Escuticociliatosis: mecanismos de evasión de la respuesta inmunitaria del rodaballo y desarrollo de vacunas

La escuticociliatosis producida por Philasterides dicentrarchi genera mortalidades en rodaballos de cultivo provocando pérdidas económicas. Para evitarlo, es necesario desarrollar medidas profilácticas basadas en la utilización de vacunas, a partir de la búsqueda de antígenos que generen protección, y/o fármacos que inhiban mecanismos vitales para la supervivencia del ciliado. Por tanto, la identificación de antígenos protectores y de moléculas implicadas en el metabolismo, las cuales permiten al escuticociliado sobrevivir en el hospedador, han sido los objetivos principales del presente estudio. Hemos descubierto que el parásito posee dos familias de proteínas (leishmanolisinas y proteínas variables de superficie (VSPs)) asociadas a la membrana celular e involucradas en la evasión de la respuesta inmunitaria del hospedador. También hemos identificado y caracterizado proteínas de secreción producidas por los extrusomas, capaces de generar cápsulas de mucopolisacáridos que protegen del ataque de los factores humorales citotóxicos y superóxido dismutasas, que permiten al ciliado inactivar radicales libres de oxígeno tóxicos asociados al estrés oxidativo generado durante la infección. Para solventar los problemas actuales relacionados con el uso de vacunas inactivadas formuladas en adyuvantes oleosos, también hemos desarrollado un adyuvante biodegradable (hidrogel de quitosano) que presenta capacidad inmunoestimulante similar a los oleosos y resulta menos dañino para el pez. La producción de proteínas recombinantes en levadura, como potenciales antígenos para generar vacunas recombinantes, nos permite incluir los cambios aminoacídicos que se producen entre las diferentes cepas, obteniendo así un antígeno inmunogénico universal que genere respuesta frente a todas ellas. Para conseguir una mayor protección es necesario incluir en la vacuna varios antígenos empleando proteínas quiméricas recombinantes a partir de péptidos antigénicos procedentes de varias proteínas. Estas formulaciones vacunales de quitosano han generado elevados niveles de protección en ensayos realizados en rodaballo, indicando que esta estrategia podría ser útil para obtener una vacuna universal eficaz frente a la escuticociliatosis

Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite

This is the accepted manuscript of the following article: Folgueira I, Lamas J, De Felipe AP, Sueiro RA, Leiro JM. (2019). Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite. Fish Shellfish Immunol. 2019 Jul 5;92:802-812. doi: 10.1016/j.fsi.2019.07.008Like other ciliates, Philasterides dicentrarchi, the scuticociliate parasite of turbot, produces a feeding-only or growing stage called a trophont during its life cycle. Exposure of the trophonts to heat-inactivated serum extracted from the turbot host and containing specific antibodies that induce agglutination/immobilization leads to the production of a mucoid capsule from which the trophonts later emerge. We investigated how these capsules are generated, observing that the mechanism was associated with the process of exocytosis involved in the release of a matrix material from the extrusomes. The extruded material contains mucin-like glycoproteins that were deposited on the surface of the cell and whose expression increased with time of exposure to the heat-inactivated immune serum, at both protein expression and gene expression levels. Stimulation of the trophonts with the immune serum also caused an increase in discharge of the intracellular storage compartments of calcium necessary for the exocytosis processes in the extrusomes. The results obtained suggest that P. dicentrarchi uses the extrusion mechanism to generate a physical barrier protecting the ciliate from attack by soluble factors of the host immune system. Data on the proteins involved and the potential development of molecules that interfere with this exocytic process could contribute to improving the prevention and control of scuticociliatosis in turbotThis study was financially supported by grants from the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional -FEDER- (European Union) (AGL2017-83577-R) and from the Xunta de Galicia (ED431C2017/31) and also by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429S

Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia)

This is the accepted manuscript of the following article: Folgueira, I., de Felipe, A.P., Sueiro, R.A., Lamas, J. & Leiro, J. (2018). Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia). Cryobiology, 80, 77-83. doi: 10.1016/j.cryobiol.2017.11.010Philasterides dicentrarchi is a free-living marine ciliate that can become an endoparasite that causes a severe disease called scuticociliatosis in cultured fish. Long-term maintenance of this scuticociliate in the laboratory is currently only possible by subculture, with periodic passage in fish to maintain the virulence of the isolates. In this study, we developed and optimized a cryopreservation protocol similar to that used for the long-term storage of scuticociliates of the genus Miamiensis. The cryogenic medium comprised ATCC medium 1651 and a combination of 11% dimethylsulfoxide and 5% glycerol. We have verified that the most important factor ensuring the efficiency of the cryopreservation procedure is the growth phase of the culture, and that ciliates should be cryopreserved at the stationary phase (around the sixth day of culture). The cryopreservation protocol described here can be used for all strains of P. dicentrarchi as well as commercial strains of Miamiensis and enables the virulence of the strains to be maintained. Finally, this cryopreservation protocol has been shown to be more effective than others routinely applied to scuticociliates, yielding a higher survival rate with a lower initial concentration of ciliates. The results obtained indicate that the cropreservation protocol enables the long-term storage of scuticociliate parasites while maintaining the virulence of the isolates. The protocol is therefore suitable for use in vaccine production and related studiesThis work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL), by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain)S

Identification and Molecular Characterization of Superoxide Dismutases Isolated From A Scuticociliate Parasite: Physiological Role in Oxidative Stress

Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fsh. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity apable of inactivating the superoxide anion (•O2−) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34–44kDa and with very diferent AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2 − is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stressThis study was financially supported by grant AGL2017-83577-R awarded by the Ministerio de Economía y Competitividad (Spain) and Fondo Europeo de Desarrollo Regional -FEDER- (European Union), by grant ED431C2017/31 from the Xunta de Galicia (Spain), and by PARAFISHCONTROL project, which received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429S

New data on flatfish scuticociliatosis reveal that Miamiensis avidus and Philasterides dicentrarchi are different species

This article has been published in a revised form in Parasitology [http://doi.org/10.1017/S0031182017000749]. This version is free to view and download for private research and study only. Not for re-distribution, re-sale or use in derivative works. © 2017 Cambridge University PressScuticociliatosis is a severe disease in farmed flatfish. However, the causative agent is not always accurately identified. In this study, we identified two isolates of scuticociliates from an outbreak in cultured fine flounder Paralichthys adspersus. Scuticociliate identification was based on morphological data, examination of life stages and the use of molecular approaches. The isolates were compared with a strain of Philasterides dicentrachi from turbot Scophthalmus maximus and with a strain deposited in the American Type Culture Collection as Miamiensis avidus ATCC® 50180™. The use of morphological, biological and molecular methods enabled us to identify the isolates from the fine flouder as P. dicentrarchi. Comparison of P. dicentrachi isolates and M. avidus revealed some differences in the buccal apparatus. Unlike P. dicentrarchi, M. avidus has a life cycle with three forms: macrostomes (capable of feeding on P. dicentrarchi), microstomes and tomites. Additionally, we found differences in the 18S rRNA and α- and β-tubulin gene sequences, indicating that P. dicentrarchi and M. avidus are different species. We therefore reject the synonymy/conspecificity of the two taxa previously suggested. Finally, we suggest that a combination of morphological, biological, molecular (by multigene analysis) and serological techniques could improve the identification of scuticociliates parasites in fishThis work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL), by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain)S

Combined antiparasitic and anti‐inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis

This is the peer reviewed version of the following article: Mallo, N. , DeFelipe, A. P., Folgueira, I. , Sueiro, R. A., Lamas, J. and Leiro, J. M. (2017), Combined antiparasitic and anti‐inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis. J Fish Dis, 40: 205217. doi:10.1111/jfd.12503, which has been published in final form at https://doi.org/10.1111/jfd.12503. This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsThe histiophagous scuticociliate Philasterides dicentrarchi is the aetiological agent of scuticociliatosis, a parasitic disease of farmed turbot. Curcumin, a polyphenol from Curcuma longa (turmeric), is known to have antioxidant and anti-inflammatory properties. We investigated the in vitro effects of curcumin on the growth of P. dicentrarchi and on the production of pro-inflammatory cytokines in turbot leucocytes activated by parasite cysteine proteases. At 100 μm, curcumin had a cytotoxic effect and completely inhibited the growth of the parasite. At 50 μm, curcumin inhibited the protease activity of the parasite and expression of genes encoding two virulence-associated proteases: leishmanolysin-like peptidase and cathepsin L-like. At concentrations between 25 and 50 μm, curcumin inhibited the expression of S-adenosyl-L-homocysteine hydrolase, an enzyme involved in the biosynthesis of the amino acids methionine and cysteine. At 100 μm, curcumin inhibited the expression of the cytokines tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) produced in turbot leucocytes activated by parasite proteases. Results show that curcumin has a dual effect on scuticociliatosis: an antiparasitic effect on the catabolism and anabolism of ciliate proteins, and an anti-inflammatory effect that inhibits the production of proinflammatory cytokines in the host. The present findings suggest the potential usefulness of this polyphenol in treating scuticociliatosisThis work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL) and by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-RS

Vaccine-induced modulation of gene expression in turbot peritoneal cells. A microarray approach

This is the accepted manuscript of the following article: Fontenla, F., Blanco-Abad, V., Pardo, B.G., Folgueira, I., Noia, M., Gómez-Tato, A., Martínez, P., Leiro, J.M. & Lamas J. (2016). Vaccine-induced modulation of gene expression in turbot peritoneal cells. A microarray approach. Molecular Immunology, 75, 188-99. doi: 0.1016/j.molimm.2016.06.001We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan- PVMMA microspheres, Freund´s complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cellvaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccinationThis work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL), by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain)S

The coagulation system helps control infection caused by the ciliate parasite Philasterides dicentrarchi in the turbot Scophthalmus maximus (L.)

This is the accepted manuscript of the following article: Blanco-Abad, V., Noia, M., Valle, A., Fontenla, F., Folgueira, I., & De Felipe, A. et al. (2018). The coagulation system helps control infection caused by the ciliate parasite Philasterides dicentrarchi in the turbot Scophthalmus maximus (L.). Developmental & Comparative Immunology, 87, 147-156. doi: 10.1016/j.dci.2018.06.001Many studies have shown that coagulation systems play an important role in the defence against pathogens in invertebrates and vertebrates. In vertebrates, particularly in mammals, it has been established that the coagulation system participates in the entrapment of pathogens and activation of the early immune response. However, functional studies investigating the importance of the fish coagulation system in host defence against pathogens are scarce. In the present study, injection of turbot (Scopthalamus maximus) with the pathogenic ciliate Philasterides dicentrarchi led to the formation of macroscopic intraperitoneal clots in the fish. The clots contained abundant, immobilized ciliates, many of which were lysed. We demonstrated that the plasma clots immobilize and kill the ciliates in vitro. To test the importance of plasma clotting in ciliate killing, we inhibited the process by adding a tetrapeptide known to inhibit fibrinogen/thrombin clotting in mammals. Plasma tended to kill P. dicentrarchi slightly faster when clotting was inhibited by the tetrapeptide, although the total mortality of ciliates was similar. We also found that kaolin, a particulate activator of the intrinsic pathway in mammals, accelerates plasma clotting in turbot. In addition, PMA-stimulated neutrophils, living ciliates and several ciliate components such as cilia, proteases and DNA also displayed procoagulant activity in vitro. Injection of fish with the ciliates generated the massive release of neutrophils to the peritoneal cavity, with formation of large aggregates in those fish with live ciliates in the peritoneum. We observed, by SEM, numerous fibrin-like fibres in the peritoneal exudate, many of which were associated with peritoneal leukocytes and ciliates. Expression of the CD18/CD11b gene, an integrin associated with cell adhesion and the induction of fibrin formation, was upregulated in the peritoneal leukocytes. In conclusion, the findings of the present study show that P. dicentrarchi induces the formation of plasma clots and that the fish coagulation system may play an important role in immobilizing and killing this parasiteThis work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL), by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain)S