Bachelor’s Degree in Microbiology . Final Project 2015

maracarmonafarfan@gmail.com

AQUACULTURE & MICROBIOLOGY VIRUS

IHNV (Infectious Haematopoietic Necrosis

Virus)

VHSV (Viral Hemorrhagic Septicemia Virus)

ISA virus (Infectious Salmon Anemia virus)

SVCV (Spring Viraemia of Carp Virus)

BACTERIA

Yersinia ruckeri

Aeromonas spp.

Pseudomonas aeruginosa

Vibrio spp.

Lacotoccus garvieae

Streptococcus spp.

PROTOZOA Uronema marinum

CONTROL DISEASE IN AQUACULTURE

Table 1. Main viral bacterial and protozoa pathogens in aquaculture.

50% of world seafood production comes from aquaculture.

Aquaculture is the farming of aquatic organisms (fish, shellfish

and plants) in freshwater and marine environments. In recent

years this sector has increased its economical relevance as a

massive production system (≈50% of the total world fish

captures; FAO 2013) but presence of pathogens (Table 1) lead to

the appearance of diseases provoking important economical

losses in fish farms. The infrastructure’s properties such as

temperature and density allow the spread of these pathogens.

In order to avoid the spread of pathogens in fish farms different methods have been applied, being the use of antibiotics the most used until recently. Due to the appearance of resistances more stringent

policies regarding antibiotic use have been applied. Nowadays efforts in this field have been focused in prophylactic methods such as vaccination and immunostimulation. These methods have been improved

through the use of emerging technologies such micro-and nanotechnologies. The implementation of these new technologies represents an advance with great potential to enhance therapeutic effectiveness

of immunostimulants and vaccines.

IMMUNOSTIMULANTS Levamisome

LPS DNA & RNA

β-glucan Vitamines Others…

VACCINES DNA-based vaccines

Bacterins Life-attenuated vaccines

10 nm 1 nm 100 nm 1 μm 10 μm 100 μm 1 mm

Micro- and nanoparticles used in aquaculture are

based in polymeric, polysaccharide and phospholipid

structures relevant due to their biodegradable

properties.

Encapsulation of immunostimulants and vaccines in

these structures allow to improve the biodistribution,

increase their permanence and can be modified for an

efficient delivery in specific organs, tissues or cells.

RESULTS OVERVIEW

Liposome Sphere Capsule

Nanoscale Microscale

PLGA

QUITOSAN

ALGINATE

PHOSPOLIPID

J. Skermo et al. (1995)

Alginate micro-spheres containing high mannuromic

acid content.

Artemia nauplii

d0 d2

Challenge with Vibrio anguillarum (dose: 105

CFU mL-1)

1 week post-challenge

Scophtalmus maximus (turbot)

Inmunostimulation protected against V.

anguillarum and reduced mortality by

48% in comparison with control group

(non stimulated and challenge with V.

anguillarum)

RESULTS

MATERIALS & METHODS

T. Behera et al. (2010)

CONCLUSIONS

PLGA microparticles

Aeromonas hydrophila

Outer membrane proteins (OMP)

isolation

encapsulation

Inactivation (1%formalin 4ºC) Whole cell

bacteria

MATERIALS & METHODS

Labeo rohita (rohu)

i.p. (intraperitoneal

injection)

d0 21 d post-injection 42 post-injection

RESULTS

IMMUNE RESPONSE STUDIES

Myeloperoxidase activity Respiratory burst assay

Bacterial aglutinattion activity Haemagluttination

Triplr antibody indirect ELISA

i.p. (intraperitoneal injection)

All parameters, including antibody titres, in the PLGA-OMP treated group were higher than

in the control.

M. Adomako et al. (2012)

PLGA nanoparticles (PLGA-PCDNA-G)

Oncorhynchus mykiss (rainbow trout)

4 groups n=26

IHNV Infectious haematopoietic

necrosis virus

G (glycoprotein)

gene sequence

encapsulation

d0

6 weeks post-vaccination

21 d post-challenge

n=15 Challenge with

IHNV (dose: 2.0 x 103 /0,1mL

pfu) n=15

Organs & muscle tissue dissection (RT-PCR)

MATERIALS & METHODS

G gene expression was low in foregut,

kidney and spleen and non detected in muscle. Low anti-IHNV titres

only in two treated fishes.

No significat differences in RPS

(relative percent survival) between

control and treated groups after challenge.

RESULTS

n=15 Antibody test

Survival analysis

Survival analysis

Survival analysis

Ferosekhan S. et al. (2014)

RNA (source:yeast)

Quitosan nanoparticles (CtNPs)

Labeo rohita (rohu) 5 groups

n=36

d0 60 d

i.p. (intraperitoneal injection)

n=14 Challenge with 0,2 mL

Aeromonas hydrophila (dose: 108 CFU mL-1)

Survival analysis

15 d post-challenge

Blood collection &

Organs dissection (liver, viscera)

Oral administration

OMP

MATERIALS & METHODS

Immunostimulation with RNA increases

parameters such white blood cells,

respiratory burst activity and lysozyme

activity.

Survival increased to 67% (0,2% RNA-CtNPs) and to 83% (0,4% RNA-

CtNPs) compared to control group.

RESULTS

Ramasamy H. et al. (2012)

Uronema marinum

Epinephelus bruneus

(longtooth grouper) 3 groups

n=25

PLGA microspheres

enca

psu

lati

on

killed cells(1%formalin)

(i-antigen /vaccine (V))

i.p. (intraperitoneal

injection)

d0 4 weeks

post-vaccination

i.p. (intraperitoneal injection)

Challenge with 100µL PBS +U. marinum (3,2

x107 mL-1)

Survival analysis

30 d post-challenge

MA

TE

RIA

LS

& M

ET

HO

DS

4 weeks post-vaccination

respiratory burst, lysozyme activity and complement activity

increased. 20% of mortality in

PLGA+V treated group in comparison with

control groups (90% of mortality

RESULTS Serum collection

Elok N. F. et al. (2014)

Cinnamaldehyde

encap

sulatio

n

Liposome (L)

Danio rerio (zebrafish)

3 groups n=20

75 μL/L empty liposome (LE) (inmersion)

75 μL/L liposome encapsulating cinnamaldehyde (LEC)

d0 Challenge (i.p. injection ) with 10 µL of :

Aeromonas hydrophila (106 CFU mL-1)

Vibrio vulnificus (106 CFU mL-1) Streptococcus agalactiae (107 CFU mL-1)

8 d post -challenge

Survival analysis

With LEC treatment survival increased :

31% (S.agalactiae) 35% (A. hydrophila) 57% (V. vulnificus)

compared to control group

(100% mortality in all challenges)

RESULTS

MATERIALS & METHODS

REFERENCES

Ruyra A. et al. (2014)

Micro- and nanoencapsulation systems based in biodegradable structures have shown to be effective for

immunostimulant and vaccine administration. Their use enhance the immune system response conferring protection against pathogens.

Recent findings show that the use of liposomes encapsulating more than one substance could be a good strategy to treat diseases in aquaculture.

Further studies are necessary in order to ensure their effectiveness and their safety.

1. Skjermo J, Defoort T, Dehasque M, Espevikt T, Olsen Y, Skjak-Braaeks g, Sogerloos P VO. (1995). Immunostimulation of juvenile turbot (Scophthalmus maximus L.) using an alginate with high mannuronic acid content administered via the live food organism Artemia. Fish Shellfish Immunol. 5:521–534.

2. Behera T, Nanda PK, Mohanty C, Mohapatra D, Swain P, Das BK, Routray P, Mishra BK, Sahoo SK. (2010). Parenteral immunization of fish, Labeo rohita with Poly D, L-lactide-co-glycolic acid (PLGA) encapsulated antigen microparticles promotes innate and adaptive immune responses. Fish Shellfish Immunol. 28:320–5.

3. M Adomako, S St-Hilarie, Y Zheng, J Eley, R D Marcum, W Sealey, B C Donahower, S LaPatra , P P Sheridan. (2012). Oral DNA vaccination of rainbow trout, Onchorhunchus mykiss (Walbaum), against infectious haematopoietic necrosis virus using PLGA[Poly(D,L-Lactic-Co-Glycolic Acid) nanoparticles. Journal of Fish Diseases. 35:203-214

4. Ramasamy Harikrishnan, Chellam Balasundaram, Moon-Soo Heo. (2012). Poly D,L-lactide-co-glycolic acid(PLGA)-encapsulated vaccine on immune system in Epinephelus bruneus against Uronema marinum. Experimental Parasitology. 131:325-332

5. Ferosekhan S, Gupta S, Singh A, Rather M, Kumari R, Kothari D, Pal A, Jadhao S. (2014). RNA-Loaded Chitosan Nanoparticles for enhanced Growth, Immunostimulation and Disease Resistance in Fish. Curr. Nanosci. 10:453–464.

6. Faikoh EN, Hong Y-H, Hu S-Y. 2014. Liposome-encapsulated cinnamaldehyde enhances zebrafish (Danio rerio) immunity and survival when challenged with Vibrio vulnificus and Streptococcus agalactiae. Fish Shellfish Immunol. 38:15–24.

7. Ruyra A, Cano-Sarabia M, García-Valtanen P, Yero D, Gibert I, Mackenzie S a, Estepa A, Maspoch D, Roher N. 2014. Targeting and stimulation of the zebrafish (Danio rerio) innate immune system with LPS/dsRNA-loaded nanoliposomes. Vaccine 32:3955–62.

Danio rerio (zebrafish)

Nanoliposome (NLc) Lypopolysaccharide

(LPS)

Polyonisinic- polycytidylic acid

[Poly (I:C)]

encapsulation

10 μL mixture of free immunostimulants (8,2 mg/Kg Poly (I:C) and 4,1 mg/Kg LPS)

i.p. (intraperitoneal injection)

MATERIALS & METHODS

10 μL empty nanoliposomes (NLs) and 10 μL loaded

nanoliposomes (8,2 mg/Kg Poly (I:C) and 4,1 mg/Kg LPS)

immersion

10 μL empty nanoliposomes (NLs) and 10 μL loaded

nanoliposomes (16,6 mg/Kg Poly (I:C) and 8,3 mg/Kg LPS)

10 μL mixture of free immunostimulants (16,6 mg/Kg

Poly (I:C) and 8,3 mg/Kg LPS)

d0 7 d post – injection/immersion

(4 groups n=36) Challenge with LD50 of

Pseudomonas aeruginosa (PAO1) (3,2 x107 ∼ 2,5 x108

cfu)

(4 groups n=15) Challenge with Spring Viraemia

of Carp Virus (7,1 x107 pfu/mL)

7d post-challenge

13 d post-challenge

Survival analysis

RE

SU

LTS