1. Growth Response and Acquired Resistance of Nile Tilapia Following Infection or Vaccination with Streptococcus iniae Craig Shoemaker, Chhorn Lim, Mediha Yildirim-Aksoy, Tom Welker, Phillip Klesius and Joyce Evans Aquatic Animal Health Research Laboratory, USDA-ARS, Auburn, AL, USA

2. Introduction Streptococcus iniae is a disease of worldwide importance. Streptococcus iniae is a disease of worldwide importance. 22 species of fish affected - cultured, wild and marine. 22 species of fish affected - cultured, wild and marine. US losses are estimated at $10 million, annually. US losses are estimated at $10 million, annually.

3. Introduction Most growth performance studies to date involve diet manipulation and/or probiotic or prebiotic supplementation. Most growth performance studies to date involve diet manipulation and/or probiotic or prebiotic supplementation. Disease resistance assessed by challenge. Disease resistance assessed by challenge.

4. Introduction Little or no information is available on the growth response of tilapia following infection and/or vaccination with S. iniae. Little or no information is available on the growth response of tilapia following infection and/or vaccination with S. iniae. Producers suggest that infected fish do not perform well and the poor performance results in lost profits. Producers suggest that infected fish do not perform well and the poor performance results in lost profits. Cook (1999) estimated at least $500 million annually lost due to immune-induced growth suppression in terrestrial animals. Cook (1999) estimated at least $500 million annually lost due to immune-induced growth suppression in terrestrial animals.

5. Introduction Reports suggest improved growth of vaccinated eels (Esteve-Gassent et al. 2004). Reports suggest improved growth of vaccinated eels (Esteve-Gassent et al. 2004). Administration of a modified live Edwardsiella ictaluri vaccine has resulted in increased performance ($1700/acre over non-vaccinated fish). Administration of a modified live Edwardsiella ictaluri vaccine has resulted in increased performance ($1700/acre over non-vaccinated fish).

6. Objectives Determine growth performance and acquired resistance of Nile tilapia following infection or vaccination. Determine growth performance and acquired resistance of Nile tilapia following infection or vaccination.

7. Study I - Materials and Methods 1200 Nile tilapia, Oreochromis niloticus (11g) were stocked at 120 fish/tank in ten 208-L aquarium. Duplicate tanks were used to generate survivors at day 14. Treatment Challenge dose (CFU/fish) Cumulative Mortality (%) Low challenge 8.8 X 10 3 29.6 a Medium challenge 8.8 X 10 4 36.3 a,b High challenge 8.8 X 10 5 45.0 b ControlNon-injected 3.4 c 3.4 c ControlBroth-injected 0.8 c 0.8 c Pooled SEM 2.7 2.7

8. Study I - Materials and Methods Survivors without clinical signs of disease were stocked at 30 fish per 57-L aquarium (triplicate). Survivors without clinical signs of disease were stocked at 30 fish per 57-L aquarium (triplicate). A 32 % crude protein commercial diet was fed to S. iniae-recovered tilapia twice daily to apparent satiation for 8-weeks. A 32 % crude protein commercial diet was fed to S. iniae-recovered tilapia twice daily to apparent satiation for 8-weeks.

9. Study I - Materials and Methods Weight gain (g), feed intake (g dry matter/fish), feed efficiency ratio [FER = weight gain (g) / dry feed fed (g)] and survival were recorded after the 8 week period. Weight gain (g), feed intake (g dry matter/fish), feed efficiency ratio [FER = weight gain (g) / dry feed fed (g)] and survival were recorded after the 8 week period. Agglutinating antibody titer was determined by the method of Klesius et al. (2000). Agglutinating antibody titer was determined by the method of Klesius et al. (2000).

10. Study I - Materials and Methods Following the 8 week performance period fish were challenged with 1 X 10 6 CFU/fish by i.p. injection. Following the 8 week performance period fish were challenged with 1 X 10 6 CFU/fish by i.p. injection. Mortality was monitored for 21 days and the % mortality calculated. Mortality was monitored for 21 days and the % mortality calculated.

11. Study II - Materials and Methods A 2 X 4 factorial experiment was conducted – triplicate tanks of 25 fish each. A 2 X 4 factorial experiment was conducted – triplicate tanks of 25 fish each. Group A – Non-vaccinated with 0 mg/kg β-glucan. Group A – Non-vaccinated with 0 mg/kg β-glucan. Group B – Vaccinated with 0 mg/kg diet β-glucan. Group B – Vaccinated with 0 mg/kg diet β-glucan. Group C – Non-vaccinated with 50 mg/kg diet β-glucan. Group C – Non-vaccinated with 50 mg/kg diet β-glucan. Group D – Vaccinated with 50 mg/kg diet β-glucan. Group D – Vaccinated with 50 mg/kg diet β-glucan. Group E - Non-vaccinated with 100 mg/kg diet β-glucan. Group E - Non-vaccinated with 100 mg/kg diet β-glucan. Group F - Vaccinated with 100 mg/kg diet β-glucan. Group F - Vaccinated with 100 mg/kg diet β-glucan. Group G - Non-vaccinated with 200 mg/kg diet β-glucan. Group G - Non-vaccinated with 200 mg/kg diet β-glucan. Group H - Vaccinated with 200 mg/kg diet β-glucan. Group H - Vaccinated with 200 mg/kg diet β-glucan.

12. Study II - Materials and Methods Fish were vaccinated by IP injection of 100 µl killed S. iniae vaccine (Klesius et al. 2000) and booster vaccinated at day 21. Fish were vaccinated by IP injection of 100 µl killed S. iniae vaccine (Klesius et al. 2000) and booster vaccinated at day 21. Weight gain (g), feed intake (g dry matter/fish), feed efficiency ratio [FER = weight gain (g) / dry feed fed (g)] and survival were recorded after a 10 week period. Weight gain (g), feed intake (g dry matter/fish), feed efficiency ratio [FER = weight gain (g) / dry feed fed (g)] and survival were recorded after a 10 week period.

13. Study II - Materials and Methods Following the 10 week performance period fish were challenged with 1 X 10 7 CFU/fish by i.p. injection. Following the 10 week performance period fish were challenged with 1 X 10 7 CFU/fish by i.p. injection. Mortality was monitored for 14 days and the relative percent survival (Amend 1981) calculated. Mortality was monitored for 14 days and the relative percent survival (Amend 1981) calculated.

14. Results -

15. Study I - Average weight gain, dry matter intake, feed efficiency ratio (FER) and survival of S. iniae-recovered tilapia fed for 8-weeks (Shoemaker et al. 2006) Treatment Weight gain (g) Feed intake (g DM/fish) FERSurvival Low challenge 27.049.10.5576.7 Medium challenge 27.947.80.5881.1 High challenge 26.344.90.5884.7 Control(non-injected)27.946.00.6088.9 Control(broth-injected)27.845.20.6195.6 Pooled SEM 1.6 1.6 1.8 1.80.02 4.7 4.7

16. Study I - Agglutinating antibody titer of S. iniae- survived tilapia and mean cumulative mortality at 21 days post challenge with S. iniae of S. iniae- survived tilapia (Shoemaker et al. 2006) Treatment Ab-titer 1 (log 10 ) Ab-titer 2 (log 10 ) Mortality (%) 3 Low challenge 1.0 a 0.2 a,b 0.2 a,b 7.4 a Medium challenge 1.1 a 0.4 a 3.3 a High challenge 1.8 b 0.4 a 8.3 a Control(non-injected) 0.0 c 0.0 b 41.7 b Control(broth-injected) 0.0 c 0.0 b 43.3 b Pooled SEM 0.20.26.0 1 Prior to 8-week feeding period. 2 Following 8-week feeding period. 3 P < 0.001.

17. Study II - Mean weight gain, feed efficiency ratio (FER) and survival of vaccinated and control tilapia fed diets containing β-glucan for 10-weeks (Whittington et al. 2005) Treatment Β-glucan (mg/kg) Weight gain (g) FER Survival (%) Vaccinated052.10.67 98.3 98.3 5055.50.71 93.3 93.3 10058.20.63100.0 20055.10.64 97.5 97.5 Control055.50.67 96.7 96.7 5054.70.67 95.0 95.0 10059.00.63 96.7 96.7 20052.00.62100.0 Pooled SEM 2.1 2.10.05 1.2 1.2

18. Study II - Agglutinating antibody titer of vaccinated and non-vaccinated tilapia and relative percent survival following challenge (Whittington et al. 2005) 1 21 day post boost. 2 RPS=relative percent survival. Treatment Β-glucan (mg/kg) Ab-titer 1 (log 10 ) RPS 2 Vaccinated01.595 501.8100 1001.7100 2001.191 Control00.1-- 500.00.2 1000.033 2000.712 Pooled SEM 0.052.8

19. Discussion Studies examining growth performance in fish have historically been conducted using apparently healthy animals (Lim and Webster 2001). Studies examining growth performance in fish have historically been conducted using apparently healthy animals (Lim and Webster 2001). We examined the performance of fish that recovered (survived) from S. iniae infection or following vaccination. We examined the performance of fish that recovered (survived) from S. iniae infection or following vaccination.

20. Discussion Following the 8 week feeding period, no differences were observed in growth, feed intake and feed efficiency ratio of S. iniae- recovered tilapia. Following the 8 week feeding period, no differences were observed in growth, feed intake and feed efficiency ratio of S. iniae- recovered tilapia. Following the 10 week feeding period, no differences were observed in growth, feed efficiency ratio and survival of vaccinated tilapia. Following the 10 week feeding period, no differences were observed in growth, feed efficiency ratio and survival of vaccinated tilapia.

21. Discussion The study demonstrated acquired resistance to S. iniae in the S. iniae–recovered and vaccinated tilapia. The study demonstrated acquired resistance to S. iniae in the S. iniae–recovered and vaccinated tilapia. Passive immunization and vaccine studies have demonstrated the importance of antibody in S. iniae immunity (Klesius et al. 2000; Shelby et al. 2002; Eldar et al. 1997; Whittington et al. 2005). Passive immunization and vaccine studies have demonstrated the importance of antibody in S. iniae immunity (Klesius et al. 2000; Shelby et al. 2002; Eldar et al. 1997; Whittington et al. 2005). Agglutinating antibody titer was demonstrated in S. iniae recovered and vaccinated tilapia. Agglutinating antibody titer was demonstrated in S. iniae recovered and vaccinated tilapia.

22. Conclusion Results suggest that vaccinated tilapia or tilapia that survive S. iniae infection seroconvert and produce an acquired immune response with little or no effect on growth performance of 10-20 g fish. Results suggest that vaccinated tilapia or tilapia that survive S. iniae infection seroconvert and produce an acquired immune response with little or no effect on growth performance of 10-20 g fish.

23. Management implications Removal of tilapia showing overt signs of disease (i.e., difficulty in consuming feed) may improve the overall production efficiency of water re-use systems (Shoemaker et al. 2006). Removal of tilapia showing overt signs of disease (i.e., difficulty in consuming feed) may improve the overall production efficiency of water re-use systems (Shoemaker et al. 2006). Removal of dead/moribund fish will reduce disease transmission (Shoemaker et al. 2000). Removal of dead/moribund fish will reduce disease transmission (Shoemaker et al. 2000). Vaccination can significantly reduce mortality and is a viable strategy to prevent streptococcal disease (Klesius et al. 2000). Vaccination can significantly reduce mortality and is a viable strategy to prevent streptococcal disease (Klesius et al. 2000).

24. References Klesius, P.H., C.A. Shoemaker, J.J. Evans. 2000. Efficacy of single and combined S. iniae isolate vaccine administered by intraperitoneal or intramuscular routes in tilapia (O. niloticus). Aquaculture 188:237-246. Klesius, P.H., C.A. Shoemaker, J.J. Evans. 2000. Efficacy of single and combined S. iniae isolate vaccine administered by intraperitoneal or intramuscular routes in tilapia (O. niloticus). Aquaculture 188:237-246. Shoemaker, C.A., C. Lim, M. Yildirim-Aksoy, T.L. Welker, P.H. Klesius. 2006. Growth response and acquired resistance of Nile tilapia, O. niloticus (L.), that survived S. iniae infection. Aquaculture Research 37:1238-1245. Shoemaker, C.A., C. Lim, M. Yildirim-Aksoy, T.L. Welker, P.H. Klesius. 2006. Growth response and acquired resistance of Nile tilapia, O. niloticus (L.), that survived S. iniae infection. Aquaculture Research 37:1238-1245. Whittington R., C. Lim, P.H. Klesius. 2005. Effect of dietary beta-glucan levels on the growth response and efficacy of S. iniae vaccine in Nile tilapia, O. niloticus. Aquaculture 248:217-225. Whittington R., C. Lim, P.H. Klesius. 2005. Effect of dietary beta-glucan levels on the growth response and efficacy of S. iniae vaccine in Nile tilapia, O. niloticus. Aquaculture 248:217-225.