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      Mycobacterium pseudoshottsii in Mediterranean Fish Farms: New Trouble for European Aquaculture?

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          Abstract

          Mycobacterium pseudoshottsii, a slow-growing mycobacterium closely related to M. marinum, has been isolated only in wild fish in the United States and in Japanese fish farms to date. Here, we report cases of mortality in three farmed fish species ( Dicentrarchus labrax, Sparus aurata, and Sciaenops ocellatus) caused by M. pseudoshottsii in Italy. Samples underwent necropsy, histology, and culture with pathogen identification based on PCR and sequencing of housekeeping genes (16S rRNA, hsp65, rpoB). Multifocal to coalescing granulomatous and necrotizing inflammation with acid-fast bacilli were observed in the parenchymatous organs, from which M. pseudoshottsii was isolated and identified. Phylogenetic analysis confirmed the results of gene sequencing and allowed subdivision of the isolates into three distinct groups. M. pseudoshottsii poses a potential threat for Mediterranean aquaculture. Its origin in the area under study needs to be clarified, as well as the threat to the farmed fish species.

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          Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis.

          A method for the rapid identification of mycobacteria to the species level was developed on the basis of evaluation by the polymerase chain reaction (PCR) of the gene encoding for the 65-kDa protein. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria. Using two restriction enzymes, BstEII and HaeIII, medically relevant and other frequent laboratory isolates were differentiated to the species or subspecies level by PCR-restriction enzyme pattern analysis. PCR-restriction enzyme pattern analysis was performed on isolates (n = 330) from solid and fluid culture media, including BACTEC, or from frozen and lyophilized stocks. The procedure does not involve hybridization steps or the use of radioactivity and can be completed within 1 working day.
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            rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria.

            Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial beta subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited 3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM.
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              A review of the main bacterial fish diseases in mariculture systems

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                Author and article information

                Journal
                Pathogens
                Pathogens
                pathogens
                Pathogens
                MDPI
                2076-0817
                27 July 2020
                August 2020
                : 9
                : 8
                : 610
                Affiliations
                [1 ]Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, 10154 Torino, Italy; katia.varello@ 123456izsto.it (K.V.); paolo.pastorino@ 123456izsto.it (P.P.); vasco.menconi@ 123456izsto.it (V.M.); elena.bozzetta@ 123456izsto.it (E.B.); simona.zoppi@ 123456izsto.it (S.Z.); alessandro.dondo@ 123456izsto.it (A.D.); marino.prearo@ 123456izsto.it (M.P.)
                [2 ]Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum, 40064 Ozzano dell’Emilia (BO), Italy; andrea.gustinelli2@ 123456unibo.it (A.G.); daniela.florio@ 123456unibo.it (D.F.); marialeti.fioravanti@ 123456unibo.it (M.L.F.)
                Author notes
                [* ]Correspondence: davide.mugetti@ 123456izsto.it ; Tel.: +39-0112686251
                Author information
                https://orcid.org/0000-0002-0486-2121
                https://orcid.org/0000-0002-0585-1168
                https://orcid.org/0000-0003-1176-6995
                https://orcid.org/0000-0001-9949-4423
                https://orcid.org/0000-0002-9236-5933
                Article
                pathogens-09-00610
                10.3390/pathogens9080610
                7459456
                32726963
                959e579e-e2e6-4291-8100-4d880cb48b56
                © 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
                : 06 July 2020
                : 24 July 2020
                Categories
                Article

                atypical mycobacteria,slow-growing mycobacteria,mycobacterium marinum complex,emerging diseases,european sea bass,gilthead sea bream,red drum,granulomas,gene sequencing

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