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

Many 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 parasite.

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

Philasterides 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 studies.