Surveillance studies of Lymphocystis disease virus in farmed gilthead seabream (Sparus aurata) by real-time PCR

Lymphocystis disease (LCD) is the main viral infection reported to affect cultured gilthead seabream (Sparus aurata) in Southern Atlantic and Mediterranean aquaculture. Its etiological agent is the Lymphocystis disease virus (LCDV), a member of the family Iridoviridae (genus Lymphocystivirus). The only adequate measures for LCD prevention in the aquaculture systems are general prophylactic practices, such as the control of fish to be introduced in the farm facilities in order to detect carrier fish. These animals may pose a risk for the introduction of LCDV in fish farms, as direct contact between fish specimens is considered the main route of LCDV spreading. More recently, asymptomatic carrier breeders, as well as virus contaminated-live food, have been involved in LCDV transmission to fish larvae. The detection of subclinical viral infections in carrier fish requires the use of sensitive diagnostic methods. In this context, the objective of this study was to establish the applicability of a real-time PCR assay for LCDV diagnosis in surveillance studies. In addition, the assay has been evaluated with samples from a gilthead seabream hatchery, in order to prove its utility to trace the origin of LCDV in fish farms. Juvenile fish were collected at four farms with different background regarding to LCD. LCDV was detected in all farms, and 30 to 100% of fish were identified as LCDV-infected. Estimated viral load in caudal fin of asymptomatic fish was two to five orders of magnitude lower than in diseased fish. Carrier fish were also identified in the broodstock from a farm with LCD records by analysing caudal fin samples by qPCR. In this farm, the q-PCR assay developed in this study allowed the quantitative detection of LCDV in all samples collected in the hatchery, including fertilized eggs, larvae and fingerlings, and also rotifer cultures and artemia metanauplii and cysts used for larval rearing.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Course of infection with Lymphocystis disease virus in gilthead seabream (Sparus aurata)

Lymphocystis disease virus (LCDV) is the etiological agent of lymphocystis disease (LCD), a pathology that affects a wide variety of fish species. Data about LCDV pathogenesis are very short, and mainly limited to histopathological studies of skin lesions. Recent studies on viral genome detection (both by PCR or DNA-DNA in situ hybridization) suggest that LCDV establish a systemic and persistent infection in gilthead seabream (Sparus aurata), but further studies are necessary to prove if this infection is productive or not. In the present study viral quantification and viral mRNA detection (by qPCR and RT-qPCR) have been used to investigate LCDV multiplication in different organs of juvenile gilthead seabream. In addition, a histopathological study was carried out. Animals were collected from two commercial farms in Southwestern Spain. In one farm, where no LCD outbreaks have been recorded, apparently healthy fish were collected, whereas in the other farm, diseased and recovered (two months after LCD symptoms disappearance) fish were sampled. All the animals were LCDV-infected, and viral gene expression was detected in every organ analysed (caudal fin, intestine, liver, spleen, kidney and brain). In asymptomatic animals, both apparently healthy and recovered, a low-titre infection was observed, with the highest viral copy numbers detected in brain and kidney. In diseased fish, viral loads were significantly higher than in subclinical infected animals, being maximal in caudal fin, where lymphocysts were present in the dermis. Different histological alterations were observed in the internal organs from diseased fish analysed, although no hypertrophied cells were detected in any of them. In recovered fish, most of the organs examined presented similar histological features to those in healthy animals. Thus, pathological changes were only detected in the intestine and liver, although they were less severe than those observed in diseased fish. The results presented showed that LCDV establishes a systemic infection in juvenile gilthead seabream, which can be subclinical. In addition, although the disease is self-limiting, the virus is not removed after disease recovery, but produces a persistent infection.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Persistencia del virus de la enfermedad de linfocistis (LCDV) en agua

El virus de la enfermedad de linfocistis (LCDV), perteneciente al género Lymphocystivirus (familia Iridoviridae), es el agente etiológico de la enfermedad de linfocistis (LCD), principal patología de origen vírico descrita en doradas en la acuicultura marina mediterránea. Aunque esta enfermedad es muy frecuente, las vías de transmisión de su agente etiológico no se han dilucidado completamente, considerándose el contacto directo entre peces o la ruta hídrica las principales vías de entrada del virus en los sistemas acuícolas. Aunque el LCDV puede transmitirse vía hídrica, al menos de forma experimental, no existen resultados sobre su persistencia en agua de mar, lo que es esencial para determinar la importancia del agua como ruta de transmisión del virus en las piscifactorías marinas. En el presente trabajo se ha abordado el estudio de la capacidad de persistencia del LCDV en agua de mar sometida a diferentes tratamientos, observándose el efecto de los factores bióticos y abióticos sobre dicha persistencia. Para el estudio se utilizó un stock vírico de la cepa de colección ATCC VR-342, usando agua de mar artificial y agua de mar sometida a tratamientos (esterilización por calor húmedo y filtración) para eliminar microorganismos y materia orgánica presentes en el agua, evaluándose el efecto de estos tratamientos, así como la temperatura (18°C y 22°C) y la carga viral, en la infectividad del virus. Se determinó el título infectivo de las suspensiones víricas en las muestras a diferentes tiempos mediante ensayo en cultivos celulares, aplicando el método de dilución final de efecto citopático (TCID50) y ensayo ICC-RT-PCR (Integrated Cell Culture-RT-PCR). Los resultados obtenidos indican que los factores abióticos presentes en el agua sometida a esterilización por filtración y calor no afectan significativamente a la persistencia del virus. Sin embargo, se ha demostrado que tanto la temperatura como la carga vírica son factores determinantes de la persistencia del LCDV en agua.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Desarrollo y evaluación de una vacuna DNA contra el virus de la enfermedad de linfocistis (LCDV)

El virus de la enfermedad de linfocistis (LCDV) infecta tanto a peces marinos como dulceacuícolas, produciendo una patología que supone un grave problema para la acuicultura debido a las pérdidas económicas asociadas a su alta morbilidad. En el presente estudio se ha desarrollado un plásmido recombinante con objeto de ser utilizado como vacuna DNA para limitar la incidencia de dicha enfermedad en el cultivo de dorada (Sparus aurata). Para ello, se clonó el ORF de la proteína principal de la cápside (MCP) del LCDV de dorada (LCDV-Sa) en el plásmido de expresión pcDNA3.1/NT-GFP-TOPO, y se utilizó para transfectar la línea celular de dorada SAF-1. Los resultados muestran la correcta expresión del plásmido vacunal en estas células mediante la detección de GFP por microscopía de fluorescencia e inmunodetección de la MCP viral. Posteriormente, se realizó un experimento de vacunación por inyección intramuscular (0,15 µg/g pez) en doradas juveniles, estudiándose la distribución y expresión del plásmido vacunal. Los análisis de PCR y RT-PCR realizados muestran que la vacuna se distribuye de forma sistémica, expresándose en los diferentes tejidos analizados. También se evaluó la protección conferida por la vacuna frente a la infección por LCDV inyectado intraperitonealmente (105 TCID50/g pez) a los 21 d post-vacunación, analizándose la presencia de genoma viral a los 10 d post-inoculación en peces vacunados y controles. Sólo se detectó el genoma viral en los grupos controles, demostrándose así que existe una protección frente a la infección por LCDV en los peces vacunados.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Detección y cuantificación del virus de la enfermedad de linfocistis mediante ensayo ICC-RT-PCR (integrated cell culture-RT-PCR)

La enfermedad de linfocistis es la enfermedad de etiología viral más frecuentemente detectada en la acuicultura marina europea, siendo la principal patología de origen vírico descrita en doradas cultivadas. El agente etiológico de esta enfermedad es el virus de la enfermedad de linfocistis (LCDV), miembro del género Lymphocystivirus, perteneciente a la familia Iridoviridae. Se han desarrollado diversos protocolos de PCR y PCR a tiempo real que permiten la detección y cuantificación del LCDV en diversas muestras, si bien no aportan ninguna indicación de la infectividad de los virus detectados. La detección de partículas víricas infectivas requiere la utilización de cultivos celulares, pero en el caso del LCDV la observación de efectos citopáticos (CPE) es difícil y a menudo está sujeta a subjetividad, especialmente en muestras con baja carga vírica. Por este motivo, en el presente trabajo se ha desarrollado un ensayo de ICC-RT-PCR (Integrated Cell Culture-RT-PCR) que permite la detección de partículas infectivas del LCDV. Este ensayo se ha aplicado en combinación con el método del número más probable (NMP) para la determinación del título infectivo en cultivos celulares. El protocolo de ICC-RT-PCR desarrollado permitió la detección de mRNA viral a partir de células SAF-1 inoculadas con un título infectivo de LCDV de 0,1 TCID50/ml, procesadas a los 5 d p.i, mientras que el límite de detección mediante observación de CPE fue de 10 TCID50/ml a 14 d p.i. La sensibilidad de la técnica he permitido la detección de partículas infectivas del LCDV en ejemplares de dorada asintomáticos, donde no se observaron CPE en cultivos celulares inoculados en paralelo y mantenidos hasta 14 d p.i. Este protocolo también se ha aplicado para la determinación del título infectivo de diferentes aislados víricos obtenidos a partir de peces enfermos, reduciéndose de forma considerable el tiempo necesario para realizar la titulación en comparación con el método de la dosis infectiva 50% en cultivos celulares (TCID50) (5 d versus 14-21 d, respectivamente). Así mismo, se han titulado stocks víricos obtenidos en cultivos celulares, donde la carga vírica es inferior al límite de detección del ensayo de observación de CPE. En conclusión, el protocolo de ICC-RT-PCR desarrollado es una técnica sensible, rápida y útil para la detección y cuantificación de LCDV infectivos, lo que la convierte en una herramienta adecuada para el estudio de esta patología viral.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Desarrollo de una vacuna DNA para el control de la enfermedad de linfocistis

Leiva, R., Borrego, J.J., Castro, D. y Labella, A.M. 2018. Desarrollo de una vacuna DNA para el control de la enfermedad de linfocistis. En: La Acuicultura en el Litoral Suratlántico. IX Jornadas de Acuicultura en el Litroral Suratlántico, p 96. Instituto de Investigación y Formación Agraria y Pesquera (IFAPA), Consejería de Agricultura, Pesca y Desarrollo Rural, Junta de Andalucía.El virus de la enfermedad de linfocistis (LCDV) es el agente etiológico de la enfermedad de linfocistis, que afecta tanto a peces marinos como dulceacuícolas. Esta enfermedad supone un grave problema para el sector de la acuicultura, provocando pérdidas económicas debido a su alta morbilidad. En el presente estudio se ha desarrollado un plásmido recombinante con objeto de ser utilizado como vacuna de DNA para limitar la incidencia de dicha enfermedad en el cultivo de dorada (Sparus aurata). El gen codificante de la proteína principal de la cápside (MCP) del LCDV de dorada (LCDV-Sa) se clonó en primer lugar en el vector de expresión pGEX-6P-3, con el fin de expresar la MCP viral como proteína de fusión con GST (glutathione S-transferase) en Escherichia coli BL-21. La expresión de la proteína de fusión MCP-GST en células bacterianas se comprobó mediante Western blot e inmunoensayo dot-blot con anticuerpos específicos dirigidos contra la MCP y la GST. Esta MCP recombinante se empleará como antígeno de captura para la detección mediante ELISA de anticuerpos anti-LCDV en peces vacunados. Por otra parte, el gen codificante de la MCP se subclonó en el vector de expresión pcDNA3.1/NT-GFP-TOPO, obteniéndose así el plásmido vacunal. Tras su amplificación en E. coli TOP 10, el plásmido se purificó y se utilizó para transfectar la línea celular de dorada SAF-1, evaluándose dos métodos de transfección: Nucleofector Kit y lipofectamina. La expresión de la MCP viral en células SAF-1 transfectadas con el plásmido vacunal se ha demostrado mediante detección de la GFP (green fluorescent protein) por microscopía de epifluorescencia, y por inmunodetección de la MCP viral utilizando anticuerpos específicos. El plásmido vacunal obtenido se inyectará intramuscularmente en ejemplares de dorada para determinar la distribución y expresión temporal de la vacuna, así como para detectar anticuerpos anti-LCDV en los peces vacunados.Proyecto de Excelencia de la Junta de Andalucía (P12-RNM-2261). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

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