Fish Pathogens

The genus Tenacibaculum encompasses several species pathogenic for marine fish. Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" (Quotation marks denote species that have not been validly named.) were retrieved from skin lesions of farmed fish such as European sea bass or Atlantic salmon. They cause a condition referred to as tenacibaculosis and severe outbreaks and important fish losses have been reported in Spanish, Norwegian, and Chilean marine farms. We report here the draft genomes of the T. dicentrarchi and "T. finnmarkense" type strains. These genomes were compared with draft genomes from field isolates retrieved from Chile and Norway and with previously published Tenacibaculum genomes. We used Average Nucleotide Identity and core genome-based phylogeny as a proxy index for species boundary delineation. This work highlights evolution of closely related fish-pathogenic species and suggests that homologous recombination likely contributes to genome evolution. It also corrects the species affiliation of strain AYD7486TD claimed by .

The validity and distinctiveness of Vibrio anguilla rum (= Listonella anguillara), v: (= Photo bacterium) damsela, v: ordalii and v: salmonicida was confirmed. However, strains received as v: cholerae and v: splendidus were heterogeneous. Ribotyping, phenotypic (BIOLOG-GN fingerprints and API 20E profiles), chemotaxonomic (lipopolysaccharide [LPS] and outer membrane proteins [OMP]), serogrouping and plasmid profiling data were not always congruent. v: anguillarum isolates were recovered in a single ribotype cluster, but many serogroups. There was little variation in OMP profiles, but not so for LPS profiles and plasmid composition. Heterogeneity was recorded in the phenotypic characters, particularly with the API 20E rapid identification system. v: damsela displayed heterogeneity by ribotyping, but homogeneity by BIOLOG-GN fingerprints and API 20E profiles. Four serogroups were defined, but only one LPS profile was recognised. v: ordalii was homogeneous by ribotyping, serogrouping and plasmid profiling, was accommodated in two LPS groups, but was more heterogeneous by BIOLOG-GN and API 20E. Despite its more exacting cultural requirements, v: salmonicida autoagglutinated and could not be serogrouped, but was accommodated in a single LPS group showing a profile associated with rough strains, and contained plasm ids of 4.7 and 42 kb. Heterogeneity was recorded with the API 20E rapid identification system.

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The efficacy of cellular components of probiotics Kocuria SM1 and Rhodococcus SM2 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against vibriosis was assessed. Groups of fish (average weight = 10-15 g) were immunized intraperitoneally (i.p.) with 0.1 ml of subcellular materials, i.e. 0.2 ± 0.05 mg protein per fish, comprising extracellular proteins (ECPs), cell wall proteins (CWPs) and whole cell proteins (WCPs) of SM1 and SM2, respectively, or with 0.1 ml of phosphate-buffered saline (PBS) to serve as the control. Seven days after administration, fish from each group were challenged i.p. with 0.1 ml of a suspension in PBS of 3 × 10 5 cells ml −1 per fish of Vibrio anguillarum. Use of CWPs and WCPs demonstrated significantly (P < 0.05) better protection against V. anguillarum insofar as mortalities were reduced to 11-17% [relative percent survival (RPS) = 80-87%], although ECPs fared less well (mortalities = 33-38%; RPS = 56-62%; P > 0.05), compared to 86% mortalities of the controls. The mode of action reflected activation of innate immune factors by CWPs and WCPs, demonstrating significantly (P < 0.05) increased expression of respiratory burst (optical density; OD 550nm) from 0.039 to 0.043-0.045, peroxidase (OD 550nm) from 0.26 to 0.37-0.55, and bacterial killing activities (i.e. percentage of surviving bacteria reduced from 79% to 56-57% for SM2). Moreover, an elevation of leucocyte number (from 1.93% to 1.98-2.93%; P > 0.05) and immunoglubolin level (from 27 mg ml −1 to 28.5-33 mg ml −1 ; P > 0.05) were observed with the experimental groups. These results indicate that cell components of the probiotics stimulate an immune response.

Fifty two isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and geographical locations, were heterogeneous in terms of molecular and phenotypic characteristics, and represented taxa which could not be accommodated by the current classification of four subspecies. Generally, there was incongruence between the molecular (PCR, RAPD and ribotyping) and phenotypic methods in terms of cluster membership. By PCR, 6 groups were described of which Group 1 encompassed 12 isolates including the type strain of A. salmonicida subsp. smithia. Group 2 accommodated 23 isolates including the reference cultures of subspecies achromogenes and masoucida. The named culture of Haemophilus piscium was recovered in Group 6. By ribotyping and RAPD, the reference cultures were recovered in separate groups. All methods pointed to the uniqueness of subspecies smithia. Most isolates contained 2-6 plasmids, of 2.3 to 150 kb in length. Nevertheless, all isolates possessed certain key characteristics, including Gram-negativity, and the absence of motility.

Studies on the bacteriocins of Bacillus species are increasing due to enhanced research interests and benefits for diverse applications ranging from bio-preservatives to probiotics, providing natural antimicrobial functions in fish farming and related aquaculture production needs. These benefits are due to their broad spectrum of antimicrobial activity and mode of action, their ability to secrete these important peptides in large amounts (g/L level) in a culture medium, and the ubiquitous nature of producing strains. It is well known that fish farming industries are challenged with pathogenic infection, indiscriminate antibiotic use, and now linked to antibiotic resistance of pathogens due to poor sewage disposals and persistence in nature. To overcome these challenges and the quest to stop the unregulated use of conventional antibiotics in fish farming industries has encouraged studies toward more eco-friendly options such as probiotics and associated bacteriocins with antimicrobial potential. Therefore, this review summarized and discussed recent studies on the effectiveness of Bacillus strains probiotic application in fish farming, emphasizing the importance of their proper characterization, extended field trials and the need for specifications in the selection criteria of the potential Bacillus probiotic strains of interest before further investigations, approvals and registrations.

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in ...

Background: Marine invertebrates are provided as a first feed for marine fish larvae because of their strict nutritional requirements, despite also being a potential source of infectious agents. Aim: To assess horizontal transmission of a nervous necrosis virus reassortant strain (NNV) to sole larvae via Artemia and rotifers. Materials and methods: Rotifer (Brachionus plicatilis) and Artemia (Artemia salina) nauplii cultures were bath infected with a reassortant (RGNNV/SJNNV) NNV strain isolated from gilthead sea bream and viral internalisation was confirmed by IFA. Senegalese sole (Solea senegalensis) larvae were fed on infected Artemia and disease signs and mortality were recorded. In addition, NNV viability was checked in cultures of either unfed invertebrates or invertebrates fed on phytoplankton and in the supernatant of microalgae cultures. All samples were tested by RT-qPCR and inoculation in cell culture. Results: Both rotifers and Artemia internalised NNV. Experimental transmission to sole larvae was achieved using infected Artemia and subsequently 60% mortality was recorded. At 24 h post-infection, orally infected individuals contained 9.34 Â 10 4 copies of viral RNA, whereas the bath infection yielded 2.05 Â 10 6 RNA copies larvae À1. Viral presence in both invertebrates was detected up to 8 days post infection but viral load decreased over time. Feeding with microalgae decreased viral detection even more and microalgae supernatants were demonstrated to significantly affect NNV viability. Conclusions: Our results demonstrate that both invertebrates can bioaccumulate NNV and that Senegalese sole larvae fed on infected Artemia might develop viral encephalopathy and retinopathy and high mortality.

The biochemical, serological and molecular characteristics of 8-pigmented isolates phenotypically related to Flavobacterium psychrophilum were determined. The bacteria were isolated in 2006 from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) cultured in the South of Chile. The F. psychrophilum type strain ATCC 49418 T and isolate B97026, obtained from rainbow trout in UK, were included in the study as a comparison. Their potential to cause pathology in Atlantic salmon was also investigated. The 8 isolates were biochemically identical, however were completely different to F. psychrophilum. Slide agglutination and Dot blot allowed us to confirm that the isolates constituted a homogeneous, but distinctive serological group to the F. psychrophilum isolates. Similar homogeneity was observed using RAPD-PCR with identical DNA patterns obtained, regardless of the source of isolation or geographic origin. Therefore, any of the above tests can be used to discri...

The genus Tenacibaculum encompasses several species pathogenic for marine fish. Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" (Quotation marks denote species that have not been validly named.) were retrieved from skin lesions of farmed fish such as European sea bass or Atlantic salmon. They cause a condition referred to as tenacibaculosis and severe outbreaks and important fish losses have been reported in Spanish, Norwegian, and Chilean marine farms. We report here the draft genomes of the T. dicentrarchi and "T. finnmarkense" type strains. These genomes were compared with draft genomes from field isolates retrieved from Chile and Norway and with previously published Tenacibaculum genomes. We used Average Nucleotide Identity and core genome-based phylogeny as a proxy index for species boundary delineation. This work highlights evolution of closely related fish-pathogenic species and suggests that homologous recombination likely contributes to genome evolution. It also corrects the species affiliation of strain AYD7486TD claimed by Grothusen et al. (2016).

We report the first isolation, identification and characterization of a group of Chilean strains of atypical Aeromonas salmonicida isolated from freshwater farmed Atlantic salmon, Salmo salar. Affected fish showed superficial ulcers and pale liver with or without petechial haemorrhages. Outbreaks of the disease occurred in two farms in the south of Chile about 2200 km apart. Five strains were isolated in pure culture and identified by serological assays and immunofluorescence tests as belonging to Aeromonas salmonicida. Although the bacterial isolates were phenotypically homogeneous, minor differences with the reference strain A. salmonicida subsp. salmonicida ATCC 33658 were noted. Three specific primer sets and partial 16S rRNA gene sequencing allowed the identification of the Chilean isolates as atypical A. salmonicida, with A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida as their closest relatives (100% sequence similarity). Molecular typing indicated that...

In the present study, we have designed and synthesized a short compositionally simple peptide RY12WY having potent antimicrobial activity. The molecular docking study results showed that peptide has a strong affinity towards two protein targets of A. sobria; aerolysin and outer membrane protein (OMP). The MIC values ranged from 0.98 to 500 μM and MBC values ranged from 4 to 650 μM against the selected bacterial and fungal pathogens. The intense antimicrobial activity of RY12WY is reported against A. sobria, A. hydrophila, E. tarda, S. aureus, V. parahaemolyticus, P. aeruginosa and E.coli at low concentration.The peptide also showed good activity against A. salmonicida and S. parasitica zoospores. The peptide retained its antimicrobial activity at higher temperatures. Besides, it was active in the presence of physiological salts and serum.The peptide showed negligible haemolytic activity at 125 μM and HC 50 was found to be 1437.10 μM. The DNA binding assay indicated that peptide can bind with the genetic material of the bacteria and may inhibit its replication. The bacterial viability assay reported that the peptide interferes with bacterial membrane integrity. To conclude, the results suggest that RY12WY could be a promising therapeutic agent in aquaculture and has possible application in food processing industry which warrants higher temperatures.

Vibrio anguillarum causes a hemorrhagic septicemia that affects cold-and warm-water adapted fish species. The main goal of this work was to determine the temperaturedependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25 • C, the degree of virulence of V. anguillarum RV22 is higher at 15 • C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15 • C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm-or cold-water temperatures. The comparative analysis of transcriptomes at high-and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25 • C than at 15 • C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15 • C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25 • C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

From 194 faecal dropping samples of common house geckos collected from offices (60), houses (88), integrated farm units (IFS,18) and hostels, guest houses, and dining rooms of different canteen/mess (HGM, 28), 326 bacterial isolates of enteric bacteria belonging to 17 genera and 34 species were detected. Escherichia coli were the most frequently (39) isolated followed by Citrobacter freundii (33), Klebsiella pneumonia (27), Salmonella indica (12), Enterobacter gergoviae (12), and Ent. agglomerans (11). Other important bacteria isolated from gecko droppings were Listonella damsela (2), Raoultella terrigena (3), S. salamae (2), S. houtenae (3), Edwardsiella tarda (4), Edwardsiella hoshinae (1), and Klebsiella oxytoca (2). Of the 223 isolates tested for antimicrobial drug sensitivity, 27 (12.1%) had multiple drug resistance (MDR). None of the salmonellae or edwardsiellae had MDR however, MDR strains were significantly more common among Escherichia spp. (= 1.9 × 10 −5) and isolates from IFS units (= 3.58 × 10 −23). The most effective herbal drug, Ageratum conyzoides extract, inhibited growth of only 27.8% of strains tested followed by ethanolic extract of Zanthoxylum rhetsa (13.9%), eucalyptus oil (5.4%), patchouli oil (5.4%), lemongrass oil (3.6%), and sandalwood oil (3.1%), and Artemisia vulgaris essential oil (3.1%).

The Isatis species has antibacterial, anticancer and antiviral properties and these important endemic plants grow widely in various parts of Anatolian-Turkey. In the present study, the antioxidant activities of Isatis floribunda Boiss. ex Bornm. flower and root extracts were determined with total antioxidant activity, free radical scavenging activity, ferric ion reducing power, and cupric ion reducing antioxidant capacity assay. The total phenolic compounds and flavonoids were also examined for the extracts. The antimicrobial activities of the extracts were investigated by using the disc diffusion and microdilution-broth methods against human and fish pathogen microorganisms. The phenolic composition of I. floribunda root methanolic extract was analyzed by high performance liquid chromatography (HPLC). The major component in the extract was cholorogenic acid (1980.20 µg/g). The cytotoxic effect of the methanolic root extract was also tested on human breast cell (MCF12A). The results...

The present study was carried out to evaluate the moderating properties of oregano (Origanum vulgare) (ORG) extract on the toxic effects of organophosphate pesticide, diazinon on growth and some metabolism associated components of rainbow trout, Oncorhynchus mykiss. In non-diazinon-exposed fish, the triiodothyronine (T3) levels in serum and body weight index (BWI %) and specific growth rate (SGR) values were higher in fish supplemented with 2, 6 and 10 g ORG/kg diet compared to control (non-ORG-supplemented fish) and fish fed 14 g ORG/kg diet after 60 days feeding trial (P<0.05). Furthermore, the serum thyroxine (T4) levels increased in fish fed 6 and 10 g ORG/kg diet compared to control and those supplemented with 2 and 14 g ORG/kg diet (P<0.05). Fish supplemented with 14 g/kg ORG diet showed the lowest BWI %, SGR and serum thyroid hormones (P<0.05). The lowest FCR values were observed in fish supplemented with 6 and 10 g ORG/kg diet (P<0.05). In diazinon exposed fish, thyroid hormones, BWI % and SGR significantly declined during 60 days exposure to diazinon in control and ORG-supplemented fish (P<0.05). However, these parameters were found to be higher in fish supplemented with 6 and 10 g ORG/kg diet compared to other exposed fish (P<0.05). FCR values significantly increased in control and fish supplemented with 2, 6 and 14 g ORG/kg diet after exposure (P<0.05). The levels of cortisol significantly elevated during exposure to diazinon in control and fish supplemented with 2 and 14 g ORG/kg diet (P<0.05). The glucose levels significantly increased in fish fed 6 and 10 g ORG/kg diet (P<0.5). During exposure period, diazinon induced ranges of histological lesions in liver, which the severity of theses lesions was lower in ORG-supplemented fish (P<0.05). In conclusion, ORG at optimum dietary levels (6-10 g/kg diet) could moderate the growth suppressing effects, stress and tissue lesions induced by diazinon. In addition, ORG at high dietary levels had toxic effects on fish metabolism.

To investigate the impacts of rearing density and oregano essential oil (OEO) supplementation on growth, behavioral and stress response of Nile tilapia, a factorial design (3 Â 3 Â 2) was assembled using 18 (100 L) glass aquaria where each aquarium had a rearing density and an OEO supplemental level. Six aquaria had a density of 10 fish aquarium À1 (low rearing density; LRD), another six had a density of 20 fish aquarium À1 (medium rearing density; MRD) and the remaining six aquaria had a density of 40 fish aquarium À1 (high rearing density; HRD). For each rearing density, fish were fed three diets with variable OEO levels (0.0, 1.0, and 2 ml kg À1 diet) and were assayed in two replicas for 10 weeks. OEO improved the growth performance of fish maintained at LRD and MRD. Tilapia exhibited behavioral changes in all densities with a reduced aggression in the HRD group. The OEO reduced the oxidative stress and the aggressiveness of juvenile tilapia as deduced from stress and welfare indicators' levels (cortisol antioxidant enzymes). In addition, a density-stress related effect as well as a nonspecific immune stimulant effect of OEO was detected from the increased antioxidant and NO activity in this study.

Bacteriocins are ribosomal-synthesized peptides or proteins produced by bacterial strains and can inhibit pathogenic bacteria. Numerous factors influence the potential activity of bacteriocins in food matrices. For example, food additives usage, chemical composition, physical conditions of food, and sensitivity of proteolytic enzymes can constrain the application of bacteriocins as beneficial food preservatives. However, novel bacteriocin nanoencapsulation has appeared as an encouraging solution. In this review, we highlight the bacteriocins produced by Gram-negative bacteria and Gram-positive bacteria including lactic acid bacteria that have shown positive results as potential food preservatives. In addition, this review encompasses the major focus on bacteriocins encapsulation with nanotechnology to enhance the antimicrobial action of bacteriocins. Several strategies can be employed to encapsulate bacteriocins; however, the nanotechnological approach is one of the most effective s...

Aquaculture is a farming activity under constant expansion. The sector has practically doubled its annual production since 2000 (FAO, 2018). Brazil is one of the major producers of freshwater fish in the world and the fourth largest producer of Nile tilapia (Oreochromis niloticus) (IBGE, 2016). The species was introduced in Brazil in 1970, and due to its African origins, Nile tilapia has adapted well to the tropical climatic conditions of the country (Melo & Stipp, 2001). An important characteristic of the species is its ability to withstand water salinity levels, which renders its culture an attractive alternative, mainly in estuarine and coastal regions (Pereira et al., 2016). The growing expansion of aquaculture led to its intensification, which has been achieved through a reduction in production areas and an increase in fish stocking density. These practices ultimately result in decreased animal performance (Sánchez-Muros et al., 2016), which has in turn led to indiscriminate use of antibiotics as growth promoters. As a consequence, a number of problems have arisen regarding food safety in animal nutrition such as selection

Fish transportation is a regular farming and experimental practice that mainly results in stress conditions that may extend to induce a high mortality rate. In the present experimental simulation, Oreochromis niloticus were transported for 2 h and maintained for another 6 h in transportation containers to assess the effect of tranquilization with tricaine methanesulfonate (MS-222) and/or the addition of iodine on mortality rate and restoring the normal physiological functions. Experimental fish were divided into four groups: group 1 (control) was transported in farm water without MS-222 or iodine, group (2) was transported in farm water supplemented with 40 mg/L of MS-222, fish in group (3) were transported in farm water supplemented with 40 mg/L of MS-222 + 10 ppm iodine, and fish in group (4) were transported in farm water containing 10 ppm iodine. Blood samples were collected for the determination of serum cortisol and glucose, while skin mucus was collected for assaying lysozyme...

This study aimed to determine the effect of dietary oregano (Origanum vulgare) essential oil (EO) on the growth performance, digestive enzyme activity, intestinal histomorphology, and intestinal microbiota of the Black Sea salmon juvenile (Salmo labrax). Fish were fed diets different levels of oregano EO such as 50, 100, 200, and 400 mg kg−1. For this purpose, a total of 675 fish were distributed randomly in triplicate into 5 experiment groups in 15 experiment tanks. Fish with average initial weights of 3.52±0.01 g were fed by hand at 3% of live weight for 90 days. At the end of the study, feeding with oregano EO supplementation did not significantly affect the growth performance of fish. Except for pepsin, there were no significant differences in the activity of digestive enzymes among the control and oregano EO groups. Besides, oregano EO at the doses of 50 or 400 mg kg−1 may have the potential to increase the surface area required for digestion by increasing intestinal villi leng...

Piscirickettsia salmonis is the most persistent and threatening pathogen for Chilean aquaculture. In our search for alternatives to control the disease, we analyzed class II polymorphic genes of the Major Histocompatibility Complex (MHC) in juvenile fish trying to find alleles that may confer resistance or susceptibility to the agent in the three major salmonid species cultured in Chile: Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch. DNA from farmed fish naturally exposed to the pathogen in different aquaculture centers in Chile were analyzed via PCR for the MHC class II alpha and beta loci (DAA and DAB), and characterized by denaturant gradient gel electrophoresis (DGGE) followed by DNA sequencing. The comparison between alleles present in either healthy or diseased individuals clearly demonstrated allelic differences between the two populations, specifically for the DAB locus in the three species and only for the DAA locus in S. salar. We conclude that a thorough analysis of the allelic differences identified between fish naturally susceptible/ resistant to P. salmonis could potentiate MHC to become a powerful genetic marker for both prophylactic and breeding purposes.

Piscirickettsia salmonis is the most persistent and threatening pathogen for Chilean aquaculture. In our search for alternatives to control the disease, we analyzed class II polymorphic genes of the Major Histocompatibility Complex (MHC) in juvenile fish trying to find alleles that may confer resistance or susceptibility to the agent in the three major salmonid species cultured in Chile: Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch. DNA from farmed fish naturally exposed to the pathogen in different aquaculture centers in Chile were analyzed via PCR for the MHC class II alpha and beta loci (DAA and DAB), and characterized by denaturant gradient gel electrophoresis (DGGE) followed by DNA sequencing. The comparison between alleles present in either healthy or diseased individuals clearly demonstrated allelic differences between the two populations, specifically for the DAB locus in the three species and only for the DAA locus in S. salar. We conclude that a thorough analysis of the allelic differences identified between fish naturally susceptible/ resistant to P. salmonis could potentiate MHC to become a powerful genetic marker for both prophylactic and breeding purposes.

The elder ( spp.) tree has a number of uses in traditional medicine. Previous studies have demonstrated the antimicrobial properties of elderberry liquid extract against human pathogenic bacteria and also influenza viruses. These properties have been mainly attributed to phenolic compounds. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. Here, we studied peptide extracts from flowers of L. The mass spectrometry analyses determined peptides of 3 to 3.6 kDa, among them, cysteine-rich peptides were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Chilean aquaculture. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and SYTOX Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that these peptides exert their action by destroying the bacterial membrane.

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in ...

Vibrio anguillarum causes a hemorrhagic septicemia that affects cold-and warm-water adapted fish species. The main goal of this work was to determine the temperaturedependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25 • C, the degree of virulence of V. anguillarum RV22 is higher at 15 • C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15 • C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm-or cold-water temperatures. The comparative analysis of transcriptomes at high-and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25 • C than at 15 • C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15 • C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25 • C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

This study aimed to evaluate the influence of increasing concentrations of dietary dry oregano leaves (DOL), ranging from 0 to 0.15%, on different performance parameters of Nile tilapia. Hence, two separate experimental trials testing growth factors (Trial 1) and innate immunity and disease resistance against Streptococcus agalactiae infection (Trial 2) were conducted with a total of 464 tilapia juveniles and two to four replicates. As a result, dietary inclusion of DOL did not induce significant effects on growth performance, hematological parameters, and fillet composition. However, there were significant differences in lysozyme activity between groups of DOL at 0.075% and the control (0%). Moreover, the bactericidal index of the positive control and groups supplemented with DOL were significantly higher than baseline treatment groups. For disease resistance, there was a reduction of 15% in the relative risk of streptococcosis in fish juveniles after dietary inclusion of oregano at 0.15%. In conclusion, the inclusion of DOL in tilapia diets resulted in no significant gains in terms of growth; nevertheless, the immune system and resistance of tilapia against S. agalactiae infection were significantly improved.

Highlights • First published study monitoring water quality and fish health (physiology and behaviour) through a commercial ornamental fish supply chain. • Water quality was high overall but significant changes in water chemistry occurred between different stages of the supply chain. • Potentially pathogenic microbes were detected in all samples but frequency of detection changed throughout the supply chain. • Of the measured health indicators, behaviour was found to change the most between different stages of transport, and was also species-specific.

Piscirickettsia salmonis is the most persistent and threatening pathogen for Chilean aquaculture. In our search for alternatives to control the disease, we analyzed class II polymorphic genes of the Major Histocompatibility Complex (MHC) in juvenile fish trying to find alleles that may confer resistance or susceptibility to the agent in the three major salmonid species cultured in Chile: Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch. DNA from farmed fish naturally exposed to the pathogen in different aquaculture centers in Chile were analyzed via PCR for the MHC class II alpha and beta loci (DAA and DAB), and characterized by denaturant gradient gel electrophoresis (DGGE) followed by DNA sequencing. The comparison between alleles present in either healthy or diseased individuals clearly demonstrated allelic differences between the two populations, specifically for the DAB locus in the three species and only for the DAA locus in S. salar. We conclude that a thorough analysis of the allelic differences identified between fish naturally susceptible/ resistant to P. salmonis could potentiate MHC to become a powerful genetic marker for both prophylactic and breeding purposes.

Plantaricin BM-1 is a class IIa bacteriocin with a strong bactericidal effect on gram-positive bacteria. Although plantaricin BM-1 also inhibits the growth of some gram-negative bacteria, including Escherichia coli, the mechanism is not clear. In this study, we used tandem mass tag-based quantitative proteomics analysis to examine the inhibitory mechanism of plantaricin BM-1 against E. coli K12, and evaluated the morphological effects by electron microscopy. The results demonstrated that plantaricin BM-1 inhibits the growth of E. coli K12 by bacteriostatic action, mainly acting on the surface of the cell wall, leading to its collapse. Proteomic analysis identified 976 differentially expressed proteins (>1.2-fold change, p < 0.05) under treatment with plantaricin BM-1, including 490 up-regulated proteins and 486 down-regulated proteins. These proteins were mainly involved in peptidoglycan synthesis and energy metabolism pathways, including amino acid, glyoxylate and dicarboxylate, ABC transporter, and quorum-sensing pathways. Specifically, plantaricin BM-1 treatment significantly improved peptidoglycan synthesis and enhanced the tricarboxylic acid cycle in E. coli K12, and altered the expression of cell membrane proteins. These results provide new insight into the inhibition mechanism of class IIa bacteriocins on gram-negative bacteria, which can lay the foundation for its broader use as an alternative to conventional antibiotics.

Bacteriocins are emerging as a viable alternative to antibiotics due to their ability to inhibit growth or kill antibiotic resistant pathogens. Herein, we evaluated the ability of the bacteriocin Garvicin KS (GarKS) produced by Lactococcus garvieae KS1546 isolated from cow milk to inhibit the growth of fish and foodborne bacterial pathogens. We found that GarKS inhibited the growth of five fish L. garvieae strains isolated from infected trout and eels. Among fish pathogens, GarKS inhibited the growth of Streptococcus agalactiae serotypes Ia and Ib, and Aeromonas hydrophila but did not inhibit the growth of Edwardsiella tarda. In addition, it inhibited the growth of A. salmonicida strain 6421 but not A. salmonicida strain 6422 and Yersinia ruckeri. There was no inhibition of three foodborne bacterial species, namely Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli. In vitro cytotoxicity tests using different GarKS concentrations showed that the highest concentration o...

The purpose of this study was to assess the in vitro susceptibility of 64 Vietnamese isolates of Edwardsiella ictaluri, the causal agent of the infectious disease Bacillus Necrosis Pangasius in Pangasianodon hypophthalmus, using the agar dilution technique. All isolates originated from different farms and were collected between 2002 and 2005. None of the isolates displayed acquired resistance to amoxicillin, amoxicillin-clavulanic acid, chloramphenicol, florfenicol, gentamicin, kanamycin, neomycin, and nitrofurantoin. Acquired resistance to streptomycin was detected in 83%, to oxytetracycline in 81%, and to trimethoprim in 71% of the isolates, as indicated by a bimodal distribution of the minimal inhibitory concentrations (MICs) of these antimicrobials. The MICs of enrofloxacin displayed a monomodal distribution with tailing toward the higher MIC values, possibly indicating reduced susceptibility of a minority of isolates (3 out of the 64). For the quinolone antimicrobial agents flumequin and oxolinic acid, acquired resistance was encountered in 8% and 6% of the strains, respectively. All strains were intrinsically resistant to the polypeptide antimicrobial agent colistin. Seventy-three percent of the isolates were shown to have acquired resistance to at least three antimicrobial agents. The results of this study emphasize the strict need to control both the prophylactic and curative use of antimicrobial agents in Vietnamese aquaculture.

The purpose of this study was to assess the in vitro susceptibility of 64 Vietnamese isolates of Edwardsiella ictaluri, the causal agent of the infectious disease Bacillus Necrosis Pangasius in Pangasianodon hypophthalmus, using the agar dilution technique. All isolates originated from different farms and were collected between 2002 and 2005. None of the isolates displayed acquired resistance to amoxicillin, amoxicillin-clavulanic acid, chloramphenicol, florfenicol, gentamicin, kanamycin, neomycin, and nitrofurantoin. Acquired resistance to streptomycin was detected in 83%, to oxytetracycline in 81%, and to trimethoprim in 71% of the isolates, as indicated by a bimodal distribution of the minimal inhibitory concentrations (MICs) of these antimicrobials. The MICs of enrofloxacin displayed a monomodal distribution with tailing toward the higher MIC values, possibly indicating reduced susceptibility of a minority of isolates (3 out of the 64). For the quinolone antimicrobial agents flumequin and oxolinic acid, acquired resistance was encountered in 8% and 6% of the strains, respectively. All strains were intrinsically resistant to the polypeptide antimicrobial agent colistin. Seventy-three percent of the isolates were shown to have acquired resistance to at least three antimicrobial agents. The results of this study emphasize the strict need to control both the prophylactic and curative use of antimicrobial agents in Vietnamese aquaculture.

The present study was aimed to isolate, characterize and examine the probiotic properties of Bacillus infantis (KADR2) from Labeo rohita. The total of seven strains isolated from gastrointestinal tract of Labeo rohita, one of them KADR2 showed higher antagonistic effect against fish pathogens. The strain was evaluated under in vitro intestinal condition based on resistance to bile tolerance, low pH, hydrophobicity, catalase activity and antibiotics susceptibilities. Partial 16S rRNA gene sequence of this strain KADR2 were blasted and showed homology with Bacillus infantis(99%) supported by morphological and physiological characterization. Consequently, the positive results of this study suggested that further studies in challenge experiments in fish to explore their probiotic effects having great scope for being used as a potential probiotic in aquaculture

Seven fresh bacterial isolates were isolated from disease affected fish species such as Labeo rohita, Cyprinus carpio, H. molitrix, and Puntius sarana from different carp farms and market sources. Bacterial isolates were collected from different tissues or organ such as skin, gill, spot lesions on the skin, abdomen, kidney etc. Several tests were conducted for characterization of bacteria. Susceptibility of Aeromonas spp. bacteria to Streptomycin (10 μg/200 mL) antibiotic were 150 colony and in control 320 colony in R-1. Where in R-2, 142 colony and in control 296 colony and finally in R-3, 145 colony and in control 311 colony were found in T-4. Where resistance of Aeromonas spp. bacteria to Streptomycin (30 μg/200 mL) antibiotic were 1 colony and in control 318 colony in R-1. Where in R-2, 0 colony and in control 309 colony and finally in R-3 also 0 colony and in control 314 colony were found in T-4. All the treatments ware performed through serial dilution method at 310×10-4 bacte...

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to i...

Piscirickettsia salmonis is the most persistent and threatening pathogen for Chilean aquaculture. In our search for alternatives to control the disease, we analyzed class II polymorphic genes of the Major Histocompatibility Complex (MHC) in juvenile fish trying to find alleles that may confer resistance or susceptibility to the agent in the three major salmonid species cultured in Chile: Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch. DNA from farmed fish naturally exposed to the pathogen in different aquaculture centers in Chile were analyzed via PCR for the MHC class II alpha and beta loci (DAA and DAB), and characterized by denaturant gradient gel electrophoresis (DGGE) followed by DNA sequencing. The comparison between alleles present in either healthy or diseased individuals clearly demonstrated allelic differences between the two populations, specifically for the DAB locus in the three species and only for the DAA locus in S. salar. We conclude that a thorough analysis of the allelic differences identified between fish naturally susceptible/ resistant to P. salmonis could potentiate MHC to become a powerful genetic marker for both prophylactic and breeding purposes.

Piscirickettsia salmonis is the most persistent and threatening pathogen for Chilean aquaculture. In our search for alternatives to control the disease, we analyzed class II polymorphic genes of the Major Histocompatibility Complex (MHC) in juvenile fish trying to find alleles that may confer resistance or susceptibility to the agent in the three major salmonid species cultured in Chile: Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch. DNA from farmed fish naturally exposed to the pathogen in different aquaculture centers in Chile were analyzed via PCR for the MHC class II alpha and beta loci (DAA and DAB), and characterized by denaturant gradient gel electrophoresis (DGGE) followed by DNA sequencing. The comparison between alleles present in either healthy or diseased individuals clearly demonstrated allelic differences between the two populations, specifically for the DAB locus in the three species and only for the DAA locus in S. salar. We conclude that a thorough analysis of the allelic differences identified between fish naturally susceptible/ resistant to P. salmonis could potentiate MHC to become a powerful genetic marker for both prophylactic and breeding purposes.

The transmission of puffy skin disease (PSD) to rainbow trout Oncorhynchus mykiss Walbaum was tested in the laboratory by conducting co-habitation challenges with puffy skin (PS)-affected fish (Trojans) collected from the field. Two separate challenges were conducted using Trojans sourced from two different sites and diploid (first trial) or triploid (second trial) naïve fish. PSD-specific clinical signs were observed in both groups of naïve fish, with 66% of the fish sampled during the challenges showing signs of varying severity. The first clinical features of PSD were presented as white oval skin patches on one or both flanks 15-21 days post-challenge (dpc). The extent of the lesions ranged from 10 to 90% of the body surface, depending on the severity of the lesion. Both the severity and number of affected fish increased during the challenge. Macroscopically, oedema of the skin and multifocal petechial haemorrhaging were observed towards the end of the trials. Abnormal fish behaviour consisting of "flashing" and excessive mucous production was noted from 15 dpc onwards. Fish with severe PSD lesions also displayed inappetence and associated emaciation. Rodlet cells were observed in 41% of the fresh skin scrapes analysed from the second trial. Histologically epidermal oedema was observed in 31% of the naive fish showing gross pathology, with additional 12% displaying epidermal hyperplasia, mostly observed at the end of the challenge. Other concomitant features of the PSD lesions in challenged fish were epithelial erosion and sloughing, and occasionally mild or focal inflammation. No consistent pathology of internal organs was observed. The parasites Ichthyophthirius multifiliis and Ichthyobodo necator were observed in skin samples of a proportion of naïve challenged fish and in Trojans but not in control fish. The presence of these and other known fish pathogens in the skin of PSD-fish was confirmed by high-throughput sequencing analysis. In summary, we have demonstrated that PSD is a transmissible condition. However, even though a number of known fish pathogens were identified in the skin tissues of PSD-fish, the actual causative infectious agent(s) remain(s) unknown.

Piscirickettsia salmonis is the most persistent and threatening pathogen for Chilean aquaculture. In our search for alternatives to control the disease, we analyzed class II polymorphic genes of the Major Histocompatibility Complex (MHC) in juvenile fish trying to find alleles that may confer resistance or susceptibility to the agent in the three major salmonid species cultured in Chile: Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch. DNA from farmed fish naturally exposed to the pathogen in different aquaculture centers in Chile were analyzed via PCR for the MHC class II alpha and beta loci (DAA and DAB), and characterized by denaturant gradient gel electrophoresis (DGGE) followed by DNA sequencing. The comparison between alleles present in either healthy or diseased individuals clearly demonstrated allelic differences between the two populations, specifically for the DAB locus in the three species and only for the DAA locus in S. salar. We conclude that a thorough analysis of the allelic differences identified between fish naturally susceptible/ resistant to P. salmonis could potentiate MHC to become a powerful genetic marker for both prophylactic and breeding purposes.

The world-wide emergence of resistant pathogenic strains against the currently available antibiotics and the toxicity associated with them has compelled scientific community to increase their quests in finding other natural antimicrobial alternatives. In this regard recent discoveries on antimicrobial arsenals secreted by bacterial species against competing strains are gaining utmost importance not only because of their antimicrobial but also due to their anti-cancer potential. These natural bacterial arsenals are ribosomally synthesized low-molecular weight, heat stable, membrane active, proteolytically degradable and pore-forming, cationic peptides known as “Bacteriocins”. Owing to their anti-bacterial, anti-viral, anti-fungal, and anti-biofilm potential, bacteriocins seem to be the immediately available promising option for mitigation of AMR (antimicrobial Resistance) crisis and minimization of toxic effects of antibiotics on Host‟s microbiome. The non-toxic and Generally Regarde...

This study aimed to determine the effect of dietary oregano (Origanum vulgare) essential oil (EO) on the growth performance, digestive enzyme activity, intestinal histomorphology, and intestinal microbiota of the Black Sea salmon juvenile (Salmo labrax). Fish were fed diets different levels of oregano EO such as 50, 100, 200, and 400 mg kg−1. For this purpose, a total of 675 fish were distributed randomly in triplicate into 5 experiment groups in 15 experiment tanks. Fish with average initial weights of 3.52±0.01 g were fed by hand at 3% of live weight for 90 days. At the end of the study, feeding with oregano EO supplementation did not significantly affect the growth performance of fish. Except for pepsin, there were no significant differences in the activity of digestive enzymes among the control and oregano EO groups. Besides, oregano EO at the doses of 50 or 400 mg kg−1 may have the potential to increase the surface area required for digestion by increasing intestinal villi leng...

Fishes counteract certain microbial attacks in water by producing antimicrobial proteins/peptides in their skin surface. The present study focused on screening the bactericidal activity of skin and skin mucus extracts of Catla catla and Channa striatus. The bactericidal activity was assessed against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, Aeromonas hydrophila, Staphylococcus aureus and Bacillus coagulans by disc diffusion method. The minimal inhibitory concentration was also determined. Protein profiles in skin and skin mucus extracts were analyzed by SDS-PAGE. Samples from both fishes showed antibacterial activity. Detailed analysis of individual protein and peptide would throw light on their medicinal importance to be used against pathogenic microbes.

The elder ( spp.) tree has a number of uses in traditional medicine. Previous studies have demonstrated the antimicrobial properties of elderberry liquid extract against human pathogenic bacteria and also influenza viruses. These properties have been mainly attributed to phenolic compounds. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. Here, we studied peptide extracts from flowers of L. The mass spectrometry analyses determined peptides of 3 to 3.6 kDa, among them, cysteine-rich peptides were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Chilean aquaculture. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and SYTOX Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that these peptides exert their action by destroying the bacterial membrane.