Trials to improve the response of Orechromis niloticus to Aeromonas hydrophila vaccine using immunostimulants ( garlic , Echinacea ) and probiotics ( Organic Green TM and Vet-Yeast TM )

1 Department of Pathology, Faculty of Veterinary Medicine/Fish Farming and Technology Institute, Suez Canal University, Ismailia, Egypt. 2 Department of Medical Laboratory, Faculty of Applied Medical Sciences, Qassim University, KSA. 3 National Institute of Oceanography and Fisheries, Suez, Egypt/Post-Doctoral Fellow, Aquaculture and Genetic Improvement WorldFish, Egypt. 4 Animal Health Research Institute, Ismailia Lab, Dokki Giza, Egypt.


INTRODUCTION
Aquaculture is a promising sector of fish industry in the world with about 80 million tones being produced annually (Kolkovski and Kolkovski, 2011).The development of aquaculture faced several constraints; among these are diseases constituting the most limiting factors.Bacterial infections, pose one of the most significant threat to successful fish production throughout the world (Rahman et al., 2009).In Egypt, bacterial diseases induce heavy mortality in farm fish (Aly, 2013).Moreover, winter season in Egypt stress tilapia causing low survival and render hatcheries unable to produce fry to stock in the ponds during winter which limit the length of the production season until June with a huge economic loss for the inability to use the fish farm during that period.Recently, the control of bacterial infection in fish is managed through the use of vaccines or via biological control using safe microorganisms (Aly et al., 2015;Aly and Mohamed, 2010).
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(Aly et al., , 2008a, b), 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 lymphocytesynthesis, 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.

Fish
Nile tilapia fry, Orechromis niloticus (Fifteen thousand, initial weight 0.02 g) were divided into five equal groups, each of three equal replicates that are reared in 15 cement ponds (5 x 2.5 x 1 m).Ponds were filled with tap water and partially renewed during the experiment.The water quality was optimal throughout the period of experiment, except temperature which was lower than optimal range for tilapia culture because of winter season (November-March).The water was exchanged 20% daily to maintain its quality within an acceptable range for the survival of tilapia {NO3 (0.20 mg/L,), total ammonia nitrogen (0.2 mg/L), Chl a (42.27 mg/L), and orthophosphate (0.02 mg/L)}.Water temperatures during the experiment ranged from 10 to 20°C.

Preparation of diets
Dietary ingredients of 35% protein were prepared in the World Fish Center as pellets [21].Ingredients were prepared by grinding the corn to granules (0.5 mm mesh size) (Thomes-Willey Laboratory Mill Model 4, Swedesboro, NJ 08085 U.S.A).Ingredients were mixed by mixer (Hobart model D300T, U.S.A.) at a low speed for 30 min.Oil (vegetable and cod liver) was added gradually to ensure the homogeneity of the ingredients.
The immunostimulants {Garlic (Allium sativum L), Echinacea (Echinacea purpurea)} and probiotics {Organic Green TM and Vet-Yeast TM ) were procured from the local market of Egypt.Echinacea (E.purpurea) extract contains 1.5% chicoric acid grade.Organic Green TM contains 1 x 10 11 bacterial cells each from Lactobacillus acidophilus, Bacillus subtilis, Saccharomyces cervisiae and Aspergillus oryzae.One gram (1 g) of Vet-Yeast TM product contains 1 x 10 9 Saccharomyces cervisiae dried cells according to the manufacturers.
The tested immunostimulants and probiotics prepared and mixed with formulated balanced diet where garlic (A.sativum L) used at a rate of 40 g of garlic kg -1 feed, Echinacea (E.purpurea) extract at 4 g of Echinacea kg -1 feed, Organic Green TM at 4 g Organic Green TM kg -1 feed while Vet-Yeast TM at 4 g Vet-Yeast TM kg -1 feed.The feed were prepared each two weeks and the pellets were left for 24 h to air dry, and stored in a refrigerator (4°C).

Bacterial pathogen
A pathogenic A. hydrophila was obtained as a reference strain from *Corresponding author.E mail: salahaly@hotmail.com.Tel: 00201212789838.

Creative Commons Attribution
Author(s) agree that this article remains permanently open access under the terms of the License 4.0 International License Fish Health Management Division of the WorldFish Center, which was isolated from infected tilapia species and was identified by polymerase chain reaction (PCR).The isolate was used in the vaccination trial to test response of the overwintered vaccinated-fry.

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)

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 (10 8 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).

RESULTS
The antibody titer was not different among all the Capital letters compare between columns and small letters compare between rows.Columns with the same letter are not significant different.
Table 3. Relative level of protection of overwintered vaccinated tilapia after challenge infection of all the groups at the end of the 6 th , 8 th and 10 th week PV.(5) Vet-yeast (4 g/kg feed) 50.9 Aa ± 0.67 50.4 Aa ± 0.55 45.8 Ab ± 1.49

Group
Capital letters compare between columns and small letters compare between rows.
Columns with the same letter are not significant different.
treatment groups (Table 2).The antibody titer of the vaccinated overwintered tilapia of the control group initially did not significantly differ but later started to increase at the 6 th -8 th week PV.In the garlic and Echinacea supplemented group, a statistically significant increase began during the 4 th -8 th week PV, while in the organic green and Vet-yeast supplemented groups, a significant increase was seen earlier than in the other groups (2 nd -10 th week PV). A. hydrophila challenge infection of the overwintered-vaccinated tilapia induced the highest mortality in the non-vaccinated control group, followed by the vaccinated control.Lowest mortality was seen in the Vet-yeast supplemented group at the three challenge periods (6 th , 8 th and 10 th week PV).The other groups showed variable results for the three periods of challenge (Table 3).

DISCUSSION
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 nonspecific 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 nonspecific 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., 1996Verlhac et al., , 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.

Table 1 .
Design of the vaccination experiment of overwintering tilapia and challenge following vaccination.

Table 2 .
Antibody titer of overwintered tilapia from the 1 st till the end of the 10 th week PV (Mean ± SE).