Control of enteric parasitic diseases of farmed gilthead sea bream: New insights into Enteromyxum leei (Myxozoa) and Enterospora nucleophila (Microsporidia) infections

Tesis por compendio[ES] La producción en acuicultura se ha visto menguada por aparición de enfermedades en los sistemas de cría de peces. En concreto, en la dorada (Sparus aurata), hay dos parásitos destacados: Enteromyxum leei (Myxozoa) y Enterospora nucleophila (Microsporidia). Hasta la fecha, para ninguno de los dos se ha establecido un cultivo in vitro, y solo para E. leei se ha conseguido establecer un modelo de mantenimiento de la infección in vivo. La presente tesis pretende incrementar el conocimiento sobre estos parásitos y sus relaciones con el hospedador, sentando las bases para generar soluciones que puedan ser aplicadas en la acuicultura. El objetivo con E. leei fue estudiar la inmunidad adquirida inducida en la dorada y la posibilidad de generar herramientas de diagnóstico y vacunas frente a esta enfermedad. Para ello, primero se demostró la resistencia del pez al parásito tras una segunda exposición, la cual duró hasta 16 meses. Además, la resistencia parece estar correlacionada con altos niveles de inmunoglobulina (Ig) M específica en sangre, y una alta expresión de Igs, incluso antes de la re-exposición al parásito. El siguiente paso fue afinar el protocolo de infección con E. leei. Los resultados mostraron que una semana es suficiente para transmitir la infección de E. leei por efluente, independientemente de la temperatura. Tras la demostración de la respuesta adaptativa eficaz frente a E. leei, y al disponer de un modelo de infección refinado, se realizó un ensayo de inmunización pasiva. Aquí, los resultados mostraron que los anticuerpos especi'ficos efectivamente consigue ralentizar la invasión del intestino por el parásito y disminuir los síntomas de la enfermedad. Paralelamente, el resultado del análisis del repertorio de las regiones variables de la IgM e IgT del intestino peces resistentes mostró la inducción de una respuesta policlonal en las ce'lulas B. En base a estos resultados, se realizó una búsqueda de antígenos de E. leei que pudieran ser utilizados como candidatos para la producción de vacunas (análisis proteómico) o herramientas de diagnóstico (análisis in silico). Para ello, se ensambló un transcriptoma de novo utilizando una muestra mixta de intestino de dorada y parásito. Los resultados dieron lugar a 7 y 12 candidatos en la búsqueda in silico y proteómica, respectivamente. En los estudios de E. nucleophila, debido a que fue descrita muy recientemente, el punto de partida fue más básico. Las muestras de este parásito solo se pueden obtener de brotes naturales en piscifactorias. Por ello, primero se realizó un estudio de caracterización de la patología de la infección a partir de peces infectados naturalmente. En etapas tempranas de la infección, el parásito se localiza principalmente en el intestino, pero meses después, la prevalencia en intestino baja e incrementa en los órganos hematopoyéticos y el esto'mago. Los signos clínicos de la infección consistieron en una reducción significativa del crecimiento, emaciación, y palidez de las paredes intestinales. A nivel celular, en los casos ma's graves se observó hipercelularidad en el epitelio intestinal y proliferación de ce'lulas rodlet, un elevado número de linfocitos en la base del epitelio e infiltración de granulocitos acidófilos en el epitelio intestinal. Finalmente se probaron varias formas de transmisión horizontal de E. nucleophila (cohabitación, efluente, intubación oral y anal) con para desarrollar un modelo de mantenimiento in vivo. Se consiguió la transmisión el parásito por todas las vías, pero con una disminución de prevalencia a lo largo del tiempo. Variables como la temperatura, la dosis, y el estado de los peces donantes parecen ser más determinantes que la ruta seleccionada para la transmisión. Entre las rutas probadas, la intubación anal parece ser la más prometedora, pero ninguna de ellas fue capaz de reproducir los signos clínicos observados en las infecciones naturales.[CA] La producció en aqüicultura s'ha vist minvada per aparició de malalties en els sistemes de cria de peixos. En concret, en l'orada (Sparus aurata), hi ha dos paràsits destacats: Enteromyxum leei (Myxozoa) i Enterospora nucleophila (Microsporidia). Fins avui, per a cap dels dos s'ha establert un cultiu in vitro, i només per a E. leei s'ha aconseguit establir un model de manteniment de la infecció in vivo. La present tesi pretén incrementar el coneixement sobre aquests paràsits i les seves relacions amb l'hoste, establint les bases per a generar solucions que puguin ser aplicades en l'aqüicultura. L'objectiu amb E. leei va ser estudiar la immunitat adquirida induïda en l'orada i la possibilitat de generar eines de diagnòstic i vacunes enfront d'aquesta malaltia. Per a això, primer es va demostrar la resistència del peix al paràsit després d'una segona exposició, la qual va durar fins a 16 mesos. A més, la resistència sembla estar correlacionada amb alts nivells d'immunoglobulina (Ig) M específica en sang, i una alta expressió de Igs, fins i tot abans de la re-exposició al paràsit. El següent pas va ser afinar el protocol d'infecció amb E. leei. Els resultats van mostrar que una setmana és suficient per a transmetre la infecció de E. leei per efluent, independentment de la temperatura. Després de la demostració de la resposta adaptativa eficaç enfront de E. leei, i en disposar d'un model d'infecció refinat, es va realitzar un assaig d'immunització passiva. Aquí, els resultats van mostrar que els anticossos específics efectivament aconsegueix alentir la invasió de l'intestí pel paràsit i disminuir els símptomes de la malaltia. Paral·lelament, el resultat de l'anàlisi del repertori de les regions variables de la IgM i IgT de l'intestí peixos resistents va mostrar la inducció d'una resposta policlonal en les cèl·lules B. Sobre la base d'aquests resultats, es va realitzar una cerca d'antígens de E. leei que poguessin ser utilitzats com a candidats per a la producció de vacunes (anàlisis proteómico) o eines de diagnòstic (anàlisi in silico). Per a això, es va assemblar un transcriptoma de novo utilitzant una mostra mixta d'intestí d'orada i paràsit. Els resultats van donar lloc a 7 i 12 candidats en la cerca in silico i proteòmica, respectivament. En els estudis de E. nucleophila, pel fet que va ser descrita molt recentment, el punt de partida va ser més bàsic. Les mostres d'aquest paràsit només es poden obtenir de brots naturals en piscifactorias. Per això, primer es va realitzar un estudi de caracterització de la patologia de la infecció a partir de peixos infectats naturalment. En etapes primerenques de la infecció, el paràsit es localitza principalment en l'intestí, però mesos després, la prevalença en intestí baixa i incrementa en els òrgans hematopoètics i l'estómac. Els signes clínics de la infecció van consistir en una reducció significativa del creixement, emaciació, i pal·lidesa de les parets intestinals. A nivell cel·lular, en els casos més greus es va observar hipercelularidad en l'epiteli intestinal i proliferació de cèl·lules rodlet, un elevat nombre de limfòcits en la base de l'epiteli i infiltració de granulòcits acidòfils en l'epiteli intestinal. Finalment es van provar diverses formes de transmissió horitzontal de E. nucleophila (cohabitació, efluent, intubació oral i anal) amb per a desenvolupar un model de manteniment in vivo. Es va aconseguir la transmissió el paràsit per totes les vies, però amb una disminució de prevalença al llarg del temps. Variables com la temperatura, la dosi, i l'estat dels peixos donants semblen ser més determinants que la ruta seleccionada per a la transmissió. Entre les rutes provades, la intubació anal sembla ser la més prometedora, però cap d'elles va ser capaç de reproduir els signes clínics observats en les infeccions naturals.[EN] Aquaculture production is hampered by the emergence of parasite diseases in fish farming systems. Among them, in Sparus aurata, there are two important enteric parasites described: Enteromyxum leei (Myxozoa) Enterospora nucleophila (Microsporidia). To date, no in vitro culture has been established for either parasite, and only for E. leei was it possible to establish a model for maintaining the infection in vivo. The aim of this thesis is to gain new knowledge about these parasites and their relationship with the host, also the basic foundations for generating solutions that can be applied in aquaculture. The general objective for E. leei was to study the acquired immunity induced in gilthead bream and the possibility of generating diagnostic tools and vaccines against this disease. To this end, resistance against the parasite was assessed with a second exposure against the parasite, which showed a resistance for at least 16 months. Besides resistance seemed to be correlated with high levels of specific immunoglobulin (Ig) M in blood, and a high expression of Igs, in particular, the soluble forms, even before re-exposure to the parasite. The next step was refining the protocol for effluent infection with E. leei by studying infection at different exposure time points, temperatures and population densities. The results showed that one week of exposure is sufficient to spread E. leei infection by effluent, regardless of temperature. After demonstrating the resistance against E. leei, and with a refined infection model, a passive immunization assay was performed. The results showed that the serum with specific antibodies effectively slows down the invasion of the gut by the parasite and reduces the symptoms of the disease. At the same time, the analysis of the repertoire of the variable regions of intestinal IgM and IgT showed an induction of a polyclonal response in B cells. On the basis of these results, a research was carried out for E. leei antigens that could have use as candidates for the production of vaccines (proteomic study) or diagnostic tools (in silico study) using the parasite transcriptomic data. To do this, a de novo transcriptome was assembled using a mixed sample of gilthead sea bream and parasite, with a posterior filtrate of the sequences. The In silico and proteomic analysis search resulted in 7 and 12 transcripts, respectively, which are being used for diagnostic and vaccine production. The starting point was more basic in E. nucleophila studies, since this is a recently described disease. The samples of this parasite can only be obtained from natural outbreaks in fish farms. Therefore, first study was carried out to characterize the pathology of the infection of naturally infected fish. In the early stages of the infection, the parasite is mainly located in the intestine, but months later, the prevalence is lower in the intestine and increases in the hematopoietic organs and the stomach. Clinical signs of infection were significant reduction in growth, wasting, and intestinal walls paleness. At the cellular level, in the most severe cases hypercellularity in the intestinal epithelium, proliferation of rodlet cells, high number of lymphocytes at the base of the epithelium and infiltration of acidophilic granulocytes in the intestinal epithelium were observed. Finally, horizontal transmission of E. nucleophila was tried using different transmission methods: cohabitation, effluent, and oral and anal intubation. Transmission of the parasite was achieved with all routes, but there was a decrease in prevalence over time in all cases except for the anal route. Variables such as temperature, dose, and the status of the donor fish appear to be more important than the selected route. Among the routes tested, anal intubation seemed to be the most promising, as it was sustained over a longer period of time, but none of them was able to reproduce the same clinical signs of infection observed in natural infections.The authors kindly acknowledge the collaboration of anonymous fish farming companies allowing access to the animals during the disease outbreaks. We thank J. Monfort and L. Rodríguez (IATS-CSIC) for the technical assistance on histological processing.This work has been carried out with financial support from the European Union and the Spanish Ministry of Economy and Competitiveness (MINECO) under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project. Primer sequences and access to the gilthead sea bream transcriptomic database were kindly provided by Prof. J. Pérez-Sánchez of the IATS- Nutrigenomics group. The authors thank I. Vicente for fish maintenance and technical assistance during samplings. The authors thank P. Boudinot (INRAE) for his help in designing and interpreting the immunoglobulin repertoire study and results, J. Pérez-Sánchez (IATS-CSIC) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis.This work was funded by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM).Picard Sánchez, MA. (2021). Control of enteric parasitic diseases of farmed gilthead sea bream: New insights into Enteromyxum leei (Myxozoa) and Enterospora nucleophila (Microsporidia) infections [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167035TESISCompendi

Disruption of gut integrity and permeability contributes to enteritis in a fish‑parasite model: a story told from serum metabolomics

Background In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality. Methods This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits. Results The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits. Conclusions The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei

Serum metabolomics tells the story of disease degree in a fish enteritis model

Comunicación presentada en la 19th International Conference on Diseases of Fish and Shellfish, celebrada en Oporto (Portugal) del 9 al 12 de septiembre de 2019.[Introduction]: In animal production, enteritis is responsible for serious economic losses, being intestinal parasitism a major stress factor leading to malnutrition and lowered performance and production efficiency. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) that impairs nutrient absorption causing anorexia, cachexia, growth impairment, reduced marketability and increased mortality. This study aimed to outline the gut failure produced in this fish-parasite model using a multifaceted approach and to find and validate serum non-lethal markers of gut barrier dysfunction.[Methodology]: Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp-1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits.[Results]: The expression of the tight junction proteins Tjp-1 and Cldn3 was significantly lower in parasitized fish along all the intestine, while no differences were found in E-cadherin labeling. Some parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology studies showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile when compared to the normal absorptive profile of the control animals. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses (PLS-DA), and even separated groups by intensity of infection. Of these compounds, inosine and creatine were identified as relevant and tested with commercial kits in serum samples.[Conclusion]: This study demonstrates the loss of barrier function induced by the enteric parasite E. leei and underlines key markers to differentiate control and infected fish. The untargeted serum metabolomics approach did not reveal specific effects by the parasite, but more a profile typical of absorption dysfunction and anorexia, which are, of course, part of the disease signs.Funding: ParaFishControl H2020 project (634429), Aquaexcel2020 (652831, TNA AE10004-INTEBREAM), AGL2013-48560-R

Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians

© 2021 by the authors.The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.This research was funded by the Ministry of Education, Youth, and Sports of the Czech Republic, grant number LTAUSA17201; by the European Commission under the H2020 Programme—ParaFishControl, grant number 634429; by the Czech Science Foundation, grant number 19-28399X (to A. S. Holzer, G. Alama-Bermejo, and J. Kyslík) and 21-16565S and by the Czech Academy of Sciences and Hungarian Academy of Sciences, grant number MTA 19-07. This publication reflects the views of the authors only; the European Commission cannot be held responsible for any use which may be made of the information contained therein

Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians

Acknowledgments: We thank to Baveesh Pudhuvai (BC CAS, Budweis, Czech Republic) for help in the PCR verification of Buddenbrockia stefin. We also thank Ivan Fiala (BC CAS, Budweis, Czech Republic) for providing suggestions to improve the manuscript and for sharing M. lieberkuehni and N. pickii transcriptomic data. We are grateful to Hanna Hartikainen (ETH Zurich, Switzerland) for sharing T. bryosalmonae genome data. Funding: This research was funded by the Ministry of Education, Youth, and Sports of the Czech Republic, grant number LTAUSA17201; by the European Commission under the H2020 Programme— ParaFishControl, grant number 634429; by the Czech Science Foundation, grant number 19-28399X (to A. S. Holzer, G. Alama-Bermejo, and J. Kyslík) and 21-16565S and by the Czech Academy of Sciences and Hungarian Academy of Sciences, grant number MTA 19-07. This publication reflects the views of the authors only; the European Commission cannot be held responsible for any use which may be made of the information contained therein.Peer reviewedPublisher PD

Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.This work has been carried out with financial support from the European Union and the Spanish MINECO under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project, RDP was contracted under the PTA-Program from the Spanish Ministry of Science, Innovation and Universities (PTA2018-015315-I), and MCP under CSIC PIE Project No. 201740E013. NHA was the recipient of a fellowship from the Egyptian Cultural Office in Spain.Peer reviewe

Passive Immunization delays disease outcome in Gilthead sea bream infected with Enteromyxum leei (Myxozoa), despite the moderate changes in IgM and IgT repertoire

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, Sparus aurata) that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that E. leei induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial role of spAb responses against E. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture.This work was funded by EU H2020 program through ParaFishControl Project (634429) and by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM). This publication reflects only the authors’ view and the European Union cannot be held responsible for any use that may be made of the information contained herein. MP was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I/AEI/10.13039/501100011033 & ESF), IE under APOSTD/2016/037 grant by the “Generalitat Valenciana” and RP was contracted under the PTA-Program from the Spanish Ministry of Science, Innovation and Universities (PTA2018-015315-I). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Does time matter in Enteromyxum leei (Myxozoa) fish-to-fish experimental transmission?

Comunicación presentada en la 19th International Conference on Diseases of Fish and Shellfish, celebrada en Oporto (Portugal) del 9 al 12 de septiembre de 2019.[Introduction]: Enteromyxum leei is a myxozoan histozoic parasite that infects the intestine of several teleostean fish species. In the gilthead sea bream (Sparus aurata) it provokes a chronic disease, with anorexia, delayed growth with weight loss, cachexia, reduced marketability and mortality. Direct fish-to-fish transmission has been demonstrated for E. leei via effluent, cohabitation, oral and anal routes. Effluent transmission trials in GSB are set up for long times of exposure (70-120 day) to water from tanks holding infected fish (donors, D). However, the minimum time of exposure to infect fish has not been established, nor the possible effect on the fish immune response. [Methodology]: A D tank was setup to effluent water to two equal recipient (R) tanks, each with 50 naïve fish. R1 tank was kept with the same number of fish all the trial, whereas in R2 tank, some fish were left all the time (R2-13) and 10 fish were removed at 1 (R2-1), 3 (R2-3), 5 (R2-5) and 7 (R2-7) weeks post exposure (wpe) and placed in separated tanks with non-infected water. At 9 wpe, a non-lethal sampling was done to evaluate the progression of the infection and a final sampling at 13 wpe to obtain intestinal and serum samples. [Results]: No effect of time of exposure was detected on prevalence of infection, as it varied between 100% (R2-1) and 80% (R2-3). Although no significant differences were found in weight, length and condition factor among R groups, the weight decrease typical of the infection was lowest in R2-1. The percentage of fish with specific antibodies against E. leei varied between 50 and 100%. [Conclusion]: From the significant correlations found among the different variables, we can conclude that 1) the earlier the infection is achieved, the higher amount of Abs are produced, 2) the longer exposure times, the higher impact on biometrical values, 3) the higher reduction of growth, the higher extension of the infection along the intestine and the higher percentage of fish with antibodies. Further trials have to be performed under lower temperatures and exposure times shorter than one week.Funding: ParaFishControl H2020 (634429)

Can passive immunization prevent disease outcome in gilthead sea bream exposed to Enteromyxum leei?

Trabajo presentado en 3rd International Conference on Fish and Shellfish Immunology (ISFSI), celebrado en Gran Canaria (España), del 16 al 20 de junio de 2019Passive immunization is used in humans for treating or preventing some infectious diseases, but it also constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Intracoelomically-injected antibodies can be detected in fish sera within the first 8 h and their half-life ranges from 7 to 22 days post-injection, depending on the species. The fish models studied so far used fast-acting pathogens such as virus, bacteria or ciliate parasites. The current work aimed to determine if passive immunization could help to prevent enteromyxosis in gilthead sea bream (GSB, Sparus aurata). Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that GSB that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific serum antibodies. In the current study, we evaluated whether injection with sera from resistant animals would protect naïve fish when challenged by effluent exposure to the parasite. Serum from a pool of resistant (R) and naïve (N) animals (intact or heat inactivated, 10 ¿l/g BW) was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). At 23 dpc, the different groups were allocated in separate tanks and the effluent exposure was terminated. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for hematology, circulating antibodies, histological and molecular diagnosis and gene expression. Groups injected with R sera had lower prevalence and intensity of infection than those with N sera, both in the intermediate and final samplings. At 100 dpc, the prevalence of infection in the PBS and N groups was 70%, whereas in R group it only reached 55%. Condition factor (CF) and specific growth rate (SGR), key parameters affected by enteromyxosis, were higher in R group. There was a significant correlation between prevalence of infection and SGR and CF. Immunohistochemistry and gene expression studies will reveal whether this partial protection was due to higher presence of specific antibodies or specific cell populations. These results show that, even with this long term disease, passive immunization can confer some degree of protection. The administration of specific antibodies during exposure, probably provided fish with time to activate the specific defenses before the parasite proliferated.This work was funded by EU H2020 program through ParaFishControl Project (634429) and Spanish MINECO project AGL2013-48560. M.C.P. was contracted under CSIC PIE project no. 201740E013 and FPDI-2013-15741 and I.E. under APOSTD/2016/037 grant by the “Generalitat Valenciana”

Filtering and characterization of Enteromyxum spp. (Myxozoa) transcriptomes from RNAseq data

Trabajo presentado en la 18th International Conference on Diseases of Fish and Shellfish, celebrada en Belfast, del 4 al 8 de septiembre de 2017Enteromyxum spp. are obligate parasites closely related to Cnidarians. They are responsible of serious emaciative diseases in marine aquacultured fish worldwide. Differently to other myxozoans with a 2-host cycle, which develop myxospores and actinospores, Enteromyxum spp. are only known to develop in fish intestines, dwelling in the paracellullar space in an intimate relationship with host cells. Spore production may be quite limited or inexistent, depending on the individual fish-Enteromyxum sp. model. Recent studies have focused on the obtention of transcriptome data from myxozoans, mostly from spore stages that can be easily isolated in large quantities. Although useful for gene hunting and phylogenomic studies, spores presumably contain a limited amount and repertory of expressed genes for functional studies. The goal of this work is to obtain a snapshot of genes expressed by developmental stages of Enteromyxum spp. in the course of their trophic development, invasion and pathogenic action in their hosts. This strategy required specifically designed tools to sort myxozoan RNAseq sequences from those of the hosts. An algorithm based on the transcripts¿ dinucleotide frequencies, and their Blastn score against specific fish/cnidarian datasets, was designed and tutored with curated test data. This resulted in the determination of the algorithm¿s error rate and the assignment of a p value to each filtering decision. As a result, 26,071 out of 281,264 transcripts were assigned to Enteromyxum spp. (p>0.99) while 30,551 were assigned to the hosts (p>0.99) and 57,507 were classified in a ¿greyzone¿ in between. The Enteromyxum filtered dataset was enriched with 4,500 additional transcripts initially classified as ¿fish¿ or ¿greyzone¿ [p(myxo)<0.99] after a repechage round with a curated Myxobolus pendula transcriptome dataset (Foo et al., 2015). The final dataset contains over 30,000 sequences with very low levels of contaminating fish transcripts. Functional annotation and characterization of the dataset, including comparison between E. scophthalmi and E. leei offers a basis for the identification of key molecules related to the development and pathogeny of these parasites. Additional comparative analyses with other myxozoans and free-living cnidarians can enlighten some of the unique adaptions of myxozoans to their parasitic lifestyles.This study was partially supported by EU H2020 program through the ParaFishControl project (634429).Peer reviewe