeri, Flavobacterium branchiophilum, F. psychrophilum, and Ichthyophthirius multifiliis, after an immersion-dilution based exposure to Lactococcus garvieae carried out in controlled experimental conditions. Twenty-six resistant and twenty-six susceptible female rainbow trout (mean body weight 80 g, 9 months aged, F5 generation) were obtained from an intensive farm considered L. garvieae free and were exposed to the pathogen. Moreover, 10 resistant and 10 susceptible fish were used as uninfected controls. After 5 days, blood and tissue samples were collected for immunological analyses. A significantly higher serum and mucus lysozyme activity was recorded in resistant rainbow trout compared to susceptible fish (P ≤ 0.05), both before and after exposure to L. garvieae. Similarly, respiratory burst activity of head kidney leukocytes resulted more intense in resistant fish (P ≤ 0.05), suggesting that phagocytes could more quickly activate their microbicidal mechanisms to counteract the bacterial spread. Resistant group displayed also an up-regulation of immunoglobulins M (IgM), major histocompatibility complex II (MHC-II) and interleukin 8 (IL-8) gene expression (P ≤ 0.05) and a significantly higher blood lymphocytes count (P ≤ 0.05), highlighting their potential better ability to trigger the recruitment of defensive cells and the initiation of specific immune processes such as antigen presentation to CD4+ T lymphocytes and IgM synthesis. The results herein presented might be useful for the identification of immunological markers to be used as indirect indicators in rainbow trout selective breeding programs.

The rapid expansion of the ornamental aquaculture industry over the past decades has resulted in a concomitant increase in the use of antibiotics to combat infectious diseases. The aim of this study was to assess the possible role of ornamental fish in the translocation of antibiotic resistant bacteria, with possible consequences for aquarium and public health. We assessed the prevalence of bacterial infections and the antibiotic resistance profile of bacteria isolated from 134 ornamental fish imported into northwest Italy during two years of monitoring. Ornamental fish analyzed were imported mainly from Singapore (40%) and Israel (20%), followed by Thailand (13%), Sri Lanka (12%), Czech Republic (7%), Vietnam (5%) and Indonesia (3%). The most commonly imported fish were freshwater species, particularly those belonging to the Poeciliidae family. Bacteriological exam was positive in 68% of the fish examined. The most frequently isolated bacterium was Aeromonas sobria (37%). Bacteria showed resistance against lincomycin, ampicillin, oxytetracycline and tetracycline. Sensitivity was found for florfenicol, chloramphenicol, gentamicin, and trimethoprim/sulfamethoxazole. Odds ratio (OR) values were calculated as a measure of the association between antibiotic resistance of A. sobria and selected factors (country of origin, fish family and fish species), considering Thailand, Poeciliidae and Poecilia reticulata as control cases. Higher values were found for Vietnam (OR 5.6) and Xiphophorus helleri and X. maculatus (OR 3.0 and 3.7 respectively). Our findings underline the need to improve targeted surveillance of antimicrobial resistance and prevent the translocation of resistant or multi-resistant bacterial strains in ornamental fish, especially in fish imported from countries where surveillance is limited or lacking.

This study aimed to assess the biological and sanitary conditions of alien fish in a high-mountain lake (Balma Lake) located in the Cottian Alps. A single fish sampling session (August 2018) using gillnets collected 90 specimens of brook trout (Salvelinus fontinalis). Sex and age were determined (59 females and 31 males, age class 0+ to 4+). Regression analysis showed no difference in total weight and total length between males and females (ANCOVA: F = 0.453; p = 0.954). The mean condition factor (K-mean) decreased with increasing age for males and females. Terrestrial insects were the main prey found in the fish stomachs. The parasitological exam was negative, and the bacteriological exam was positive for Carnobacterium maltaromaticum and C. divergens in 33% of specimens. The total mercury, cadmium, and lead concentration in muscle tissue was within the maximum limit established by the European Commission for human consumption. The brook trout population was found to be well structured; these findings may help local administrations in the implementation of eradication measures.

In this study we investigated the occurrence of larval ascaridoid nematodes in Illex coindetii, a commercially relevant cephalopod, via a cross-sectional prevalence study (January 2015 to February 2018) in two fishing areas (Savona and Piombino) in the Ligurian Sea (northwest Mediterranean Sea). A total of 745 specimens of I. coindetii were caught by local fisherman through bottom trawling. Parasitological examination revealed ascaridoid nematodes (L3) larvae (n = 9): Hysterothylacium sp. (n = 6) and Anisakis spp. (n = 3). All larvae morphologically referred to the genus Anisakis were identified by PCR-RFLP as A. pegreffii. The prevalence of Hysterothylacium sp. infestation was 0.88% for the Savona and 0.56% for the Piombino sampling site; the prevalence of A. pegreffii was 0.18% for Savona and 1.11% for Piombino and did not differ between the two sampling sites (chi-square test; p = 0.0848). Better knowledge of the distribution of ascaridoid nematodes, especially of Anisakis species is essential for defining their epidemiological role and the potential risk for consumer health.

Twelve biochemical parameters were measured in serum blood samples from rainbow trout (Oncorhynchus mykiss) maintained under controlled conditions. Forty-five samples were taken every 3 months (T1–T4) over the course of one year to define baseline values. The effect of fish total weight (TW) was also evaluated. Principal component analysis showed a clear separation between T4 and T1, and T2 and T3, indicating an increase in certain biochemical parameters with weight. Linear regression analysis showed how TW significantly explained 11–67% of the variability observed for nine parameters out of 12. Pearson’s correlation matrix showed a significant positive correlation (p < 0.05) between TW and albumin, alkaline phosphatase, alanine-aminotransferase, creatinine, gamma-glutamyl transferase, magnesium, phosphorus and total protein. Furthermore, significant correlations (p < 0.05) between the majority of the biochemical parameters were found, indicating that growth can influence several parameters at the same time. The present study provides several useful baseline values for assessing the health of O. mykiss, indicating that fish weight is an important factor for interpreting the blood biochemical profile.

Lactococcosis is a fish disease of major concern in Mediterranean countries caused by Lactococcus garvieae. The most susceptible species is the rainbow trout (Oncorhynchus mykiss), suffering acute disease associated with elevated mortalities compared to other fish species. References reported that other salmonids are also susceptible to the disease, but no mortality outbreak has been described to date. The aim of this study was to present a mortality outbreak that occurred in brook trout (Salvelinus fontinalis) farmed in northwestern Italy during the summer of 2018. Fish exhibited clinical signs, such as exophthalmos, diffused hemorrhages localized in the ocular zone, hemorrhagic enteritis, and enlarged spleen. L. garvieae was isolated in all fish. Molecular and epidemiological characterization of the isolates, through Pulsed Field Gel Electrophoresis (PFGE), confirmed the initial hypothesis of water as vehicle of infection favoring transmission between rainbow trout farmed in upstream compartments and brook trout located in downstream tanks. Moreover, several environmental conditions affected and promoted the outbreak, among them the high-water temperature, which probably induced a physiological stress in brook trout, being way above the optimal temperature for this species, increasing the susceptibility to infection.

A case of mycobacterial infection in silver arowana (Osteoglossum bicirrhosum) is described in the present work. An adult male silver arowana exhibited granulomas in the liver and spleen with acid-fast bacilli identified as Mycobacteria fortuitum. To our knowledge this is the first report of mycobacterial infection in this fish species.

Sturgeon aquaculture production has increased worldwide, particularly in Italy, yet there is little information about diseases that affect sturgeons. With this study we describe the results of screening for bacterial infections during monitoring campaigns carried out from 2014 to 2017. A total of 402 sturgeons from six sturgeon farms with one or more reared sturgeon species among five species and two hybrids were analysed. Bacteriological exam was positive in 93 individuals (23%) and negative in the remaining 309 (77%). Generally, no external or internal macroscopic lesions were present, though some specimens showed skin lesions, liver anaemia or splenomegaly, which are non-pathognomonic signs of bacterial infection. The majority of the isolated bacteria were opportunistic. This knowledge base is important to help create a bacterial profile of sturgeons reared in Italy.

Insect protein meals are considered as a promising alternative feedstuff in aquafeeds, but until now the effects induced by its dietary inclusion on histological features, gut mucin composition and oxidative stress in fish are still not assessed. In the present study, we describe these effects after inclusion of a partially defatted Hermetia illucens (HI) larvae meal in Oncorhynchus mykiss diets. Three isoproteic, isolipidic and isoenergetics diets were formulated containing HI larvae meal in partial substitution of fish meal: 0% (HI0, control diet), 25% (HI25) and 50% (HI50), corresponding to dietary inclusion levels of 0, 20% and 40% of HI. Fish were fed for 78 days with the experimental diets and at the end of the growing trial, histological and histochemical evaluations were performed on spleen, liver and gut samples of control and treatment groups. Oxidative stress biomarkers, such as superoxide dismutase, catalase, glutathione peroxidases, ethoxyresorufin O-deethylase, glutathione S-transferase, glutathione reductase, total glutathione and malondialdehyde were investigated in liver and kidney of experimental groups. Histopathological examination of liver, spleen and gut revealed no adverse effects following increasing levels of insect meal inclusions. Hermetia illucens consumption stimulated higher production of neutral than acidic mucins. Altered values of several antioxidant biomarkers indicated an unbalance of oxidative homeostasis in liver and mainly in kidney, linked to the feeding conditions. While lowering of glutathione peroxidase activity may reflect slight adverse effect of HI meals in both tissues, strengthening in levels of ethoxyresorufin O-deethylase, glutathione S-transferase and total glutathione may indicate the shielding of HI meals-mediated oxidative process. Although the administration of both inclusion levels of insect meal did not negatively affect the histological features in fish, the changes of oxidative stress biomarkers advise for an HI dietary inclusion lower than 20%.

Buccal swabbing is a minimally invasive method to obtain DNA and biological material from humans and animals, including fish. Reports on buccal swabbing in fish are few and only for a limited number of species. Rainbow trout (Oncorhynchus mykiss) is an important animal model and because the yield of DNA may vary among and within different species in individuals of different sizes, it was selected as useful to optimize the buccal DNA collection in this species. Different storage methods were evaluated, aimed at DNA preservation by limiting DNA degradation and bacterial growth, using commonly available and inexpensive reagents. DNA quality was also tested by amplification of a single-copy nuclear gene and a mitochondrial gene. The results suggest that ethanol is the best storage choice for buccal swab sampling in fish genetic studies, as well as suitable for small-bodied rainbow trout.

Infectious Hematopoietic Necrosis (IHN) and Viral Haemorrhagic Septicaemia (VHS) are two systemic infections of several species of salmonids; whose reporting is mandatory in accordance with Directive 67/1991 EC. In order to obtain and preserve the free status at farm level, the Italian legislation (D.L.vo 148/2008) adopted a surveillance program involving all freshwater farms raising salmonids to be released on watercourses. This study provides a description of the epidemiology of IHN and VHS in the Piedmont Region, during the period 2000-2015. In fact in the last 16 years the surveillance was extended to all fisheries. We firstly described the disease distribution by year and by geographical area., then the potential role of several farm charcteristics was tested using unvariate and multivariate analysis.

We examined the time-course stress responses in the liver of rainbow trout exposed to cuprous chloride (CuCl) and cupric chloride (CuCl2). The treatment groups received a single intraperitoneal injection of CuCl or CuCl2 (both at a dose of 0.01 and 0.05 mg/kg); the control group received only the physiologic solution vehicle. Liver tissue samples were analyzed for total copper, superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glyoxalases, and lactate dehydrogenase at 3, 6, and 9 days post-injection. Total glutathione, metallothionein, and malondialdehyde levels were also measured. The time course of metal accumulation differed between the groups; no dose-response relationship for metal load was found. Both copper species elicited significant changes in oxidative stress markers and in metal trapping. Copper underwent adaptive shifts in glutathione and metallothionein concentrations. The defense strategy primarily versus CuCl2 first involved glutathione, with a peak in metallothionein levels at day 6 for CuCl2 (at both doses) and for CuCl (0.05 mg/kg). Early stimulation of lipid peroxidation was noted after treatment with the higher copper dose and at day 9 after treatment with the lower dose of both CuCl and CuCl2. Antioxidant enzyme activity was impaired due to a more or a less severe oxidative stress condition in relation to the copper species and exposure time. Copper dynamics, in terms of metal accumulation and homeostatic regulation, is noticeably complex. The present findings may advance our understanding of the effects of both copper species on the antioxidant response of rainbow trout.