Chiara Bulfon

Innovative vaccination protocol against vibriosis in Dicentrarchus labrax (L.) juveniles: Improvement of immune parameters and protection to challenge

The effect of vaccination on immune parameters of European sea bass, Dicentrarchus labrax, is not fully established, as well as surveyed throughout rearing till the commercial size. Furthermore, available information on the possible role of booster treatments is scarce. Sea bass juveniles were vaccinated against Listonella anguillarum using a commercial bivalent formulation administered by immersion (priming: 95 dph; booster: 165 dph) or by immersion (priming: 95 dph; booster: 165 dph) and subsequent i.p. injection (booster: 233 dph). Serum specific IgM and numbers of IgM(+) cells in head kidney and spleen evidenced B-cell responses mainly after the immersion booster, accompanied by increased TcR-β transcripts and leucocyte respiratory burst. Immune enhancement was confirmed by the protection towards i.p. challenges with a virulent strain. RPS accounted for >70% in fish immersion-boosted and near 100% in fish further boosted i.p. Differently from usual farm practices, this innovative vaccination protocol proved to be highly effective. Booster treatments are therefore strongly recommended.

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1β, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Expression of infection-related immune response in European sea bass (Dicentrarchus labrax) during a natural outbreak from a unique dinoflagellate Amyloodinium ocellatum

In the Mediterranean area, amyloodiniosis represents a major hindrance for marine aquaculture, causing high mortalities in lagoon-type based rearing sites during warm seasons. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish species living within its ecological range. In the present study, A. ocellatum was recorded and collected from infected European sea bass (Dicentrarchus labrax) during a summer 2017 outbreak in north east Italy. Histological observation of infected ESB gill samples emphasized the presence of round or pear-shaped trophonts anchored to the oro-pharingeal cavity. Molecular analysis for small subunit (SSU) rDNA of A. ocellatum from gill genomic DNA amplified consistently and yielded 248 bp specific amplicon of A. ocellatum, that was also confirmed using sequencing and NCBI Blast analysis. Histological sections of ESB gill samples were addressed to immunohistochemical procedure for the labelling of ESB igm, inos, tlr2, tlr4, pcna and cytokeratin. Infected gills resulted positive for igm, inos, pcna and cytokeratin but negative to tlr-2 and tlr-4. Furthermore, ESB immune related gene response (innate immunity, adaptive immunity, and stress) in the course of A. ocellatum infection using quantitative polymerase chain reaction (qpcr) for infected gills and head kidney was analysed. Among the twenty three immune related gene molecules tested, cc1, il-8, il-10, hep, cox-2, cla, cat, casp9, and igt were significantly expressed in diseased fish. Altogether, these data on parasite identification and expression of host immune-related genes will allow for a better understanding of immune response in European sea bass against A. ocellatum and could promote the development of effective control measures.

Computational modeling of immune system of the fish for a more effective vaccination in aquaculture

Motivation: A computational model equipped with the main immunological features of the sea bass (Dicentrarchus labrax L.) immune system was used to predict more effective vaccination in fish. The performance of the model was evaluated by using the results of two in vivo vaccinations trials against L. anguillarum and P. damselae. Results: Tests were performed to select the appropriate doses of vaccine and infectious bacteria to set up the model. Simulation outputs were compared with the specific antibody production and the expression of BcR and TcR gene transcripts in spleen. The model has shown a good ability to be used in sea bass and could be implemented for different routes of vaccine administration even with more than two pathogens. The model confirms the suitability of in silico methods to optimize vaccine doses and the immune response to them. This model could be applied to other species to optimize the design of new vaccination treatments of fish in aquaculture. Availability and implementation: The method is available at http://www.iac.cnr.it/˜filippo/c‐immsim/ Contact: nromano@unitus.it Supplementary information: Supplementary data are available at Bioinformatics online.