Epidemiological cut-off values for Yersina ruckeri disc diffusion data generated by a standardised method.

In order to establish the meaning of data generated in antimicrobial agent susceptibility tests, it is necessary to develop internationally harmonised interpretive criteria. Currently, such criteria have not been developed for data generated in studies of the susceptibility of the fish pathogen Yersinia ruckeri. This work generated the data that would be required to set epidemiological cut-off values for the susceptibility data of this species that had been generated using a standardised disc diffusion method that specified the use of Mueller Hinton agar and incubation at 22°C for 24-28 h. Using this method, sets of inhibition zones data for 4 antimicrobial agents were generated by 3 independent laboratories. The data from these laboratories were aggregated and analysed using the statistically based normalised resistance interpretation. For ampicillin, florfenicol, oxytetracycline and trimethoprim-sulfamethoxazole the cut-off values calculated by this analysis were ≥16, ≥23, ≥24 and ≥30 mm, respectively. Evidence is presented demonstrating that the data for these 4 agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for Y. ruckeri.

Setting epidemiological cut-off values for Vibrio harveyi relevant to MIC data generated by a standardised microdilution method.

The lack of internationally harmonised criteria for interpreting the data generated by standardised susceptibility testing methods presents a serious obstacle for the development of prudent use of antimicrobials in aquaculture. The data required to set epidemiological cut-off values for minimum inhibitory concentrations for antibiotic agents against Vibrio harveyi was determined using a standard microdilution method that specified the use of cation-adjusted Mueller Hinton broth and incubation at 28°C for 24 to 28 h. In total, 120 observations were made in 4 independent laboratories from 109 unique isolates. The aggregated data from these laboratories were analysed by the normalised resistance method and by ECOFFinder to calculate epidemiological cut-off values. The data for chloramphenicol, meropenem and sulfamethoxazole were not considered as suitable for analysis. The data for ampicillin indicated that this species is innately resistant to this agent. No acceptable ranges for quality control strains have been set for ceftazidime and, therefore, only provisional cut-off values could be generated for this agent. The epidemiological cut-off values were, however, calculated for the other 6 agents. These values were ≤0.5 µg ml-1 for enrofloxacin, ≤1 µg ml-1 for florfenicol, oxolinic acid and oxytetracycline, ≤4 µg ml-1 for gentamicin and ≤0.5/9.5 µg ml-1 for trimethoprim/sulfamethoxazole. Evidence is presented demonstrating that the data for these 6 antimicrobial agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for V. harveyi.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

Severe gastroenteropathy associated with Clostridium perfringens isolation in starving juvenile sturgeons.

In November 2020 a mortality episode (30%) in juvenile Siberian and Russian sturgeons (Acipenser baerii, Brandt, and A. gueldenstaedtii, Brandt & Ratzeburg) and GUBA hybrid sturgeons (A. gueldenstaedtii × A. baerii) occurred in a hatchery in Northern Italy, associated with severe coelomic distension and abnormal reverse surface swimming. The fish were reared in concrete tanks supplied by well water, fed at 0.4% of body weight (b.w.) per day. Thirty sturgeon specimens were collected for necropsy, histological, bacteriological and virological examination. Macroscopic findings included diffuse and severe bloating of gastrointestinal tracts due to foamy contents with thinning and stretching of the gastrointestinal walls. Histological analysis revealed variable degrees of sloughing and necrosis of the intestinal epithelium, and the presence of bacterial aggregates. Anaerobic Gram-positive bacteria were investigated, and Clostridium perfringens was isolated from the gut. Specific PCRs identified the toxinotype A and the ß2 toxin gene. The daily feed administration was increased to 1.5% b.w. and after 5 days, the mortality ceased. A new animal cohort from the same groups was examined after 12 weeks, showing neither gut alterations nor isolation of C. perfringens. The imbalance of intestinal microbiota, presumably caused by underfeeding, favoured C. perfringens overgrowth and severe gas formation. The diet increase possibly restored the normal microbiota.

Carp edema virus-related mortality in wild adult common carp (cyprinus carpio) in Italy.

Mortality in wild fish populations represents a challenging issue for public fish health inspectors. When a single fish species is involved, an infective aetiology is frequently suspected, with focus on viral notifiable diseases. However, other viral agents not subjected to regulation and causing mortality in common carp have been reported such as carp edema virus (CEV). In mid-June 2020, a severe common carp mortality was observed in an artificial lake in north-east of Italy. Sleepy fish were noted some days before the beginning of the mortality itself, which lasted several days and involved over 340 adult specimens. During the outbreak, water temperature was around 15°C, water quality was normal, and no adverse meteorological events were reported in the area. Four specimens, which showed severe cutaneous hyperaemia and increased mucus production on skin and gills, were tested by bacteriological methods and virological analysis targeting the main carp pathogens. Molecular analysis performed on gills, kidney and brains from all the fish analysed resulted positive for CEV, which, based on anamnestic information and laboratory findings, was considered the responsible for the mortality event herein described.

Viral encephalopathy and retinopathy is endemic in wild groupers (genus Epinephelus spp.) of the Algerian coast.

This work describes betanodavirus infection in two species of groupers (family Serranidae) from the Algerian coast: the dusky grouper Epinephelus marginatus and the golden grouper Epinephelus costae. At necropsy, characteristic clinical signs, external injuries, clouded eyes and brain congestion, generally associated with viral encephalopathy and retinopathy (VER) infection were observed. The partial sequences of RNA1 and RNA2 from two viral strains were obtained, and the phylogenetic analysis revealed the presence of the red-spotted grouper nervous necrosis virus (RGNNV) genotype closely related to strains previously detected in groupers in the same geographic area. Results obtained in this study support the hypothesis that VER disease is endemic in the Algerian grouper population.

Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA.

Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups A and B in UK waters.

Viral encephalopathy and retinopathy (VER) disease in Epinephelus marginatus from the Balearic Islands marine protected areas.

This is the first description of a betanodavirus infection in the dusky grouper Epinephelus marginatus within the marine protected areas (MPAs) of the Balearic Islands. Histopathology techniques were employed to describe neurological lesions in infected fish. Abnormal swimming, mortality, and neurological lesions were detected in all analysed grouper individuals. Virus particles were observed by means of transmission electron microscopy. Reverse transcription of RNA1 and RNA2 followed by cDNA amplification and sequencing allowed viral classification. Phylogenetic analysis showed the isolates from wild E. marginatus of the Balearic Islands MPAs to be closely related to Dicentrarchus labrax and Mullus barbatus strains from Cyprus and Italy. Although vertical transmission from infected spawners has been described as the major route for nodavirus infection, we point out in this work that horizontal transmission among sub-clinical fishes after migration or commercial import for aquaculture production could play a major role in the spreading of the disease in MPAs.

Mycobacterium gordonae infecting redclaw crayfish Cherax quadricarinatus.

The redclaw crayfish Cherax quadricarinatus (von Martens, 1868) is one of the most commonly exploited freshwater crayfish species worldwide. Redclaw crayfish are susceptible to a number of pathogens but none have been linked to widespread epizootics. Mycobacterial infections have been sporadically reported in crayfish. In the case described, histopathology and bacterial identification confirmed an opportunistic infection caused by Mycobacterium gordonae in a hatchery of C. quadricarinatus in Israel. Intranuclear inclusion bodies, recorded in cells of the tubular epithelium of the hepatopancreas by histopathology, indicate a co-infection with a viral agent, referable to C. quadricarinatus bacilliform virus (CqBV). To the best of our knowledge this is the first description of mycobacteriosis in redclaw crayfish.

Lumpfish (Cyclopterus lumpus, Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus.

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail-biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV-infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.

New genotyping method for the causative agent of crayfish plague (Aphanomyces astaci) based on whole genome data.

The oomycete Aphanomyces astaci causes crayfish plague, the most important disease of European freshwater crayfish species. Presumably introduced into Europe 150 years ago with the import of North American crayfish, A. astaci is highly pathogenic to European crayfish species. Five genotypes (A, B, C, D, and E) have been defined based on random amplified polymorphic DNA analysis (RAPD-PCR) from A. astaci pure cultures. The distinction of genotypes is an essential tool to conduct molecular epidemiological studies on crayfish plague and it has been used to clarify and better understand the history and spread of this disease in Europe. Whereas RAPD-PCR requires DNA from pure culture isolates, the development of genotyping tools that can be applied to DNA extracted from clinical samples allows a much wider application of genotyping studies, including revisiting historic samples. In this study, we present a new approach that adds to currently available methods for genotyping A. astaci strains directly from clinical crayfish samples. Whole-genome sequencing of A. astaci strains representing all currently known genotypes was employed, genomic regions unique to the respective genotype identified, and a PCR-based genotyping assay designed, which focuses on the presence/absence of PCR product after amplification with the genotype-specific primers. Our diagnostic methodology was tested using DNA extracts from pure A. astaci cultures, other Aphanomyces species and additional oomycetes, samples from a recent Italian crayfish plague outbreak and additional historical samples available in the Centre for Environment, Fisheries and Aquaculture Science laboratory. The new markers were reliable for pure culture and clinical samples from a recent outbreak and successfully discriminated genotype A, B, D, and E. The marker for genotype C required an additional sequencing step of the generated PCR product to confirm genotype.

Ultrastructural and molecular characterization of Vairimorpha austropotamobii sp. nov. (Microsporidia: Burenellidae) and Thelohania contejeani (Microsporidia: Thelohaniidae), two parasites of the white-clawed crayfish, Austropotamobius pallipes complex (Decapoda: Astacidae).

The microsporidiosis of the endangered white-clawed crayfish Austropotamobius pallipes complex has generally been attributed to only one species, Thelohania contejeani, the agent of porcelain disease. Species identification was mostly assessed by macroscopic examination or microscopic evaluation of muscle samples rather than by molecular or ultrastructural analyses. A survey conducted on A. pallipes complex populations in Northern Italy highlighted the presence of two different microsporidia causing similar muscular lesions, T. contejeani and an undescribed octosporoblastic species Vairimorpha austropotamobii sp. nov. Mature spores and earlier developmental stages of V. austropotamobii sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, which gave rise to a rosette-shaped plasmodium, and eight uninucleate spores were produced within the persistent SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and an average of 3.9 × 2.2 µm in size. The polar filament was coiled 11-14 times, lateral to the posterior vacuole. The small subunit ribosomal RNA gene (SSU rRNA) and the large subunit RNA polymerase II gene (RPB1) of V. austropotamobii sp. nov. were sequenced and compared with other microsporidia. The highest sequence identity of SSU rRNA (99%) and RPB1 (74%) genes was with the amphipod parasite Nosema granulosis and subsequently with V. cheracis, which infects the Australian yabby Cherax destructor. In our work we discuss about the reasons for placing this new species in the genus Vairimorpha. In addition, we provide for T. contejeani a RPB1 gene sequence, supplemental sequences of SSU rRNA gene and ultrastructural details of its sporogony in the host A. pallipes complex.

Detection and characterization of a rhabdovirus causing mortality in black bullhead catfish, Ameiurus melas.

This study fully describes a severe disease outbreak occurred in 2016 in black bullhead catfish farmed in Italy. Affected fish showed nervous clinical signs as well as emaciations and haemorrhagic petechiae on the skin at the fin bases, abdomen and gills. Viral isolation in cell culture allowed the subsequent identification of a rhabdovirus, tentatively named ictalurid rhabdovirus (IcRV), through electron microscopy, immunofluorescence and whole genome sequencing (WGS). The newly isolated virus, together with 14 additional viral strains stored in our repository and detected during similar mortality episodes in the period 1993-2016, was phylogenetically analysed on the basis of the nucleoprotein and the glycoprotein nucleotide and amino acid sequences. The genetic distances among Italian IcRV strains were also estimated. Our results show that all the IcRV strains belong to the genus Sprivivirus and are closely related to the tench rhabdovirus (TenRV). Italian catfish production is constantly decreasing, mainly due to viral infections, which include the newly characterized IcRV. Data presented in this work will assist to investigate the molecular epidemiology and the diffusive dynamics of this virus and to develop adequate surveillance activities.

Hikui disease in nine koi carp (Cyprinus carpio): first description of a cutaneous perivascular wall tumour.

BACKGROUND: Hikui disease is a well known disfiguring disease of koi carp (Cyprinus carpio) primarily affecting fish with red pigmentation. It causes light orange to golden yellow, multifocal to coalescing raised patches, starting from the red cutaneous areas. Some cases respond to surgery or topical treatment, but recurrence is common. OBJECTIVES: To describe the clinical and pathological presentation of Hikui disease and its cause. ANIMALS: Nine affected koi carp belonging to private hobbyists. METHODS: Eight fish underwent surgery or biopsy; one was euthanized. Tissues were submitted for histology, immunohistochemistry and transmission electron microscopy. RESULTS: Five fish showed typical lesions of Hikui disease, whereas four fish showed an atypical presentation characterized by focal or multifocal, oedematous, dark red cutaneous plaques or nodules. Histology showed unencapsulated, infiltrating and densely cellular neoplasms composed of spindle cells arranged in bundles, rows and whorls frequently centred on capillaries. Immunohistochemistry for smooth muscle actin labelled neoplastic cells in all cases. Ultrastructure showed neoplastic cells with slender cytoplasmic processes encircling the capillaries, a thin basal membrane and occasional plasmalemmal vesicles. CONCLUSIONS AND CLINICAL IMPORTANCE: All of the data supported a neoplastic process producing perivascular wall tumours. Immunoreactivity to smooth muscle actin and the ultrastructural features were indicative of a pericyte origin (haemangiopericytoma). This is the first report dealing with Hikui disease that has achieved a conclusive diagnosis. The neoplastic nature of this condition suggests the potential usefulness of a surgical approach in the clinical management of less severe cases.

Mortality occurrence and pathogen detection in Crassostrea gigas and Mytilus galloprovincialis close-growing in shallow waters (Goro lagoon, Italy).

The complex interactions occurring between farmed bivalves and their potential pathogens in the circumstances of global climate changes are current matter of study, owing to the recurrent production breakdowns reported in Europe and other regions of the world. In the frame of Project FP7-KBBE-2010-4 BIVALIFE, we investigated the occurrence of mortality and potential pathogens during the Spring-Summer transition in Crassostrea gigas and Mytilus galloprovincialis cohabiting in the shallow waters of one northern Italian lagoon (Sacca di Goro, Adriatic Sea) and regarded as susceptible and resistant species, respectively. In 2011, limited bivalve mortality was detected in the open-field trial performed with 6-12 month old spat whereas subsequent trials with 2-3 month old spat produced almost complete (2012) and considerable (2013) oyster mortality. Macroscopical examination and histology excluded the presence of notifiable pathogens but, in the sampling preceding the massive oyster spat mortality of 2012, a µdeleted variant of OsHV-1 DNA was found in wide-ranging amounts in all analyzed oysters in conjunction with substantial levels of Vibrio splendidus and Vibrio aestuarianus. The large oyster spat mortality with borderline OsHV-1 positivity recorded in 2013 supports the multi-factorial etiology of the syndrome. This is the first report of a OsHV-1 (under a form interpreted as the variant µVar) in the Goro lagoon. Transcriptional host footprints are under investigation to better understand the bivalve response to environmental factors, included viral and bacterial pathogens, in relation to the observed mortalities.

Betanodavirus meningoencephalitis in an Atlantic blue marlin.

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans.

Invasive parasites and global change: Evidence for the recent and rapid spillover of a potential pathogen of tilapias with a complex, three-host life cycle.

Biological invasions pose a serious threat to local flora and fauna and have negative impacts on ecosystems. Invasive parasites can also cause severe losses in aquaculture. In this article, we provide evidence of the recent spillover of an African parasite with a complex, three-host life cycle that has rapidly and successfully established itself in the Middle East, most likely due to the recent migration of its final hosts (great cormorant) from Africa. This case of parasite introduction into a country with intensive aquaculture is also important from an economic point of view, since large (up to 2 cm long) larvae of this parasite, the cyclophyllidean tapeworm Amirthalingamia macracantha (Cestoda) localised in the liver, can be pathogenic to their fish hosts, including farmed and wild fish, as shown by our histopathological examination of heavily infected fish. Since its first detection in Israel in November 2020, the parasite has spread rapidly and is currently found in both migratory (great cormorant, Phalacrocorax carbo) and non-migratory birds (pygmy cormorant, Microcarbo pygmaeus), as well as in fish intermediate hosts, including farmed tilapia in several farms in Israel and wild cichlids. There are numerous examples of the spillover of introduced parasites, including those that parasitise fish of commercial importance, but have a direct life cycle or use only a single intermediate host. Tilapines are the second most important group of farmed fish in the world after carps and are produced mainly in Southeast Asia, Central and South America. The global spread of great cormorants and the early evidence that pygmy cormorant may also harbour A. macracantha pose the risk of further spread of this invasive parasite to other countries and areas. In addition, global warming and reductions in foraging and resting areas near these waters may allow the parasite to complete its life cycle in new hosts.

Identification of Mycobacterium pseudoshottsii in the Eastern Mediterranean.

Among the numerous pathogenic nontuberculous mycobacteria (NTM), which may cause disease in both poikilothermic and homoeothermic organisms, members of the unique clade Mycobacterium ulcerans/Mycobacterium marinum (MuMC) may cause disease in both fish and humans. Here, we describe the emergence of Mycobacterium pseudoshottsii, one of the four MuMC members, in Israel. For many years, M. marinum was the dominant NTM that was diagnosed in Israel as a fish pathogen. To the best of our knowledge, this is the first isolation and genomic characterization of M. pseudoshottsii infecting edible fish from two different fish species farmed in offshore sea cages in the eastern Mediterranean as well as in a recirculating aquaculture system in Israel. We compared the M. pseudoshottsii whole-genome sequences to all available genomic sequences of MuMC in free, publicly accessible databases. IMPORTANCE Mycobacterium pseudoshottsii was first detected in 1997 in the USA, infecting wild striped bass (Morone saxatilis). Since then, several reports from different countries worldwide have shown its capacity to become established in new regions as well as its pathogenicity to saltwater and euryhaline finfish of different genera. Our phylogenetic analysis revealed that the Mycobacterium ulcerans/Mycobacterium marinum clade (MuMC) is divided into two main branches: one that includes M. marinum and M. pseudoshottsii, and the second, which includes other M. marinum isolates as well as two isolates of M. shottsii. Our results reinforce the proposition that the geographical distribution of M. pseudoshottsii is much more extensive than is commonly believed. The emergence of M. pseudoshottsii in different parts of the world and its pathogenic traits that affect finfish of different genera may be a cause for concern among fish farmers, researchers, and environmental organizations.

Effect of TiO2 Nanoparticle on Bioaccumulation of ndl-PCBs in Mediterranean Mussels (Mitilus galloprovincialis).

The interaction of nanomaterials with pollutants in the marine environment might alter bioavailability, as well as toxicity, of both nanomaterials and pollutants, representing a risk, not only for marine organisms, but also for consumers through the marine food chain.The aim of this study was to evaluate the effect of titanium dioxide nanoparticles (TiO2NPs) in terms of bioaccumulation and toxicity on Mediterranean mussels (Mytilus galloprovincialis) exposed to six-indicator non-dioxin-like polychlorinated biphenyls (ndl-PCBs). Mussels were exposed to ndl-PCBs (20 µg/mL) (groups 3-4) or to a combination of ndl-PCBs (20 µg/mL) and TiO2NPs (100 µg/mL) (groups 5-6) for four consecutive days. TiO2NPs was detected in groups 5-6 (3247 ± 567 and 1620 ± 223 µg/kg respectively), but their presence did not affect ndl-PCBs bioaccumulation in mussels. In fact, in groups 3-4, the concentration of ndl-PCBs (ranging from 3818.4 ± 166.0-10,176 ± 664.3 µg/kg and 2712.7 ± 36.1-9498.0 ± 794.1 µg/kg respectively) was not statistically different from that of groups 5-6 (3048.6 ± 24.0-14,635.9 ± 1029.3 and 5726.0 ± 571.0-9931.2 ± 700.3 µg/kg respectively). Histological analyses showed alterations to the structure of the gill tissue with respect to the control groups, with more severe and diffuse dilatation of the central hemolymphatic vessels of the gill lamellae in groups 5-6 (treated with TiO2NPs and ndl-PCBs concurrently) compared to groups 3-4 (ndl-PCBs only). Finally, in mussels submitted to a seven-day depuration process, most TiO2NPs were eliminated, and NPs had a synergistic effect on ndl-PCBs elimination; as a matter of fact, in groups 5-6, the percentage of concentration was statically inferior to the one observed in groups 3-4. In any case, consumers might be exposed to TiO2NPs and ndl-PCBs (both concurrently and separately) if edible mussels, harvested in a contaminated environment, are consumed without a proper depuration process.