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.

Particle size and traffic of phagocytes between the turbot peritoneal cavity and lymphoid organs.

New adjuvants based on microparticles are being developed for use in fish vaccines. The size of the microparticles may affect the immune response generated, as the adjuvant can either be retained at the site of injection or transported to lymphoid organs. The objectives of this study were to evaluate the maximum size of particles that can be exported out of the cavity, to determine the phagocytosis kinetics and to establish the routes whereby particle-containing cells move from the peritoneal cavity after injection. Fish were injected intraperitoneally with fluorescent cyclodextrins or with fluorescent particles of different size (0.1-10 µm). Phagocytes containing beads of size 4 µm or larger did not reach lymphoid organs, although some were able to cross the peritoneal mesothelium. The number of free peritoneal neutrophils and macrophage-like cells containing beads peaked at 6 and 24 h respectively, and the numbers then decreased quickly, indicating migration of cells to the peritoneum or other body areas. Migration of cells containing beads mainly occurs through the visceral peritoneum. These cells were found on the latero-ventral surfaces of the peritoneal folds that connect the visceral organs. Except for some vascularised areas, the surfaces of liver, stomach and intestine were devoid of particle-containing cells. Some cells containing beads were also found attached to the parietal peritoneum, although in lower numbers than in the visceral peritoneum. Such cells were also found in high numbers in the spleen and kidney 6 h post injection. Because cells containing phagocytosed material quickly become attached to the peritoneum or migrate to lymphoid organs, the immune response generated by a vaccine or by an inflammatory stimulus should probably be evaluated in attached cells as well as in free peritoneal cells.

Evaluation of different procedures for the optimized detection of Vibrio parahaemolyticus in mussels and environmental samples.

Vibrio parahaemolyticus is a marine bacterium with a worldwide distribution and is frequently associated with human outbreaks of infection. Detection and isolation of V. parahaemolyticus from natural sources is often problematical because of limitations in the analytical procedures. We evaluated a combination of conventional and molecular protocols previously described for the investigation of V. parahaemolyticus, with the aim of identifying the best procedures for improved detection of this organism in environmental matrixes. A total of 259 samples of zooplankton (103), mussels (48) and seawater (108) were investigated by an Absence-Presence method (A/P), whereas 118 samples of zooplankton (70) and mussels (48) were analyzed by the Most Probable Number (MPN) method. All samples were processed by a two-step enrichment procedure, firstly with APW broth and then with SPB as selective secondary broth. Detection of V. parahaemolyticus was by direct-PCR and by plate culture on TCBS and CHROMagar Vibrio, after sample enrichment in APW and SPB. With the A/P method, V. parahaemolyticus was detected in 23.6% samples by direct-PCR, whereas only 11.2% samples were positive with the plate culture method. With the MPN method, V. parahaemolyticus was detected in 54.2% and 27.1% of the samples by direct-PCR and plate culture respectively; this indicated the existence of 31% false negative results with the A/P method. No significant differences between the use of a single (APW) or two-step enrichment (APW+SPB) were observed by direct-PCR with A/P or MPN, although a significant higher presence of V. parahaemolyticus was detected by plate culture in both protocols with the two-step enrichment procedure. In conclusion, direct-PCR after sample enrichment in APW broth was the most successful method for detection of V. parahaemolyticus with the A/P procedure and enumeration by MPN. Better detection was obtained with MPN than with the A/P protocol. Conversely, the plate culture procedure showed better results with the two-step enrichment protocol in which CHROMagar Vibrio was used as the selective agar.