Vaccines to prevent Vaccines to prevent Streptococcus Streptococcus iniaeiniae and and S. agalactiaeS. agalactiae disease in disease in

tilapia, tilapia, Oreochromis niloticusOreochromis niloticus

P.H. Klesius, J.J. Evans, C.A. Shoemaker P.H. Klesius, J.J. Evans, C.A. Shoemaker

and D.J. Pasnikand D.J. Pasnik

Aquatic Animal Health Research Laboratory, Aquatic Animal Health Research Laboratory, Auburn, AL and Chestertown, MDAuburn, AL and Chestertown, MD

pklesius@ars.usda.govpklesius@ars.usda.gov

Fluorescent-Chromophore CalceinFluorescent-Chromophore Calcein

A calcein solution (500 mg/l) was used to non-A calcein solution (500 mg/l) was used to non-invasively mark sham-vaccinated tilapia invasively mark sham-vaccinated tilapia (control) by bath immersion for 4 h.(control) by bath immersion for 4 h.

Calcein stains (arrows) the calcified skeletal Calcein stains (arrows) the calcified skeletal structures of the head and fins.structures of the head and fins.

Fluorescent-Chromophore CalceinFluorescent-Chromophore Calcein

Dead fish (twice daily) were examined for Dead fish (twice daily) were examined for fluorescent staining using a hand-held fluorescent staining using a hand-held portable UV lamp set at 365 nm.portable UV lamp set at 365 nm.

Materials and MethodsMaterials and Methods

Nile Tilapia (Nile Tilapia (Oreochromis niloticusOreochromis niloticus) 15±2 g.) 15±2 g.

Aquarium- Flow-through 57 l supplied with 0.5 l Aquarium- Flow-through 57 l supplied with 0.5 l water/h.water/h.

Streptococcus iniaeStreptococcus iniae ARS #60. ARS #60.

Calcein- Sigma Chemical Co, 500 mg/l dechloridated Calcein- Sigma Chemical Co, 500 mg/l dechloridated water, dissolved oxygen 3.5±0.56 mg/l, 26.1±0.63°C water, dissolved oxygen 3.5±0.56 mg/l, 26.1±0.63°C and pH 7.9±0.05.and pH 7.9±0.05.

Vaccine- Vaccine- S. iniaeS. iniae modified killed (Klesius et al. 1999 modified killed (Klesius et al. 1999 BEAFP 19:39-41), by IP injection and bath immersion BEAFP 19:39-41), by IP injection and bath immersion with diluted vaccine.with diluted vaccine.

Vaccine efficacy- % cumulative mortality and Relative Vaccine efficacy- % cumulative mortality and Relative Percent Survival (RPS).Percent Survival (RPS).

Vaccination and challenge modelVaccination and challenge model

20 cohabited tilapia in a aquarium for 30 d. or more

10 Sham-vaccinated calcein marked

10 Vaccinated non-marked

Immersion or IP challenged with virulent S. iniae and a RPS at 15 d PC.

RPS = (1/8 -1) x 100 = 87.5

Experimental DesignExperimental Design

MarkingMarking TreatmentTreatment # CM # CM & NM & NM fishfish

# CM & NM# CM & NM

Cohabited/Cohabited/

aquariumaquarium

ReplicatesReplicates

CalceinCalcein

MarkedMarked

(CM)(CM)

Sham-Sham-

VaccinatedVaccinated

(CMSV)(CMSV)

160160 2020

88

NonmarkeNonmarkedd

(NM)(NM)

VaccinatedVaccinated

(NMV)(NMV)160160 2020

30 d cohabited and then challenged with by IP injection at doses of S. iniae at 3.5 × 107 , 6.8 × 107 , 10.0 × 107 , or 15.0 × 107 CFU/fish. % cumulative mortality and RPS calculated at 15 PC.

Percent Cumulative Mortality*Percent Cumulative Mortality*

S. iniae CFU/fish

# Fish challenged

CMSV NMV Probability

3.5 × 107

6.8 × 107

40

40

50

63

2.5

17.5

0.0316

0.0517

10.0 × 107

15.0 × 107

40

40

55

65

15

12.5

<0.0001

0.0365

All doses 160 58.1 11.9 0.0003*15 d PC

RPS*RPS*

S. iniae CFU/fish

# Fish challenged

RPS

3.5 × 107

6.8 × 107

40

40

95

72

10.0 × 107

15.0 × 107

40

40

73

81

All doses 160 80

*15 d PC

Table 1. Efficacy of formalin-killed Table 1. Efficacy of formalin-killed Streptococcus iniaeStreptococcus iniae cells, cells, extracellular product (ECP) and complete vaccine (extracellular product (ECP) and complete vaccine (S. iniaeS. iniae cells + ECP) diluted 1:500 administrated by bath immersion cells + ECP) diluted 1:500 administrated by bath immersion for 10 sec to Nile tilapia (15 g) and followed by experimental for 10 sec to Nile tilapia (15 g) and followed by experimental challenge infection with challenge infection with S. iniaeS. iniae at 14 d post-vaccination. at 14 d post-vaccination.

Vaccine Vaccine componentscomponents

Percent cumulative Percent cumulative mortality in mortality in cohabited cohabited vaccinatesvaccinates

Percent cumulative Percent cumulative mortality in mortality in

cohabited non-cohabited non-vaccinatesvaccinates

RPSRPS

CellsCells 1010 7070 85.785.7

ECPECP 4040 2525 00

Cells + ECPCells + ECP 55 5050 9090

Relative percent survival at 14 d post-challenge with 1 X 107 CFU/fish of Relative percent survival at 14 d post-challenge with 1 X 107 CFU/fish of S. iniaeS. iniae

Table 2. Efficacy of Table 2. Efficacy of Streptococcus Streptococcus vaccine (Cells + ECP) vaccine (Cells + ECP) dilutions administrated by bath immersion for 10 and 20 sec dilutions administrated by bath immersion for 10 and 20 sec to Nile tilapia (15 g) and followed by experimental challenge to Nile tilapia (15 g) and followed by experimental challenge infection with infection with S. iniaeS. iniae at 14 d post-vaccination. at 14 d post-vaccination.

Dilutions + Dilutions + duration of duration of exposureexposure

Percent cumulative Percent cumulative mortality in mortality in cohabited cohabited vaccinatesvaccinates

Percent cumulative Percent cumulative mortality in mortality in

cohabited non-cohabited non-vaccinatesvaccinates

RPSRPS

1:100 10 sec1:100 10 sec

1:100 20 sec1:100 20 sec1010

20202020

1001005050

7070

1:100 10 sec1:100 10 sec

1:500 20 sec1:500 20 sec55

25252525

65654040

61.561.5

1:1000 10 sec1:1000 10 sec

1:1000 20 sec1:1000 20 sec1515

30304040

505062.562.5

4040

Relative percent survival at 14 d post-challenge with 1 X 10Relative percent survival at 14 d post-challenge with 1 X 107 7 CFU/fish of CFU/fish of S. iniaeS. iniae

Table 3. Mean specific antibody titers in Nile tilapia (15 g) Table 3. Mean specific antibody titers in Nile tilapia (15 g) administrated the 1:100 and 1:500 dilutions of administrated the 1:100 and 1:500 dilutions of Streptococcus Streptococcus vaccine (Cells + ECP) dilutions by bath immersion for 20 sec vaccine (Cells + ECP) dilutions by bath immersion for 20 sec before and after experimental challenge infection with before and after experimental challenge infection with S. iniaeS. iniae at 30 d post-vaccination.at 30 d post-vaccination.

Dilutions + Dilutions + duration of duration of exposureexposure

Mean specific Mean specific antibody titer ± SE antibody titer ± SE after vaccination 30 after vaccination 30

dd

Mean specific Mean specific antibody titer ±SE antibody titer ±SE 14 d post-challenge14 d post-challenge

RPSRPS

Sham- vaccineSham- vaccine 203 ± 42203 ± 42 4880 ± 8584880 ± 858 --

1:1001:100 2304 ± 462304 ± 46 3840 ± 8103840 ± 810 7070

1:5001:500 1120 ± 4291120 ± 429 7680 ± 6197680 ± 619 61.561.5

Relative percent survival at 14 d post-challenge with 1 X 10Relative percent survival at 14 d post-challenge with 1 X 107 7 CFU/fish of CFU/fish of S. iniaeS. iniae

Table 4. Efficacy of Table 4. Efficacy of Streptococcus Streptococcus vaccine (Cells + ECP) vaccine (Cells + ECP) diluted 1:500 administrated by bath immersion for 20 sec to diluted 1:500 administrated by bath immersion for 20 sec to Nile tilapia and followed by experimental challenge infection Nile tilapia and followed by experimental challenge infection with with S. iniaeS. iniae at 14 d post-vaccination. at 14 d post-vaccination.

Tilapia weight Tilapia weight (g)(g)

Percent cumulative Percent cumulative mortality in mortality in

cohabited vaccinescohabited vaccines

Percent cumulative Percent cumulative mortality in mortality in

cohabited non-cohabited non-vaccinesvaccines

RPSRPS

0.50.5

1.0*1.0*3535

7070100100

1001006565

3030

3.0*3.0*

5.05.05050

3535100100

1001005050

6565

10.010.0

15.015.01515

55100100

50508585

9090

Relative percent survival at 14 d post-challenge with 1 X 10Relative percent survival at 14 d post-challenge with 1 X 107 7 CFU/fish of CFU/fish of S. iniaeS. iniae

ConclusionsConclusions

• Solubility of calcein may vary withinSolubility of calcein may vary within different sources of water, may require buffering.different sources of water, may require buffering.

• Calcein has been demonstrated to be effective in Calcein has been demonstrated to be effective in marking many species of fish at different life marking many species of fish at different life stages.stages.

• Diluted Diluted S. iniaeS. iniae vaccine protection against vaccine protection against S. iniaeS. iniae infection in 0.5 and large weight tilapia except for infection in 0.5 and large weight tilapia except for

1.0-3.0 g weights.1.0-3.0 g weights.

• Specific serum antibody response to vaccination Specific serum antibody response to vaccination appears to provide protection.appears to provide protection.

• Multi-immersion vaccination at different tilapia Multi-immersion vaccination at different tilapia weights appears to a successful strategy to weights appears to a successful strategy to prevent prevent S. iniaeS. iniae infection in tilapia. infection in tilapia.

ReferencesReferencesEvans et al. 2006. Efficacy of Evans et al. 2006. Efficacy of Streptococcus agalactiaeStreptococcus agalactiae

(Group B) vaccine in tilapia ((Group B) vaccine in tilapia (Oreochromis niloticusOreochromis niloticus) ) by intraperitoneal and bath immersion by intraperitoneal and bath immersion administration. Vaccines 22: 3769-3773.administration. Vaccines 22: 3769-3773.

Klesius et al. 1999. Efficacy of a killed Klesius et al. 1999. Efficacy of a killed Streptococcus Streptococcus iniaeiniae vaccine in tilapia ( vaccine in tilapia (Oreochromis niloticusOreochromis niloticus) . ) . Bulletin of European Association of Fish Pathologists Bulletin of European Association of Fish Pathologists 19:1-3.19:1-3.

Klesius et al. 2006. A vaccination and challenge model Klesius et al. 2006. A vaccination and challenge model using calcein marked fish. Fish & Shellfish Immunol. using calcein marked fish. Fish & Shellfish Immunol. 20:20-28.20:20-28.

Shoemaker et al. 2006. Efficacy of Shoemaker et al. 2006. Efficacy of Streptococcus iniaeStreptococcus iniae modified bacterin delivered using Oralject modified bacterin delivered using Oralject technology in Nile tilapia (technology in Nile tilapia (Oreochromis niliticusOreochromis niliticus) ) Aquaculture 255:151-156.Aquaculture 255:151-156.