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1.
Front Cell Neurosci ; 18: 1406839, 2024.
Article in English | MEDLINE | ID: mdl-38933177

ABSTRACT

Introduction: Human cerebral organoids (hCOs) derived from pluripotent stem cells are very promising for the study of neurodevelopment and the investigation of the healthy or diseased brain. To help establish hCOs as a powerful research model, it is essential to perform the morphological characterization of their cellular components in depth. Methods: In this study, we analyzed the cell types consisting of hCOs after culturing for 45 days using immunofluorescence and reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) assays. We also analyzed their subcellular morphological characteristics by transmission electron microscopy (TEM). Results: Our results show the development of proliferative zones to be remarkably similar to those found in human brain development with cells having a polarized structure surrounding a central cavity with tight junctions and cilia. In addition, we describe the presence of immature and mature migrating neurons, astrocytes, oligodendrocyte precursor cells, and microglia-like cells. Discussion: The ultrastructural characterization presented in this study provides valuable information on the structural development and morphology of the hCO, and this information is of general interest for future research on the mechanisms that alter the cell structure or function of hCOs.

2.
J Tissue Eng ; 15: 20417314231226027, 2024.
Article in English | MEDLINE | ID: mdl-38343770

ABSTRACT

Human cerebral organoids (hCOs) offer the possibility of deepening the knowledge of human brain development, as well as the pathologies that affect it. The method developed here describes the efficient generation of hCOs by going directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures to three-dimensional (3D) neuroepithelial tissue, avoiding dissociation and aggregation steps. This has been achieved by subjecting 2D cultures, from the beginning of the neural induction step, to dual-SMAD inhibition in combination with CHIR99021. This is a simple and reproducible protocol in which the hCOs generated develop properly presenting proliferative ventricular zones (VZs) formed by neural precursor and radial glia (RG) that differentiate to give rise to mature neurons and glial cells. The hCOs present additional cell types such as oligodendrocyte precursors, astrocytes, microglia-like cells, and endothelial-like cells. This new approach could help to overcome some of the existing limitations in the field of organoid biotechnology, facilitating its execution in any laboratory setting.

3.
Int J Mol Sci ; 24(19)2023 Sep 27.
Article in English | MEDLINE | ID: mdl-37834082

ABSTRACT

Amyloid precursor protein (APP) has been widely studied due to its association with Alzheimer's disease (AD). However, the physiological functions of APP are still largely unexplored. APP is a transmembrane glycoprotein whose expression in humans is abundant in the central nervous system. Specifically, several studies have revealed the high expression of APP during brain development. Previous studies in our laboratory revealed that a transient increase in APP expression induces early cell cycle exit of human neural stem cells (hNSCs) and directs their differentiation towards glial cells (gliogenesis) while decreasing their differentiation towards neurons (neurogenesis). In the present study, we have evaluated the intrinsic cellular effects of APP down-expression (using siRNA) on cell death, cell proliferation, and cell fate specification of hNSCs. Our data indicate that APP silencing causes cellular effects opposite to those obtained in previous APP overexpression assays, inducing cell proliferation in hNS1 cells (a model line of hNSCs) and favoring neurogenesis instead of gliogenesis in these cells. In addition, we have analyzed the gene and protein expression levels of ß-Catenin as a possible molecule involved in these cellular effects. These data could help to understand the biological role of APP, which is necessary to deepen the knowledge of AD.


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Neurogenesis , Humans , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Neural Stem Cells/metabolism , Neuroglia/metabolism , Neurons/metabolism
4.
Int J Mol Sci ; 24(16)2023 Aug 19.
Article in English | MEDLINE | ID: mdl-37629148

ABSTRACT

Numerous studies have focused on the pathophysiological role of amyloid precursor protein (APP) because the proteolytic processing of APP to ß-amyloid (Aß) peptide is a central event in Alzheimer's disease (AD). However, many authors consider that alterations in the physiological functions of APP are likely to play a key role in AD. Previous studies in our laboratory revealed that APP plays an important role in the differentiation of human neural stem cells (hNSCs), favoring glial differentiation (gliogenesis) and preventing their differentiation toward a neuronal phenotype (neurogenesis). In the present study, we have evaluated the effects of APP overexpression in hNSCs at a global gene level by a transcriptomic analysis using the massive RNA sequencing (RNA-seq) technology. Specifically, we have focused on differentially expressed genes that are related to neuronal and glial differentiation processes, as well as on groups of differentially expressed genes associated with different signaling pathways, in order to find a possible interaction between them and APP. Our data indicate a differential expression in genes related to Notch, Wnt, PI3K-AKT, and JAK-STAT signaling, among others. Knowledge of APP biological functions, as well as the possible signaling pathways that could be related to this protein, are essential to advance our understanding of AD.


Subject(s)
Alzheimer Disease , Neural Stem Cells , Humans , Amyloid beta-Protein Precursor/genetics , Phosphatidylinositol 3-Kinases , Neurogenesis/genetics , Alzheimer Disease/genetics , Signal Transduction
5.
Animals (Basel) ; 12(18)2022 Sep 19.
Article in English | MEDLINE | ID: mdl-36139336

ABSTRACT

Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. Here we show the prevalence and transmission of L. monocytogenes in dairy farms in the Cantabria region, on the northern coast of Spain. A total of 424 samples was collected from 14 dairy farms (5 organic and 9 conventional) and 211 L. monocytogenes isolates were recovered following conventional microbiological methods. There were no statistically significant differences in antimicrobial resistance ratios between organic and conventional farms. A clonal relationship among the isolates was assessed by pulsed field gel electrophoresis (PFGE) analysis and 64 different pulsotypes were obtained. Most isolates (89%, n = 187) were classified as PCR serogroup IVb by using a multiplex PCR assay. In this case, 45 isolates of PCR serogroup IVb were whole genome-sequenced to perform a further analysis at genomic level. In silico MLST analysis showed the presence of 12 sequence types (ST), of which ST1, ST54 and ST666 were the most common. Our data indicate that the environment of cattle farms retains a high incidence of L. monocytogenes, including subtypes involved in human listeriosis reports and outbreaks. This pathogen is shed in the feces and could easily colonize dairy products, as a result of fecal contamination. Effective herd and manure management are needed in order to prevent possible outbreaks.

6.
Front Cell Dev Biol ; 10: 912319, 2022.
Article in English | MEDLINE | ID: mdl-35938168

ABSTRACT

Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical ß-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active ß-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence.

7.
Front Oncol ; 12: 903033, 2022.
Article in English | MEDLINE | ID: mdl-35957902

ABSTRACT

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.

8.
Int J Mol Sci ; 23(10)2022 May 22.
Article in English | MEDLINE | ID: mdl-35628629

ABSTRACT

Amyloid-ß 40 peptides [Aß1-40 (Aß40)] are present within amyloid plaques in the brains of patients with Alzheimer's disease (AD). Even though Aß peptides are considered neurotoxic, they can mediate many biological processes, both in adult brains and throughout brain development. However, the physiological function of these Aß peptides remains poorly understood, and the existing data are sometimes controversial. Here, we analyze and compare the effects of monomeric Aß40 on the biology of differentiating human neural stem cells (human NSCs). For that purpose, we used a model of human NSCs called hNS1. Our data demonstrated that Aß40 at high concentrations provokes apoptotic cellular death and the damage of DNA in human NSCs while also increasing the proliferation and favors neurogenesis by raising the percentage of proliferating neuronal precursors. These effects can be mediated, at least in part, by ß-catenin. These results provide evidence of how Aß modulate/regulate human NSC proliferation and differentiation, suggesting Aß40 may be a pro-neurogenic factor. Our data could contribute to a better understanding of the molecular mechanisms involved in AD pathology and to the development of human NSC-based therapies for AD treatment, since these results could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.


Subject(s)
Alzheimer Disease , Neural Stem Cells , Adult , Alzheimer Disease/pathology , Amyloid beta-Peptides/chemistry , Humans , Neurogenesis , Plaque, Amyloid/pathology
9.
Int J Mol Sci ; 22(17)2021 Sep 02.
Article in English | MEDLINE | ID: mdl-34502444

ABSTRACT

Amyloid-ß 42 peptide (Aß1-42 (Aß42)) is well-known for its involvement in the development of Alzheimer's disease (AD). Aß42 accumulates and aggregates in fibers that precipitate in the form of plaques in the brain causing toxicity; however, like other forms of Aß peptide, the role of these peptides remains unclear. Here we analyze and compare the effects of oligomeric and fibrillary Aß42 peptide on the biology (cell death, proliferative rate, and cell fate specification) of differentiating human neural stem cells (hNS1 cell line). By using the hNS1 cells we found that, at high concentrations, oligomeric and fibrillary Aß42 peptides provoke apoptotic cellular death and damage of DNA in these cells, but Aß42 fibrils have the strongest effect. The data also show that both oligomeric and fibrillar Aß42 peptides decrease cellular proliferation but Aß42 oligomers have the greatest effect. Finally, both, oligomers and fibrils favor gliogenesis and neurogenesis in hNS1 cells, although, in this case, the effect is more prominent in oligomers. All together the findings of this study may contribute to a better understanding of the molecular mechanisms involved in the pathology of AD and to the development of human neural stem cell-based therapies for AD treatment.


Subject(s)
Amyloid beta-Peptides/physiology , Neural Stem Cells/physiology , Peptide Fragments/physiology , Humans , Primary Cell Culture
10.
J Food Prot ; 83(3): 443-451, 2020 Mar 01.
Article in English | MEDLINE | ID: mdl-32053831

ABSTRACT

ABSTRACT: Listeria monocytogenes can survive in food production facilities and can be transmitted via contamination of food during the various stages of food production. This study was conducted to compile the results of three independent previous studies on the genetic diversity of L. monocytogenes in a poultry production company in Spain and to determine the potential virulence and sanitizer resistance of the strains by using both genotype and phenotype analyses. L. monocytogenes was detected at three production stages: a broiler abattoir, a processing plant, and retail stores marketing fresh poultry products from the same company. These three stages spanned three locations in three provinces of Spain. A set of 347 L. monocytogenes isolates representing 39 subtypes was obtained using pulsed-field gel electrophoresis (PFGE). A total of 28 subtypes (68%) had a full-length internalin A gene, and two subtypes had a phenotype with low potential for virulence because of a mutation in the prfA gene. A total of 32 subtypes (82%) were classified as benzalkonium chloride resistant (BAC-R) and contained the resistance determinant bcrABC (21 subtypes, 54%) or the resistance gene qacH (11 subtypes, 28%). A total of 13 persistent BAC-R subtypes (minimum of 3 months between the first and last sample from with the isolate was recovered) were identified at the abattoir and processing plant. The three production stages shared a unique subtype (PFGE type 1), which had the mutation in the prfA gene and the bcrABC resistance determinant. Whole genome sequencing revealed this subtype to be sequence type 31. Limited genetic diversity was noted in the isolates studied, including some subtypes that were persistent in the environment of the investigated facilities. Given the high prevalence of BAC-R subtypes, these results support the association between resistance to biocides and persistence of L. monocytogenes.


Subject(s)
Benzalkonium Compounds/pharmacology , Food Handling/methods , Listeria monocytogenes , Animals , Chickens , Electrophoresis, Gel, Pulsed-Field , Food Contamination/analysis , Food Contamination/prevention & control , Food Microbiology , Listeria monocytogenes/pathogenicity , Poultry , Spain , Virulence
11.
Microbiol Resour Announc ; 8(11)2019 Mar 14.
Article in English | MEDLINE | ID: mdl-30938320

ABSTRACT

Here, we present the draft genome sequences of seven Listeria monocytogenes strains isolated during three independent studies carried out in three stages of a poultry meat production chain. The genome sequences of these strains obtained from different stages can help to understand the possible transmission of L. monocytogenes.

12.
Front Microbiol ; 7: 638, 2016.
Article in English | MEDLINE | ID: mdl-27199964

ABSTRACT

The persistence of certain strains of Listeria monocytogenes, even after the food processing environment has been cleaned and disinfected, suggests that this may be related to phenomena that reduce the concentration of the disinfectants to subinhibitory levels. This includes (i) the existence of environmental niches or reservoirs that are difficult for disinfectants to reach, (ii) microorganisms that form biofilms and create microenvironments in which adequate concentrations of disinfectants cannot be attained, and (iii) the acquisition of resistance mechanisms in L. monocytogenes, including those that lead to a reduction in the intracellular concentration of the disinfectants. The only available data with regard to the resistance of L. monocytogenes to disinfectants applied in food production environments refer to genotypic resistance to quaternary ammonium compounds (QACs). Although there are several well-characterized efflux pumps that confer resistance to QACs, it is a low-level resistance that does not generate resistance to QACs at the concentrations applied in the food industry. However, dilution in the environment and biodegradation result in QAC concentration gradients. As a result, the microorganisms are frequently exposed to subinhibitory concentrations of QACs. Therefore, the low-level resistance to QACs in L. monocytogenes may contribute to its environmental adaptation and persistence. In fact, in certain cases, the relationship between low-level resistance and the environmental persistence of L. monocytogenes in different food production chains has been previously established. The resistant strains would have survival advantages in these environments over sensitive strains, such as the ability to form biofilms in the presence of increased biocide concentrations.

13.
Appl Environ Microbiol ; 82(1): 308-17, 2016 01 01.
Article in English | MEDLINE | ID: mdl-26497458

ABSTRACT

The aim of this study was to investigate the basis of the putative persistence of Listeria monocytogenes in a new industrial facility dedicated to the processing of ready-to-eat (RTE) Iberian pork products. Quaternary ammonium compounds, which included benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance to disinfectants was associated with persistence. Of the 14 isolates obtained from product contact and non-product contact surfaces, only five different pulsed-field gel electrophoresis (PFGE) types were identified during the 27-month study period. Two of these PFGE types (S1 and S10-1) were previously identified to be persistent and BAC-resistant (BAC(r)) strains in a geographically separate slaughterhouse belonging to the same company. The remaining three PFGE types, which were first identified in this study, were also BAC(r). Whole-genome sequencing and in silico multilocus sequence typing (MLST) analysis of five BAC(r) isolates of the different PFGE types identified in this study showed that the isolate of the S1 PFGE type belonged to MLST sequence type 31 (ST31), a low-virulence type characterized by mutations in the inlA and prfA genes. The isolates of the remaining four PFGE types were found to belong to MLST ST121, a persistent type that has been isolated in several countries. The ST121 strains contained the BAC resistance transposon Tn6188. The disinfection-resistant L. monocytogenes population in this RTE pork product plant comprised two distinct genotypes with different multidrug resistance phenotypes. This work offers insight into the L. monocytogenes subtypes associated with persistence in food processing environments.


Subject(s)
Disinfectants/pharmacology , Food Contamination/prevention & control , Listeria monocytogenes/drug effects , Listeria monocytogenes/genetics , Meat Products/microbiology , Meat/microbiology , Abattoirs , Animals , Benzalkonium Compounds/pharmacokinetics , Comparative Genomic Hybridization , Disinfection/statistics & numerical data , Electrophoresis, Gel, Pulsed-Field , Food Handling , Food Microbiology , Listeria monocytogenes/classification , Listeria monocytogenes/isolation & purification , Molecular Sequence Data , Multilocus Sequence Typing , Mutation , Spain , Swine
14.
Health Inf Sci Syst ; 3(Suppl 1 HISA Big Data in Biomedicine and Healthcare 2013 Con): S5, 2015.
Article in English | MEDLINE | ID: mdl-25960872

ABSTRACT

OBJECTIVE: To describe the importance of bioinformatics tools to analyze the big data yielded from new "omics" generation-methods, with the aim of unraveling the biology of the pathogen bacteria Lactococcus garvieae. METHODS: The paper provides the vision of the large volume of data generated from genome sequences, gene expression profiles by microarrays and other experimental methods that require biomedical informatics methods for management and analysis. RESULTS: The use of biomedical informatics methods improves the analysis of big data in order to obtain a comprehensive characterization and understanding of the biology of pathogenic organisms, such as L. garvieae. CONCLUSIONS: The "Big Data" concepts of high volume, veracity and variety are nowadays part of the research in microbiology associated with the use of multiple methods in the "omic" era. The use of biomedical informatics methods is a requisite necessary to improve the analysis of these data.

15.
Genome Announc ; 3(2)2015 Mar 05.
Article in English | MEDLINE | ID: mdl-25745005

ABSTRACT

We announce the draft genome sequences of five Listeria monocytogenes strains from two Iberian pork-processing plants located in Spain that were distinguished by their resistance to benzalkonium chloride. These strains seem highly adapted to the meat-processing environment according to their persistence and transmission capabilities.

16.
Stud Health Technol Inform ; 202: 20-3, 2014.
Article in English | MEDLINE | ID: mdl-25000005

ABSTRACT

There is an open controversy in the use of the terms personalised and precision medicine and what they refer to in different contexts. In the present work we have considered the data types managed by each of them rather than their application and we have been able to identify commonalities but also differences in the types of data addressed in both approaches that would ultimately lead to include, from a data perspective, personalised medicine within the broader precision medicine term.


Subject(s)
Chromosome Mapping/methods , DNA Mutational Analysis/methods , Genetic Predisposition to Disease/genetics , Genome, Human/genetics , Polymorphism, Single Nucleotide/genetics , Precision Medicine/methods , Humans , Reproducibility of Results , Sensitivity and Specificity
17.
PLoS One ; 8(11): e79692, 2013.
Article in English | MEDLINE | ID: mdl-24223997

ABSTRACT

Lactococcus garvieae is an important fish and an opportunistic human pathogen. The genomic sequences of several L. garvieae strains have been recently published, opening the possibility of global studies on the biology of this pathogen. In this study, a whole genome DNA microarray of two strains of L. garvieae was designed and validated. This DNA microarray was used to investigate the effects of growth temperature (18°C and 37°C) on the transcriptome of two clinical strains of L. garvieae that were isolated from fish (Lg8831) and from a human case of septicemia (Lg21881). The transcriptome profiles evidenced a strain-specific response to temperature, which was more evident at 18°C. Among the most significant findings, Lg8831 was found to up-regulate at 18°C several genes encoding different cold-shock and cold-induced proteins involved in an efficient adaptive response of this strain to low-temperature conditions. Another relevant result was the description, for the first time, of respiratory metabolism in L. garvieae, whose gene expression regulation was temperature-dependent in Lg21881. This study provides new insights about how environmental factors such as temperature can affect L. garvieae gene expression. These data could improve our understanding of the regulatory networks and adaptive biology of this important pathogen.


Subject(s)
Gene Expression Profiling , Lactococcus/genetics , Temperature , Lactococcus/growth & development , Lactococcus/pathogenicity , Oligonucleotide Array Sequence Analysis , Transcription, Genetic
18.
Article in English | MEDLINE | ID: mdl-23920716

ABSTRACT

There are many challenges for Biomedical Informatics (BMI) to meet the promises of Translational Medicine. Several of the challenges have to do with the re-use of clinical data to progress towards 4P Medicine (personalized, preventive, participatory and predictive) and using epidemiology and clinical research as well as the translation of data and knowledge obtained in the laboratory into useful information for the clinical setting. The INBIOMEDvision EU funded project tackled these challenges in a series of events with expert scientists in different related disciplines. As a result, some consensus ideas were reached about the BMI challenges in the research areas of Genotype-Phenotype integration, Translational Systems Biology and Re-use of Clinical information in the Electronic Health Records (EHR) for research and what is needed to meet them.


Subject(s)
Forecasting , Health Information Systems/trends , Medical Informatics/trends , Medical Records Systems, Computerized/trends , Translational Research, Biomedical/trends , European Union
19.
Article in English | MEDLINE | ID: mdl-23920753

ABSTRACT

The development of personalized medicine is tightly linked with the correct exploitation of molecular data, especially those associated with the genome sequence along with these use of genomic data there is an increasing demand to share these data for research purposes. Transition of clinical data to research is based in the anonymization of these data so the patient cannot be identified, the use of genomic data poses a great challenge because its nature of identifying data. In this work we have analyzed current methods for genome anonymization and propose a one way encryption method that may enable the process of genomic data sharing accessing only to certain regions of genomes for research purposes.


Subject(s)
Algorithms , Computer Security , Confidentiality , Databases, Genetic , Electronic Health Records , Genome-Wide Association Study/methods , Health Records, Personal , Genomics/methods , Information Dissemination/methods , Information Storage and Retrieval/methods , Software
20.
J Bacteriol ; 193(16): 4263-4, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21685280

ABSTRACT

Lactococcus garvieae is the etiological agent of lactococcosis, one of the most important disease threats to the sustainability of the rainbow trout farming industry. Here, we present the draft genome sequence of Lactococcus garvieae strain 8831, isolated from diseased rainbow trout, which is composed of 2,087,276 bp with a G+C content of 38%.


Subject(s)
Disease Outbreaks/veterinary , Fish Diseases/microbiology , Genome, Bacterial , Gram-Positive Bacterial Infections/veterinary , Lactococcus/genetics , Oncorhynchus mykiss , Animals , Base Sequence , Fish Diseases/epidemiology , Gram-Positive Bacterial Infections/epidemiology , Gram-Positive Bacterial Infections/microbiology , Lactococcus/classification , Molecular Sequence Data , Spain/epidemiology
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