Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
ORAL VACCINATION AGAINST INFECTIOUS SALMON ANEMIA IN ATLANTIC SALMON (Salmo salar) INDUCES SPECIFIC IMMUNITY AND PROVIDES PROTECTION AGAINST INFECTIOUS SALMON ANEMIA VIRUS CHALLENGE. Jaime A. Tobar1*, Francisco C. Contreras1, Yelena Betz2, Catalina Bravo1, Mario Caruffo1, Sofía Jerez1, Thomas Goodrich2, and Arun K. Dhar2*. 1 Centrovet Ltda, Av. Los Cerrillos 602, 9201291 Cerrillos, Santiago, Chile. [email protected] 2 Advanced BioNutrition, 7155-H Columbia Gateway Drive, Columbia, MD 21046, USA. [email protected] ABSTRACT Infectious salmon anemia (ISA) caused by the infectious salmon anemia virus (ISAv) arose in Chile in 2007 and it has become the major cause for Atlantic salmon mortality. The disease affects post-smolt salmon (Salmo salar) regardless of the age of the fish and causes major economic looses to the Chilean salmon industry. Although several ISA vaccines are available, all of them are currently applied by injection route in freshwater, a procedure which albeit ensures fish-by-fish application, may also induce stress-related effects on fish, such as immunosuppression, reduction in feeding rate and mortality. Moreover, injectable vaccines do not represent a practical choice when fish will need to be boostered, a procedure intended to enhance or prolong acquired anti-ISAv immune response. We describe here a novel oral ISAv vaccine from Centrovet Laboratory using their unique ISAv-derived antigens expressed in yeast, and encapsulated with a proprietary encapsulation technology, MicroMatrix™, of Advanced BioNutrition Corp. Fish fed with MicroMatrix™ encapsulated ISAv vaccine showed specific anti-ISA antibody (IgM) titers detectable up to 1200 day-degrees after vaccination compared to control treatment. Additionally, when fish were challenged with a virulent ISAV strain using a cohabitation challenge model, vaccinated fish showed a Relative Percent Survival (RPS) of 80, and fish survival was correlated with IgM titer. In addition, oral vaccine also induced local immunity, showing mucosal inflammation and antibody generation. Finally, vaccination of fish using MicroMatrix™ encapsulated technology appeared to be safe for fish, since overfeeding with up to 5x dose did not interfere with fish weight gain. These results suggest that anti-ISA immunity can be successfully induced by oral route and the ISAv vaccine containing Centrovet recombinant ISAv antigens, and encapsulated using MicroMatrix™ technology, protects Atlantic salmon efficiently upon ISAv challenge. INTRODUCTION Infectious salmon anemia (ISA), caused by the infectious salmon anemia virus (ISAv) is a highly contagious disease of Atlantic salmon (Salmo salar) that was first reported in Norway (Kibenge et al., 2004). The disease was subsequently reported from Scotland, Canada and the United States. In 2007, the disease was reported in Chile and since then it has caused a major economic loss estimated at US $100M. In Chile, besides biosafety measures including early harvest and compulsatory slaughter of infected stock, there are several commercially available vaccines against ISA, which are applied by injection in freshwater stage. Although this procedure ensures vaccine application, it has several limitations including immunosuppression and reduction in feeding rate after vaccine application. Furthermore, vaccine protection usually does not cover the entire farming period, rendering fish susceptible to infection at higher sizes. Although a revaccination (booster) could overcome this problem, to inject fish at higher sizes exposes them to infectious agents and rises up vaccine-associated costs. Thus, the availability of an alternative delivery strategy such as an oral vaccination represents attractive candidates to evaluate. We report here a novel oral ISAv vaccine from Centrovet Laboratory (Chile) using their unique ISAv-derived antigen expressed in yeast, and encapsulated with a proprietary encapsulation technology, MicroMatrix™, of Advanced BioNutrition Corp., Columbia, Maryland, USA. We showed that vaccinated fish produced specific ISAv antibodies, and the antibody titer is correlated with protection upon challenge with a virulent ISAv strain. Materials & Methods ORAL - ISA - VACCINE INDUCES MUCOSAL ANTIBODIES AND RECRUITS LYMPHOCYTES TO THE HINDGUT. n= 180 ISAv Injectable vaccine n= 60 ISAv Oral vaccine n= 60 • ISAv-specific antibodies were induced in mucus from fish hindgut (Fig. 2A), and it correlated with the presence of a higher number of inflammatory cells in intestinal tissue (Figure 2B). ORAL ISAv VACCINE DOES NOT INTERFERE WITH WEIGHT GAIN WHEN ADMINISTERED UP TO 5-FOLD CONCENTRATION. • Fish vaccinated using 1x and 5x of vaccine dose showed no significant differences in growth among control (15.40 g ± 0,33), 1x (15.44 g ± 0,49), and 5x (15.43 g ± 0,46) groups. • The data suggested that inflammation induced by oral vaccine did not affect nutrient absorption parameters, and subsequently growth performance. Control Tratment n= 60 Collection of serum 180, 300, 450, 600, 900 and 1200 dd for ELISA, ISAv neutralizing assay and histology. ISAv challenge using cohabitation model at 310, 650, and 830 dd post - vaccination. Evaluating ISAv Oral vaccine safety at 1X and 5X vaccine dose. Figure 2A. Determination of the levels of anti-ISAv specific antibodies on intestinal mucus induced by oral vaccine. B. Histological sections (H&E) of control and vaccinated fish taken at 300 dd after vaccination. Arrows show lymphocyte cell in lamina propia of intestinal vili. Figure 4. Determination of the safety of ISA oral vaccine. Fish were vaccinated with three different oral vaccine lots at the recommended dosage (1x) or over dosage (5x) of vaccine and weight gain was measured at 300 dd after vaccination. THE ANTI - ISAv - SPECIFIC ANTIBODY LEVEL CORRELATES WITH PROTECTION DEGREE UPON CHALLENGE. CONCLUSIONS • Mortality up to 95% was observed in nonvaccinated fish whereas vaccinated fish had 17% mortality (Fig. 2A). RESULTS ORAL ISA-VACCINE ANTIBODIES WITH ABILITIES. ISAv incubated with serum from healthy fish but not from vaccinated fish could infect SHK-1 cells. The arrows show ISA-derived CPE on SHK-1 cells. INDUCES ISAV-SPECIFIC ISAv - NEUTRALIZING • Anti-ISAv antibody was detected at 300 dd post-vaccination reaching peak between 450 and 600 dd (Fig. 1A). • Anti-ISAv antibodies were virus-specific and were able to neutralize the infectious virus (Fig. 1B). • Moribund or dead fish showed typical ISAv clinical signs, such as haemorrhagic liver and intestine, ascetic fluid, petechia in caecum and visceral fat tissue (Fig. 2B). • ISA protection evaluated using three different vaccine lots showed that it lasted at least 830 dd after vaccination (Fig. 2C). IgM (O.D. 450 nm Anti - ISAv IgM Figure 1. Determining the anti-ISAv antibody response in Atlantic salmon following oral vaccination. A. The anti ISAv antibody response was measured by ELISA at 150, 300, 450, 600, 900 and 1200 degree days in vaccinated and non-vaccinated (control) fish . B. In vitro assay to determine the anti-ISAv neutralizing abilities of serum obtained from vaccinated fish. Figure 3. A. ISAv induced mortality pattern in Atlantic salmon vaccinated via oral route. Vaccinated (pink) and non-vaccinated (blue) fish were challenged by cohabitation using a virulent ISAv strain (HRP 7b region). B. Typical clinical signs of ISAv infection in dead fish. C. The relative percent survival (RPS) values of fish vaccinated with three different lots of ISAv-oral vaccine (blue, red and green bars) and challenged at 310, 650 and 830 day-degrees after vaccination. ACKNOLEDGEMENTS We thank Dr. Moti Harel, Advanced BioNutrition,Columbia, MD for his helpful discussions about ISAv-oral vaccine formulations. - ISAv-Oral vaccine induces both systemic and mucosal immunity starting at 300 dd and remains until 800 dd postvaccination. -Serum from vaccinated fish has ISAv neutralizing ability since it inhibited ISAv infection of SHK-1 cells in in vitro assay. - The anti-ISAv antibody response induced by vaccination correlated with the protection upon ISAv challenge with a virulent strain. - The vaccine is safe for application, causing no adverse effects when applied up to 5x-fold higher than the recommended dose.