Publications by authors named "Amedeo Manfrin"

13 Publications

  • Page 1 of 1

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

J Fish Dis 2021 Feb 16. Epub 2021 Feb 16.

Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Legnaro, 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.
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http://dx.doi.org/10.1111/jfd.13353DOI Listing
February 2021

Development of molecular and histological methods to evaluate stress oxidative biomarkers in sea bass (Dicentrarchus labrax).

Fish Physiol Biochem 2020 Aug 9;46(4):1577-1588. Epub 2020 May 9.

Istituto Zooprofilattico Sperimentale delle Venezie, National Reference Laboratory for Fish, Mollusc and Crustacean Diseases, Viale Università, 10, 35020, Legnaro, Italy.

In aquaculture, fish species may experience stressful episodes caused by poor farming conditions. The exponential increase of global aquaculture has raised the number of research studies aimed at demonstrating the sensitivity of aquatic animals in confined environments. The development of a real-time PCR and immunohistochemistry methods were investigated to evaluate the presence, localization, and quantity of biomarkers of oxidative stress in European sea bass (Dicentrarchus labrax). In particular, stress tests such as manipulation and temperature changes were conducted through molecular methods to identify the expression level of heat shock protein 70 (HSP70) in stressed animals compared with a control group. The immunohistochemical technique was also applied to locate and study the trends-levels of nitrotyrosine (NT), heat shock protein 70 (HSP70), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in different tissues from stressed animals and control group. The presence of the rodlet cell (RCs) was evaluated by histology in both a control and stressed group. Our results show that the real-time PCR method developed is specific for the evaluated target gene and that manipulation and temperature increase are strong stressors for animals. Relative quantification data revealed a gene expression increase of HSP70 in the stressed group of animals compared to the control group. The antibodies used for the immunohistochemical staining were efficient, and it was possible to appreciate the increase of immunoprecipitates in European sea bass either manipulated or stressed by temperature increase. The present study can be a starting point to allow the quantification of HSP70 and the identification of other stress biomarkers in D. labrax.
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http://dx.doi.org/10.1007/s10695-020-00811-xDOI Listing
August 2020

Behavioral and physiological responses to stocking density in sea bream (Sparus aurata): Do coping styles matter?

Physiol Behav 2019 12 15;212:112698. Epub 2019 Oct 15.

COISPA Tecnologia & Ricerca, Bari, Italy.

Stocking density is considered a stress factor for fish and is therefore one of the numerous concerns about fish welfare in an aquaculture context. Stress coping styles (SCS) are defined as a coherent set of individual physiological and behavioral differences in stress responses that are consistent across time and context and appear to be promising for improving fish welfare in aquaculture. The aim of the present study was to describe the physiological and zootechnical performances of gilthead sea bream (Sparus aurata) at different stocking densities (low density, LD: 15 kg/m and high density, HD: 30 kg/m), depending on individual SCS. To do so, the fish SCS were first screened by measuring boldness (prior to the experiment). Three consecutive samplings were performed over the experiment to measure several blood parameters, including hematocrit (Hct), red blood cell count (RBCC), hemoglobin (Hb), cortisol, adrenalin, noradrenalin, glucose, lactate, and lysozyme, to infer the consequence of the SCS profile on the welfare condition in response to stocking density. Finally, swimming activity was recorded in a subsample of individuals (9 BOLD and 9 SHY individuals per density), and BOLD individuals displayed higher swimming activity than SHY ones at HD, while the opposite pattern was observed at LD. According to principal component analysis, physiological parameters are linked to the SCS profile, mostly at the beginning of the experiment, while density effects on physiology remain during the entire experiment duration. In conclusion, regarding all the variables observed, fish SCS appeared to be promising criteria to select the most adaptive individuals relating to rearing conditions and therefore improve welfare.
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http://dx.doi.org/10.1016/j.physbeh.2019.112698DOI Listing
December 2019

Occurrence of nodular gill disease in farmed brown trout (Salmo trutta L.).

J Fish Dis 2019 09 3;42(9):1315-1320. Epub 2019 Jun 3.

Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy.

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http://dx.doi.org/10.1111/jfd.13027DOI Listing
September 2019

Systemic polymicrobic infection involving Shewanella putrefaciens group in koi.

J Fish Dis 2018 Dec 26;41(12):1929-1931. Epub 2018 Sep 26.

Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy.

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http://dx.doi.org/10.1111/jfd.12892DOI Listing
December 2018

Induced expression of cathelicidins in trout (Oncorhynchus mykiss) challenged with four different bacterial pathogens.

J Pept Sci 2018 Jul 28;24(7):e3089. Epub 2018 May 28.

Department of Life Sciences, University of Trieste, Via L. Giorgieri 5, 34127, Trieste, Italy.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.
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http://dx.doi.org/10.1002/psc.3089DOI Listing
July 2018

Genomic Diversity and Evolution of the Fish Pathogen .

Front Microbiol 2018 7;9:138. Epub 2018 Feb 7.

Unité Mathématiques et Informatique Appliquées du Génome à l'Environnement (MaIAGE), Institut National de la Recherche Agronomique, Université Paris-Saclay, Jouy-en-Josas, France.

, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the species is analyzed using a set of 41 genomes, including 30 newly sequenced isolates. These were selected on the basis of available MLST data with the two-fold objective of maximizing the coverage of the species diversity and of allowing a focus on the main clonal complex (CC-ST10) infecting farmed rainbow trout () worldwide. The results reveal a bacterial species harboring a limited genomic diversity both in terms of nucleotide diversity, with ~0.3% nucleotide divergence inside CDSs in pairwise genome comparisons, and in terms of gene repertoire, with the core genome accounting for ~80% of the genes in each genome. The pan-genome seems nevertheless "open" according to the scaling exponent of a power-law fitted on the rate of new gene discovery when genomes are added one-by-one. Recombination is a key component of the evolutionary process of the species as seen in the high level of apparent homoplasy in the core genome. Using a Hidden Markov Model to delineate recombination tracts in pairs of closely related genomes, the average recombination tract length was estimated to ~4.0 Kbp and the typical ratio of the contributions of recombination and mutations to nucleotide-level differentiation (r/m) was estimated to ~13. Within CC-ST10, evolutionary distances computed on non-recombined regions and comparisons between 22 isolates sampled up to 27 years apart suggest a most recent common ancestor in the second half of the nineteenth century in North America with subsequent diversification and transmission of this clonal complex coinciding with the worldwide expansion of rainbow trout farming. With the goal to promote the development of tools for the genetic manipulation of , a particular attention was also paid to plasmids. Their extraction and sequencing to completion revealed plasmid diversity that remained hidden to classical plasmid profiling due to size similarities.
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http://dx.doi.org/10.3389/fmicb.2018.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808330PMC
February 2018

First report of a fish kill episode caused by pyrethroids in Italian freshwater.

Forensic Sci Int 2017 Dec 6;281:176-182. Epub 2017 Nov 6.

Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, Legnaro (PD), 35020, Italy.

Introduction: Fish kills are events of strong emotional impact on the population because of the frequent suspicion that they can be the result of serious pollution accidents. As a matter of fact, they are often due to natural occurrences, such as low levels of dissolved oxygen in the water, but in many cases the causes remain unknown. Fish are particularly sensitive to pesticides and pyrethroids are reported to be the most ecotoxicologically active in the aquatic environment. Nevertheless, the reported cases of massive wild fish mortalities due to these toxicants are very few. This paper describes a fish kill episode occurred in the Padua Province (Veneto Region - North Eastern Italy) which involved several fish species and for which it was possible to identify the cause in the presence of pyrethroids in the water.

Case Presentation: When a whitish liquid coming from the rainwater drain of an industrial area was seen to be spilling into a drainage channel, a fish massive mortality was noticed and investigated. The collected water samples showed the presence of relevant concentrations of cypermethrin, permethrin, deltamethrin and tetramethrin. Analyses on the fish tissues revealed the presence of cypermethrin and permethrin at a concentration range of 476-2834μg/kg and 346-2826μg/kg on a lipid basis, respectively.

Discussion: According to the results of the performed analyses, we can reasonably state that the described episode had been caused by the exposure of biota to high concentrations of pyrethroids. The present case report significantly contributes to the limited literature available on pesticides-related fish kills. Moreover, it highlights the importance of sharing protocols for fish kill management at a national level, as this would help to better define the roles of the different institutions involved and to improve the investigation and the reporting of these events.
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http://dx.doi.org/10.1016/j.forsciint.2017.10.040DOI Listing
December 2017

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).

J Invertebr Pathol 2018 01 6;151:59-75. Epub 2017 Nov 6.

Department of Earth and Environmental Sciences, University of Pavia, Via Adolfo Ferrata 7, I-27100 Pavia, Italy.

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.
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http://dx.doi.org/10.1016/j.jip.2017.11.002DOI Listing
January 2018

Genomic Sequencing of Ranaviruses Isolated from Edible Frogs ().

Genome Announc 2017 Sep 21;5(38). Epub 2017 Sep 21.

Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

Ranaviruses were isolated from wild edible frogs () during epizootics in Denmark and Italy. Phylogenomic analyses revealed that these isolates are closely related and belong to a clade of ranaviruses that includes the ranavirus (ADRV), common midwife toad ranavirus (CMTV), ranavirus (THRV), and pike-perch iridovirus (PPIV).
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http://dx.doi.org/10.1128/genomeA.01015-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609427PMC
September 2017

Occurrence and molecular characterisation of Vibrio parahaemolyticus in crustaceans commercialised in Venice area, Italy.

Int J Food Microbiol 2016 Mar 24;220:39-49. Epub 2015 Dec 24.

Istituto Zooprofilattico Sperimentale delle Venezie, Viale Dell'Università 10, 35020 Legnaro, Padua, Italy.

Infections due to the pathogenic human vibrios, Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus, are mainly associated with consumption of raw or partially cooked bivalve molluscs. At present, little is known about the presence of Vibrio species in crustaceans and the risk of vibriosis associated with the consumption of these products. The aim of the present study was to evaluate the prevalence and concentration of the main pathogenic Vibrio spp. in samples of crustaceans (n=143) commonly eaten in Italy, taking into account the effects of different variables such as crustacean species, storage conditions and geographic origin. Subsequently, the potential pathogenicity of V. parahaemolyticus strains isolated from crustaceans (n=88) was investigated, considering the classic virulence factors (tdh and trh genes) and four genes coding for relevant proteins of the type III secretion systems 2 (T3SS2α and T3SS2β). In this study, the presence of V. cholerae and V. vulnificus was never detected, whereas 40 samples (28%) were positive for V. parahaemolyticus with an overall prevalence of 41% in refrigerated products and 8% in frozen products. The highest prevalence and average contamination levels were detected in Crangon crangon (prevalence 58% and median value 3400 MPN/g) and in products from the northern Adriatic Sea (35%), with the samples from the northern Venetian Lagoon reaching a median value of 1375 MPN/g. While genetic analysis confirmed absence of the tdh gene, three of the isolates contained the trh gene and, simultaneously, the T3SS2β genes. Moreover three possibly clonal tdh-negative/trh-negative isolates carried the T3SS2α apparatus. The detection of both T3SS2α and T3SS2β apparatuses in V. parahaemolyticus strains isolated from crustaceans emphasised the importance of considering new genetic markers associated with virulence besides the classical factors. Moreover this study represents the first report dealing with Vibrio spp. in crustaceans in Italy, and it may provide useful information for the development of sanitary surveillance plans to prevent the risk of vibriosis in seafood consumers.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2015.12.007DOI Listing
March 2016

Synergistic effect of high hydrostatic pressure (HHP) and marination treatment on the inactivation of hepatitis a virus in mussels (Mytilus galloprovincialis).

Food Environ Virol 2015 Mar 25;7(1):76-85. Epub 2014 Oct 25.

Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, via Bianchi 7/9, 25124, Brescia, Italy,

Consumption of raw or insufficiently cooked mussels contaminated with hepatitis A virus (HAV) is a major cause of infection to humans. The origin of mussels commonly used for the preparation of marinated seafood salads is often unknown, since different producers worldwide undergo a precooking treatment at the original collection site with methods and parameters not always indicated. These treatments could be insufficient for the inactivation of HAV, which is characterized by a high temperature resistance. Both high hydrostatic pressure (HHP) and marinade treatments have been shown to affect HAV vitality. In this study, two treatments (HHP and marinating) were combined in order to assess a potential synergistic effect on the virus vitality. A kinetic test was conducted by subjecting the experimentally-contaminated mussels (HAV titre: 10(6)/ml TCID50) to marinating, and to different HHP treatment (4,000; 5,000; and 6,000 bar for 1, 5, and 9 min). Virus post-treatment vitality was assessed by its ability to grow on cell cultures and by quantitative real-time RT-PCR to evaluate virus resistance under such conditions. Marinating treatment alone (final pH 4.3, and NaCl 2 %) did not inactivate the virus. On the other hand, the use of HHP treatment alone on non-marinated HAV-contaminated mussels was effective only above 5,000 bar for 5 min. The results of the present study elucidate the synergistic effect of a combination between marination and HHP treatments on the inactivation of the virus.
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http://dx.doi.org/10.1007/s12560-014-9167-zDOI Listing
March 2015

Determination of microbial diversity of Aeromonas strains on the basis of multilocus sequence typing, phenotype, and presence of putative virulence genes.

Appl Environ Microbiol 2011 Jul 3;77(14):4986-5000. Epub 2011 Jun 3.

Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padova, Agripolis, Viale dell'Università 16, 35020 Legnaro, Padua, Italy.

The genus Aeromonas has been described as comprising several species associated with the aquatic environment, which represents their principal reservoir. Aeromonas spp. are commonly isolated from diseased and healthy fish, but the involvement of such bacteria in human infection and gastroenteritis has frequently been reported. The primary challenge in establishing an unequivocal link between the Aeromonas genus and pathogenesis in humans is the extremely complicated taxonomy. With the aim of clarifying taxonomic relationships among the strains and phenotypes, a multilocus sequencing approach was developed and applied to characterize 23 type and reference strains of Aeromonas spp. and a collection of 77 field strains isolated from fish, crustaceans, and mollusks. All strains were also screened for putative determinants of virulence by PCR (ast, ahh1, act, asa1, eno, ascV, and aexT) and the production of acylated homoserine lactones (AHLs). In addition, the phenotypic fingerprinting obtained from 29 biochemical tests was submitted to the nonparametric combination (NPC) test methodology to define the statistical differences among the identified genetic clusters. Multilocus sequence typing (MLST) achieved precise strain genotyping, and the phylogenetic analysis of concatenated sequences delineated the relationship among the taxa belonging to the genus Aeromonas, providing a powerful tool for outbreak traceability, host range diffusion, and ecological studies. The NPC test showed the feasibility of phenotypic differentiation among the majority of the MLST clusters by using a selection of tests or the entire biochemical fingerprinting. A Web-based MLST sequence database (http://pubmlst.org/aeromonas) specific for the Aeromonas genus was developed and implemented with all the results.
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http://dx.doi.org/10.1128/AEM.00708-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147379PMC
July 2011