Streptococcus Iniae Vaccine in Sea Bass
The Safety and Efficacy of a Streptococcus iniae Vaccine in Asian Sea Bass
Published: January 12, 2011
By: Nantarika Chansue, Jirasak Tangtongpiros and John S. Clark
A European system of immersion followed by injection vaccination is indicated as the strategy of choice in the control of this pathogen.
Asian sea bass (Lates calcarifer), also known as barramundi, is becoming increasingly popular as a food fish in export markets such as the US and EU. As demand increases, more and more farms are appearing in the South East Asian region to supply these growing markets. Concomitantly with the rise in production is increased disease risk. In hatcheries, sea bass larvae are prone to Vibrio infections and to Viral Nervous Necrosis (VNN); in the nursery culture phase, Vibrio, S. iniae and Iridovirus are problematic. In both these phases of culture, protozoan ecto-parasites such as scuticociliates are prevalent. Risk is greatest, however, during the grow-out phase of culture. Ecto-parasites such as capsalid flukes and sea lice are prevalent and conventional bathing treatments are ineffectual. There is a need, currently being addressed, for in-feed treatments to control and eradicate these parasites.
Such parasites, feeding on skin, mucous and blood of the host, leave many portals of entry for pathogens such as S. iniae, a commercially important pathogen in sea bass culture. Antibiotic treatments for this pathogen are limited in use due to the ability of the pathogen to "hide" within the body. Vaccination would appear to be the only viable route to protect cultured fish from such a pathogen. The aim of the following research work was to established safety and efficacy data for immersion and injection forms of a S. iniae vaccine for application to Asian sea bass culture in Thailand and the Asian region.
Experimental details
In this experiment, 1,000 juvenile sea bass approximately 5-7 cm body length were used. These fish were purchased from a private farm. Fish were acclimated in 2,000 litre concrete tanks for 14 days prior to beginning of the experiment. Three replicate tanks of 20 fish each were used in all immunization and infection experiments. Aeration was efficiently supplied by air stones. Fifty percent water was changed every 3 days. The fish were fed at 5% body weight daily. Feed used was commercial feed (INVE, Thailand).
To verify the S. iniae free status of the fish two fish were sacrificed and samples were obtained for bacterial culture by inoculation loop from kidney and brain. The samples were streaked directly on TSA (Tryptic Soy Agar) and sheep blood agar and then were incubated at 30°C for 24 hours. PCR used for final confirmation of pathogen presence or absence.
The temperature and water quality were monitored daily. Water pH, hardness, ammonia, nitrite and alkalinity were determined using a commercial test kit (AQUA-VBC, Thailand). In all trials, the temperature was stable at 27°C, pH was 8.5-9, hardness was 1,200-1,400 ppm, ammonia was 0.01 ppm and nitrite was 0.2-0.25 ppm, salinity was 34 ppt, and alkalinity was 90-110 ppm.
Healthy juvenile sea bass from a private farm in Rayong
Rearing tanks of 50 litres with individual water filter pumps
Bacteria
S. iniae isolated from moribund adult sea bass was provided by VMARC (Chulalongkorn University, Thailand). The isolate was plated onto supplemented TSA and harvested after 24 hours of growth. Bacteria were presumptively identified as S. iniae by their transparent colonies and coccal shaped cell morphology, Gram-positive stain, negative catalase production and bacterial species were confirmed by using API 20 (BioMerieux, Madrid, Spain) and the API profiles compared with the API database (Apilab Plus, version 3.3.3.; BioMerieux).The pure isolate was kept in stock media at room temperature. PCR was used for final confirmation.
The bacterial isolate used for virulence assays was inoculated into 420 juvenile sea bass via intra-peritonial injection (IP). The culture was adjusted to an optical density of 540 nm using a spectrophotometer to give S. iniae doses ranging in concentration from 103 to 108 CFU/ml. Each concentration in a volume of 0.01 ml was used to establish pathogen doses that would kill 50% (50% lethal dose; LD50) of injected fish within 72 hours. Each dose had 3 replicates with 20 fish in each replicate.
Control groups were injected with sterile 0.9 % (v/v) normal saline. After injection of the pathogen, fish were observed daily. Mortalities were recorded on a daily basis, and symptoms associated with the ensuing pathogen attack were recorded and photographed. Samples of liver, kidney, gill, digestive tract and brain were preserved for histological examination, and a photographic record was maintained.
Figure 1. Determination of LD50 for S.iniae in juvenile sea bass.
The mean lethal dose (LD 50) was calculated by the Reed and Muench (1938) procedure for establishing optimal pathogen dose. The dose response curves for this particular isolate of S. iniae clearly indicate 1x105 CFU/ml to be used during the efficacy stage of the trial (Figures 1 and 3).
Vaccination
The two types of vaccine used were injection and immersion vaccine forms.
Injection
Three hundred juveniles were divided into 5 groups as follows: 0.01 ml vaccine injection (T2), large volume 0.02 ml vaccine injection (T3) and 3 groups of non-immunized controls (injected with sterile normal saline at a volume of 0.02 ml (CN), no injection (C) and non-vaccinated, injected with LD50 dose of S. iniae).
Three replicates were conducted per group of 20 fish each. Fish were not sedated to minimise the effects of over handling. The vaccine was administered intra-peritoneally (IP) to each fish. Fish were then returned to their designated tanks.
Immersion
Sixty juveniles were use in this trial at three replicates of 20 fish each. One litre of vaccine was maintained at 4°C. The vaccine was shaken thoroughly then diluted with 9 litres of clean, well-oxygenated fresh water and mixed thoroughly. The vaccine bath was well oxygenated during the vaccination of the fish to minimize stress. Fish are placed in a fine mesh, soft net then were immersed for a period of 1 minute in the vaccine bath. After vaccination (T1), the fish were returned to their respective tanks.
Immunized and control fish were held for 21 days before challenge. All groups were monitored for mortality on a daily basis. In this manner, vaccine safety could be established.
Challenge tests
At 21 days post-vaccination, the positive control and all test groups received a 0.01 ml aliquot of S. iniae at 50% lethal dose concentration or 1x105 cfu/ml administered IP (determined by previous study). The non-immunized controls were injected with normal saline 0.01ml (CN). The fish were monitored for mortality for 72 hours post-challenge.
Fish mortalities were observed and recorded on a daily basis and symptoms of the ensuing pathogen attack were recorded and photographed. Tissue samples of liver, kidney, gill, digestive tract and brain were preserved for histological examination, again with a corresponding photographic record. The percentage mortality rates of each vaccine dose were calculated against the relevant controls. In this way, the efficacy of both vaccine doses could be established.
Dead fish were removed immediately when observed and upon post-mortem examination, specimens were obtained aseptically from kidney, liver and brain sites for examination of S. iniae infection.
Specimens were cultured directly onto TCBS at 30°C for 24 hours and biochemically identified by API 20 Strep. The mortality data for each group was analyzed by two-way analysis of variance using a SPSS program. Significant differences were determined at P<0.05.
Plate 1. Remarkable hepatic cell swelling and vacuolation with severe panlobular fatty degeneration with mutifocal melano-macrophages of the liver (x100)
Table 1. Mean % mortality of juvenile sea bass at day 21 after vaccination (safety study).
*Values are not significantly different at P<0.05.
S. iniae was not isolated from the randomly selected sea bass, indicating that fish held at the start of the experiment were clear of S. iniae infection.
In the safety study of vaccine determined by a 21 day period after vaccination, pre-challenge. Table 1 and Figure 1 showed no significant difference in the percentage mortality rate in all groups when compared to the control groups (P>0.05). There is an indication that the higher injection volume of 0.02ml was in fact more stressful for fish and this should be taken into consideration when planning a vaccination strategy.
Table 2. Mortality rate of juvenile sea bass after challenged with S. iniae (efficacy study).
In this efficacy test of vaccination, all vaccine treated groups had significantly lower mortality rates than the control group with normal saline injection (CN) and with the negative control (NC). The highest rate of protection against S. iniae was observed at 0.01 ml vaccinated fish (IS) which had significantly lower percentage mortality rates (4.91%) in other groups (P<0.05), excluding only the positive control group (C). The immersion group (T1) displayed 14.16% mortality rate, which was regarded as excellent for an immersion route vaccine. Table 2 and Figures 2- 4 summarise the results of safety, efficacy and RPS.
The dead fish from this challenge experiment were confirmed positive for S.iniae infection by re-culture and re-identification using API 20 Strep and PCR techniques.
Clinical signs observed in infected fish included symptoms such as stationary or quiescent behaviour at the bottom of the aquaria, loss of equilibrium, frequent sinking and rising, lethargy, slow acceptance or refusal of food. Dark skin colouration, exophthalmia and corneal opacity were noted in moribund fish. The majority of these fish died over the course of the next 72 hours after sampling.
Histopathological findings included vacuolar degeneration in the liver tissue. Numerous melano-macrophages infiltrated in the kidney, spleen and liver (Plate 1). The brain showed meningio-encephalitis with thick outer layers of tissue and leucocyte infiltration (Plate 2).
Conclusion
From the vaccine safety study, the vaccine, regardless of route of administration, showed high safety to juvenile experimental fish. There was no significant difference in mortality rate of experimental groups compared with the control groups.
Plate 2. Meninges showed swollen outer layer tissue and leucocyte infiltration (x400).
The results from the efficacy study showed vaccinations have the potential to reduce fish mortality by S. iniae, and that intraperitoneal administration via 0.01 ml injection is the optimal route.
The injection of 0.01 ml (T2) conferred significantly lower mortality rate then other treatments. It should be noted that, immersion (T1) also induced higher survival rates, approaching 90%. Even if T1 groups had significantly higher mortality rates than T2, this is not viewed as negative, in that in European vaccination strategies an immersion vaccine primer is usually followed by an injectable booster dose. This may be applicable for vaccine used in commercial farms (Dunn et al., 1990) in Asian rearing conditions. Commercially, fish have been vaccinated by immersion at 1g size, then injection vaccinated at 5g or 20g depending on farm preference. Injection volume in such cases is 0.05ml and 0.1ml respectively.
This experiment proved that this vaccine was effective in reducing mortality rate of juvenile sea bass infected with S. iniae and was safe to use on the commercial production of juvenile sea bass.
Figure 2. The 21 day safety of control (not injected), control (saline injected, immersion vaccinated (test 1) and vaccine injected (tests 2 and 3) in juvenile sea bass.
Figure 3. The efficacy of control (not injected), control (saline injected), immersion vaccinated (test 1) and vaccine injected (tests 2) in juvenile sea bass.
Figure 4. RPS of control (not injected), control (saline injected), immersion vaccinated (test 1) and vaccine injected (tests 2) in juvenile sea bass.
Reference
Dunn, J., Polk, A., Scarrett, J., Olivier, G., Lall S. and Goosen. F.A. 1990. Vaccine in aquaculture: the search for an efficient delivery system. Aquaculture 9: 23-32.
Asian sea bass (Lates calcarifer), also known as barramundi, is becoming increasingly popular as a food fish in export markets such as the US and EU. As demand increases, more and more farms are appearing in the South East Asian region to supply these growing markets. Concomitantly with the rise in production is increased disease risk. In hatcheries, sea bass larvae are prone to Vibrio infections and to Viral Nervous Necrosis (VNN); in the nursery culture phase, Vibrio, S. iniae and Iridovirus are problematic. In both these phases of culture, protozoan ecto-parasites such as scuticociliates are prevalent. Risk is greatest, however, during the grow-out phase of culture. Ecto-parasites such as capsalid flukes and sea lice are prevalent and conventional bathing treatments are ineffectual. There is a need, currently being addressed, for in-feed treatments to control and eradicate these parasites.
Such parasites, feeding on skin, mucous and blood of the host, leave many portals of entry for pathogens such as S. iniae, a commercially important pathogen in sea bass culture. Antibiotic treatments for this pathogen are limited in use due to the ability of the pathogen to "hide" within the body. Vaccination would appear to be the only viable route to protect cultured fish from such a pathogen. The aim of the following research work was to established safety and efficacy data for immersion and injection forms of a S. iniae vaccine for application to Asian sea bass culture in Thailand and the Asian region.
Experimental details
In this experiment, 1,000 juvenile sea bass approximately 5-7 cm body length were used. These fish were purchased from a private farm. Fish were acclimated in 2,000 litre concrete tanks for 14 days prior to beginning of the experiment. Three replicate tanks of 20 fish each were used in all immunization and infection experiments. Aeration was efficiently supplied by air stones. Fifty percent water was changed every 3 days. The fish were fed at 5% body weight daily. Feed used was commercial feed (INVE, Thailand).
To verify the S. iniae free status of the fish two fish were sacrificed and samples were obtained for bacterial culture by inoculation loop from kidney and brain. The samples were streaked directly on TSA (Tryptic Soy Agar) and sheep blood agar and then were incubated at 30°C for 24 hours. PCR used for final confirmation of pathogen presence or absence.
The temperature and water quality were monitored daily. Water pH, hardness, ammonia, nitrite and alkalinity were determined using a commercial test kit (AQUA-VBC, Thailand). In all trials, the temperature was stable at 27°C, pH was 8.5-9, hardness was 1,200-1,400 ppm, ammonia was 0.01 ppm and nitrite was 0.2-0.25 ppm, salinity was 34 ppt, and alkalinity was 90-110 ppm.
Healthy juvenile sea bass from a private farm in Rayong
Rearing tanks of 50 litres with individual water filter pumps
Bacteria
S. iniae isolated from moribund adult sea bass was provided by VMARC (Chulalongkorn University, Thailand). The isolate was plated onto supplemented TSA and harvested after 24 hours of growth. Bacteria were presumptively identified as S. iniae by their transparent colonies and coccal shaped cell morphology, Gram-positive stain, negative catalase production and bacterial species were confirmed by using API 20 (BioMerieux, Madrid, Spain) and the API profiles compared with the API database (Apilab Plus, version 3.3.3.; BioMerieux).The pure isolate was kept in stock media at room temperature. PCR was used for final confirmation.
The bacterial isolate used for virulence assays was inoculated into 420 juvenile sea bass via intra-peritonial injection (IP). The culture was adjusted to an optical density of 540 nm using a spectrophotometer to give S. iniae doses ranging in concentration from 103 to 108 CFU/ml. Each concentration in a volume of 0.01 ml was used to establish pathogen doses that would kill 50% (50% lethal dose; LD50) of injected fish within 72 hours. Each dose had 3 replicates with 20 fish in each replicate.
Control groups were injected with sterile 0.9 % (v/v) normal saline. After injection of the pathogen, fish were observed daily. Mortalities were recorded on a daily basis, and symptoms associated with the ensuing pathogen attack were recorded and photographed. Samples of liver, kidney, gill, digestive tract and brain were preserved for histological examination, and a photographic record was maintained.
Figure 1. Determination of LD50 for S.iniae in juvenile sea bass.
The mean lethal dose (LD 50) was calculated by the Reed and Muench (1938) procedure for establishing optimal pathogen dose. The dose response curves for this particular isolate of S. iniae clearly indicate 1x105 CFU/ml to be used during the efficacy stage of the trial (Figures 1 and 3).
Vaccination
The two types of vaccine used were injection and immersion vaccine forms.
Injection
Three hundred juveniles were divided into 5 groups as follows: 0.01 ml vaccine injection (T2), large volume 0.02 ml vaccine injection (T3) and 3 groups of non-immunized controls (injected with sterile normal saline at a volume of 0.02 ml (CN), no injection (C) and non-vaccinated, injected with LD50 dose of S. iniae).
Three replicates were conducted per group of 20 fish each. Fish were not sedated to minimise the effects of over handling. The vaccine was administered intra-peritoneally (IP) to each fish. Fish were then returned to their designated tanks.
Immersion
Sixty juveniles were use in this trial at three replicates of 20 fish each. One litre of vaccine was maintained at 4°C. The vaccine was shaken thoroughly then diluted with 9 litres of clean, well-oxygenated fresh water and mixed thoroughly. The vaccine bath was well oxygenated during the vaccination of the fish to minimize stress. Fish are placed in a fine mesh, soft net then were immersed for a period of 1 minute in the vaccine bath. After vaccination (T1), the fish were returned to their respective tanks.
Immunized and control fish were held for 21 days before challenge. All groups were monitored for mortality on a daily basis. In this manner, vaccine safety could be established.
Challenge tests
At 21 days post-vaccination, the positive control and all test groups received a 0.01 ml aliquot of S. iniae at 50% lethal dose concentration or 1x105 cfu/ml administered IP (determined by previous study). The non-immunized controls were injected with normal saline 0.01ml (CN). The fish were monitored for mortality for 72 hours post-challenge.
Fish mortalities were observed and recorded on a daily basis and symptoms of the ensuing pathogen attack were recorded and photographed. Tissue samples of liver, kidney, gill, digestive tract and brain were preserved for histological examination, again with a corresponding photographic record. The percentage mortality rates of each vaccine dose were calculated against the relevant controls. In this way, the efficacy of both vaccine doses could be established.
Dead fish were removed immediately when observed and upon post-mortem examination, specimens were obtained aseptically from kidney, liver and brain sites for examination of S. iniae infection.
Specimens were cultured directly onto TCBS at 30°C for 24 hours and biochemically identified by API 20 Strep. The mortality data for each group was analyzed by two-way analysis of variance using a SPSS program. Significant differences were determined at P<0.05.
Plate 1. Remarkable hepatic cell swelling and vacuolation with severe panlobular fatty degeneration with mutifocal melano-macrophages of the liver (x100)
Table 1. Mean % mortality of juvenile sea bass at day 21 after vaccination (safety study).
*Values are not significantly different at P<0.05.
S. iniae was not isolated from the randomly selected sea bass, indicating that fish held at the start of the experiment were clear of S. iniae infection.
In the safety study of vaccine determined by a 21 day period after vaccination, pre-challenge. Table 1 and Figure 1 showed no significant difference in the percentage mortality rate in all groups when compared to the control groups (P>0.05). There is an indication that the higher injection volume of 0.02ml was in fact more stressful for fish and this should be taken into consideration when planning a vaccination strategy.
Table 2. Mortality rate of juvenile sea bass after challenged with S. iniae (efficacy study).
In this efficacy test of vaccination, all vaccine treated groups had significantly lower mortality rates than the control group with normal saline injection (CN) and with the negative control (NC). The highest rate of protection against S. iniae was observed at 0.01 ml vaccinated fish (IS) which had significantly lower percentage mortality rates (4.91%) in other groups (P<0.05), excluding only the positive control group (C). The immersion group (T1) displayed 14.16% mortality rate, which was regarded as excellent for an immersion route vaccine. Table 2 and Figures 2- 4 summarise the results of safety, efficacy and RPS.
The dead fish from this challenge experiment were confirmed positive for S.iniae infection by re-culture and re-identification using API 20 Strep and PCR techniques.
Clinical signs observed in infected fish included symptoms such as stationary or quiescent behaviour at the bottom of the aquaria, loss of equilibrium, frequent sinking and rising, lethargy, slow acceptance or refusal of food. Dark skin colouration, exophthalmia and corneal opacity were noted in moribund fish. The majority of these fish died over the course of the next 72 hours after sampling.
Histopathological findings included vacuolar degeneration in the liver tissue. Numerous melano-macrophages infiltrated in the kidney, spleen and liver (Plate 1). The brain showed meningio-encephalitis with thick outer layers of tissue and leucocyte infiltration (Plate 2).
Conclusion
From the vaccine safety study, the vaccine, regardless of route of administration, showed high safety to juvenile experimental fish. There was no significant difference in mortality rate of experimental groups compared with the control groups.
Plate 2. Meninges showed swollen outer layer tissue and leucocyte infiltration (x400).
The results from the efficacy study showed vaccinations have the potential to reduce fish mortality by S. iniae, and that intraperitoneal administration via 0.01 ml injection is the optimal route.
The injection of 0.01 ml (T2) conferred significantly lower mortality rate then other treatments. It should be noted that, immersion (T1) also induced higher survival rates, approaching 90%. Even if T1 groups had significantly higher mortality rates than T2, this is not viewed as negative, in that in European vaccination strategies an immersion vaccine primer is usually followed by an injectable booster dose. This may be applicable for vaccine used in commercial farms (Dunn et al., 1990) in Asian rearing conditions. Commercially, fish have been vaccinated by immersion at 1g size, then injection vaccinated at 5g or 20g depending on farm preference. Injection volume in such cases is 0.05ml and 0.1ml respectively.
This experiment proved that this vaccine was effective in reducing mortality rate of juvenile sea bass infected with S. iniae and was safe to use on the commercial production of juvenile sea bass.
Figure 2. The 21 day safety of control (not injected), control (saline injected, immersion vaccinated (test 1) and vaccine injected (tests 2 and 3) in juvenile sea bass.
Figure 3. The efficacy of control (not injected), control (saline injected), immersion vaccinated (test 1) and vaccine injected (tests 2) in juvenile sea bass.
Figure 4. RPS of control (not injected), control (saline injected), immersion vaccinated (test 1) and vaccine injected (tests 2) in juvenile sea bass.
Reference
Dunn, J., Polk, A., Scarrett, J., Olivier, G., Lall S. and Goosen. F.A. 1990. Vaccine in aquaculture: the search for an efficient delivery system. Aquaculture 9: 23-32.
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