Small, charged proteins in salmon louse (Lepeophtheirus salmonis) secretions modulate Atlantic salmon (Salmo salar) immune responses and coagulation.

Little is known about glandular proteins secreted from the skin- and blood-feeding ectoparasite salmon louse (Lepeophtheirus salmonis). The labial gland has ducts extending into the oral cavity of the lice, and the present study aimed to identify novel genes expressed by this gland type and to investigate their role in modulation of host parameters at the lice feeding site. Five genes associated with labial gland function were identified and named Lepeophteirus salmonis labial gland protein (LsLGP) 1-4 and 1 like (LsLGP1L). All LsLGPs were predicted to be small charged secreted proteins not encoding any known protein domains. Functional studies revealed that LsLGP1 and/or LsLGP1L regulated the expression of other labial gland genes. Immune dampening functions were indicated for LsLGP2 and 3. Whereas LsLGP2 was expressed throughout the parasitic life cycle and found to dampen inflammatory cytokines, LsLGP3 displayed an increased expression in mobile stages and appeared to dampen adaptive immune responses. Expression of LsLGP4 coincided with moulting to the mobile pre-adult I stage where hematophagous feeding is initiated, and synthetic LsLGP4 decreased the clotting time of Atlantic salmon plasma. Results from the present study confirm that the salmon louse secretes immune modulating and anti-coagulative proteins with a potential application in new immune based anti-salmon louse treatments.

Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss.

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i.p.) injection. The fish were exposed to virulent A. salmonicida 7 weeks after immunization. To assess the efficacy of the subunit vaccines we evaluated the immune response in fish after immunization and challenge infection by measuring the antibody levels and monitoring the survival of fish in different groups. The survival of fish at 3 weeks after challenge infection showed that all 3 groups of fish immunized with 3 different protein combinations exhibited significantly lower mortalities (17-30%) compared to the control groups (48% and 56%). The ELISA results revealed significantly elevated antibody levels in fish against several protein antigens, which in some cases were positively correlated to the survival.

Comparative evaluation of infection methods and environmental factors on challenge success: Aeromonas salmonicida infection in vaccinated rainbow trout.

When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.

Comparative evaluation of administration methods for a vaccine protecting rainbow trout against Yersinia ruckeri O1 biotype 2 infections.

Numerous outbreaks of enteric red mouth disease (ERM) caused by Yersinia ruckeri O1 biotype 2 in rainbow trout farms are currently being recorded despite established vaccination procedures against this disease. This could indicate that the currently used application of single immersion vaccination (using a commercial vaccine AquaVac(®) RELERA™) does not provide full protection. We elucidated by a controlled duplicated experiment if different vaccine administration methods can improve level and extent of protection. Rainbow trout, Oncorhynchus mykiss were vaccinated by: (1) a single immersion in bacterin diluted 1:10 for 30s (only primary vaccination); (2) two times 30s immersion (primary immersion vaccination followed by booster immersion vaccination 1 month later); (3) a single i.p. injection (only primary vaccination); (4) immersion vaccination followed by injection booster 1 month later; (5) a single 1h bath in bacterin diluted 1:2000; and (6) immersion (30s, 1:10) plus booster (1h in diluted 1:2000 vaccine) 5 months later). Injection challenge experiments were performed 3, 5 and 7 months post primary vaccination with 8.5×10(6) CFU/fish, 10.6×10(6) CFU/fish and 1×10(8) CFU/fish, respectively. In the first challenge trial, control fish exhibited a mortality of 76%, one time immersion vaccination had a mortality of 37%, two times immersion vaccinated fish had a 4% mortality, the one-time injection vaccinated group showed a mortality of 2% and the immersion plus injection boostered fish showed no mortality at all. When rainbow trout were challenged 5 months post primary vaccination, 26% mortality occurred in control fish, 21% in one time immersion group, 12% in two times immersion group, 5% in the one-time injection vaccinated group whereas immersion plus injection boostered fish again showed no mortality at all. When challenged 7 months post vaccination, one-time immersion vaccinated were not protected at all compared to the control group whereas injection vaccinated fish showed lower mortality (17%) compared to booster immersed fish (32% mortality) which was still better than un-vaccinated controls (44% mortality). It was noteworthy that a diluted bacterin (1:2000 for 1h after 5 months post primary vaccination) booster showed the same effect as a booster with 1:10 bacterin dilution for 30s applied 1 month after primary vaccination. Antibody levels showing significant elevations 28 days post challenge in vaccinated fish point to this immune parameter as a protective element. The superior and extended protection offered by booster vaccination or simply injection is noteworthy and may be applied in future vaccination strategies at farm level.

Tissue specific uptake of inactivated and live Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss): visualization by immunohistochemistry and in situ hybridization.

Understanding of uptake and invasion routes of Yersinia ruckeri, causing Enteric Red Mouth Disease (ERM) in rainbow trout (Oncorhynchus mykiss), is essential for improved understanding of the pathogenicity and immune response mechanisms associated this disease. The present work shed light on areas of invasion in rainbow trout by the use of immunohistochemistry and in situ hybridization techniques. Fish were exposed to live or formalin inactivated bacteria and samples were subsequently taken for histology from various outer and inner surfaces. We applied a specific monoclonal antibody and specific oligonucleotide probes binding to Y. ruckeri (serotype O1, biotype 2) in tissue sections and were able to demonstrate a tissue specific uptake of this bacterium (both formalin inactivated and live form). Uptake and subsequent translocation dynamics at various surfaces demonstrated different site specific propensities between the formalin inactivated and live bacterial organisms. Lateral lines, dorsal fin, epidermis and gastro-intestinal tract mucosal tissue were the primary areas where bacterial uptake was demonstrated readily after exposure. The fate of internalized bacterial organisms within the host suggested that central immune organs are involved in the final antigen processing.