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12 pages, 457 KiB

Open AccessArticle

Screening of New Potential Probiotics Strains against Photobacterium damselae Subsp. piscicida for Marine Aquaculture

by Ana Gutiérrez Falcón, Daniel Padilla, Fernando Real, María José Ramos Sosa, Begoña Acosta-Hernández, Andrés Sánchez Henao, Natalia García-Álvarez, Inmaculada Rosario Medina, Freddy Silva Sergent, Soraya Déniz and José Luís Martín-Barrasa

Animals 2021, 11(7), 2029; https://doi.org/10.3390/ani11072029 - 7 Jul 2021

Cited by 5 | Viewed by 3731

Abstract

On intensive fish farms, 10% of the population dies exclusively from pathogens, and Photobacterium damselae subsp. Piscicida (Ph. damselae subsp. Piscicida), the bacteria causing pasteurellosis in marine aquaculture, is one of the major pathogens involved. The objective of this study was to [...] Read more.

On intensive fish farms, 10% of the population dies exclusively from pathogens, and Photobacterium damselae subsp. Piscicida (Ph. damselae subsp. Piscicida), the bacteria causing pasteurellosis in marine aquaculture, is one of the major pathogens involved. The objective of this study was to obtain new probiotic strains against pasteurellosis in order to limit the use of chemotherapy, avoiding the environmental repercussions generated by the abusive use of these products. In this study, 122 strains were isolated from the gills and intestines of different marine fish species and were later evaluated in vitro to demonstrate the production of antagonistic effects, the production of antibacterial substances, adhesion and growth to mucus, resistance to bile and resistance to pH gradients, as well as its harmlessness and the dynamic of expression of immune-related genes by real-time PCR after administration of the potential probiotic in the fish diet. Only 1/122 strains showed excellent results to be considered as a potential probiotic strain and continue its characterization against Ph. damselae subsp. piscicida to determine its protective effect and elucidating in future studies its use as a possible probiotic strain for marine aquaculture. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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19 pages, 3610 KiB

Open AccessArticle

Evaluation of Gilthead Seabream (Sparus aurata) Immune Response after LCDV-Sa DNA Vaccination

by Rocío Leiva-Rebollo, Dolores Castro, Patricia Moreno, Juan J. Borrego and Alejandro M. Labella

Animals 2021, 11(6), 1613; https://doi.org/10.3390/ani11061613 - 29 May 2021

Cited by 3 | Viewed by 4341

Abstract

Lymphocystis disease is the main viral pathology reported in gilthead seabream. Its etiological agent is Lymphocystis disease virus 3 (LCDV-Sa), genus Lymphocystivirus, family Iridoviridae. There are no effective treatments or vaccines for LCDV control, thus the main aim of this study was [...] Read more.

Lymphocystis disease is the main viral pathology reported in gilthead seabream. Its etiological agent is Lymphocystis disease virus 3 (LCDV-Sa), genus Lymphocystivirus, family Iridoviridae. There are no effective treatments or vaccines for LCDV control, thus the main aim of this study was to develop a DNA vaccine, and to evaluate both the protection conferred against LCDV-Sa infection and the immune response in vaccinated fish. The vaccine was constructed by cloning the mcp gene (ORF LCDVSa062R) into pcDNA3.1/NT-GFP-TOPO. Two independent vaccination trials were conducted. In the first one, 5–7 g fish were intramuscularly injected with the vaccine (pcDNA-MCP) or the empty-plasmid, and the distribution and expression of the vaccine was investigated. Furthermore, vaccinated fish were challenged with LCDV-Sa in order to access the protective capacity of the vaccine. In the second trial, 70–100 g fish were vaccinated as specified, and the immune response was evaluated analyzing the expression of 23 immune-related genes and the production of specific antibodies. The results showed that the vaccine triggers an immune response characterized by the overexpression of genes relating to the inflammatory process, but not the innate antiviral immunity relating to type I IFN (interferon), and also induces the production of specific neutralizing antibodies, which could explain the protection against LCDV-Sa in vaccinated fish. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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21 pages, 918 KiB

Open AccessArticle

Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards

by José G. Olveira, Sandra Souto, Isabel Bandín and Carlos P. Dopazo

Animals 2021, 11(4), 1100; https://doi.org/10.3390/ani11041100 - 12 Apr 2021

Cited by 11 | Viewed by 2497

Abstract

The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediterranean countries. Fast and accurate diagnosis is essential to control it, and viral quantification is required to predict the level of risk of new viral detections in field samples. [...] Read more.

The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediterranean countries. Fast and accurate diagnosis is essential to control it, and viral quantification is required to predict the level of risk of new viral detections in field samples. For both, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) is used by diagnostic laboratories. In the present study, we developed an RT-qPCR procedure for the diagnosis and simultaneous quantification of NNV isolates from any of the four genotypes. The method proved to be highly sensitive in terms of crude virus titer: 5.56–9.88 TCID₅₀/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID₅₀/mL or 0.08 TCID₅₀/reaction. Other standards also yielded very low detection limits: 16.3 genome copies (cps) of purified virus per mL, 2.36 plasmid cps/mL, 7.86 in vitro synthetized RNA cps/mL, and 3.16 TCID₅₀/mL of virus from infected tissues. The diagnostic parameters evaluated in fish samples were much higher in comparison to cell culture isolation and nested PCR. In addition, the high repeatability and reproducibility of the procedure, as well as the high coefficient of determination (R²) of all the calibration curves with any type of standard tested, ensure the high reliability of the quantification of NNV using this RT-qPCR procedure, regardless of the viral type detected and from the type of standard chosen. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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16 pages, 1834 KiB

Open AccessArticle

Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV)

by Carmen López-Vázquez, Isabel Bandín and Carlos P. Dopazo

Animals 2021, 11(3), 841; https://doi.org/10.3390/ani11030841 - 16 Mar 2021

Viewed by 1541

Abstract

The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. [...] Read more.

The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5–50 TCID₅₀/_mL), analytical specificity (intra and intergroup), efficiency (E = 100.0–101.1) and reliability (repeatability and reproducibility with CV < 5%, and standard curves with R² < 0.95) with strains from any VHSV genotype have been widely demonstrated. The procedure is based on the ‘binary multiplex RT-qPCR system (bmRT-qPCR)’ previously reported by the same team, applied to arrays of 96-well PCR strip tubes plates, which can be stored at −25 °C for three months and up to one year before their use, without significant loss of efficiency. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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19 pages, 3065 KiB

Open AccessArticle

A Seasonal Study of Koi Herpesvirus and Koi Sleepy Disease Outbreaks in the United Kingdom in 2018 Using a Pond-Side Test

by Irene Cano, John Worswick, Brian Mulhearn, David Stone, Gareth Wood, Jacqueline Savage and Richard Paley

Animals 2021, 11(2), 459; https://doi.org/10.3390/ani11020459 - 9 Feb 2021

Cited by 7 | Viewed by 2734

Abstract

Fluorescence real-time LAMP assays were designed for the orf43 gene of CyHV-3 European genotype and the p4a gene of the CEV genogroup I. A third LAMP assay to detect the ef1a gene of the host common carp was designed as an internal control. [...] Read more.

Fluorescence real-time LAMP assays were designed for the orf43 gene of CyHV-3 European genotype and the p4a gene of the CEV genogroup I. A third LAMP assay to detect the ef1a gene of the host common carp was designed as an internal control. The limit of detection was 10² and 10³ viral copies under 25 min for CyHV-3 and CEV, respectively. The specificity of the CyHV-3 LAMP assay was 95.6% of 72 fish herpesviruses tested. Sixty-three non-lethal common carp mucus swabs were collected across 16 sites during disease investigations. DNA extractions were performed in under 10 min using the QuickExtract™ digestion buffer. The LAMP amplification of CyHV-3 DNA in mucus swabs from clinical cases was detected from 4 to 13 min in 13 sites, while a co-infection of CyHV-3 and CEV was confirmed by LAMP in a single site. The LAMP results agreed with the results of the reference laboratory. The common carp ef1a was amplified only in 61% of the mucus swabs collected, preventing its use as a robust internal control to distinguish false negatives from invalid tests. After further optimization, these tests could be implemented for border inspection posts surveillance and decentralizing testing during disease outbreaks. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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26 pages, 5204 KiB

Open AccessArticle

Steps of the Replication Cycle of the Viral Haemorrhagic Septicaemia Virus (VHSV) Affecting Its Virulence on Fish

by Carmen López-Vázquez, Isabel Bandín, Valentina Panzarin, Anna Toffan, Argelia Cuenca, Niels J. Olesen and Carlos P. Dopazo

Animals 2020, 10(12), 2264; https://doi.org/10.3390/ani10122264 - 1 Dec 2020

Cited by 7 | Viewed by 2118

Abstract

The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying [...] Read more.

The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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15 pages, 2630 KiB

Open AccessArticle

First Identification and Characterization of Lactococcus garvieae Isolated from Rainbow Trout (Oncorhynchus mykiss) Cultured in Mexico

by Cesar Ortega, Rute Irgang, Benjamín Valladares-Carranza, Constanza Collarte and Ruben Avendaño-Herrera

Animals 2020, 10(9), 1609; https://doi.org/10.3390/ani10091609 - 9 Sep 2020

Cited by 20 | Viewed by 4419

Abstract

Lactococcosis is a hyperacute hemorrhagic septicemia disease caused by Lactococcus garvieae, which is an emerging pathogen in global fish farming. Between 2016 and 2018, rainbow trout (Oncorhynchus mykiss) from five farms that presented outbreaks were sampled as part of a [...] Read more.

Lactococcosis is a hyperacute hemorrhagic septicemia disease caused by Lactococcus garvieae, which is an emerging pathogen in global fish farming. Between 2016 and 2018, rainbow trout (Oncorhynchus mykiss) from five farms that presented outbreaks were sampled as part of a Mexican surveillance program for the detection of fish diseases. Fourteen L. garvieae isolates were recovered from sampled fish, as confirmed by biochemical tests, 16S rRNA gene sequencing, and clinical and histological insights. The biochemical and protein profiles of the isolates obtained were homogeneous. Repetitive extragenic palindromic—(REP)—and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) analyses established weak genetic heterogeneity. Rainbow trout challenged with two of the isolates used at different bacterial concentrations (10⁻² and 10⁻⁴ CFU/mL) showed melanosis, and hemorrhages were observed in the fins, liver, kidney, and spleen. Isolates were obtained from all of the organs sampled, including from surviving fish, as either pure or mixed cultures. The present study is the first to confirm the presence of L. garvieae as the agent of severe lactococcosis outbreaks in the two primary Mexican states for trout farming. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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14 pages, 1744 KiB

Open AccessArticle

Possible Mechanisms of Action of Two Pseudomonas fluorescens Isolates as Probiotics on Saprolegniosis Control in Rainbow Trout (Oncorhynchus mykiss Walbaum)

by Concepción González-Palacios, Juan-Miguel Fregeneda-Grandes and José-Miguel Aller-Gancedo

Animals 2020, 10(9), 1507; https://doi.org/10.3390/ani10091507 - 26 Aug 2020

Cited by 7 | Viewed by 2851

Abstract

Probiotics have been proposed as one of the alternatives to the chemical treatments currently used in aquaculture. Recently, the possible usefulness of certain microorganisms, mainly bacteria, has been highlighted as a potential biocontrol for saprolegniosis. In the present work we investigated the possible [...] Read more.

Probiotics have been proposed as one of the alternatives to the chemical treatments currently used in aquaculture. Recently, the possible usefulness of certain microorganisms, mainly bacteria, has been highlighted as a potential biocontrol for saprolegniosis. In the present work we investigated the possible mechanisms of action of two isolates of Pseudomonas fluorescens (LE89 and LE141) with proven ability to reduce Saprolegnia parasitica infection in rainbow trout under experimental conditions when they are added to the tank water. The stimulation of the innate immune response and the production of siderophores and bioactive substances inhibiting S. parasitica present in cells and supernatants of LE89 and LE141 were studied. Regarding the immune response the only noteworthy points were the increase in the phagocytic activity of macrophages and the concentration of serum proteins when LE141 was administered. Both bacteria produced siderophores. When analyzing the protein substances present in supernatants, it was observed that in both isolates the proteins with inhibitory activity present might be siderophores. In LE141, besides siderophores, a protein of 66 kDa was identified in the fraction responsible for inhibition. To sum up, the two P. fluorescens isolates might be usable for biocontrol of saprolegniosis and that the mode of action of these bacteria is likely to be related to the production of siderophores. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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10 pages, 286 KiB

Open AccessArticle

In Vitro Antibacterial Potential of Salix babylonica Extract against Bacteria that Affect Oncorhynchus mykiss and Oreochromis spp.

by Lenin Rangel-López, Adrian Zaragoza-Bastida, Benjamín Valladares-Carranza, Armando Peláez-Acero, Carolina G. Sosa-Gutiérrez, Helal F. Hetta, Gaber El-Saber Batiha, Ali Alqahtani and Nallely Rivero-Perez

Animals 2020, 10(8), 1340; https://doi.org/10.3390/ani10081340 - 3 Aug 2020

Cited by 11 | Viewed by 3535

Abstract

Aquaculture development is limited by bacteria associated with several diseases; antibiotics are used for the treatment of these affections, but bacteria have developed resistance to these drugs. It is important to develop effective treatments that allow the production of antibiotic-free food. The aim [...] Read more.

Aquaculture development is limited by bacteria associated with several diseases; antibiotics are used for the treatment of these affections, but bacteria have developed resistance to these drugs. It is important to develop effective treatments that allow the production of antibiotic-free food. The aim of the present study is to evaluate the in vitro antibacterial effects of Salix babylonica hydro-alcoholic extract (SbHE) against Aeromonas hydrophila, Listonella anguillarum, Edwarsiella tarda, and Streptococcus iniae, bacteria that affect Oncorhynchus mykiss and Oreochromis spp. production. SbHE was obtained through the maceration technique. Reference strains were used and their sensitivity to antibiotics was determined. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of SbHE were determined. Results showed that three of four evaluated bacteria were multidrug resistant, except S. iniae. SbHE showed antibacterial activity against all bacteria. Results indicate an MIC of 1.56 to 25 mg/mL and an MBC of 3.12 to 100 mg/mL. The greatest inhibitory activity occurred against L. anguillarum obtaining a MIC of 1.56 mg/mL and an MBC of 3.12 mg/mL. Results indicate that SbHE has bactericidal activity against A. hydrophila, L.anguilalurm, and S. iniae as well as bacteriostatic activity against E. tarda and could be an alternative treatment against these bacteria. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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17 pages, 2622 KiB

Open AccessArticle

Microbial Communities Associated with Farmed Genypterus chilensis: Detection in Water Prior to Bacterial Outbreaks Using Culturing and High-Throughput Sequencing

by Arturo Levican, Jenny C. Fisher, Sandra L. McLellan and Ruben Avendaño-Herrera

Animals 2020, 10(6), 1055; https://doi.org/10.3390/ani10061055 - 18 Jun 2020

Cited by 6 | Viewed by 2714

Abstract

The red conger eel (Genypterus chilensis, Guichenot) is a native species included in the Chilean Aquaculture Diversification Program due to high commercial demand. In the context of intensified farming, prior reports link two disease outbreaks with emerging pathogens in the Vibrio [...] Read more.

The red conger eel (Genypterus chilensis, Guichenot) is a native species included in the Chilean Aquaculture Diversification Program due to high commercial demand. In the context of intensified farming, prior reports link two disease outbreaks with emerging pathogens in the Vibrio and Tenacibaculum genera. However, the roles remain unclear for the bacterial community and each specific bacterium is associated with the rearing environment for healthy specimens. The success of red conger eel farming therefore warrants research into the bacterial composition of aquaculture conditions and the antimicrobial susceptibilities thereof. This study used culturing methods and high-throughput sequencing to describe the bacterial community associated with water in which G. chilensis was farmed. With culturing methods, the predominant genera were Vibrio (21.6%), Pseudolteromonas (15.7%), Aliivibrio (13.7%), and Shewanella (7.8%). Only a few bacterial isolates showed amylase, gelatinase, or lipase activity, and almost all showed inhibition zones to commonly-used antibiotics in aquaculture. By contrast, high-throughput sequencing established Paraperlucidibaca, Colwellia, Polaribacter, Saprospiraceae, and Tenacibaculum as the predominant genera, with Vibrio ranking twenty-seventh in abundance. High-throughput sequencing also established a link between previous outbreaks with increased relative abundances of Vibrio and Tenacibaculum. Therefore, monitoring the presence and abundance of these potential pathogens could be useful in providing prophylactic measures to prevent future outbreaks. Full article

(This article belongs to the Special Issue Infectious diseases in aquaculture and fisheries, new and emerging pathogens. Disease control methods)

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