Download Oral Vaccination Against Infectious Salmon Anemia in Atlantic Salmon

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.