Beware of dogs! Domestic animals as a threat for wildlife conservation in Alpine protected areas.

Diseases are natural regulating factors of wildlife populations, but some pathogens may become an important threat in wildlife conservation, especially for endangered species. The presence of domestic animals may foster the spread of diseases in natural population, although their role in the dynamic of infections in wildlife is not clear. In this study, we investigated the presence and prevalence of a range of multi-host pathogens in wild species (red fox, Eurasian badger, beech marten, pine marten, stoat for a total of 89 carcasses analysed) and domestic animals (n = 52 shepherd and n = 25 companion dogs) living in a protected area of the Alps (the Gran Paradiso National Park) and discussed the role of domestic dogs as possible source of infection for wild species. Our results showed that domestic dogs are potential shedder of three important pathogens: Canine distemper virus, Toxoplasma sp. and Neospora caninum. In particular, shepherd dogs seem to represent a threat for wildlife as they are exposed to multiple pathogens because of free-roaming, scavenging lifestyles and close proximity to livestock. However, also companion dogs more subject to veterinary care may foster the spread of pathogens. Our results highlight the importance of regulating the access of domestic dogs to protected areas that aim at preserving biodiversity and enhancing the conservation of endangered species.

Isolation of Arcobacter species and other neglected opportunistic agents from aborted bovine and caprine fetuses.

BACKGROUND: Infectious abortion in ruminants is a problem in animal husbandry worldwide. It is important to obtain a diagnosis, to make sure that proper control measures can be instituted, but most abortion cases remain without an etiologic diagnosis. This report describes the presence of Arcobacter species and several neglected opportunistic abortifacient agents in ruminant abortion cases showing or not co-infections among at least one of the major recognized protozoal, fungal, bacterial and viral abortifacient agents. RESULTS: A total of 67 fetuses (55 cattle and 12 goats) and just one placenta (cattle) were considered. Among the most common abortive agents, Neospora caninum (19,4%), followed by Chlamydophila abortus (4,5%), Listeria monocytogenes 1/2a (2,98%), Bovine Viral Diarrhea Virus type 1b (2,98%), Bovine herpesvirus 4 (2,98%), and Aspergillus spp. (2,98%) were detected. The isolated neglected opportunistic bacteria include Escherichia coli, Acinetobacter lwoffii, Staphylococcus spp., Streptococcus spp., Streptococcus uberis, Streptococcus suis, Trueperella pyogenes, Mannheimia haemolytica, Bacillus cereus and Nocardia spp. Other bacterial species, not associated with abortion by literature, but described as causes of diseases occurring sporadically both in humans and animals, were also detected. Three Arcobacter strains, namely two A. skirrowii and one A. cryaerophilus, were isolated from 3 bovine aborted fetuses, and A. butzleri was isolated from the placenta. CONCLUSIONS: A not negligible isolation of Arcobacter species and other neglected abortifacient agents has to be mentioned, with prevalences that seem to be emerging and replacing or co-placing the major infectious players in bovine and caprine reproductive failure due to abortion disease, even if further studies investigating the aetiological power and transmission routes are needed in order to define the role of these microrganisms in ruminant abortion.

Occlusive mycotic tracheobronchitis and systemic Alphaherpesvirus coinfection in a free-living striped dolphin Stenella coeruleoalba in Italy.

A juvenile female striped dolphin Stenella coeruleoalba live stranded on 4 March 2016 at Alassio, western Ligurian Sea coast, Italy. The dolphin died shortly after stranding, and a complete postmortem examination was performed. Necropsy revealed severe tracheal occlusion and unilateral bronchial stenosis with luminal accumulation of abundant green-yellow mucous-gelatinous material. Histological features suggestive of tracheobronchial aspergillosis were observed. Cultures of lung tissue and tracheo-bronchial exudate isolated Aspergillus fumigatus, identified by a Microseq D2 LSUrDNA fungal sequencing kit. A pan-Herpesvirus nested-PCR assay on frozen samples obtained from multiple organs was positive. Phylogenetic analysis on the partial DNA polymerase gene revealed that the striped dolphin isolate was closely related to known cetacean Alphaherpesvirus sequences from the same host species. Attempted virus isolation was unsuccessful. The tissue levels of different persistent organic pollutants and the toxicological stress, evaluated using a theoretical model, showed a severely impaired immune response. This study reports the first case of occlusive mycotic tracheobronchitis in a free-living cetacean and the first molecular identification of an Alphaherpesvirus in a free-ranging striped dolphin stranded on the coast of Italy.

Coronavirus and paramyxovirus in bats from Northwest Italy.

BACKGROUND: Bat-borne virus surveillance is necessary for determining inter-species transmission risks and is important due to the wide-range of bat species which may harbour potential pathogens. This study aimed to monitor coronaviruses (CoVs) and paramyxoviruses (PMVs) in bats roosting in northwest Italian regions. Our investigation was focused on CoVs and PMVs due to their proven ability to switch host and their zoonotic potential. Here we provide the phylogenetic characterization of the highly conserved polymerase gene fragments. RESULTS: Family-wide PCR screenings were used to test 302 bats belonging to 19 different bat species. Thirty-eight animals from 12 locations were confirmed as PCR positive, with an overall detection rate of 12.6% [95% CI: 9.3-16.8]. CoV RNA was found in 36 bats belonging to eight species, while PMV RNA in three Pipistrellus spp. Phylogenetic characterization have been obtained for 15 alpha- CoVs, 5 beta-CoVs and three PMVs; moreover one P. pipistrellus resulted co-infected with both CoV and PMV. A divergent alpha-CoV clade from Myotis nattereri SpA is also described. The compact cluster of beta-CoVs from R. ferrumequinum roosts expands the current viral sequence database, specifically for this species in Europe. To our knowledge this is the first report of CoVs in Plecotus auritus and M. oxygnathus, and of PMVs in P. kuhlii. CONCLUSIONS: This study identified alpha and beta-CoVs in new bat species and in previously unsurveyed Italian regions. To our knowledge this represents the first and unique report of PMVs in Italy. The 23 new bat genetic sequences presented will expand the current molecular bat-borne virus databases. Considering the amount of novel bat-borne PMVs associated with the emergence of zoonotic infections in animals and humans in the last years, the definition of viral diversity within European bat species is needed. Performing surveillance studies within a specific geographic area can provide awareness of viral burden where bats roost in close proximity to spillover hosts, and form the basis for the appropriate control measures against potential threats for public health and optimal management of bats and their habitats.

A new genotype of border disease virus with implications for molecular diagnostics.

Border disease virus (BDV) is a (+) single-stranded RNA pestivirus affecting mainly sheep and goats worldwide. Genetic typing of BDV has led to the identification of at least seven major genotypes. This study reports the detection of a BDV strain from a goat in northwestern Italy during routine investigations. Sequence analysis revealed mutations in the 5'-UTR of the virus with implications for BDV molecular diagnostics. Moreover, subsequent phylogenetic analysis based on the combined 5'-UTR and Npro/partial C genes, showed divergence from known BDV genotypes, revealing the detection of a novel pestivirus group, for which we propose the name BDV genotype 8.

Meningoencephalitis and Listeria monocytogenes, Toxoplasma gondii and Brucella spp. coinfection in a dolphin in Italy.

Listeria monocytogenes, Toxoplasma gondii and Brucella spp. can infect a wide range of species, including humans. In cetaceans, meningoencephalitis has been associated with T. gondii and Brucella spp. infection, whereas to our knowledge, L. monocytogenes infection has not previously been reported. Meningoencephalitis and L. monocytogenes, T. gondii and Brucella spp. were identified by means of both direct and indirect laboratory techniques in an adult female striped dolphin Stenella coeruleoalba found stranded in January 2015 on the Ligurian Sea coast, northwestern Italy. The animal was emaciated, and histopathology disclosed severe meningoencephalitis. The nature of the inflammatory response and intra-lesional protozoa were consistent with a mixed infection by L. monocytogenes, T. gondii and Brucella spp. We believe this is an unprecedented case of infection by 3 zoonotic pathogens and also the first bacteriologically confirmed case report of neurolisteriosis in cetaceans. Cerebral toxoplasmosis and neurobrucellosis may have led to the animal's disorientation and stranding, with L. monocytogenes having likely exacerbated the coinfection leading to the demise of this dolphin.

Ruminant rhombencephalitis-associated Listeria monocytogenes strains constitute a genetically homogeneous group related to human outbreak strains.

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Estimates of dairy herd health indicators of mastitis, ketosis, inter-calving interval, and fresh cow replacement in the Piedmont region, Italy.

Test-day milk analysis has largely been used to study health and performance parameters in dairy cows. In this study, we estimated four health indicators of dairy cows using test-day data. Our purpose was to estimate (1) mastitis incidence rate, prevalence, and the probability of recovery; (2) the incidence proportion of ketosis; (3) the duration of inter-calving interval; and (4) the risk of a fresh cow being replaced, in a large cohort of dairy herds in the Piedmont region (Italy). We retrospectively analysed test day records of 261,121 lactating cows and 1315 herds during five years (2015-2020). Mastitis was defined by somatic cell count and ketosis by fat-to-protein ratio. Calving dates were used to calculate ICI and to estimate the removal of a fresh cow from the herd. Mixed-effect generalized linear models were used to adjust for unmeasured herd-level risk factors. The risk of mastitis increased by 120% with parity (Odds ratio [OR] = 2.20, confidence interval [CI]: 2.17 - 2.23), by 7% by months in milking (OR = 1.07, CI: 1.07 - 1.07), and even more if the cow was already affected during the same lactation (OR = 8.74, CI: 8.67 - 8.82). Lactose concentration on the previous test day was the best positive prognostic factor for mastitis recovery (OR = 1.12, CI: 1.08 - 1.17). Ketosis risk was the highest between 3rd and 4th lactations and itself increased the risk of having ICI longer than 440 days (OR = 1.12, CI: 1.02 - 1.22), and fresh-cow removal (OR = 1.75, CI: 1.58 - 1.93). Also, the removal of fresh cows was more likely when mastitis (OR = 1.31, CI: 1.19 - 1.45) or long ICI (OR = 1.34, CI: 1.22 - 1.48) occurred. For each health indicator, herd-level risk factors had an important role (18-56% of within-herd covariance). Our results indicate that milk analysis could be also useful for predicting mastitis, its cure rate, and ketosis. Cow-level risk factors are not enough to explain the risk of these issues. By studying a large population over a long period, this study provides an updated estimate of dairy cow health indicators in Piedmont (north-western Italy), useful for benchmarking dairy herds.

Evaluation of the Immune Response to Transport Stress in the Aosta Valley Breed.

Transportation is a recurring event in a farm animal's life, and it is considered one of the main stressors with possible negative repercussions for both the health and welfare of farm animals. The objective of the present study was to examine the effect of transportation on some blood variables of 45 young bulls moved from their original farms to a livestock collection centre. Transportation took no more than 8 h and was carried out between January and March 2021. Blood samples were taken before transportation (T0), upon arrival at the collection centre (T1), and 7 days after arrival (T2). Samples were processed for blood cell count, clinical chemistry analyses, serum protein electrophoresis, and the evaluation of innate immunity parameters. The results showed a typical stress leukogram with neutrophilia and changes in the neutrophil:lymphocyte ratio. No significant alterations were observed in either serum proteins or pro-inflammatory cytokines. Significant, albeit transient, alterations were observed in some clinical chemistry parameters after transportation, which could be accounted for by stressful conditions such as the transportation itself and handling and mixing with other animals. Our results indicated that the adopted transportation conditions only slightly affected the blood variables under study with no significant impact on animal welfare.

Toward Sustainability: An Overview of the Use of Green Hydrogen in the Agriculture and Livestock Sector.

The agro-livestock sector produces about one third of global greenhouse gas (GHG) emissions. Since more energy is needed to meet the growing demand for food and the industrial revolution in agriculture, renewable energy sources could improve access to energy resources and energy security, reduce dependence on fossil fuels, and reduce GHG emissions. Hydrogen production is a promising energy technology, but its deployment in the global energy system is lagging. Here, we analyzed the theoretical and practical application of green hydrogen generated by electrolysis of water, powered by renewable energy sources, in the agro-livestock sector. Green hydrogen is at an early stage of development in most applications, and barriers to its large-scale deployment remain. Appropriate policies and financial incentives could make it a profitable technology for the future.

An Epidemiological Update on Anisakis Nematode Larvae in Red Mullet (Mullus barbatus) from the Ligurian Sea.

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.

Is the Introduction into a New Environment Stressful for Young Bulls?

Several events in an animal's life are considered stressful. Among them, the most studied and significant are transportation, weaning, and adaptation to climate change. Moreover, other events, such as the separation from the dam, moving from the original farm to another, management practices, such as regrouping with other animals, and new hierarchical conditions, represent routine conditions in the bovine's life, which can influence the animal's homeostasis. The purpose of this study is to evaluate the changes in blood parameters of 45 calves introduced into a new environment from their original farms. Blood samples were collected upon arrival at a genetic center (T1), 7 (T2), 30 (T3), and 120 (T4) days after arrival. Blood count, protein electrophoresis, clinical chemistry, and innate immunity parameters were performed on the samples. Significant alterations in some clinical chemistry parameters were related to liver function in the serum protein and the values of IL-6 and TNF-α; the main cytokines mediating the stress response emerged from the results. The evidence indicates the mild response to adaptation stress by calves raised in close association with people after their introduction into a new environment.

Brucella ceti Infection in Striped Dolphins from Italian Seas: Associated Lesions and Epidemiological Data.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 (N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis (N = 9), meningitis (N = 6), and meningoencephalomyelitis (N = 5), which was also associated with typical lesions in other tissues (N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) (N = 21) and, in a few cases, ST49 (N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Occurrence of microplastics in the gastrointestinal tract of benthic by-catches from an eastern Mediterranean deep-sea environment.

Concern about microplastic pollution little is known about levels in deep-sea species; to fill this knowledge gap, levels of microplastics in the gastrointestinal (GI) tracts of 34 fish from eight different deep-sea by-catches: blackmouth catshark, lesser spotted dogfish, and velvet belly, armless snake eel, hollowsnout grenadier, phaeton dragonet, royal flagfin, and slender snipe eel were measured. All were collected at the same site (east Sardinia, Mediterranean Sea; 40°10'12.49″N, 9°44'12.31″E) using a bottom gillnet at depths between -820/250 and -1148 ft./350 m. Microplastics (MPs) were retrieved in 16 out of 34 fish. At least one microplastic item was found in 48% (33%, E. spinax - 75%, G. melastomus) of the samples. The most frequent was polyethylene (PE), with nine items (filaments, films, fragments) found in five specimens. This preliminary study of by-catches adds new data on MPs ingestion by species inhabiting a deep-sea environment of the Mediterranean.

Renal carcinoma with metastatic spread in a tiger (Panthera tigris): morphological and immunohistochemical study.

A 12­year­old intact male Panthera tigris presented with pain and weight loss was euthanatized. Necroscopical examination revealed a neoplastic mass expanding to the left renal pelvis with metastatic dissemination to local lymph node, adrenal gland, and lung. Immunohistochemical characterization was performed revealing co­expression of both cytokeratin and vimentin and negativity for both PAX8 and c­KIT. Considering histochemical and immunohistochemical results the tumour was classified as renal cell carcinoma with metastatic spread. This report provides insights into the morphological and immunohistochemical features of renal cell carcinoma in Panthera tigris.

Non-Native Turtles (Chelydridae) in Freshwater Ecosystems in Italy: A Threat to Biodiversity and Human Health?

Marketed globally, freshwater turtles are popular pets. Two species of the Chelydridae family are increasingly reported in Italy: the snapping turtle (Chelydra serpentina) and the alligator snapping turtle (Macrochelys temminckii). Both pose potential threats to public safety and habitat biodiversity. This update reports on their distribution and impact on biodiversity and human health. The recent increase in the number of C. serpentina in urban and rural areas suggests illegal importation into the country. Findings are reported for the north (35% and 100% for C. serpentina and M. temminckii, respectively) and the central-northern regions (60% for C. serpentina), predominantly Umbria and Latium, and the Tiber River catchment area in particular. Because omnivorous, Chelydridae species can affect native biodiversity; because they are carriers of pathogens, they endanger public health. Monitoring plans need to take account of this neglected threat.

Modulation of Antioxidant Defense in Farmed Rainbow Trout (Oncorhynchus mykiss) Fed with a Diet Supplemented by the Waste Derived from the Supercritical Fluid Extraction of Basil (Ocimum basilicum).

Phytotherapy is based on the use of plants to prevent or treat human and animal diseases. Recently, the use of essential oils and polyphenol-enriched extracts is also rapidly increasing in the aquaculture sector as a means of greater industrial and environmental sustainability. Previous studies assessed the antibacterial and antiparasitic effects of these bioactive compounds on fish. However, studies on the modulation of oxidative stress biomarkers are still scant to date. Thus, in this study, the modulation of antioxidant defense against oxidative stress exerted by fish diets supplemented with a basil supercritical extract (F1-BEO) was assessed in rainbow trout Oncorhynchus mykiss. The F1-BEO extracted with supercritical fluid extraction was added to the commercial feed flour (0.5, 1, 2, 3% w/w) and mixed with fish oil to obtain a suitable compound for pellet preparation. Fish were fed for 30 days. The levels of stress biomarkers such as superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glyoxalase I, glyoxalase II, lactate dehydrogenase, glutathione and malondialdehyde showed a boost in the antioxidant pathway in fish fed with a 0.5% F1-BEO-supplemented diet. Higher F1-BEO supplementation led to a failure of activity of several enzymes and the depletion of glutathione levels. Malondialdehyde concentration suggests a sufficient oxidative stress defense against lipid peroxidation in all experimental groups, except for a 3% F1-BEO-supplemented diet (liver 168.87 ± 38.79 nmol/mg prot; kidney 146.86 ± 23.28 nmol/mg prot), compared to control (liver 127.76 ± 18.15 nmol/mg prot; kidney 98.68 ± 15.65 nmol/mg prot). Our results suggest supplementing F1-BEO in fish diets up to 0.5% to avoid potential oxidative pressure in farmed trout.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

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.

Ecotoxicity of Copper(I) Chloride in Grooved Carpet Shell (Ruditapes decussatus).

Copper (Cu) is a ubiquitous trace element in the aquatic environment, and is usually found at low levels. Copper environmental concentrations can be altered as a result anthropogenic activities. Shellfish are useful bioindicators to ensure adequate environmental monitoring. Thus, the aim of the present study was as follows: (a) determine the LC50 of copper(I) chloride in grooved carpet shell (Ruditapes decussatus) collected in the Santa Gilla lagoon (Sardinia, Italy), and (b) analyze the antioxidant biomarkers in digestive gland and gills of same specimens exposed to different concentrations of the above-mentioned metal (0.045, 0.45, and 0.90 mg/L) for 96 h. A withdrawal period of 96 h was considered for the treated clam, carrying out the same biochemical analyses, superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GPx), glutathione S-transferases (GSTs), and total glutathione (GSH+2GSSG) in the two tissues. Different time and dose responses of the antioxidant biomarkers were recorded in the digestive glands and gills. Oxidative stress biomarkers highlighted the ability of Cu to induce oxidative stress in R. decussatus. Clam, following the withdrawal period of 96 h, has not been able to achieve the control levels of all biochemical markers in the digestive gland and gills. R. decussatus can be a suitable model to assess the ecotoxicity of copper in aquatic ecosystems. These findings may advance knowledge on the role and the effects of copper on oxidative stress biomarkers in grooved carpet shell. The metal ecotoxicity response can be useful to perform accurate biomarker-based monitoring programs using this bivalve species.