Research in Veterinary Science 97 (2014) 271–273
Contents lists available at ScienceDirect
Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Efficacy of lipopolysaccharide antigen of Yersinia ruckeri in rainbow trout by intraperitoneal and bath immersion administration Unal Ispir a,*, Mustafa Dorucu b a b
Fisheries Faculty, Inonu University, Malatya, Turkey Faculty of Fisheries, Firat University, Elazig, Turkey
A R T I C L E
I N F O
Article history: Received 25 March 2014 Accepted 25 July 2014 Keywords: Lipopolysaccharide Yersinia ruckeri Intraperitoneal vaccine Bath immersion vaccine Response Rainbow trout
A B S T R A C T
In this study, Intraperitoneal (IP) and bath immersion (BI) vaccine trials were conducted in fish with a mean weight of 6.3 g. Rainbow trout vaccinated with lipopolysaccharide (LPS) was 50 mg/L protein concentration and challenged by IP injection with 9.8 × 106 cell/ml of Yersinia ruckeri at 45 days postimmunization had a relative percent survival (RPS). To obtain an effective bath immersion vaccine against yersiniosis, LPS preparation was obtained from the Y. ruckeri and with the LPS antigen. After 28 and 60 days vaccinated fish with first and second immunizations by LPS were challenged via intraperitoneal injection with 9.8 × 106 cell/ml of Y. ruckeri for evaluating the mortality rates and calculating the relative percentage of survival (RPS). RPS value of experimental groups, which was significantly (P < 0.05) larger than that of the control group. © 2014 Elsevier Ltd. All rights reserved.
Y. ruckeri is the causative agent of enteric redmouth disease (ERM), an infectious disease which produces high mortalities and severe economic losses on fish farms, particularly in rainbow trout and in Atlantic salmon (Tobback et al., 2007). Vaccination as a means of controlling ERM and yersiniosis is one of the most significant and successful health practices within the aquaculture industry, proving that the use of antibiotics to control bacterial diseases is likely unnecessary. Injection or immersion vaccination with heat, formalininactivated bacterins, extracellular products and O antigens also provides some protection against Y. ruckeri (Cagirgan and Tanrıkul, 1998; Ispir and Dorucu, 2010; Ispir et al., 2009). Protection of rainbow trout after immersion vaccination with extracellular products vaccine of Y. ruckeri was reported by Ispir and Dorucu (2010). LPS is derived from Gram-negative bacteria and consists of lipid A, core polysaccharide and O-specific chain. The lipid A portion of an LPS is known as an endotoxin and is responsible for most of the immunomodulatory effects of LPS. Fish immunized either intramuscularly or intraperitoneally with vaccine showed protection against challenges (Dehghani et al., 2012). The purpose of this study was to investigate the effects of LPS from Y. ruckeri in the protective immunity of rainbow trout after experimental infection with live bacteria. The rainbow trout with mean weights of 6.3 g were acclimated in de-chlorinated water for 14 days prior to experiments. To verify the Y. ruckeri-free status the rainbow trout, bacteriologic samples
* Corresponding author. Tel./fax: +90 422 846-1265. E-mail address: [email protected] (U. Ispir). http://dx.doi.org/10.1016/j.rvsc.2014.07.020 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
were obtained for bacterial culture from kidney. The samples were incubated at 25 °C for 48 h. Y. ruckeri was not isolated from selected rainbow trout. The LPS antigen was prepared according to the hot aqueous phenol extraction method of Westphal and Jann (1965). Final purified LPS product was lyophilized and stored at 4 °C. The protein concentrations in LPS antigen samples were determined by the method of Lowry (Lowry et al., 1951). To determine whether the LPS antigen was protective against Y. ruckeri (intraperitoneal and immersion administrations), rainbow trout with mean weight of 6.3 g were divided into 9 tanks of 40 fish each, including non-immunized control. Intraperitoneal injections (0.1 ml) of the LPS antigen preparation (the protein concentration was 50 mg/L) were performed in trial 1 and 2 (assay groups), whereas trial 1 and 2 (control groups) intraperitoneally received 0.1 ml of sterile phosphate buffer saline (PBS). Fishes of trial 2 were again intraperitoneally immunized with the same quantity of LPS antigen 20 days after the first injection to evaluate the occurrence of a booster effect. LPS antigen immersion trials (trials 3 and 4) were performed. For trial 3, fish were divided into four groups. Control rainbow trout were immersed in sterile PBS for 2 min. In trial 4, trouts immunized 2 times by LPS with an interval of 20 days (booster group) were challenged by an intraperitoneal injection (0.1 ml) of 9.8 × 106 Y. ruckeri cells diluted in PBS, 28 and 60 days after the last immunization with the LPS antigen preparation. Control fishes (n = 40) were not vaccinated. At 45 days post-vaccination for IP administration, the groups of vaccinates and non-vaccinates were IP challenged with 0.1 ml of a virulence Y. ruckeri isolate at cell concentrations from 9.8 × 106 cell/ ml and monitored daily for clinical signs and mortality for 14 days. It groups as immersion of LPS antigen, the challenge with virulent
272
U. Ispir, M. Dorucu/Research in Veterinary Science 97 (2014) 271–273
Y. ruckeri was performed both 28 and 60 days post-immunization after which the relative percent survival (RPS) was calculated by relating the number of diseased fish in the vaccinated groups with the corresponding number in the control group. Dead fish were removed and bacterial samples were obtained aseptically from the anterior kidney of dead fish to confirm the presence of Y. ruckeri. The mean percent mortality and mean percent cumulative mortality of vaccinated and non-vaccinated rainbow trout for each trial was determined over a 14-day period. The RPS was calculated according to Amend (1981). A one-way ANOVA test was used to determine statistical differences in the mortality among the different groups. In IP experiments, the protective indexes in the primo-vaccinated and boosted groups were 77.4% and 83.8% on the 45th day. In BI experiments, the protective indexes in the primo-vaccinated and boosted groups were 77.4% and 83.8% on the 28th day and 77.7% and 85.1% on the 60th day, respectively; the mortality rate recorded in the control groups were 77.5% and 67.5% (Figs. 1 and 2). Some of the dead fish showed one or more typical signs of yersiniosis, including hemorrhage around the mouth and tongue, dark skin, slight growth in the spleen, hemorrhages in the liver and occasionally in the intestinal tract. Figures A significantly lower mortality was achieved in all vaccinated groups (primo-vaccinated and boosted groups of intraperitoneal and bath
Cumulative mortality (%)
120 100 80 primo-vac.
60
booster
40
control
20
immersion) than in the non vaccinated controls. This result shows that the Y. ruckeri LPS is a protective immunogenic compound for rainbow trout, at least at the assayed concentration, and this is in agreement with previous studies which reported that LPS-based vaccines can offer protection against bacterial diseases in fish (Velji et al., 1990). In the same way, Erdal (1989) also reported a strong increase of the relative percentage of survival reaching 79% in the Atlantic salmon (Salmo salar) treated with Y. ruckeri membrane antigen and challenged with bacteria 44 days later. It was also described that different serotypes of the ERM vaccines enhanced resistance against yersiniosis, the RPS ranging from 67.1% to 95.1% (Erdal, 1990). The RPS obtained during exposure to a virulent challenge 60 days after a single or a booster vaccination with Y. ruckeri LPS antigens in the present study were similar (even slightly higher) with these previous data. Many studies within the three past decades have shown positive effects against several bacteria infections of LPS vaccine. Saeed and Plumb (1986), demonstrated that LPS-vaccine stimulates percent survival against E. ictaluri in channel catfish. The results show that mortality rate was reduced to 3.3% and 36.7% compared to control groups with mortality rate of 70%. Sun et al. (2011), investigated the immune protection and disease resistance in grass carp (Ctenopharyngodon idella) during Aeromonas hydrophila infection with LPS as a vaccine. The RPS rate was reduced by 55.6–83.3% in group vaccine with LPS when compared to in the control groups. Dehghani et al. (2012) studied the efficacy of formalin killed, heat killed and LPS vaccines against A. veroni and A. hydrophila in rainbow trout. The results showed the percentage of RPS as 34% for the LPS vaccine. Mao et al. (2013) used LPS-vaccine for control of Pseudomonas putida infection in large yellow croaker, Pseudosciaena crocea. The experimental groups showed a significant increase of RPS. The results in this study are in agreement with the results in previous investigations. The results of this study indicate that the Y. ruckeri-LPS vaccine preparation yields excellent efficacy against Y. ruckeri infection by a single IP injection in rainbow trout. Bath immersion of this vaccine preparation in fish provided a degree of protection that may be satisfactory in nursery and production systems where the fingerling and juvenile size fish can be vaccinated against Y. ruckeri. However, the immune defense mechanisms of fish against Y. ruckeri and the possibility that these bacteria can overcome some of these mechanisms have to be investigated in much more detail to be able to develop new approaches in the protection of fish.
0 1
2
3
4
5
6
7
8
9 10 11 12 13 14
References
days Fig. 1. Mean percent cumulative mortality of rainbow trout administered Yersinia ruckeri lipopolysaccharide antigen after 45 days by intraperitoneal injection and IP challenge with Y. ruckeri.
Cumulative mortality (%)
120 100 primo-vac/28day
80
booster/28 day
60
primo-vac/60 day
40
booster/60 day control/28 day
20
control/60 day
0
days 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig. 2. Mean percent cumulative mortality of rainbow trout administered Yersinia ruckeri lipopolysaccharide antigen after 28 and 60 days by immersion bath and IP challenge with Y. ruckeri.
Amend, D.F., 1981. Potency testing of fish vaccines. Developments in Biological Standardization 49, 447–454. Cagirgan, H., Tanrıkul, T., 1998. Testing the effectiveness of a Yersinia ruckeri in infected and chemically treated juvenile rainbow trout (Oncorhynchus mykiss). Journal of Applied Ichthyology 14, 239–243. Dehghani, S., Akhlaghi, M., Dehghani, M., 2012. Efficacy of formalin-killed, heat-killed and lipopolysaccharide vaccines against motile aeromonads infection in rainbow trout (Oncorhynchus mykiss). Global Veterinaria 9, 409–415. Erdal, J.I., 1990. Protective immunity and antibody response after vaccination of Atlantic salmon (Salmo salar) with vaccines on different serovars of Yersinia ruckeri. Norsk Veterinaertidsskrift 101, 45–61. Erdal, J.L., 1989. Vaccination of Atlantic salmon (Salmo salar L.) against different serovar of Yersinia ruckeri. In: The IV Conference of EAFP, Diseases of Fish and Shellfish. 78. Ispir, U., Dorucu, M., 2010. Effect of immersion booster vaccination with Yersinia ruckeri extracellular products (ECP) on rainbow trout Oncorhynchus mykiss. International Aquatic Research 2, 127–130. Ispir, U., Gokhan, B., Ozcan, M., Dorucu, M., Saglam, N., 2009. Immune response of rainbow trout (Oncorhynchus mykiss) to selected antigens of Yersinia ruckeri. Acta Veterinaria Brno 78, 45–150. Lowry, O.H., Rosenberough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement with Folin phenol reagent. Journal of Biochemistry 193, 265–275. Mao, Z., Ye, J., Li, M., Xu, H., Chen, J., 2013. Vaccination efficiency of surface antigens and killed whole cell of Pseudomonas putida in large yellow croaker (Pseudosciaena crocea). Fish & Shellfish Immunology 35, 375–381. Saeed, M.O., Plumb, J.A., 1986. Immune response of channel catfish to lipopolysaccharide and whole cell Edwardsiella ictaluri vaccines. Diseases of Aquatic Organisms 2, 21–26.
U. Ispir, M. Dorucu/Research in Veterinary Science 97 (2014) 271–273
Sun, J., Wang, Q., Qiao, Z., Bai, D., Sun, J., Qiao, X., 2011. Effect of Lipopolysaccharide (LPS) and Outer Membrane Protein (OMP) Vaccines on Protection of Grass Carp (Ctenopharyngodon idella) against Aeromonas hydrophila. The Israeli Journal of Aquaculture – Bamidgeh IIC:63.2011.655. Tobback, E., Decostere, A., Hermans, K., Haesebrouck, F., Chiers, K., 2007. Yersinia ruckeri infections in salmonid fish. Journal of Fish Diseases 30, 257– 268.
273
Velji, M.I., Albrigh, L.J., Evelyn, T.P.T., 1990. Protective immunity in juvenile coho salmon Oncorhynchus kisutch following immunization with Vibrio ordalii lipopolysaccharide or from exposure to live V. ordalii cells. Diseases of Aquatic Organisms 9, 25–29. Westphal, O., Jann, K., 1965. Bacterial lipopolysaccharide extraction with phenol-water and further applications of the procedure. In: Whistler, R.L. (Ed.), Methods in Carbohydrate Chemistry, vol. 5. New York, pp. 83–96.
Contents lists available at ScienceDirect
Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Efficacy of lipopolysaccharide antigen of Yersinia ruckeri in rainbow trout by intraperitoneal and bath immersion administration Unal Ispir a,*, Mustafa Dorucu b a b
Fisheries Faculty, Inonu University, Malatya, Turkey Faculty of Fisheries, Firat University, Elazig, Turkey
A R T I C L E
I N F O
Article history: Received 25 March 2014 Accepted 25 July 2014 Keywords: Lipopolysaccharide Yersinia ruckeri Intraperitoneal vaccine Bath immersion vaccine Response Rainbow trout
A B S T R A C T
In this study, Intraperitoneal (IP) and bath immersion (BI) vaccine trials were conducted in fish with a mean weight of 6.3 g. Rainbow trout vaccinated with lipopolysaccharide (LPS) was 50 mg/L protein concentration and challenged by IP injection with 9.8 × 106 cell/ml of Yersinia ruckeri at 45 days postimmunization had a relative percent survival (RPS). To obtain an effective bath immersion vaccine against yersiniosis, LPS preparation was obtained from the Y. ruckeri and with the LPS antigen. After 28 and 60 days vaccinated fish with first and second immunizations by LPS were challenged via intraperitoneal injection with 9.8 × 106 cell/ml of Y. ruckeri for evaluating the mortality rates and calculating the relative percentage of survival (RPS). RPS value of experimental groups, which was significantly (P < 0.05) larger than that of the control group. © 2014 Elsevier Ltd. All rights reserved.
Y. ruckeri is the causative agent of enteric redmouth disease (ERM), an infectious disease which produces high mortalities and severe economic losses on fish farms, particularly in rainbow trout and in Atlantic salmon (Tobback et al., 2007). Vaccination as a means of controlling ERM and yersiniosis is one of the most significant and successful health practices within the aquaculture industry, proving that the use of antibiotics to control bacterial diseases is likely unnecessary. Injection or immersion vaccination with heat, formalininactivated bacterins, extracellular products and O antigens also provides some protection against Y. ruckeri (Cagirgan and Tanrıkul, 1998; Ispir and Dorucu, 2010; Ispir et al., 2009). Protection of rainbow trout after immersion vaccination with extracellular products vaccine of Y. ruckeri was reported by Ispir and Dorucu (2010). LPS is derived from Gram-negative bacteria and consists of lipid A, core polysaccharide and O-specific chain. The lipid A portion of an LPS is known as an endotoxin and is responsible for most of the immunomodulatory effects of LPS. Fish immunized either intramuscularly or intraperitoneally with vaccine showed protection against challenges (Dehghani et al., 2012). The purpose of this study was to investigate the effects of LPS from Y. ruckeri in the protective immunity of rainbow trout after experimental infection with live bacteria. The rainbow trout with mean weights of 6.3 g were acclimated in de-chlorinated water for 14 days prior to experiments. To verify the Y. ruckeri-free status the rainbow trout, bacteriologic samples
* Corresponding author. Tel./fax: +90 422 846-1265. E-mail address: [email protected] (U. Ispir). http://dx.doi.org/10.1016/j.rvsc.2014.07.020 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
were obtained for bacterial culture from kidney. The samples were incubated at 25 °C for 48 h. Y. ruckeri was not isolated from selected rainbow trout. The LPS antigen was prepared according to the hot aqueous phenol extraction method of Westphal and Jann (1965). Final purified LPS product was lyophilized and stored at 4 °C. The protein concentrations in LPS antigen samples were determined by the method of Lowry (Lowry et al., 1951). To determine whether the LPS antigen was protective against Y. ruckeri (intraperitoneal and immersion administrations), rainbow trout with mean weight of 6.3 g were divided into 9 tanks of 40 fish each, including non-immunized control. Intraperitoneal injections (0.1 ml) of the LPS antigen preparation (the protein concentration was 50 mg/L) were performed in trial 1 and 2 (assay groups), whereas trial 1 and 2 (control groups) intraperitoneally received 0.1 ml of sterile phosphate buffer saline (PBS). Fishes of trial 2 were again intraperitoneally immunized with the same quantity of LPS antigen 20 days after the first injection to evaluate the occurrence of a booster effect. LPS antigen immersion trials (trials 3 and 4) were performed. For trial 3, fish were divided into four groups. Control rainbow trout were immersed in sterile PBS for 2 min. In trial 4, trouts immunized 2 times by LPS with an interval of 20 days (booster group) were challenged by an intraperitoneal injection (0.1 ml) of 9.8 × 106 Y. ruckeri cells diluted in PBS, 28 and 60 days after the last immunization with the LPS antigen preparation. Control fishes (n = 40) were not vaccinated. At 45 days post-vaccination for IP administration, the groups of vaccinates and non-vaccinates were IP challenged with 0.1 ml of a virulence Y. ruckeri isolate at cell concentrations from 9.8 × 106 cell/ ml and monitored daily for clinical signs and mortality for 14 days. It groups as immersion of LPS antigen, the challenge with virulent
272
U. Ispir, M. Dorucu/Research in Veterinary Science 97 (2014) 271–273
Y. ruckeri was performed both 28 and 60 days post-immunization after which the relative percent survival (RPS) was calculated by relating the number of diseased fish in the vaccinated groups with the corresponding number in the control group. Dead fish were removed and bacterial samples were obtained aseptically from the anterior kidney of dead fish to confirm the presence of Y. ruckeri. The mean percent mortality and mean percent cumulative mortality of vaccinated and non-vaccinated rainbow trout for each trial was determined over a 14-day period. The RPS was calculated according to Amend (1981). A one-way ANOVA test was used to determine statistical differences in the mortality among the different groups. In IP experiments, the protective indexes in the primo-vaccinated and boosted groups were 77.4% and 83.8% on the 45th day. In BI experiments, the protective indexes in the primo-vaccinated and boosted groups were 77.4% and 83.8% on the 28th day and 77.7% and 85.1% on the 60th day, respectively; the mortality rate recorded in the control groups were 77.5% and 67.5% (Figs. 1 and 2). Some of the dead fish showed one or more typical signs of yersiniosis, including hemorrhage around the mouth and tongue, dark skin, slight growth in the spleen, hemorrhages in the liver and occasionally in the intestinal tract. Figures A significantly lower mortality was achieved in all vaccinated groups (primo-vaccinated and boosted groups of intraperitoneal and bath
Cumulative mortality (%)
120 100 80 primo-vac.
60
booster
40
control
20
immersion) than in the non vaccinated controls. This result shows that the Y. ruckeri LPS is a protective immunogenic compound for rainbow trout, at least at the assayed concentration, and this is in agreement with previous studies which reported that LPS-based vaccines can offer protection against bacterial diseases in fish (Velji et al., 1990). In the same way, Erdal (1989) also reported a strong increase of the relative percentage of survival reaching 79% in the Atlantic salmon (Salmo salar) treated with Y. ruckeri membrane antigen and challenged with bacteria 44 days later. It was also described that different serotypes of the ERM vaccines enhanced resistance against yersiniosis, the RPS ranging from 67.1% to 95.1% (Erdal, 1990). The RPS obtained during exposure to a virulent challenge 60 days after a single or a booster vaccination with Y. ruckeri LPS antigens in the present study were similar (even slightly higher) with these previous data. Many studies within the three past decades have shown positive effects against several bacteria infections of LPS vaccine. Saeed and Plumb (1986), demonstrated that LPS-vaccine stimulates percent survival against E. ictaluri in channel catfish. The results show that mortality rate was reduced to 3.3% and 36.7% compared to control groups with mortality rate of 70%. Sun et al. (2011), investigated the immune protection and disease resistance in grass carp (Ctenopharyngodon idella) during Aeromonas hydrophila infection with LPS as a vaccine. The RPS rate was reduced by 55.6–83.3% in group vaccine with LPS when compared to in the control groups. Dehghani et al. (2012) studied the efficacy of formalin killed, heat killed and LPS vaccines against A. veroni and A. hydrophila in rainbow trout. The results showed the percentage of RPS as 34% for the LPS vaccine. Mao et al. (2013) used LPS-vaccine for control of Pseudomonas putida infection in large yellow croaker, Pseudosciaena crocea. The experimental groups showed a significant increase of RPS. The results in this study are in agreement with the results in previous investigations. The results of this study indicate that the Y. ruckeri-LPS vaccine preparation yields excellent efficacy against Y. ruckeri infection by a single IP injection in rainbow trout. Bath immersion of this vaccine preparation in fish provided a degree of protection that may be satisfactory in nursery and production systems where the fingerling and juvenile size fish can be vaccinated against Y. ruckeri. However, the immune defense mechanisms of fish against Y. ruckeri and the possibility that these bacteria can overcome some of these mechanisms have to be investigated in much more detail to be able to develop new approaches in the protection of fish.
0 1
2
3
4
5
6
7
8
9 10 11 12 13 14
References
days Fig. 1. Mean percent cumulative mortality of rainbow trout administered Yersinia ruckeri lipopolysaccharide antigen after 45 days by intraperitoneal injection and IP challenge with Y. ruckeri.
Cumulative mortality (%)
120 100 primo-vac/28day
80
booster/28 day
60
primo-vac/60 day
40
booster/60 day control/28 day
20
control/60 day
0
days 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig. 2. Mean percent cumulative mortality of rainbow trout administered Yersinia ruckeri lipopolysaccharide antigen after 28 and 60 days by immersion bath and IP challenge with Y. ruckeri.
Amend, D.F., 1981. Potency testing of fish vaccines. Developments in Biological Standardization 49, 447–454. Cagirgan, H., Tanrıkul, T., 1998. Testing the effectiveness of a Yersinia ruckeri in infected and chemically treated juvenile rainbow trout (Oncorhynchus mykiss). Journal of Applied Ichthyology 14, 239–243. Dehghani, S., Akhlaghi, M., Dehghani, M., 2012. Efficacy of formalin-killed, heat-killed and lipopolysaccharide vaccines against motile aeromonads infection in rainbow trout (Oncorhynchus mykiss). Global Veterinaria 9, 409–415. Erdal, J.I., 1990. Protective immunity and antibody response after vaccination of Atlantic salmon (Salmo salar) with vaccines on different serovars of Yersinia ruckeri. Norsk Veterinaertidsskrift 101, 45–61. Erdal, J.L., 1989. Vaccination of Atlantic salmon (Salmo salar L.) against different serovar of Yersinia ruckeri. In: The IV Conference of EAFP, Diseases of Fish and Shellfish. 78. Ispir, U., Dorucu, M., 2010. Effect of immersion booster vaccination with Yersinia ruckeri extracellular products (ECP) on rainbow trout Oncorhynchus mykiss. International Aquatic Research 2, 127–130. Ispir, U., Gokhan, B., Ozcan, M., Dorucu, M., Saglam, N., 2009. Immune response of rainbow trout (Oncorhynchus mykiss) to selected antigens of Yersinia ruckeri. Acta Veterinaria Brno 78, 45–150. Lowry, O.H., Rosenberough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement with Folin phenol reagent. Journal of Biochemistry 193, 265–275. Mao, Z., Ye, J., Li, M., Xu, H., Chen, J., 2013. Vaccination efficiency of surface antigens and killed whole cell of Pseudomonas putida in large yellow croaker (Pseudosciaena crocea). Fish & Shellfish Immunology 35, 375–381. Saeed, M.O., Plumb, J.A., 1986. Immune response of channel catfish to lipopolysaccharide and whole cell Edwardsiella ictaluri vaccines. Diseases of Aquatic Organisms 2, 21–26.
U. Ispir, M. Dorucu/Research in Veterinary Science 97 (2014) 271–273
Sun, J., Wang, Q., Qiao, Z., Bai, D., Sun, J., Qiao, X., 2011. Effect of Lipopolysaccharide (LPS) and Outer Membrane Protein (OMP) Vaccines on Protection of Grass Carp (Ctenopharyngodon idella) against Aeromonas hydrophila. The Israeli Journal of Aquaculture – Bamidgeh IIC:63.2011.655. Tobback, E., Decostere, A., Hermans, K., Haesebrouck, F., Chiers, K., 2007. Yersinia ruckeri infections in salmonid fish. Journal of Fish Diseases 30, 257– 268.
273
Velji, M.I., Albrigh, L.J., Evelyn, T.P.T., 1990. Protective immunity in juvenile coho salmon Oncorhynchus kisutch following immunization with Vibrio ordalii lipopolysaccharide or from exposure to live V. ordalii cells. Diseases of Aquatic Organisms 9, 25–29. Westphal, O., Jann, K., 1965. Bacterial lipopolysaccharide extraction with phenol-water and further applications of the procedure. In: Whistler, R.L. (Ed.), Methods in Carbohydrate Chemistry, vol. 5. New York, pp. 83–96.
