Development of immunity in sea bass: a study towards vaccination against pseudotuberculosis

Pseudotuberculosis, caused by the halophilic gram-negative bacterium Photobacterium damselae ssp. piscicida , has been causing high mortality losses in sea bass ( Dicentrarchus labrax L.) farming. In the present thesis, basic and applied studies were performed concerning the humoral immune system of sea bass, as part of the development of an effective vaccine for this pathogen and with the aim to determine the earliest age/weight at which sea bass can be vaccinated against that disease. Therefore, monoclonal antibodies (mAb) against the heavy (±78 kDa; WDI 1) and light (±28 kDa; WDI 2 and WDI 3) chains were successfully produced and used. The Ig characterisation has shown that non-uniformly disulphide cross-linking of monomeric (M r ±210 kDa) and dimeric (M r ±420 kDa) subunits occurs in the polymerisation of the tetrameric form (M r ±840 kDa). The sequence of the Ig L chain allowed all characteristic segments (V, J and C) to be deduced, and could be considered as a L1/G isotype. In addition, it was shown to have a cluster-like genomic organisation. Due to its importance for vaccination with T dependent antigens, the ontogenetic development of sea bass T cell was studied using a mAb (DLT 15) specific for that lymphocyte subpopulation. It was shown that early T cells may originate in a different compartment, further differentiate in the thymus and subsequently migrate to peripheral lymphoid organs. Adult proportions were reached around 137 days post-hatch (dph).Studies with different combinations of the mAb against sea bass Ig did strongly suggest the existence of B cell heterogeneity, expressing at least two heavy and two light chains. The percentage of B cells in the different immunological organs was studied using all the mAb. The combination of WDI 1 and WDI 3 appeared to label all or almost all B cells. As in other teleost species, the sea bass head kidney appears to be the primary organ for B-lymphocyte differentiation. The proportion of sIg + (B) cells in the different organs increased during the ontogenetic development being higher in lymphocyte populations of the head kidney followed by spleen, PBL, gut and thymus. This sequential order changed during ontogeny until the adult distribution is reached (about 137-145 dph). Additionally, sea bass cytoplasmic Ig + (cIg + ) cells (preB cells) were detected earlier in ontogeny than sIg + cells. Cytoplasmic Ig + plasma cells appear later in ontogeny (87 dph) and might be indicative of immunological maturation, regarding the onset of adaptive humoral immune responses.In the present thesis it was clearly demonstrated that sea bass is able to mount a specific antibody response to different antigens: Vibrio anguillarum , P. damselae ssp. piscicida and DNP. The kinetics of primary and secondary responses were studied in both intraperitoneally (ip) and direct immersion (di) vaccinated fish. While the head kidney was the main ASC-producing organ after ip immunisation, in the di immunised fish it was shown that the gills (and probably the skin) are likely to play a major role in the protection against natural challenges. In addition, a clear age effect was observed in the ASC responses of immersion vaccinated fish, with significantly higher numbers of ASC and faster ASC responses in the oldest groups. More importantly, there was no evidence that direct immersion exposure to P. damselae ssp. piscicida at the earliest stages used in the present study (0.1g) is tolerogenic. The kinetics and specificity of the invasion ability of P. damselae ssp. piscicida was studied and supported the results from others suggesting an intracellular phase to the life cycle of the bacterium. The fact that heat-inactivated antiserum obtained from sea bass immunised with a P. damselae ssp. piscicida bacterin reduced the invasiveness of the bacteria, strongly suggests that the antiserum contained antibodies against factors responsible for the adhesion and invasion of the pathogen. Intramolecular-induced suppression appears to play an important role in the regulation of the antibody response in sea bass, as suggested by the extremely low variability observed in the Ig L chain sequences obtained from DNP 494 -KLH immunised sea bass.Since intermolecular-induced suppression has been shown in other teleosts, it raises the question about how these mechanisms might influence the specificity of immune responses against vaccine antigens and induction of protection. In conclusion, it may be stated that the bacterin used in this thesis contains immunogenic determinants, which may be important for conferring protection, and that sea bass are immunocompetent enough for vaccination against P. damselae ssp. piscicida from at least 137-145 dph (1g), regardless of the route of administration, or from at least 80-100 dph (0.1g) if immersion vaccination is preferred.</p

Development of immunity in sea bass: a study towards vaccination against pseudotuberculosis

Pseudotuberculosis, caused by the halophilic gram-negative bacterium Photobacterium damselae ssp. piscicida , has been causing high mortality losses in sea bass ( Dicentrarchus labrax L.) farming. In the present thesis, basic and applied studies were performed concerning the humoral immune system of sea bass, as part of the development of an effective vaccine for this pathogen and with the aim to determine the earliest age/weight at which sea bass can be vaccinated against that disease. Therefore, monoclonal antibodies (mAb) against the heavy (±78 kDa; WDI 1) and light (±28 kDa; WDI 2 and WDI 3) chains were successfully produced and used. The Ig characterisation has shown that non-uniformly disulphide cross-linking of monomeric (M r ±210 kDa) and dimeric (M r ±420 kDa) subunits occurs in the polymerisation of the tetrameric form (M r ±840 kDa). The sequence of the Ig L chain allowed all characteristic segments (V, J and C) to be deduced, and could be considered as a L1/G isotype. In addition, it was shown to have a cluster-like genomic organisation. Due to its importance for vaccination with T dependent antigens, the ontogenetic development of sea bass T cell was studied using a mAb (DLT 15) specific for that lymphocyte subpopulation. It was shown that early T cells may originate in a different compartment, further differentiate in the thymus and subsequently migrate to peripheral lymphoid organs. Adult proportions were reached around 137 days post-hatch (dph).Studies with different combinations of the mAb against sea bass Ig did strongly suggest the existence of B cell heterogeneity, expressing at least two heavy and two light chains. The percentage of B cells in the different immunological organs was studied using all the mAb. The combination of WDI 1 and WDI 3 appeared to label all or almost all B cells. As in other teleost species, the sea bass head kidney appears to be the primary organ for B-lymphocyte differentiation. The proportion of sIg + (B) cells in the different organs increased during the ontogenetic development being higher in lymphocyte populations of the head kidney followed by spleen, PBL, gut and thymus. This sequential order changed during ontogeny until the adult distribution is reached (about 137-145 dph). Additionally, sea bass cytoplasmic Ig + (cIg + ) cells (preB cells) were detected earlier in ontogeny than sIg + cells. Cytoplasmic Ig + plasma cells appear later in ontogeny (87 dph) and might be indicative of immunological maturation, regarding the onset of adaptive humoral immune responses.In the present thesis it was clearly demonstrated that sea bass is able to mount a specific antibody response to different antigens: Vibrio anguillarum , P. damselae ssp. piscicida and DNP. The kinetics of primary and secondary responses were studied in both intraperitoneally (ip) and direct immersion (di) vaccinated fish. While the head kidney was the main ASC-producing organ after ip immunisation, in the di immunised fish it was shown that the gills (and probably the skin) are likely to play a major role in the protection against natural challenges. In addition, a clear age effect was observed in the ASC responses of immersion vaccinated fish, with significantly higher numbers of ASC and faster ASC responses in the oldest groups. More importantly, there was no evidence that direct immersion exposure to P. damselae ssp. piscicida at the earliest stages used in the present study (0.1g) is tolerogenic. The kinetics and specificity of the invasion ability of P. damselae ssp. piscicida was studied and supported the results from others suggesting an intracellular phase to the life cycle of the bacterium. The fact that heat-inactivated antiserum obtained from sea bass immunised with a P. damselae ssp. piscicida bacterin reduced the invasiveness of the bacteria, strongly suggests that the antiserum contained antibodies against factors responsible for the adhesion and invasion of the pathogen. Intramolecular-induced suppression appears to play an important role in the regulation of the antibody response in sea bass, as suggested by the extremely low variability observed in the Ig L chain sequences obtained from DNP 494 -KLH immunised sea bass.Since intermolecular-induced suppression has been shown in other teleosts, it raises the question about how these mechanisms might influence the specificity of immune responses against vaccine antigens and induction of protection. In conclusion, it may be stated that the bacterin used in this thesis contains immunogenic determinants, which may be important for conferring protection, and that sea bass are immunocompetent enough for vaccination against P. damselae ssp. piscicida from at least 137-145 dph (1g), regardless of the route of administration, or from at least 80-100 dph (0.1g) if immersion vaccination is preferred