Abstract: Monovalent, killed and live attenuated vaccines of Aeromonas hydrophila and Pseudomonas putida were used in the immunization of red tilapia against Motile Aeromonad and Pseudomonad septicemias. There were 4 treatments and a 5th control group with 3 replicates per each. A 4th replicate was kept for replacement of natural mortality among the experimented fish. The 4 treatments included, Heat-killed vaccine of A. hydrophila, Live-attenuated vaccine of A. hydrophila (using herbs), Heat-killed vaccine of P. putida and Live-attenuated vaccine of P. putida. A total of 160 brood stocks of O. niloticus with 250 g average body weight were used for all treatments (8 fish per each glass aquarium). Vaccination was conducted via the Intra Peritoneal route (I/P) as an initial dose followed by 2 booster doses every 2 weeks. The last dose was applied via the immersion route. The evaluation of vaccination was carried out through periodical antibody titration of the serum of the examined fish (every 2 weeks) using direct agglutination method as well as by the experimental challenge 3 months after the initial immunization. Results revealed that there were a significant difference between the vaccinated and non vaccinated fish of the control group regarding antibody titers and Relative Percent Survival (RPS) of the challenge test. Differences in immunity levels within the vaccinated groups themselves were demonstrated.

INTRODUCTION

Fish diseases caused by Aeromonads and Pseudomonads considered to be the major bacterial problems facing the aquaculture development causing mass mortalities, reduced production and low quality of aquatic organisms (Ghittino, 1976; Abdel-Hadi, 2004; Laila et al., 2004).

Both Aeromonas sp. (A. hydrophila, A. sobria and A. caviae) and Pseudomonas sp. (P. fluorescens, P. putida and P. aeruginosa) were incorporated in severe outbreaks among O. niloticus in fish hatcheries (Ahmed and Shoreit, 2001) in intensive culture farms (Eisa et al., 1993) in earthen ponds and in floating cages (Gamal et al., 2002; Abu El-Attah, 2003; Abdel-Hadi, 2004).

Motile Aeromonas Septicemia (MAS) is one of the most economic diseases affecting fish farms in Egypt (Atallah et al., 1999).

Aeromonas hydrophila is considered among the most pathogenic organisms to both homothermic and poikilothermic hosts including tilapia species (Amin et al., 1985; Zaki, 1991). It affects not only O. niloticus but also, causes severe outbreaks in Cyprinus carpio and Mugil cephalus (Marzouk and Nawal, 1991; Eisa et al., 1993).

Pseudomonas fluorescens, P. putida, P. aeruginosa, P. chlorophis and P. anguilliseptica were recognized as the causative agents of bacterial hemorrhagic septicemia in different species of fish (Robert, 1989; Schaperclaus, 1992; Abu El-Attah, 2003; Abdel-Hadi, 2004).

Resistance of Aeromonas and Pseudomonas sp. against the most commonly used antimicrobials in aquaculture has developed greatly in the recent years (Inglis et al., 1997; Kampfer et al., 1999; Ahmed and Shoreit, 2001; Taylor, 2003; Abdel-Hadi, 2004). Those antimicrobials have been so abused by fish farmers that they have been accumulated in the edible muscles and different internal organs of the treated fish (Somsiri et al., 1997). So, other environmentally safe alternatives including vaccination are recommended (Abdel-Hadi, 2004).

Indeed, the concept of vaccinating fish has been the subject of considerable research efforts world-wide (Wiegertjes, 2001). Vaccination is an important disease management strategy used to maintain human and animal health. Vaccines developed for aquaculture have reduced antibiotic use in fish production. Work in the 1990s showed the use of various strategies to develop modified live vaccines for use in fish. Modified live vaccines are advantageous in that they can be easily delivered (i.e., by immersion to young fish) and stimulate both humoral and cellular immunity of long duration. Disadvantages include issues with modified live vaccine safety to the host and environment (Craig et al., 2009).

Thus, this study was conducted to tackle the following objectives:

MATERIALS AND METHODS

Fish: A total of 160 red tilapia fish with average body weight of 250 g were used in this study. The fish were divided into 5 groups (groups A-D and control). Each group had 3 aquaria representing 3 replicates where 8 of the examined fish were kept in each aquarium (150x45x70 cc). One group was kept as control. The fish of the 4th replicate were kept for the replacement of natural mortality among the experimented fish. Water temperature was maintained to 27°C, dissolved Oxygen to 5.5 mg L^-1 and pH to 7.2.

Preparation of vaccines: Heat-killed vaccines were prepared according to Chandran et al. (2002).

Live attenuated vaccine: Using the crude extract the natural herb of Oreganum vulgare via using bacterial colonies attenuated by the Minimal Inhibitory Concentration (MIC) of the herb.

Sterility and safety: Sterility and safety of the prepared vaccines were tested according to Ward (1982).

Treatments
I/P immunization of tilapia with heat-killed and live-attenuated vaccines: The examined fish were first anaesthetized using MS222 in the rate of 100 mg L^-1. The fish in group A and B were injected Intraperitoneally (I/P) with 0.1 mL containing 9x10⁹ cfu mL^-1 (Zaki, 1998) of heat-killed and live attenuated A. hydrophila vaccines respectively (Azad et al., 1997). Similarly, fish in groups C and D were injected I/P with 0.1 mL containing 9x10⁹ cfu mL^-1 of heat-killed and live attenuated P. putida vaccines. Two booster doses were given, 4 and 6 weeks, after the initial dose. Fish of the control group were I/P injected with 0.1 mL of Phosphate Buffer Saline (PBS).

Direct immersion vaccination: This dose was applied 8 weeks after the initial I/P immunization (and 2 weeks after the 2nd I/P booster dose) The examined fish were anaesthetized using MS222, immersed for 2 min in an ice box containing a hypertonic solution (1.5% sodium chloride) and directly immersed in 4 ice boxes containing dechlorintaed tap water with a final concentration of 1.5x10⁹ cell mL^-1 of killed, live attenuated A. hydrophila, killed and live attenuated P. putida vaccines, respectively for 2 min (Ahmed et al., 1995).

The fish of the control group were immersed in an ice box containing dechlorintaed water plus (PBS) only. The residual water-containing vaccines for both A. hydrophila and P. putida were disinfected by chlorine as a bactericidal agent to avoid any environmental pollution.

Periodical antibody titration in the sera of the examined fish: Six samples were taken in a regular basis; every 2 weeks. Blood samples were taken from the caudal vein of 3 fish per each treatment and serum samples were separated by centrifugation (Chandran et al., 2002). Antibody titers for both A. hydrophila and P. putida were detected in the sera using the direct agglutination test (10 Double-fold dilutions from 1:1 until 1:512) according to Ayub et al. (1997). Ab titers were also detected in 12 pre-immunized tilapia (6 fish for A. hydrophila and 6 for P. putida).

Activation of the bacterial isolates for the challenge test:
The A. hydrophila and Pseudomonas putida strains, which were used for the challenge test were activated (Azad et al., 1997) 10 days before the challenge by serial I/P passage through live juvenile red tilapia fish for 3 times. Thus, they restored their virulence. Eighteen fish were used for the activation (3 fish were used per each passage per isolate).

Experimental challenge: Hot strains of A. hydrophila for the fish immunized by A. hydrophila vaccines and P. putida for the fish immunized by P. putida vaccines were injected I/P into the examined fish. Ten fish from each of the 4 treatments were taken and kept in 4 aquaria.

Twenty fish were taken from the control group and were kept in 2 aquaria with 10 fish per each (1 aquarium for A. hydrophila and the other for P. putida), where they were used as controls for the challenge test. The used challenging dose was 0.1 mL containing 7x10⁷ cfu mL^-1 (Zaki, 1998). The fish were noticed for 10 days (Azad et al., 1997) and the daily mortalities were recorded.

Statistical analysis: Was carried out using SPSS Repeated Measures ANOVA.

RESULTS AND DISCUSSION

The results revealed that antibody titers of the direct agglutination method were higher in fish vaccinated with live attenuated A. hydrophila vaccine than those obtained in fish vaccinated with heat killed A. hydrophila vaccine but with no significant difference. This is logic and agreed with the same fact in other farm animals and human (Craig et al., 2009).

However, Ab titers in the vaccinated fish with both types of vaccines were significantly, higher than those of the non vaccinated or control group (Table 1 and Fig. 1).

On the contrary, antibody titers were higher in fish vaccinated with heat killed P. putida vaccine than in fish vaccinated with live attenuated P. putida vaccine but also with no significant difference and both of them were significantly, higher than those of the non vaccinated tilapia (Table 2 and Fig. 2).

The I/P injection route induced higher Ab titers than obtained by immersion route, where Ab titers decreased dramatically after application of the immersion vaccination in both A. hydrophila and P. putida vaccines (Fig. 1 and 2). However, these Ab titers induced by the immersion route were still significantly higher than non vaccinated fish of the control group.

Table 1:	Ab titers estimated by direct agglutination in sera of red tilapia vaccinated against A. hydrophila

Table 2:	Ab titers estimated by direct agglutination in the sera of red tilapia vaccinated against P. putida

Table 3:	Mortality and RPS of the examined fish challenged with A. hydrophila and P. putida
RPS = Relative Percent Survival

Similar results were recorded by Badran (1987), Ayub et al. (1997) and Rongxing et al. (2008).

On the other hand, all examined 12 pre-immunized tilapia had no Ab titers (0) against both A. hydrophila and P. putida except 1 fish with +ve Ab titer (1:4) against A. hydrophila and 1 fish with +ve Ab titer (1:1) against

Fig. 1:	Ab titers of A. hydrophila vaccines compared with the control group

Fig. 2:	Ab titers of P. putida vaccines compared with the control group

P. putida. This may be attributed to the fact that A. hydrophila is one of the natural aquatic flora, where it lives in water and gets its name referring to water. On the other hand, 1 of the pre-immunized fish had the antibodies of P. putida. This may be due to a sporadic infection.

Regarding the challenge test, results showed that heat killed A. hydrophila vaccine gave a higher level of Relative Percent Survival (RPS) (90%) for the vaccinated fish than the live attenuated vaccine (60% of RPS) and both had higher protection than fish of the control group (0% RPS). On the contrary, live attenuated P. putida vaccine produced a higher RPS (80%) for the vaccinated fish than heat killed vaccine (50%) and both had higher

Fig. 3:	RPS of A. hydrophila vaccines compared with the control group

Fig. 4:	RPS of P. putida vaccines compared with the control group

RPS than fish of the control group where RPS was 20% (Table 3, Fig. 3 and 4). This indicates the effective immunization induced by the experimented vaccines.

CONCLUSION

Heat-killed A. hydrophila and P. putida vaccines are recommended for the immunization of red tilapia against Aeromonad and Pseudomonad septicemias respectively. They are safer for the aquatic ecosystem than live-attenuated vaccines. Besides, no significant difference was found in this study between the heat-killed and live-attenuated vaccines for both bacterial species, regarding the antibody titers in the vaccinated fish.

The I/P injection is suitable for vaccination of the brood stock in fish hatcheries. However, an insulin syringe with thin needle should be strictly used to avoid excessive traumatic effects of the larger or thicker needles.

Direct immersion could be used for vaccinating frys and fingerlings in fish hatcheries especially, prior to release to the rearing ponds.

Further study on the development and evaluation of recombinant vaccines against A. hydrophila and P. putida is recommended.

ACKNOWLEDGEMENTS

Sincere gratitude is directed to the Islamic Development Bank (IDB) the sponsor of this project, to the Central Laboratory for Aquaculture Research (CLAR) in Egypt and to all staff members of the Institute of BioSciences (IBS) and Department of Microbiology, Faculty of Medicine, University of Putra Malaysia (UPM) for their kind support.