Growth Response and Acquired Growth Response and Acquired Resistance of Nile Tilapia Following Resistance of Nile Tilapia Following

Infection or Vaccination with Infection or Vaccination with Streptococcus iniaeStreptococcus iniae

Craig ShoemakerCraig Shoemaker, Chhorn Lim, Mediha Yildirim-, Chhorn Lim, Mediha Yildirim-Aksoy,Aksoy,

Tom Welker, Phillip Klesius and Joyce EvansTom Welker, Phillip Klesius and Joyce Evans

Aquatic Animal Health Research Laboratory, Aquatic Animal Health Research Laboratory, USDA-ARS, Auburn, AL, USAUSDA-ARS, Auburn, AL, USA

IntroductionIntroduction

•Streptococcus iniaeStreptococcus iniae is a disease is a disease of worldwide importance.of worldwide importance.

•22 species of fish affected - 22 species of fish affected - cultured, wild and marine.cultured, wild and marine.

•US losses are estimated at $10 US losses are estimated at $10 million, annually.million, annually.

IntroductionIntroduction

• Most growth performance studies to Most growth performance studies to date involve diet manipulation and/or date involve diet manipulation and/or probiotic or prebiotic probiotic or prebiotic supplementation.supplementation.

• Disease resistance assessed by Disease resistance assessed by challenge. challenge.

IntroductionIntroduction

• Little or no information is available on the Little or no information is available on the growth response of tilapia following growth response of tilapia following infection and/or vaccination with infection and/or vaccination with S. iniae.S. iniae.

• Producers suggest that infected fish do not Producers suggest that infected fish do not perform well and the poor performance perform well and the poor performance results in lost profits.results in lost profits.

• Cook (1999) estimated at least $500 million Cook (1999) estimated at least $500 million annually lost due to immune-induced annually lost due to immune-induced growth suppression in terrestrial animals.growth suppression in terrestrial animals.

IntroductionIntroduction

• Reports suggest improved growth of Reports suggest improved growth of vaccinated eels (Esteve-Gassent et al. vaccinated eels (Esteve-Gassent et al. 2004).2004).

• Administration of a modified live Administration of a modified live Edwardsiella ictaluriEdwardsiella ictaluri vaccine has vaccine has resulted in increased performance resulted in increased performance ($1700/acre over non-vaccinated ($1700/acre over non-vaccinated fish).fish).

ObjectivesObjectives

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

Study I - Materials and MethodsStudy I - Materials and Methods

1200 Nile tilapia, 1200 Nile tilapia, Oreochromis niloticusOreochromis niloticus (11g) were stocked (11g) were stocked at 120 fish/tank in ten 208-L aquarium. Duplicate tanks at 120 fish/tank in ten 208-L aquarium. Duplicate tanks were used to generate survivors at day 14.were used to generate survivors at day 14.

TreatmentTreatment Challenge Challenge dose dose (CFU/fish)(CFU/fish)

Cumulative Cumulative Mortality (%)Mortality (%)

Low challengeLow challenge 8.8 X 108.8 X 1033 29.629.6aa

Medium challengeMedium challenge 8.8 X 108.8 X 1044 36.336.3a,ba,b

High challengeHigh challenge 8.8 X 108.8 X 1055 45.045.0bb

ControlControl Non-injectedNon-injected 3.43.4cc

ControlControl Broth-Broth-injectedinjected

0.80.8cc

Pooled SEMPooled SEM 2.72.7

Study I - Materials and Study I - Materials and MethodsMethods

• Survivors without clinical signs of Survivors without clinical signs of disease were stocked at 30 fish per disease were stocked at 30 fish per 57-L aquarium (triplicate).57-L aquarium (triplicate).

• A 32 % crude protein commercial diet A 32 % crude protein commercial diet was fed to was fed to S. iniaeS. iniae-recovered tilapia -recovered tilapia twice daily to apparent satiation for 8-twice daily to apparent satiation for 8-weeks. weeks.

Study I - Materials and Study I - Materials and MethodsMethods

• Weight gain (g), feed intake (g dry Weight gain (g), feed intake (g dry matter/fish), feed efficiency ratio [FER = matter/fish), feed efficiency ratio [FER = weight gain (g) / dry feed fed (g)] and weight gain (g) / dry feed fed (g)] and survival were recorded after the 8 week survival were recorded after the 8 week period.period.

• Agglutinating antibody titer was Agglutinating antibody titer was determined by the method of Klesius et determined by the method of Klesius et al. (2000).al. (2000).

Study I - Materials and Study I - Materials and MethodsMethods

• Following the 8 week performance Following the 8 week performance period fish were challenged with 1 X period fish were challenged with 1 X 101066CFU/fish by i.p. injection.CFU/fish by i.p. injection.

• Mortality was monitored for 21 days Mortality was monitored for 21 days and the % mortality calculated.and the % mortality calculated.

Study II - Materials and Study II - Materials and MethodsMethods

• A 2 X 4 factorial experiment was conducted – triplicate A 2 X 4 factorial experiment was conducted – triplicate tanks of 25 fish each.tanks of 25 fish each.

• Group A – Non-vaccinated with 0 mg/kg Group A – Non-vaccinated with 0 mg/kg ββ-glucan.-glucan.

• Group B – Vaccinated with 0 mg/kg diet Group B – Vaccinated with 0 mg/kg diet ββ-glucan.-glucan. • Group C – Non-vaccinated with 50 mg/kg diet Group C – Non-vaccinated with 50 mg/kg diet ββ-glucan.-glucan.• Group D – Vaccinated with 50 mg/kg diet Group D – Vaccinated with 50 mg/kg diet ββ-glucan.-glucan.• Group E - Non-vaccinated with 100 mg/kg diet Group E - Non-vaccinated with 100 mg/kg diet ββ-glucan.-glucan.• Group F - Vaccinated with 100 mg/kg diet Group F - Vaccinated with 100 mg/kg diet ββ-glucan.-glucan.• Group G - Non-vaccinated with 200 mg/kg diet Group G - Non-vaccinated with 200 mg/kg diet ββ-glucan.-glucan.• Group H - Vaccinated with 200 mg/kg diet Group H - Vaccinated with 200 mg/kg diet ββ-glucan.-glucan.

Study II - Materials and Study II - Materials and MethodsMethods

• Fish were vaccinated by IP injection of Fish were vaccinated by IP injection of 100 µl killed 100 µl killed S. iniaeS. iniae vaccine (Klesius et vaccine (Klesius et al. 2000) and booster vaccinated at day al. 2000) and booster vaccinated at day 21.21.

• Weight gain (g), feed intake (g dry Weight gain (g), feed intake (g dry matter/fish), feed efficiency ratio [FER matter/fish), feed efficiency ratio [FER = weight gain (g) / dry feed fed (g)] = weight gain (g) / dry feed fed (g)] and survival were recorded after a 10 and survival were recorded after a 10 week period.week period.

Study II - Materials and Study II - Materials and MethodsMethods

• Following the 10 week performance Following the 10 week performance period fish were challenged with 1 X period fish were challenged with 1 X 101077CFU/fish by i.p. injection.CFU/fish by i.p. injection.

• Mortality was monitored for 14 days Mortality was monitored for 14 days and the relative percent survival and the relative percent survival (Amend 1981) calculated.(Amend 1981) calculated.

Results -Results -

Study I - Average weight gain, dry matter intake, Study I - Average weight gain, dry matter intake, feed efficiency ratio (FER) and survival of feed efficiency ratio (FER) and survival of S. S. iniaeiniae-recovered tilapia fed for 8-weeks -recovered tilapia fed for 8-weeks (Shoemaker et al. 2006)(Shoemaker et al. 2006)TreatmentTreatment Weight Weight

gain (g)gain (g)Feed Feed intake intake

(g (g DM/fish)DM/fish)

FERFER SurvivSurvivalal

Low challengeLow challenge 27.027.0 49.149.1 0.550.55 76.776.7

Medium challengeMedium challenge 27.927.9 47.847.8 0.580.58 81.181.1

High challengeHigh challenge 26.326.3 44.944.9 0.580.58 84.784.7

ControlControl

(non-injected)(non-injected)27.927.9 46.046.0 0.600.60 88.988.9

ControlControl

(broth-injected)(broth-injected)27.827.8 45.245.2 0.610.61 95.695.6

Pooled SEMPooled SEM 1.61.6 1.81.8 0.020.02 4.74.7

Study I - Agglutinating antibody titer of Study I - Agglutinating antibody titer of S. iniae-S. iniae-survived tilapia and mean cumulative mortality survived tilapia and mean cumulative mortality at 21 days post challenge with at 21 days post challenge with S. iniaeS. iniae of of S. S. iniaeiniae- survived tilapia (Shoemaker et al. 2006)- survived tilapia (Shoemaker et al. 2006)

TreatmentTreatment Ab-titerAb-titer11

(log(log1010))

Ab-titerAb-titer22

(log(log1010))

Mortality (%)Mortality (%)33

Low challengeLow challenge 1.01.0aa 0.20.2a,ba,b 7.47.4aa

Medium challengeMedium challenge 1.11.1aa 0.40.4aa 3.33.3aa

High challengeHigh challenge 1.81.8bb 0.40.4aa 8.38.3aa

ControlControl

(non-injected)(non-injected)

0.00.0cc 0.00.0bb 41.741.7bb

ControlControl

(broth-injected)(broth-injected)

0.00.0cc 0.00.0bb 43.343.3bb

Pooled SEMPooled SEM 0.20.2 0.20.2 6.06.011Prior to 8-week feeding period. Prior to 8-week feeding period. 22Following 8-week feeding period. Following 8-week feeding period. 33P < P < 0.001.0.001.

Study II - Mean weight gain, feed efficiency ratio Study II - Mean weight gain, feed efficiency ratio (FER) and survival of vaccinated and control (FER) and survival of vaccinated and control tilapia fed diets containing tilapia fed diets containing ββ-glucan for 10--glucan for 10-weeks (Whittington et al. 2005)weeks (Whittington et al. 2005)TreatmentTreatment ΒΒ-glucan-glucan

(mg/kg)(mg/kg)Weight Weight

gain (g)gain (g)FERFER Survival Survival

(%)(%)

VaccinatedVaccinated 00 52.152.1 0.670.67 98.398.3

5050 55.555.5 0.710.71 93.393.3

100100 58.258.2 0.630.63 100.0100.0

200200 55.155.1 0.640.64 97.597.5

ControlControl 00 55.555.5 0.670.67 96.796.7

5050 54.754.7 0.670.67 95.095.0

100100 59.059.0 0.630.63 96.796.7

200200 52.052.0 0.620.62 100.0100.0

Pooled SEMPooled SEM 2.12.1 0.050.05 1.21.2

Study II - Agglutinating antibody titer of Study II - Agglutinating antibody titer of vaccinated and non-vaccinated tilapia and vaccinated and non-vaccinated tilapia and relative percent survival following challenge relative percent survival following challenge (Whittington et al. 2005)(Whittington et al. 2005)

11 21 day post boost. 21 day post boost. 22RPS=relative percent survival.RPS=relative percent survival.

TreatmentTreatment ΒΒ-glucan -glucan (mg/kg)(mg/kg)

Ab-titerAb-titer1 1 (log(log1010)) RPSRPS22

VaccinatedVaccinated 00 1.51.5 9595

5050 1.81.8 100100

100100 1.71.7 100100

200200 1.11.1 9191

ControlControl 00 0.10.1 ----

5050 0.00.0 0.20.2

100100 0.00.0 3333

200200 0.70.7 1212Pooled SEMPooled SEM 0.050.05 2.82.8

DiscussionDiscussion

• Studies examining growth performance Studies examining growth performance in fish have historically been conducted in fish have historically been conducted using apparently healthy animals (Lim using apparently healthy animals (Lim and Webster 2001).and Webster 2001).

• We examined the performance of fish We examined the performance of fish that recovered (survived) from that recovered (survived) from S. iniaeS. iniae infection or following vaccination. infection or following vaccination.

DiscussionDiscussion

• Following the 8 week feeding period, Following the 8 week feeding period, no differences were observed in no differences were observed in growth, feed intake and feed efficiency growth, feed intake and feed efficiency ratio of ratio of S. iniaeS. iniae-recovered tilapia.-recovered tilapia.

• Following the 10 week feeding period, Following the 10 week feeding period, no differences were observed in no differences were observed in growth, feed efficiency ratio and growth, feed efficiency ratio and survival of vaccinated tilapia.survival of vaccinated tilapia.

DiscussionDiscussion

• The study demonstrated acquired resistance to The study demonstrated acquired resistance to S. iniaeS. iniae in the in the S. iniaeS. iniae–recovered and –recovered and vaccinated tilapia.vaccinated tilapia.

• Passive immunization and vaccine studies have Passive immunization and vaccine studies have demonstrated the importance of antibody in demonstrated the importance of antibody in S. S. iniaeiniae immunity (Klesius et al. 2000; Shelby et immunity (Klesius et al. 2000; Shelby et al. 2002; Eldar et al. 1997; Whittington et al. al. 2002; Eldar et al. 1997; Whittington et al. 2005).2005).

• Agglutinating antibody titer was demonstrated Agglutinating antibody titer was demonstrated in in S. iniaeS. iniae recovered and vaccinated tilapia. recovered and vaccinated tilapia.

ConclusionConclusion

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

Management implicationsManagement implications

• Removal of tilapia showing overt signs of Removal of tilapia showing overt signs of disease (i.e., difficulty in consuming feed) may disease (i.e., difficulty in consuming feed) may improve the overall production efficiency of improve the overall production efficiency of water re-use systems (Shoemaker et al. 2006).water re-use systems (Shoemaker et al. 2006).

• Removal of dead/moribund fish will reduce Removal of dead/moribund fish will reduce disease transmission (Shoemaker et al. 2000).disease transmission (Shoemaker et al. 2000).

• Vaccination can significantly reduce mortality Vaccination can significantly reduce mortality and is a viable strategy to prevent streptococcal and is a viable strategy to prevent streptococcal disease (Klesius et al. 2000).disease (Klesius et al. 2000).

ReferencesReferences• Klesius, P.H., C.A. Shoemaker, J.J. Evans. 2000. Efficacy Klesius, P.H., C.A. Shoemaker, J.J. Evans. 2000. Efficacy

of single and combined of single and combined S. iniaeS. iniae isolate vaccine isolate vaccine administered by intraperitoneal or intramuscular routes administered by intraperitoneal or intramuscular routes in tilapia (in tilapia (O. niloticusO. niloticus). Aquaculture 188:237-246.). Aquaculture 188:237-246.

• Shoemaker, C.A., C. Lim, M. Yildirim-Aksoy, T.L. Welker, Shoemaker, C.A., C. Lim, M. Yildirim-Aksoy, T.L. Welker, P.H. Klesius. 2006. Growth response and acquired P.H. Klesius. 2006. Growth response and acquired resistance of Nile tilapia, resistance of Nile tilapia, O. niloticusO. niloticus (L.), that survived (L.), that survived S. iniaeS. iniae infection. Aquaculture Research 37:1238-1245. infection. Aquaculture Research 37:1238-1245.

• Whittington R., C. Lim, P.H. Klesius. 2005. Effect of Whittington R., C. Lim, P.H. Klesius. 2005. Effect of dietary beta-glucan levels on the growth response and dietary beta-glucan levels on the growth response and efficacy of efficacy of S. iniaeS. iniae vaccine in Nile tilapia, vaccine in Nile tilapia, O. niloticusO. niloticus. . Aquaculture 248:217-225.Aquaculture 248:217-225.