Eficacia de un desinfectante sobre Vibrio ordalii, Vibrio anguillarum, Francisella sp. y Virus de la Necrosis Pancreática Infecciosa (IPNV), patógenos de salmón del Atlántico (Salmo salar) cultivado en Chile

Indexación: ScieloRESUMEN En el presente trabajo se evaluó la eficacia in vitro del desinfectante Duplalim®, una combinación sinérgica de glutaraldehído y sales de amonio cuaternario de cuarta generación, contra 4 patógenos de peces prevalentes de la salmonicultura chilena. Los resultados muestran que todas las concentraciones ensayadas (diluciones entre 1:200 a 1:400) fueron eficaces sobre los aislados de Vibrio ordalii y Vibrio anguillarum post-30 s de exposición, detectando niveles de reducción igual a 1.8 x 106 UFC/ml. Concentraciones superiores de Duplalim® (dilución 1:50) y un tiempo de exposición no menor a 5 min. Fueron necesarios para eliminar completamente al patógeno intracelular Francisella sp. Cuando el desinfectante fue ensayado contra el Virus de la necrosis Pancreática infecciosa (IPNV), se detectó que la dilución 1:400 tiene un efecto significativo después de 2 minutos sin importar los títulos de IPNV testeados (mayor concentración evaluada 107.6 TCID50/ml). Duplalim® se evaluó en condiciones masivas contra los miembros de la familia Vibrionaceae. En comparación a los controles (sin adición desinfectante), la dilución 1:400 de Duplalim® eliminó completamente V. ordalii y V. anguillarum después de 15 minutos de tratamiento, tanto en el agua de cultivo como en la superficie de mallas usadas en el cultivo del salmón. Así, el análisis microbiológico del agua de los controles mostró concentraciones de 1.4 ± 0.3 × 106 UFC/ml, mientras en el caso de las mallas 7.6 ± 3.2 × 105 UFC/ml1. En resumen, los antecedentes obtenidos indican que el uso del desinfectante Duplalim® es efectivo contra V. ordalii, V. anguillarum y IPNV en bajas concentraciones y cortos periodos de exposición (dilución 1:400 por 15 min.), mientras que para el patógeno intracelular se requiere una concentración mayor. Palabras clave: desinfectante, patógenos de peces, salmón del atlántico. SUMMARY The efficacy of the disinfectant Duplalim®, a synergistic blend of superquats and glutaraldehyde, was analysed in vitro against 4 fish pathogens. All concentrations tested (1:200 to 1:400 dilutions) were efficacious on killing Vibrio ordalii and Vibrio anguillarum in seawater after 30 s, being the level of reduction equal to 1.8 x 106 CFU/ml. Higher concentration of Duplalim® (1:50 dilutions) and time of exposure (at least 5 min) is needed to kill completely Francisella sp, an intracellular freshwater pathogen. When Infectious Pancreatic Necrosis Virus (IPNV) was treated with 1:400 disinfectant dilution, this concentration had a significant effect after 2 minutes, regardless of the IPNV titres employed (concentration greater than 107.6 TCID50/ml). Duplalim® was tested in large scale against Vibrionaceae members. In comparison to the controls (without the disinfectant), 1:400 dilutions of Duplalim® totally killed V. ordalii and V. anguillarum in seawater as well as on the surface of the fishing net (used in the cages of cultured salmon) after 15 min. Cultivable bacteria remained constant in the buckets without the disinfectant (1.4 ± 0.3 × 106 CFU/ml), regardless of the period sampled. In the case of the adherence on the fishing net, bacteria not exposed to the disinfectant were detected at a concentration of 7.6 ± 3.2 × 105 CFU/ml. These data indicate that the use of Duplalim® against V. ordalii, V. anguillarum and IPNV is effective in low concentration and short time of exposure (15 min at a concentration of 1:400 dilutions), while the intracellular pathogen requires higher concentration. Key words: disinfectant, Chilean fish pathogens, Atlantic salmon

Comparative genomic analysis of two Chilean Renibacterium salmoninarum isolates and the type strain ATCC 33209T

Indexación: Scopus.Two previously characterized Chilean isolates (H-2 and DJ2R), obtained from cage-cultured Atlantic salmon with clinical signs of bacterial kidney disease in southern Chile, were used (Bethke et al. 2016, 2017). The bacteria were routinely cultured in KDM-2 agar for 15–20 days at 15°C. For sequencing, genomic DNA of the two isolates was extracted using the InstaGene Purification Matrix (Bio-Rad) according to manufacturer instructions. The DJ2R genome was sequenced using an Illumina MiSeq platform with 2 ⨯ 250 paired-end reads by the AUSTRAL-omics Institute, hosted by the Universidad Austral de Chile (Valdivia, Chile). Using the same technology and parameters, H-2 genomic DNA was sequenced by the Central Support Service for Experimental Research (SCSIE, Spanish acronym) at the University of Valencia (Valencia, Spain).This work was supported by funding of the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT, Chile) [Grant Numbers FONDAP No. 15110027 and FONDECYT No. 1150695]. J.B. also acknowledges support received by CONICYT [Doctoral Scholarship No. 21140421].Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen belonging to the high C+G content Actinobacteria phylum, is the causative agent of bacterial kidney disease, a progressive granulomatous infection affecting salmonids worldwide. This Gram-positive bacterium has existed in the Chilean salmonid industry for >30 years, but little or no information is available regarding the virulence mechanisms and genomic characteristics of Chilean isolates. In this study, the genomes of two Chilean isolates (H-2 and DJ2R)were sequenced, and a search was conducted for genes and proteins involved in virulence and pathogenicity, andwecompare with the type strain ATCC 33209T genome. The genome sizes of H-2 and DJ2R are 3,155,332 bp and 3,155,228 bp, respectively. They genomes presented six ribosomal RNA, 46 transcription RNA, and 25 noncodingRNA, and both had the same 56.27% G+C content described for the type strain ATCC 33209 T. A total of 3,522 and 3,527 coding sequences were found for H-2 and DJ2R, respectively. Meanwhile, the ATCC 33209T type strain had 3,519 coding sequences. The in silico genome analysis revealed a genes related to tricarboxylic acid cycle, glycolysis, iron transport and others metabolic pathway. Also, the data indicated that R salmoninarum may have a variety of possible virulence-factor and antibiotic-resistance strategies. Interestingly, many of genes had high identities with Mycobacterium species, a known pathogenic Actin obacteria bacterium. In summary, this study provides the first insights into and initial steps towards understanding the molecular basis of antibiotic resistance, virulence mechanisms and host/environment adaptation in twoChilean R. salmoninarum isolates that contain proteins of which were similar to those of Mycobacterium. Furthermore, important information is presented that could facilitate the development of preventive and treatment measures against R. salmoninarum in Chile and worldwide. © The Author(s) 2018.https://academic.oup.com/gbe/article/10/7/1816/504777

Stress tolerance-related genetic traits of fish pathogen Flavobacterium psychrophilum in a mature biofilm

Indexación: Scopus.Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome, and hence this bacterium is placed among the most important salmonid pathogens in the freshwater aquaculture industry. Since bacteria in biofilms differ substantially from free-living counterparts, this study sought to find the main differences in gene expression between sessile and planktonic states of F. psychrophilum LM-02-Fp and NCMB1947T, with focus on stress-related changes in gene expression occurring during biofilm formation. To this end, biofilm and planktonic samples were analyzed by RNA sequencing to detect differentially expressed candidate genes (DECGs) between the two growth states, and decreasing the effects of interstrain variation by considering only genes with log2-fold changes ≤ -2 and ≥ 2 at Padj-values = 0.001 as DECGs. Overall, 349 genes accounting for ~15% of total number of genes expressed in transcriptomes of F. psychrophilum LM-02-Fp and NCMB1947T (n = 2327) were DECGs between biofilm and planktonic states. Approximately 83 and 81% of all up- and down-regulated candidate genes in mature biofilms, respectively, were assigned to at least one gene ontology term; these were primarily associated with the molecular function term "catalytic activity." We detected a potential stress response in mature biofilms, characterized by a generalized down-regulation of DECGs with roles in the protein synthesis machinery (n = 63, primarily ribosomal proteins) and energy conservation (seven ATP synthase subunit genes), as well as an up-regulation of DECGs involved in DNA repair (ruvC, recO, phrB1, smf, and dnaQ) and oxidative stress response (cytochrome C peroxidase, probable peroxiredoxin, and a probable thioredoxin). These results support the idea of a strategic trade-offbetween growth-related processes and cell homeostasis to preserve biofilm structure and metabolic functioning. In addition, LDH-based cytotoxicity assays and an intraperitoneal challenge model for rainbow trout fry agreed with the transcriptomic evidence that the ability of F. psychrophilum to form biofilms could contribute to the virulence. Finally, the reported changes in gene expression, as induced by the plankton-to-biofilm transition, represent the first transcriptomic guideline to obtain insights into the F. psychrophilum biofilm lifestyle that could help understand the prevalence of this bacterium in aquaculture settings.https://www.frontiersin.org/articles/10.3389/fmicb.2018.00018/ful

Improved understanding of biofilm development by Piscirickettsia salmonis reveals potential risks for the persistence and dissemination of piscirickettsiosis

Indexación ScopusPiscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-economic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of the medium, but both isolates formed mature biofilms from 288 h onwards. Psal-103 biofilms were sensitive to Atlantic salmon skin mucus during early formation, whereas Psal-104 biofilms were more tolerant. The ability of P. salmonis to form viable and mucus-tolerant biofilms on plastic surfaces in seawater represents a potentially important environmental risk for the persistence and dissemination of piscirickettsiosis. © 2020, The Author(s).https://www-nature-com.recursosbiblioteca.unab.cl/articles/s41598-020-68990-

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins

Indexación: Scopus.Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain. © 2017 Oliver, Hernández, Tandberg, Valenzuela, Lagos, Haro, Sánchez, Ruiz, Sanhueza-Oyarzún, Cortés, Villar, Artigues, Winther-Larsen, Avendaño-Herrera and Yáñez.https://www.frontiersin.org/articles/10.3389/fcimb.2017.00420/ful

Flexibatteriosi da Tenacibaculum maritimum sierotipo O3 in pesce capone (Chelidonichthys lucerna L.) d’allevamento: prima segnalazione in Italia. First report of Tenacibaculum maritimum serotype O3 infecton in cultured tub gurnard (Chelidonichthys lucerna) in Italy.

The present study describes the first isolation of Tenacibaculum maritimum from tub gurnard (Chelidonichthys lucernus L.), cultured in Italy. After 3 days shipment from one fish farm to our laboratory facilities, a group of 20 tub gurnard showed eroded mouth, rotten fins and severe skin ulcerative lesions. Mortality reached 90% of the population in 9 days. Traditional bacteriological and PCR analysis allowed the identification of the causative agent as T. maritimum. Serological characterization of the isolate demonstrated that it belongs to serotype O3. Histopathological examination showed severe skin necrosis, dermis congestion associated with eterophilic and macrophagic infiltration. Immunohistochemical analysis, using specific polyclonal antiserum against T. maritimum serotype O3, allowed the visualization of numerous positively stained macrophagic cells. The high mortality observed during the outbreak leads to consider the T. maritimum as a potential risk for the culture of tub gurnard

Polyembryony in Maize: A Complex, Elusive, and Potentially Agronomical Useful Trait

Polyembryony (PE) is a rare phenomenon in cultivated plant species. Since nineteenth century, several reports have been published on PE in maize. Reports of multiple seedlings developing at embryonic level in laboratory and studies under greenhouse and field conditions have demonstrated the presence of PE in cultivated maize (Zea mays L.). Nevertheless, there is a lack of knowledge about this phenomenon; diverse genetic mechanisms controlling PE in maize have been proposed: Mendelian inheritance of a single gene, interaction between two genes and multiple genes are some of the proposed mechanisms. On the other hand, the presence of two or more embryos per seed confers higher nutrimental quality because these grains have more crude fat and lysine than normal maize kernels. As mentioned above, there is a necessity for more studies about PE maize in order to establish the genetic mechanism responsible for this phenomenon; on the other hand, previous studies showed that PE has potential to generate specialized maize varieties with yield potential and grain quality

Subcellular location of Piscirickettsia salmonis heat shock protein 60 (Hsp60) chaperone by using immunogold labeling and proteomic analysis

Indexación: Scopus.Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by the T4SS, T3SS, and T6SS, with negative results. These data support the hypothesis that small amounts of Hsp60 must reach the bacterial cell surface in a manner probably not mediated by currently characterized secretion systems, and that they remain biologically active during P. salmonis infection, possibly mediating adherence and (or) invasion.https://www.mdpi.com/2076-2607/8/1/11

Arthrobacter ulcerisalmonis sp. Nov., isolated from an ulcer of a farmed atlantic salmon (salmo salar), and emended description of the genus arthrobacter sensu lato

Indexación: Scopus.A Gram-stain positive, pleomorphic, oxidase-negative, non-motile isolate from the ulcer of a farmed Atlantic salmon (Salmo salar), designated strain T11bT, was subjected to a comprehensive taxonomic investigation. A comparative analysis of the 16S rRNA gene sequence showed highest similarities to the type strains of Pseudarthrobacter siccitolerans (98.1%) and Arthro-bacter methylotrophus and Pseudarthrobacter phenanthrenivorans (both 98.0 %). The highest ANI value observed between the assembled genome of T11bT and the publicly available Pseudarthrobacter and Arthrobacter type strain genomes were 81.15 and 80.99 %, respectively. The major respiratory quinone was menaquinone MK-9(H2). The polyamine pattern contained pre-dominantly spermidine. The polar lipid profile consisted of the major lipids diphosphatidylglycerol, phosphatidylglycerol, monogalactosyl-diacylglycerol and dimannosylglyceride. Minor amouts of trimannosyldiacylglycerol and phosphatidylinositol were also detected. The peptidoglycan was of the type A3α l-Lys–l-Ser–l-Thr–l-Ala (A11.23). In the fatty acid profile, anteiso and iso branched fatty acids predominated (anteiso C15: 0, iso C16: 0, anteiso C17: 0). Moderate to low DNA–DNA similarities, physiological traits as well as unique traits in the fatty acid pattern distinguished strain T11bT from the next related species. All these data point to the fact that strain T11bT represents a novel species of the genus Arthrobacter for which we propose the name Arthrobacter ulcerisalmonis sp. nov. The type strain is T11bT (=CIP 111621T=CCM 8854T=LMG 30632T=DSM 107127T).https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijsem.0.00400