Overwintering of tilapia fry may provide sufficient fingerlings for the following season and is carried out using heated facilities, underground warm water and in green houses (Cruz and Ridha, 1994; Jiazhao, 1991). But these resources are generally not available in Egypt. Overwintering late-season fingerlings in deep hapas (3.0 m) suspended in deep ponds (3.5 m) has also been constructed (Nguyen and Little, 2000).

Aeromonas hydrophila is one of the most common bacterial pathogens in the Egyptian aquaculture. It causes septicemia with mortalities among tilapia and other fish species reared under the hatchery and farm environment in Egypt (Aly, 2013; Aly et al., 1998). The intestinal microbiota of fry plays a role in the defense against opportunistic pathogens. The application of immunostimulants and probiotics in improving fish health was tested previously (Aly et al., 2007, 2008a, b). Garlic and Echinacea improved the body gain, survival and resistance to infection in Nile tilapia (Aly and Mohamed, 2010). Garlic plays a role in the control of bacteria and fungi (Corzo-Martinez et al., 2007). The allium enhances the immune activities through promotion of lymphocyte-synthesis, cytokine release, phagocytosis and activation of natural killer cell (Kyo et al., 2001). Few data on the use of garlic extract as immune stimulant in fish in Egypt are available (Aly et al., 2008). Echinacea, also, seems to activate the macrophages and other immunological functions in laboratory animals and humans. The evidences on the role of the polysaccharidic fraction of Echinacea on immunity are reported (Bauer, 1999). On the other hand, Bacillus spp., lactic acid bacteria (LAB) and other Gram-negative bacteria have been tested as fish probiotics (Irianto and Austin, 2002; VaŁ zquez et al., 2005). Moreover, the usage of Saccharomyces cerevisae in fish is a good enhancer of fish immune system, it also improves the survival and growth rate of supplemented fish (Abd El-Tawab et al., 2008). Previous study showed the effect of some  immunostimulants  and  probiotics  on the survival rate and final weight of overwintered tilapia fry where no significant differences was recognized in the growth while significant improvement in the survival was seen in diets containing immunostimulents or probiotics (Aly et al., 2010). The current work was designed to investigate the role of selected immunostimulants (garlic and Echinacea) and probiotics (Organic Green^TM and Vet-Yeast^TM ) on improving the response of overwintered tilapia fry to vaccination with A. hydrophila vaccine.

Vaccine preparation

Formalin-killed A. hydrophila bacterin was prepared by addition of formalin (0.3%) to the bacterial culture, which had been previously incubated at 35°C for 48 h (Baba et al., 1988). The formalized bacterial culture was held at room temperature overnight, and then subjected to sterility and safety tests according to earlier study (Cardella et al., 1990). The sterility test was performed by culturing washed bacterin on TS agar. Plates were incubated at 37°C for 24 h and examined for bacterial growth. The safety test was performed by the intraperitoneal (IP) inoculation of 20 susceptible tilapia (20 ± 4 g) with the prepared bacterin cells (0.1 ml). The fish were reared for 2 weeks post-injection and then morbid fish were subjected to necropsy for re-isolation of A. hydrophila using RS media. The prepared and tested vaccine was stored in the refrigerator at 4°C. Immediately before use, formalin killed bacterial cells were washed twice with sterile saline solution and prepared to a concentration of 3 mg wet-weight/ml saline.

Experiment

Fish of group 1 (control) was fed a balanced ration without other additives. Groups 2-5 were fed on a balanced diet supplemented with 4% garlic (group 2), 4 ppt Echinacea (group 3), 4 ppt Organic Green^TM culture (group 4) or 4 ppt Vet-Yeast^TM (group 5); respectively. Fish were fed three times daily in a plastic feeder for five months (November – March). At the end of feeding experiment, the overwintered O. niloticus (n = 900) were collected; 300 fish from the control group 1 (150 for control negative and 150 for control positive subgroups a and b) and 150 fish each from the 4 treated overwintered tilapia groups were also collected. Fish were collected equally from the three replicates of the 1^st stage experiment in a random way and subdivided into 3 equal replicates groups (each of 50 fish). Fish were vaccinated following the schedule shown in Table 1. Fish were anaesthetized with 100 mg/L MS₂₂₂ (Tricain methane sulfonate; Argent Chemical Laboratories, Fisheries Division). The negative control fish of group 1 were intraperitoneally (IP) injected by 0.1 ml sterile saline solution. Fish of other treated groups (2-5) and control positive subgroup were inoculated with 0.1 ml formalin-killed A. hydrophila diluted in 0.1 ml sterile saline (Badran et al., 1993)

Laboratory evaluation

Antibody determination

By the end of 1^st, 2^nd, 3^rd, 4^th, 6^th, 8^th and 10^th week post-vaccination (PV), fish were anesthetized by immersion in water containing 0.1 ppm MS-222 and blood samples were collected. Whole blood (0.5 ml) was obtained from caudal vein of fish of experiment 2, (n = 30/group, 10/replicate), using syringes (1-ml) and 27-gauge needles and transferred to blood Eppendorf tubes without anticoagulant. The blood samples were centrifuged at 3000 x g for 15 min and the supernatant serum was collected and stored at -20°C in screw-capped glass vials until used. Specific antibody titers, in collected sera, were determined using the bacterial agglutination test (Baba et al., 1988).

Challenge after vaccination

Fish (n = 90), with average weight of 6 ± 0.3 g, were randomly obtained from the three replicates of each treatment of experiment 2, and were used for challenge test at the end of the 6^th, 8^th and 10^th weeks post-vaccination (30 fish/challenge group). Fish were splitted into 3 equal groups (each of 3 equal replicate (10 fish) being reared in a separate glass aquarium (50 x 60 x 70 cm). Fish was IP inoculated with 0.5 ml (10⁸ bacteria cells ml^-1) of culture suspension of the reference pathogenic strains of A. hydrophila (Aly et al., 2008a). Mortality was recorded and the dead fish was subjected to necropsy for bacterial re-isolation. The relative level of protection (RLP) among the vaccinated and challenged fish was determined using the following equation (Ruangroupan et al., 1986): RLP% = 100 - (percent stimulated mortality ÷ percent mortality in thecontrol group) x 100

Statistical analysis

One-way and two-way analyses of variance (ANOVA) were carried. Also, Duncan’s multiple range test (Duncan, 1955) was used to determine differences among treatments (mean at significance level of P<0.05). Standard errors were also estimated. Analysis was carried out using the SAS package (Duncan, 1955).

The authors declare that there is no conflict of interests regarding the publication of this paper.

Two important challenges face fish culture in Egypt: first making fry available throughout the year, especially during and immediately after the winter season, which retard tilapia growth and production as well as fry survival. The second challenge is to recover the time (winter and early spring) lost, when there is no investment in fish production due to shortages of fry. This study  aimed   to   improve   the   survival  and  resistance  of tilapia fry stressed during the winter season to infection through the use of immunostimulants and probiotics together with vaccination.

According to Aly et al. (2010), an improved survival among tilapia fed on diets containing immunostimulents or probiotics was recorded where garlic-supplemented diet resulted to a higher total harvest weight. It was previously stated that, immunostimulants improve the protection of fish against diseases by enhancing non-specific and humoral defense mechanisms (Sakai, 1999). Garlic has been effect against many bacteria (Ress et al., 1993), fungi (Adetumbi et al., 1986) and viruses (Weber et al., 1992).

These findings show the effect of immunostimulants on non-specific immunity by increasing the number of phagocytes or improving phagocytosis (Shoemaker et al., 1997). Garlic and other immunostimulants enhance the bactericidal activity of the fish’s phagocytic cells (Sahu et al., 2007). Moreover, garlic shows positive action against several bacteria (Ress et al., 1993). Previous studies revealed that, yeast, b-1,3 glucan enhances resistance against several major bacterial pathogens (Vibrio anguillarum, Vibrio salmonicida, Yersinia ruckeri, Edwardsiella tarda and A. hydrophila) in fish species such as carp, Cyprinus carpio (Yano et al., 1991), Atlantic salmon, Salmo salar Robertsen et al., (1990) and African catfish, Clarias gariepinus (Yoshida et al., 1995). Several researchers reported the effect of β-(1,3) D-glucan on the cellular and humoral defense mechanisms, including the function of phagocytes, bacterial killing activity of macrophages in rainbow trout, Atlantic salmon and catfish (Nikl et al., 1991). β-(1, 3) D-glucan induces the production of superoxide anion by macrophages (Dalmo and Seljelid, 1995) and accelerates the synthesis of cytokine-like molecules (Yoshida et al., 1995). The protective effect of echinacea preparations may be due to the activation of lymphocytes (Wagner et al., 1986). However, the immunomodulatory effects of echinacea are considered to be via non-specific activation of the immune system (Maass et al., 2005).

In the present study, although the antibody titer showed no significant difference among supplemented groups at the same time post-vaccination, they were significantly increased in the vaccinated overwintered tilapia at the end of the 6^th - 8^th week PV in the control group, at 4^th- 8^th week PV in garlic and echinacea supplemented groups and at 2^nd – 10^th week PV in organic green and Vet-yeast supplemented groups. Previous studies revealed that, glucan treatment in fish enhanced the expression of interleukin 1 (Fujiki et al., 2000) and complement activity (Engstad et al., 1992). Moreover, glucan accelerated the specific immune response in catfish and Atlantic salmon against vaccines for E. ictaluri and A. salmonicida, respectively (Chen and Ainsworth, 1992; Baulny et al., 1996). Earlier finding reported that, oral administration of Lactobacillus delbreckii sp. lactis and B. subtilis, alone or combined increased phagocytic activity in gilthead seabream (Sparus aurata L.) after 2 weeks of feeding (Salinas et al., 2005).

The challenge infection of the overwintered-vaccinated tilapia using A. hydrophila showed lowest mortality in the Vet-yeast supplemented group at three challenging periods (6^th, 8^th and 10^th week PV). Yeast glucans revealed increase in the macrophage-chemotactic and phagocytic activities, the lysozyme and complement activities and also stimulated cytokines (IL-1, tumor necrosis factor) release and leukocyte migration (Secombes and Fletcher, 1992). Thereby, it significantly reduces fish mortality after challenge with various pathogens (Aly et al., 2015; Abel and Czop, 1992; Verlhac et al., 1996, 1998).

It may be concluded that, immunostimulants and probiotics can improve the response of overwintering tilapia fry to vaccination and resistance to diseases. Its use is recommended together with a vaccination program in order to improve the economic outcomes and to avoid environmental contamination through the widespread application of drugs and antibiotics.

The authors express their deep thanks and appreciation to World Fish Center, CGIAR, Abbsaa, Egypt for supporting the research idea and providing Nile tilapia fish and facilities for this experiment.