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      Diagnosis of Centrocestus formosanus Infection in Zebrafish ( Danio rerio) in Italy: A Window to a New Globalization-Derived Invasive Microorganism

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          Abstract

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          Centrocestus formosanus is an invasive parasite which originated from outside the European countries. Infections by this parasite seem to be related to the movements of its hosts. However, in Europe, the presence of C. formosanus has been sporadically reported and its zoonotic potential is still underestimated. Therefore, the present study proposes a fast and inexpensive diagnostic method through molecular analyses targeting the ribosomal internal transcribed sequence 2 (ITS2) using a newly designed species-specific primer pair. Given the potential negative consequences of C. formosanus global expansion, those responsible should adopt a one health approach to control the spread of this organism.

          Abstract

          Centrocestus formosanus is a digenetic trematode with a complex life cycle, involving invertebrate and vertebrate hosts, humans included. In particular, it causes gill lesions and mortality in freshwater fish species, and gastrointestinal symptoms in infected humans. Here, we describe the occurrence of C. formosanus infection in zebrafish imported in Italy and propose a newly designed species-specific primer pair to ameliorate the diagnostic investigations for C. formosanus. Gill arches of 30 zebrafish were examined for the presence of encysted metacercariae under a stereomicroscope and processed through molecular analyses targeting the ribosomal internal transcribed sequence 2 (ITS2). Although C. formosanus distribution was originally restricted to Asia, it has been subsequently reported in new countries, revealing itself as an invasive species and raising important concerns for biodiversity, economy, scientific research, as well as animal and public health. Given the crucial role played by the ornamental fish industry in spreading this parasite, there is an urgent need for control measures to prevent the introduction and establishment of C. formosanus in non-endemic areas, including Europe. We also suggest developing new strategies in microbiology and epidemiology to better explore this new globalization-derived invasive species.

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          Most cited references42

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          Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda).

          Complete small subunit ribosomal RNA gene (ssrDNA) and partial (D1-D3) large subunit ribosomal RNA gene (lsrDNA) sequences were used to estimate the phylogeny of the Digenea via maximum parsimony and Bayesian inference. Here we contribute 80 new ssrDNA and 124 new lsrDNA sequences. Fully complementary data sets of the two genes were assembled from newly generated and previously published sequences and comprised 163 digenean taxa representing 77 nominal families and seven aspidogastrean outgroup taxa representing three families. Analyses were conducted on the genes independently as well as combined and separate analyses including only the higher plagiorchiidan taxa were performed using a reduced-taxon alignment including additional characters that could not be otherwise unambiguously aligned. The combined data analyses yielded the most strongly supported results and differences between the two methods of analysis were primarily in their degree of resolution. The Bayesian analysis including all taxa and characters, and incorporating a model of nucleotide substitution (general-time-reversible with among-site rate heterogeneity), was considered the best estimate of the phylogeny and was used to evaluate their classification and evolution. In broad terms, the Digenea forms a dichotomy that is split between a lineage leading to the Brachylaimoidea, Diplostomoidea and Schistosomatoidea (collectively the Diplostomida nomen novum (nom. nov.)) and the remainder of the Digenea (the Plagiorchiida), in which the Bivesiculata nom. nov. and Transversotremata nom. nov. form the two most basal lineages, followed by the Hemiurata. The remainder of the Plagiorchiida forms a large number of independent lineages leading to the crown clade Xiphidiata nom. nov. that comprises the Allocreadioidea, Gorgoderoidea, Microphalloidea and Plagiorchioidea, which are united by the presence of a penetrating stylet in their cercariae. Although a majority of families and to a lesser degree, superfamilies are supported as currently defined, the traditional divisions of the Echinostomida, Plagiorchiida and Strigeida were found to comprise non-natural assemblages. Therefore, the membership of established higher taxa are emended, new taxa erected and a revised, phylogenetically based classification proposed and discussed in light of ontogeny, morphology and taxonomic history.
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            Anesthesia and euthanasia in zebrafish.

            Because of the relative ease of embryonic manipulation and observation, the ability to produce a great number of genetic mutations, efficient screening methods, and the continued advance of molecular genetic tools, such as the progress in sequencing and mapping of the zebrafish genome, the use of zebrafish (Danio rerio) as a biomedical model organism continues to expand. However, studies involving zebrafish husbandry and veterinary care struggle to keep pace with scientific progress. This article outlines some of the current, acceptable methods for providing anesthesia and euthanasia and provides some examples of how performance-based approaches can be used to advance the relatively limited number of anesthetic and euthanizing techniques available for zebrafish.
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              Probiotic modulation of the microbiota-gut-brain axis and behaviour in zebrafish

              The gut microbiota plays a crucial role in the bi-directional gut–brain axis, a communication that integrates the gut and central nervous system (CNS) activities. Animal studies reveal that gut bacteria influence behaviour, Brain-Derived Neurotrophic Factor (BDNF) levels and serotonin metabolism. In the present study, we report for the first time an analysis of the microbiota–gut–brain axis in zebrafish (Danio rerio). After 28 days of dietary administration with the probiotic Lactobacillus rhamnosus IMC 501, we found differences in shoaling behaviour, brain expression levels of bdnf and of genes involved in serotonin signalling/metabolism between control and treated zebrafish group. In addition, in microbiota we found a significant increase of Firmicutes and a trending reduction of Proteobacteria. This study demonstrates that selected microbes can be used to modulate endogenous neuroactive molecules in zebrafish.
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                Author and article information

                Journal
                Animals (Basel)
                Animals (Basel)
                animals
                Animals : an Open Access Journal from MDPI
                MDPI
                2076-2615
                09 March 2020
                March 2020
                : 10
                : 3
                : 456
                Affiliations
                [1 ]Department of Veterinary Medicine and Animal Productions, Università degli Studi di Napoli Federico II, via Delpino 1, 80137 Naples, Italy; antonino.pace@ 123456unina.it (A.P.); ludovico.dipineto@ 123456unina.it (L.D.); lorena.varriale@ 123456unina.it (L.V.); laura.rinaldi@ 123456unina.it (L.R.); menna@ 123456unina.it (L.F.M.); fioretti@ 123456unina.it (A.F.)
                [2 ]Department of Biology, Università degli Studi di Napoli Federico II, via Cintia 26, 80126 Naples, Italy; serena.aceto@ 123456unina.it (S.A.); mariaconcettacensullo@ 123456gmail.com (M.C.C.); mariacarmen.valoroso@ 123456unina.it (M.C.V.)
                Author notes
                Author information
                https://orcid.org/0000-0003-0177-3219
                https://orcid.org/0000-0003-3906-4872
                https://orcid.org/0000-0002-4210-7098
                Article
                animals-10-00456
                10.3390/ani10030456
                7143865
                32182941
                807b944b-cd2a-4f7d-9022-21f788b8a4a4
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 10 February 2020
                : 07 March 2020
                Categories
                Article

                digenetic trematodes,gill fluke,invasive species,molecular diagnosis,species-specific primer pair,zoonosis

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