1. Vaccines to prevent Streptococcus iniae and S. agalactiae disease in tilapia, Oreochromis niloticus P.H. Klesius, J.J. Evans, C.A. Shoemaker and D.J. Pasnik Aquatic Animal Health Research Laboratory, Auburn, AL and Chestertown, MD pklesius@ars.usda.gov

2. Fluorescent-Chromophore Calcein A calcein solution (500 mg/l) was used to non-invasively mark sham-vaccinated tilapia (control) by bath immersion for 4 h. Calcein stains (arrows) the calcified skeletal structures of the head and fins.

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

4. Materials and Methods Nile Tilapia (Oreochromis niloticus) 15±2 g. Aquarium- Flow-through 57 l supplied with 0.5 l water/h. Streptococcus iniae ARS #60. Calcein- Sigma Chemical Co, 500 mg/l dechloridated water, dissolved oxygen 3.5±0.56 mg/l, 26.1±0.63°C and pH 7.9±0.05. Vaccine- S. iniae modified killed (Klesius et al. 1999 BEAFP 19:39-41), by IP injection and bath immersion with diluted vaccine. Vaccine efficacy- % cumulative mortality and Relative Percent Survival (RPS).

5. Vaccination and challenge model 20 cohabited tilapia in a aquarium for 30 d. or more 10 Sham-vaccinated calcein marked 10 Vaccinated non-marked

6. Immersion or IP challenged with virulent S. iniae and a RPS at 15 d PC. RPS = (1/8 -1) x 100 = 87.5

7. Experimental Design MarkingTreatment # CM & NM fish # CM & NM Cohabited/aquariumReplicates CalceinMarked(CM)Sham-Vaccinated(CMSV)160208 Nonmarked(NM)Vaccinated(NMV)16020 30 d cohabited and then challenged with by IP injection at doses of S. iniae at 3.5 × 10 7, 6.8 × 10 7, 10.0 × 10 7, or 15.0 × 10 7 CFU/fish. % cumulative mortality and RPS calculated at 15 PC.

8. Percent Cumulative Mortality* S. iniae CFU/fish # Fish challenged CMSVNMVProbability 3.5 × 10 7 6.8 × 10 7 40 50 63 2.5 17.5 0.0316 0.0517 10.0 × 10 7 15.0 × 10 7 40 55 65 15 12.5 <0.0001 0.0365 All doses16058.111.90.0003 *15 d PC

9. RPS* S. iniae CFU/fish # Fish challenged RPS 3.5 × 10 7 6.8 × 10 7 40 95 72 10.0 × 10 7 15.0 × 10 7 40 73 81 All doses16080 *15 d PC

10. Table 1. Efficacy of formalin-killed Streptococcus iniae cells, extracellular product (ECP) and complete vaccine (S. iniae cells + ECP) diluted 1:500 administrated by bath immersion for 10 sec to Nile tilapia (15 g) and followed by experimental challenge infection with S. iniae at 14 d post-vaccination. Vaccine components Percent cumulative mortality in cohabited vaccinates Percent cumulative mortality in cohabited non- vaccinates RPS Cells107085.7 ECP40250 Cells + ECP 55090 Relative percent survival at 14 d post-challenge with 1 X 107 CFU/fish of S. iniae

11. Table 2. Efficacy of Streptococcus vaccine (Cells + ECP) dilutions administrated by bath immersion for 10 and 20 sec to Nile tilapia (15 g) and followed by experimental challenge infection with S. iniae at 14 d post-vaccination. Dilutions + duration of exposure Percent cumulative mortality in cohabited vaccinates Percent cumulative mortality in cohabited non- vaccinates RPS 1:100 10 sec 1:100 20 sec 1020201005070 1:100 10 sec 1:500 20 sec 52525654061.5 1:1000 10 sec 1:1000 20 sec 1530405062.540 Relative percent survival at 14 d post-challenge with 1 X 10 7 CFU/fish of S. iniae

12. Table 3. Mean specific antibody titers in Nile tilapia (15 g) administrated the 1:100 and 1:500 dilutions of Streptococcus vaccine (Cells + ECP) dilutions by bath immersion for 20 sec before and after experimental challenge infection with S. iniae at 30 d post-vaccination. Dilutions + duration of exposure Mean specific antibody titer ± SE after vaccination 30 d Mean specific antibody titer ±SE 14 d post- challenge RPS Sham- vaccine 203 ± 42 4880 ± 858 - 1:100 2304 ± 46 3840 ± 810 70 1:500 1120 ± 429 7680 ± 619 61.5 Relative percent survival at 14 d post-challenge with 1 X 10 7 CFU/fish of S. iniae

13. Table 4. Efficacy of Streptococcus vaccine (Cells + ECP) diluted 1:500 administrated by bath immersion for 20 sec to Nile tilapia and followed by experimental challenge infection with S. iniae at 14 d post-vaccination. Tilapia weight (g) Percent cumulative mortality in cohabited vaccines Percent cumulative mortality in cohabited non- vaccines RPS 0.51.0*35701001006530 3.0*5.050351001005065 10.015.0155100508590 Relative percent survival at 14 d post-challenge with 1 X 10 7 CFU/fish of S. iniae

15. Conclusions Solubility of calcein may vary within Solubility 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 S. iniae vaccine protection against S. iniae Diluted S. iniae vaccine protection against S. iniae infection in 0.5 and large weight tilapia except for 1.0-3.0 g weights. infection in 0.5 and large weight tilapia except for 1.0-3.0 g weights. Specific serum antibody response to vaccination appears to provide protection. Specific serum antibody response to vaccination appears to provide protection. Multi-immersion vaccination at different tilapia weights appears to a successful strategy to prevent S. iniae infection in tilapia. Multi-immersion vaccination at different tilapia weights appears to a successful strategy to prevent S. iniae infection in tilapia.

16. References Evans et al. 2006. Efficacy of Streptococcus agalactiae (Group B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion administration. Vaccines 22: 3769-3773. Klesius et al. 1999. Efficacy of a killed Streptococcus iniae vaccine in tilapia (Oreochromis niloticus). Bulletin of European Association of Fish Pathologists 19:1-3. Klesius et al. 2006. A vaccination and challenge model using calcein marked fish. Fish & Shellfish Immunol. 20:20-28. Shoemaker et al. 2006. Efficacy of Streptococcus iniae modified bacterin delivered using Oralject technology in Nile tilapia (Oreochromis niliticus) Aquaculture 255:151-156.