Identification, resistance to antibiotics and biofilm formation of bacterial strains isolated from a reverse osmosis system of a drinking water treatment plant

Membrane processes such as, reverse osmosis (RO), is one option for drinking water treatment where a high product quality is desired. RO membranes are able to effectively remove organic and inorganic compounds and microorganisms from water. One of the most serious problems in this system is membrane fouling caused by the microbial biofilm formation. Biofilms contributes to RO membrane deterioration, but it also were associated with human health risks, since they can be reservoirs of human opportunistic pathogens and/or antibiotic resistant bacteria. In the present study, samples from a drinking-water RO system were analyzed to determine the bacterial composition of the different frames of RO system: feed water, rejection water and permeate water. A total of 215 strains were identified using two methodologies, one genomic (16S rRNA gene) and other proteomic (MALDI-TOF MS). The majority bacterial groups identified were Gammaproteobacteria, Firmicutes and Betaproteobacteria, whereas Alfaproteobacteria, Bacteroidetes, and Actinomycetales were the minority bacterial identified groups. Antibiotic resistance analysis of the bacterial strains to 14 antibiotics tested revealed that resistance to three or more classes of antibiotics was observed in 37.2% of the isolates, being the occurrence of multiple antibiotic resistance phenotypes mainly related to the taxonomic affiliation of the microorganisms. Regarding of the bacterial groups, the antibiotic resistance percentages among the strains were 68.2% to β-lactams, 56.8% to macrolides, 36.4% to quinolones, 34.1% to cephalosporins, 22.7% to aminoglycosides and 9.1% to tetracyclines. On the other hand, >60% of the bacterial strains produced biofilms in vitro; from them, Aeromonas caviae, Pseudomonas aeruginosa and Sphingopyxis terrae were strong biofilm former

Lymphocystis disease virus (LCDV-Sa), polyomavirus 1 (SaPyV1) and papillomavirus 1 (SaPV1) in samples of Mediterranean gilthead seabream

Lymphocystis disease, caused by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of tumour-like lesions on the skin of affected animals associated with several environmental factors and/or with stress due to the intensive culture conditions of fish farms. In a previous study, the genomes of a new LCDV species, LCDV-Sa, were detected, together with 2 previously unknown viruses, Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1). Gilthead seabream from 17 fish farms in Spain, Italy and Turkey were sampled between 2009 and 2015 to investigate the role of the newly described SaPV1 and SaPyV1 viruses in lymphocystis disease development. Our results show that in diseased fish, either or both of the new viruses are almost invariably detected together with LCDV (98%). In asymptomatic fish, these viruses were detected in a much lower percentage (28%) and mostly in concurrence with LCDV (24%). These data confirm the suspected association among the 3 different viruses during lymphocystis disease development in gilthead seabream and warrant future studies to establish their respective contributions

Evaluation of gilthead seabream (Sparus aurata) immune response after LCDV DNA vaccination

A DNA vaccine against Lymphocystis Disease Virus (LCDV) was developed and its protective efficacy in gilthead seabream (Sparus aurata) has been established. The aim of the present study is the evaluation of immune-related gene expression after vaccination to identify which genes could be relevant to control the viral infection. The vaccine was administered intramuscularly to gilthead seabream specimens (100 g weight) at 10 µg/fish dose. In addition, two control groups, injected with the empty plasmid at the same dose or PBS, were established to evaluate non-specific immune response and basal response of fish, respectively. In this study 23 genes related to the immune response (tlr5, tlr9, ifnI, irf1, irf3, irf9, pkr, mx1, mx2, mx3, isg15, tnfα, casp1, il1β, il6, il10, ck3, ck10, c3, nccrp1, mhcII, tcrβ, and ighm) and 2 reference genes (ef1α and actβ) were analysed using real-time PCR (RT-qPCR) in samples of head kidney and intestine at 1, 3, and 8 d post-vaccination. DNA-vaccination of gilthead seabream induced the differential expression of 9 genes in head kidney and 15 genes in intestine samples. Through the course of the experiment, 9 of those genes reached high level of up-regulation comparing to control groups. These genes were related to IFN type I pathway (irf9 and mx3, in head kidney), inflammation (il1β, il6, tnfα, ck10, c3 and nccrp-1, in both organs analysed), and adaptive immune response (mhcII, in intestine). Conclusion: The results obtained allow us to understand which genes could be responsible for the protection against LCDV infection conferred by the DNA vaccine in gilthead seabream. Inflammation is the biological process mainly triggered as a systemic response in vaccinated fish. Different gene expression profiles have been observed in each organ, which may indicate specialized roles relative to immune defensive mechanisms.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Immune response of gilthead seabream (Sparus aurata) after experimental infection with lymphocystis disease virus (LCDV-Sa)

Lymphocystis disease (LCD) is caused by the lymphocystis disease virus (LCDV) (family Iridoviridae), affecting more than 150 fish species from both marine and freshwater environments. A few studies have been focused on the immune defensive mechanisms of fish against LCDV, but only one was conducted during a natural LCD outbreak in gilthead seabream, which is one of the most important cultured fish species in the Mediterranean and the European Atlantic coasts. The aim of this study was the analysis of 23 genes related to the immune response in gilthead seabream specimens after experimental infection with LCDV-Sa using real-time PCR (qRT-PCR) in samples of head kidney and intestine at 1, 3, and 8 dpi. To study the progression of LCDV-Sa infection in gilthead seabreams, the number of viral DNA copies and the expression of mcp were determined in samples of caudal fin, head kidney and intestine. LCDV-Sa was detected by qPCR in all the samples from inoculated fish analysed, whereas no amplification was obtained in samples from the control group. Regarding the gene expression following LCDV-Sa infection, a total of 22 of the 23 genes studied were differentially expressed in head kidney or intestine samples at some time points analysed. Different gene expression profiles were obtained between the organs studied, detecting 18 differentially expressed genes (DEGs) in head kidney samples, four of them exclusively up- or down-regulated (nccrp1, il10, mhcII, and tnfα genes), and 5 genes with a significant change in the expression tendency from 1 to 8 dpi (irf3, isg15, il10, ck10, and c3). In the intestine, 18 DEGs were also detected (14 shared with head kidney), being mx1, casp1, ck3 and tlr9 genes exclusively detected in these samples, and mx1, mx3, irf9 and ighm differentially regulated over time. The results obtained allow us to understand which genes are essential for host-pathogen interactions and could be used as molecular markers for vaccine efficacy evaluation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech Proyecto de Excelencia Junta de Andalucía Ref. P12-RNM-226

Cytokine-like activity of European sea bass ISG15 protein on RGNNV-infected E−11 cells

IFN-I generates an antiviral state by inducing the expression of numerous genes, called IFN-stimulated genes, ISGs, including ISG15, which is the only ISG with cytokine-like activity. In a previous study, we developed the Dl_ISG15_E11 cell line, which consisted of E11 cells able to express and secrete sea bass ISG15. The current study is a step forward, analysing the effect of secreted sea bass ISG15 on RGNNV replication in E11 cells, and looking into its immunomodulatory activity in order to corroborate its cytokine-like activity. The medium from ISG15- produccing cells compromised RGNNV replication, as it has been demonstrated both, by reduction in the viral genome synthesis and, specially, in the yield of infective viral particles. The implication of sea bass ISG15 in this protection has been demonstrated by ISG15 removal, which decreased the percentage of surviving cells upon viral infection, and by incubation of RGNNV-infected cells with a recombinant sea bass ISG15 protein, which resulted in almost full protection. Furthermore, the immunomodulatory activity of extracellular sea bass ISG15 has been demonstrated, which reaffirms a cytokine-like role for this proteinThis study has been supported by projects AGL2017-84644-R (MINECO/AEI/FEDER, UE) (Spanish Government) and UMA20- FEDERJA-103 (Consejería de Economía y Conocimiento/Programa Operativo FEDER 2014–20, UE) (Junta de Andalucía). Funding for open access charge: Universidad de M ́alaga/CBUA. Authors thank I. Bandin for the viral isolate

Evaluation of immune response after LCDV-Sa infection in DNA-vaccinated gilthead seabream

The immune-related gene expression in vaccinated gilthead seabream after Lymphocystis Disease Virus 3 (LCDV-Sa) infection was analysed by using an OpenArray based on TaqMan qPCR. The DNA vaccine used in this study encodes the viral major capsid protein and confers protection against LCDV-Sa infection in juvenile gilthead seabream. Gilthead seabream juveniles were distributed into four experimental groups and intramuscularly injected with the vaccine (vaccinated group), the empty-plasmid (mock-vaccinated group), or PBS (control groups). Thirty days after vaccination, vaccinated and mock-vaccinated fish, as well as one of the control groups, were injected intraperitoneally with LCDV-Sa (106 TCID50/fish). Samples of head-kidney (HK) from 6 fish were individually collected 1 and 3 days post-infection (dpi). The relative expression levels of 49 genes related to the immune response and 4 reference genes were analysed using an OpenArray. Samples from the non-infected control group were used as calibrator. The number of genes differentially expressed (DEG) in HK at 1 dpi was higher in vaccinated fish compared with both mock-vaccinated and non-vaccinated animals. At 3 dpi, most DEG were upregulated, and the differences in their number among groups were minimized. The recombination-activating gene 1 (rag1), a mediator of development of B and T lymphocytes, was the only gene upregulated in HK samples at 1 dpi. This gene was also upregulated in non-vaccinated animals but at 3 dpi. In contrast, early mx induction was observed in non-vaccinated animals (upregulation of mx2 at 1 dpi) in comparison to vaccinated seabreams (upregulation of mx1 and mx2 at 3 dpi). The results that will be discussed could evidence the role of the DNA vaccine as regulator of the primary lymphoid tissues (HK) in gilthead seabream against LCDV-Sa infection, through downregulation of inflammation related-genes, early upregulation of rag1, and a later expression of interferon stimulated genes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Immune response of vaccinated juvenile gilthead seabream (Sparus aurata) after LCDV-Sa infection

Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream in the Mediterranean area. In our group, a DNA-vaccine has been developed based on the major capsid protein (MCP) of the Lymphocystis Disease Virus 3 (LCDV-Sa). The aim of the present study is the evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in the vaccine-induced protection. To fulfil this objective an OpenArray® platform has been developed to study 49 genes related to the immune response. Reference and viral genes were also evaluated. Gilthead seabream specimens (5 g mean weight) were distributed into 3 experimental groups, inoculated with the vaccine at 0.1 µg/g fish dose, the empty plasmid at the same dose or PBS. Thirty days post-vaccination, fish were intramuscularly injected with the virus at 106 TCID50/fish dose. Samples of head-kidney, spleen, intestine and caudal fin from 6 fish were individually collected at 1, 2 and 3-days post-injection in all groups. The quantification of viral DNA in fins of fish challenged with LCDV-Sa were carried out by a qPCR assay targeting a viral structural gene (putative myristoylated membrane protein, MMP) alternative to the mcp gene contained in the vaccine. The results obtained showed an increase of genes deregulated within the haematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a diminished immune response compared to non-vaccinated fish, being 83 and 99 genes differentially expressed through the experiment, respectively. Moreover, viral replication decreased in groups of fish previously vaccinated. The modulation of the immune response provoked by the vaccination trial seems to control the progression of the disease.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Persistence of Lymphocystis Disease Virus (LCDV) in Seawater

Lymphocystis disease virus (LCDV), the causative agent of lymphocystis disease (LCD), is a waterborne pathogen that uses the external surfaces, including the gills, as portals to gain access to fsh host. However, there are no data on LCDV persistence in the aquatic environment. In this study, the persistence of LCDV in natural (raw), treated (autoclaved and fltered) and synthetic seawater held at 22 and 18 °C has been evaluated. The estimated T99 values for LCDV in seawater ranged from 2.7 to 242 days depending on seawater type and temperature, with the highest value recorded at 22 °C in autoclaved seawater. Microbiota and temperature seem to be the main factors afecting the persistence of LCDV in seawater. The results indicated that LCDV is more stable in treated seawater than most of the fsh pathogenic viruses studied so far, supporting the relevance of this medium for the prevalence of LCD in fsh farms

Fish Iridoviridae: infection, vaccination and immune response

Each year, due to climate change, an increasing number of new pathogens are being discovered and studied, leading to an increase in the number of known diseases affecting various fish species in different regions of the world. Viruses from the family Iridoviridae, which consist of the genera Megalocytivirus, Lymphocystivirus, and Ranavirus, cause epizootic outbreaks in farmed and wild, marine, and freshwater fish species (including ornamental fish). Diseases caused by fish viruses of the family Iridoviridae have a significant economic impact, especially in the aquaculture sector. Consequently, vaccines have been developed in recent decades, and their administration methods have improved. To date, various types of vaccines are available to control and prevent Iridoviridae infections in fish populations. Notably, two vaccines, specifically targeting Red Sea bream iridoviral disease and iridoviruses (formalin-killed vaccine and AQUAVAC® IridoV, respectively), are commercially available. In addition to exploring these themes, this review examines the immune responses in fish following viral infections or vaccination procedures. In general, the evasion mechanisms observed in iridovirus infections are characterised by a systemic absence of inflammatory responses and a reduction in the expression of genes associated with the adaptive immune response. Finally, this review also explores prophylactic procedure trends in fish vaccination strategies, focusing on future advances in the field.PCM_00059 (Proyectos de I + D + i en el marco del Plan Complementario de Ciencias Marinas

Immune response of DNA vaccinated-gilthead seabream (Sparus aurata) against LCDV-Sa infection: relevance of the inflammatory process

Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream (Sparus aurata) in the Mediterranean region. Recently, we have developed a DNA vaccine based on the major capsid protein (MCP) of the Lymphocystis disease virus 3 (LCDV-Sa). The immune response triggered by either LCDV-Sa infection or vaccination have been previously studied and seem to be highly related to the modulation of the inflammatory and the IFN response. However, a comprehensive evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in vaccineinduced protection have not been carried out to date. The present study aimed to fulfill this objective by analyzing samples of head-kidney, spleen, intestine, and caudal fin from fish using an OpenArray® platform containing targets related to the immune response of gilthead seabream. The results obtained showed an increase of deregulated genes in the hematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a significant decrease (p<0.05) of viral replication in groups of fish previously vaccinated, and the expression of the following immune genes related to viral recognition (tlr9), humoral and cellular response (rag1 and cd48), inflammation (csf1r, elam, il1b, and il6), antiviral response (isg15, mx1, mx2, mx3), cell-mediated cytotoxicity (nccrp1), and apoptosis (prf1). The exclusive modulation of the immune response provoked by the vaccination seems to control the progression of the infection in the experimentally challenged gilthead seabream