Growth Response and Acquired Resistance of Nile Tilapia Oreochromis niloticus
Following Infection or Vaccination with Streptococcus iniae
Craig A. Shoemaker*, Chhorn Lim, Mediha Yildirim-Aksoy, Thomas L. Welker,
Phillip H. Klesius and Joyce J. Evans
United States Department of Agriculture-Agricultural Research Service, Aquatic Animal
Health Research Unit, P.O. Box 0952, Auburn, Alabama, 36831, USA
Email: cshoemaker@ars.usda.gov
Key words: Streptococcus iniae, tilapia, growth performance, acquired resistance
Abstract
Growth performance and acquired resistance of Nile tilapia, Oreochromis
niloticus (L.) that survived Streptococcus iniae infection was determined. Tilapia
were challenged with three doses of S. iniae (8.8 x 103, 8.8 x 104 and 8.8 x 105 CFU
fish-1 for low, medium and high challenges, respectively). Groups of non-injected
and tryptic soy broth-injected fish were maintained as controls. Significantly (P <
0.05) higher mortality (45.0 %) occurred in the high challenge treatment than in the
low challenge treatment group (29.6 %). The medium challenge group had
mortality (36.3 %) that did not differ significantly from the high or low treatment.
Few fish died in the non-injected and broth-injected treatments (3.4 and 0.8 %,
respectively). The tilapia that survived S. iniae infection and used to assess growth
performance were selected from survivors without gross clinical signs of disease.
These fish were randomly stocked at a rate of 30 fish into each 57-L aquarium in
triplicate and fed to apparent satiation twice daily for 8 weeks. No significant
differences were detected in weight gain, feed intake, feed efficiency ratio or survival
between S. iniae-survived tilapia and the control treatments following the 8-week
growth performance trial. After the 8-week feeding study, tilapia were challenged
with 1 x 106 CFU fish-1 of S. iniae to assess acquired immunity. Mean cumulative
mortality was significantly higher (P < 0.05) in the control treatments (41.7 % for
the non-injected and 43.3 % for the broth-injected fish) than in the low, medium
and high challenge treatments (7.4, 3.3 and 8.3 %, respectively). Serum protein was
significantly (P < 0.05) elevated in S. iniae-survived tilapia that were subsequently
challenged when compared to controls challenged for the first time. Agglutinating
antibody titer was significantly higher in the fish in the medium and high challenge
treatments, compared to the control fish challenged for the first time. Tilapia
surviving S. iniae challenge without showing overt disease signs performed as well
as non-infected tilapia and gained acquired resistance to homologous S. iniae
challenge.
Streptococcus iniae is an important Gram-positive bacterial pathogen of tilapia
and other cultured fish species world wide (Eldar et al. 1997; Perera et al. 1997; Bowser
et al. 1998; Zlotkin et al. 1998; Shoemaker et al. 2001; Colorni et al. 2002; Nguyen et al.
2002). Tilapia producers suggest fish infected with S. iniae do not perform well and the
poor performance results in lost profits. Some suggestion has also been made that
decreased growth in tilapia occurred following intraperitoneal (i.p. injection) vaccination;
however, little work has been conducted to address this potential problem. Immuneinduced growth suppression has been suggested (Klasing et al. 1987) in other animal
species. Cook (1999) estimated more than $500 million U.S. is lost due to stimulation of
the immune system either via vaccination or through exposure to natural microbes in
terrestrial animals that result in decreased performance. The presence of overt disease in
a population nullifies the lost performance following vaccination (Chamberlee et al.
1992). Recent work in channel catfish (Ictalurus punctatus) administered a modified live
Edwardsiella ictaluri vaccine has shown an improved profit to farmers by as much as
$700/ha over non-vaccinated catfish due to improved growth of the vaccinated catfish
(Intervet, Inc., Technical Bulletin AQ-ESC-PKA 12/04, Millsboro, DE). Edwardsiella
ictaluri is known to be present in the aquatic environment and has been reported to be a
problem for 70% of commercial catfish operations (Wagner et al. 2002).
Most growth performance studies to date in fish have involved diet manipulation
and/or prebiotic or probiotic supplementation with subsequent challenge of fish to assess
disease resistance (Whittington et al. 2003; Peres et al. 2004; Li and Gatlin 2004). Mixed
results have been obtained with regard to the addition of nutrients and/or probiotics with
respect to increasing growth and/or resistance of tilapia to streptococcal disease. LaraFlores et al. (2003) reported that the growth response of Nile tilapia (Oreochromis
niloticus) was improved following feeding of two bacterial (S. faecium and Lactobacillus
acidophilus) and one yeast (Saccharomyces cerivisiae) probiotic for 9 weeks. The fish
in their study (Lara-Flores et al. 2003) were not challenged following feeding. In
contrast, Shelby et al. (In Press) found that supplementation of tilapia fry diets with
probiotics (3 bacteria and one yeast) did not improve growth performance or disease
resistance in tilapia fry weighing 12-29 mg. These authors did show that the probiotic
bacteria persisted for at least 48 h in the digestive tract after feeding. Limited published
information is available on the growth response of tilapia following infection or
vaccination with S. iniae. This manuscript will describe recent studies conducted by our
group on growth performance and acquired resistance of Nile tilapia following infection
or vaccination with S. iniae.
Growth Response Following Infection
Tilapia were used that had survived experimental infection with S. iniae at three
challenge levels: low (8.8 X 103 CFU/fish), medium (8.8 x 104 CFU/fish) and high (8.8 x
105 CFU/fish). Groups of non-injected and tryptic soy broth-injected fish were
maintained as controls. Significantly (P < 0.05) higher mortality (45.0 %) occurred in the
high challenge treatment than in the low challenge treatment group (29.6 %). The
medium challenge group had mortality (36.3 %) that did not differ significantly from the
high or low treatment. Few fish died in the non-injected and broth-injected treatments
(3.4 and 0.8 %, respectively). The surviving fish were selected based on the absence of
gross clinical signs of streptococcal disease (i.e., spiral swimming, cloudy eyes, or body
curvature; Klesius et al. 2000). The initial stocking density of the surviving tilapia
(30/tank in triplicate) was ~ 6.2 g/L. These fish were fed a commercial diet (32 %
protein) twice daily to apparent satiation for 8 weeks and growth performance was
assessed (Table 1). No differences were recorded in weight gain, feed intake, feed
efficiency ratio or survival following the 8 week growth performance trial (Table 1).
Table 1 Average weight gain, dry matter feed intake, feed efficiency ratio (FER) and
survival of Streptococcus iniae-survived tilapia following the 8-week feeding study1
(Shoemaker et al., In Press).
Treatment
Weight gain Feed intake
FER2 Survival3
-1
(g)
(g DM fish )
(%)
Low challenge
27.0
49.1
0.55 76.7
Medium challenge
27.9
47.8
0.58 81.1
High challenge
26.3
44.9
0.58 84.7
Control (non-injected)
27.9
46.0
0.60 88.9
Control (broth-injected)
27.8
45.2
0.61 95.6
Pooled SEM
1.6
1.8
0.02
4.7
1
Values are means of three replicates per treatment. No significant differences were
determined at P > 0.05.
2
FER = weight gain (g) / dry feed fed (g).
3
Streptococcus iniae was not isolated from dead fish during the growth performance
period.
Growth response following vaccination
Whittington et al. (2005) reported on the growth response of injection-vaccinated
(primary and booster immunization) and non-vaccinated Nile tilapia (~10.5 g/L initial
density) following feeding a commercial diet (32 %) supplemented with beta-glucan at 0,
50, 100 or 200 mg/kg for 14 weeks. Their experiment (Whittington et al. 2005)
demonstrated after 10 weeks of feeding that vaccination, beta-glucan supplementation or
a combination of vaccination and immunostimulation had no effect on weight gain or
survival. Interestingly, beta-glucan supplementation above 50 mg/kg diet appeared to
negatively influence the feed efficiency ratio (FER). The FER of the vaccinated and nonvaccinated fish ranged from 0.62-0.71.
Acquired resistance following infection and/or vaccination
The S. iniae-survived tilapia exhibited agglutinating antibody titers prior to and
following the 8-week performance period (Table 2). Acquired resistance to homologous
S. iniae challenge was also demonstrated in the S. iniae-survived tilapia challenged with
1 X 106 CFU/fish following the 8-week growth performance period (Table 2). Mean
cumulative mortality was significantly higher (P < 0.05) in the control groups (i.e.,
challenged for the first time) than in the S. iniae-survived groups. Initial level of
challenge (low, medium or high) did not influence mortality patterns following the 8week growth performance period.
Table 2 Agglutinating antibody (Ab) titer of S. iniae-survived tilapia (prior to and
following the 8-week feeding period) and mean cumulative mortality at 21 days postchallenge with S. iniae of S. iniae-survived tilapia and control fish infected for the first
time (Shoemaker et al., In Press).
Initial Treatment
Agglutinating Ab1
Agglutinating Ab
Cumulative mortality2
titer (log10) (prior to titer (log10) (post 8 (%)
8 week feeding)
week feeding)
b
Low challenge
1.04
0.17a,b
7.4a
b
a
Medium challenge 1.08
0.40
3.3a
High challenge
1.78a
0.37a
8.3a
c
b
Control (non0.00
0.00
41.7b
injected)
Control (broth0.00c
0.00b
43.3b
injected)
Pooled SEM
0.17
0.1
6.0
1
Agglutination antibody (Ab) titer as determined by microagglutination method
according to Klesius et al. (2000).
2
Percent cumulative mortality was calculated following a 21-day monitoring period. All
groups were challenged with 1 X 106 CFU fish-1 by intraperitoneal injection. Different
superscript letters indicate significant differences at P < 0.001.
Whittington et al. (2005) also demonstrated significantly lower (P < 0.05)
mortality (0.0 -7.9 %) in vaccinated tilapia compared to mortality in non-vaccinated
tilapia (48.3-72.2 %) regardless of dietary supplementation with beta-glucan. Vaccinated
fish in their study exhibited significantly higher (P < 0.05) agglutinating antibody titers
when measured 21 days post-booster immunization. Mean agglutinating titer of
vaccinated fish was 1.53 (log10) compared to 0.21 for the non-vaccinated fish. The
antibody titers determined from vaccinated fish were similar to those reported from the S.
iniae-survived fish (Table 2).
Management implications
Previous studies have demonstrated the importance of antibody in the acquired
immune response of tilapia to S. iniae either indirectly by measuring antibody in
vaccinated and protected fish (Eldar et al. 1997; Klesius et al. 1999, 2000a) or directly by
passive immunization (Shelby et al. 2002). Results of the current studies suggest that
vaccinated tilapia or tilapia that survive S. iniae infection seroconvert and produce a
protective antibody response with little or no effect on the growth performance of 10-20 g
tilapia. Densities of fish used in these studies were low (~6-20 g/L) compared to
commercial recirculation aquaculture operations (30-290 g/L). Removal of tilapia
showing overt signs of infection (i.e., difficulty in consuming feed) may improve overall
production efficiency of water re-use systems. Removal of dead /moribund fish will also
reduce disease transmission as previously suggested (Shoemaker et al. 2000).
Vaccinations, albeit labor intensive and costly at present, can significantly reduce
mortality and should be considered as a viable strategy to prevent streptococcal infection
(Klesius et al. 2000b). Relative percent survival values as reported by Whittington et al.
(2005) were 91-100 in vaccinated tilapia. Studies are presently underway to develop cost
effective strategies [e.g. oral immunization (Shoemaker et al. 2006) and immersion
immunization (Klesius et al. 2006, 7th ISTA)] to mass immunize young tilapia prior to
introduction into grow out systems.
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