IMMUNOLOGICAL STUDIES ON AMMONIA CONTROL IN CULTURED SEABASS

IMMUNOLOGICAL STUDIES ON AMMONIA CONTROL IN CULTURED SEABASS

KHALIL R.H.^*; AMANY M. DIAB^** and EL-GOHARY M.S ^**

^* Department of poultry and fish diseases Faculty of Veterinary Medicine Alexandria University.

^** Department of fish diseases Animal Health Research Institute Kafr El-Sheikh branch.

Email: fishyyanew@yahoo.com                                                                 Assiut University Email: www.aun.edu.eg

INTRODUCTION

Aquaculture has become an economic activity of great importance around the world. Aquaculture practices demand intensive production in shorter times. Under the conditions of intensive production, aquatic species are subjected to high-stress conditions, increasing the incidence of diseases and causing a decrease in productivity (Patricia et al., 2012). Sea bass is a commercially important euryhaline marine fish species which is intensively cultured in the Mediterranean area. The first stages of its culture take place in on-shore tanks and the last stages in sea cages. Lately, the potential of rearing sea bass only in tanks, in extremely high stocking densities, has been investigated (Sammouth et al., 2009). Ammonia and urea are the two main nitrogenous products excreted by teleost fish. Ammonia usually representing 75 to 90 % of nitrogenous excretion. The acute and chronic toxicities of ammonia have been extensively reviewed for freshwater species but ammonia toxicity data for marine fish species or salmonids in sea water are relatively scarce(Fivelstad et al., 1995). In seawater, ammonia is measured as total ammonia nitrogen (TAN), which represents the sum of UIA-N and NH4+-N. (Lemariéa et al., 2004). Safe levels for growth, usually extrapolated from LC50 data, are reported to range from 0.05 to 0.2 mg l-1 UIA-N (Handy and Poxton, 1993), depending on species, age and environment (oxygen concentration, pH). Sub-lethal concentrations of ammonia can damage the gills and also impair immune function leading to increased susceptibility to infectious disease. The 0.26-mg l-1 UIA-N concentration can be considered as a safe long-term limit in seawater for sea bass juveniles (The EFSA Journal, 2008). Probiotics are usually live microorganisms which when administered in adequate amounts confer a health benefits on host. In last few years probiotics became an integral part of the culture practices for promoting growth and disease resistance to procure high production, certain probiotics used as water additives can also play a significant role in decomposition of organic matter, reduction of nitrogen and phosphorus level as well as control of ammonia, nitrite, and hydrogen sulfide (Sha.Ahmadvand et al., 2012). Protexin concentrate contains beneficial probiotics microorganisms (Enterococcus faecium). This product is designed for continuous use to promote efficient digestion and immunity or at   times of stress.

The objective of the current study is conducted to investigate the effect of probiotic Enterococcus faecium "protexin" on immunity and survival of cultured sea bass against ammonia stress.

MATERIALS and METHODS

1. Fish and experimental design: A total of 300 apparently healthy sea bass with an average body weight of 40 ± 5 gram were obtained from private farm. Fish were kept in full glass aquaria measuring (90 Χ 45Χ 45 cm) and maintained in aerated water at 27^oc ± 1^oc, pH 8.3 ± 0.3 and salinity 32 for 7 days prior to use in experiments. The health status was examined throughout the acclimation period during the acclimation fish fed on the pelleted basic diet only contained 45% protein twice daily. Fish were randomly divided equally to ten experimental groups. Protexin probiotic were used and mixed thoroughly with the prepared basal fish diet during its preparation. Half of the water was changed daily.

Table 1: Outline of the experimental design:

*Protexin: Commercial probiotic manufactured by International Ltd UK contain per kg: Enterococcus faecium (NCIMB 11181 E1708). Total Viable Count   2x10¹² CFU. Ingredients: Dextrose Monohydrate. Protein 0.5%. Oil  2.0 %. Fiber 1.0 %. Ash Trace

Ammonium chloride (NH₄CL), was used as a source of ammonia by dissolving ammonium chloride powder (99.5% purity) in distilled water.

UIA-N concentrations were calculated from TAN according to pH, temperature and salinity, using the equation of Bower and Bidwell (1978): %NH3 = 100/ [1 + 10^{(log Ke – pH)} ]

Where log ke as equilibrium constant, is obtained from the expression Johanson and Wedberg (1980): Log ke = (-0.467 + 0.001135S + 2877.9)/ T

where Ke is the dissociation constant, S (in gl^-1) the salinity and T the temperature (°K)

The concentration of the un-ionized ammonia may be obtained from the expression:

NH3 = % NH3 (NH3 + NH4+)

2. Blood collection: At the zero day,4^th and 8^th week of the experiment, 2ml blood samples/fish via the caudal vessels were collected from 3 fish from each group of the experiment according to (Hawak et al., 1965).One ml of blood was collected with syringe containing anticoagulant (Heparin) and used for differential leucocytes count Lucky (1977) and Schalm (1986) as well as phagocytic assay (Kawahara et al.,1991) and the another ml of blood used for serum collection for biochemical determination (Lied et al., 1975). Serum total protein was determined according to Doumas et al. (1981). Serum albumin was determined according to Reinhold (1953). Serum globulin was determined by subtract the total serum albumin from total serum protein according to (Coles, 1974 and Khalil, 2000). Albumin/ globulin ratio was determined by division of serum albumin value on serum globulin value according to (Saffinaz, 2001( Determination of cortisol in serum according to (Gilles et al., 1997). Serum bactericidal activity was determined according to (Rainger and Rowley, 1993) and the results were recorded as survival index (SI) (Word Low and Unlles, 1978). Serum lysozome activity was measured with the turbidimetric method described by Engstad et al. (1992). The result was expressed as one unit of lysozyme activity was defined as a reduction in absorbency of 0.001/min.

3. Challenge test: At the 9th week ten fish from each group were bacteriologically tested and determined to be free from bacterial infection, were artificially infected by I/p injection with 0.2ml/fish of culture suspension of pathogenic Vibrio alginolyticus  previously adjusted to 104. Specificity of death was determined by re-isolation of injected bacteria from freshly dead fish during the period of observation (one week) according to Soliman (1988).

4. Statistical analysis:

The data were statistically analyzed according to (SAS, 2000).

RESULTS

The analysis of variance indicated that although increasing ammonia concentration had a significant negative impact on the differential leucocytic count of seabass but protexin significantly improve lymphocytes and in contrary neutrophils significantly decreased in all treated groups comparing to the control but eosinophils and basophiles had no significant (Table 2) and (Fig.1). Best results were in groups fed on diet contain protexin 0.3 gm./kg feed followed by groups fed diet contain 0.2 gm./kg feed and groups fed on diet contain 0.1 gm. /kg feed respectively.

Regarding the effect of ammonia on phagocytic activity as well as phagocytic index and cortisol level of seabass (Table 3) and (Fig.2) showed, significant decrease in phagocytic activity and phagocytic index and significant increase in cortisol level of seabass by increasing ammonia concentration in comparison to the control group.  Protexin improve the previous picture to the favor of nearly maintain phagocytic activity and phagocytic index and decreasing the increasing of cortisol level with increasing ammonia concentration.

Examination of serum indicated that protexin adverse the negative effects of ammonia on serum total protein, globulin and Albumin /globulin ratio in all treated groups comparing to the control specially at the 8th week. Best results showed in groups fed on diet contain protexin 0.3 gm. /kg feed (Table 4) and (Fig.3).

Concerning to the effect of protexin supplemented diet on the negative impacts of increasing ammonia concentrations on  the serum bactericidal activity as well as serum lysozyme activity among different groups of seabass during experimental period revealed decrease in their activities in all protexin treated groups by increasing ammonia concentrations but their activities still higher comparing to the control. Best results were in in groups fed on diet contain protexin 0.3 gm. /kg feed (Table 5) and(Fig.4).

Mortalities of seabass challenged with Vibrio alginolyticus were significantly lower in all treated groups than the control but mortalities increased by increasing the ammonia concentrations moreover the lowest mortality percent was recorded in groups fed on diet contain 0.3 gm./kg feed of protexin followed by groups fed diet contain 0.2 gm./kg feed then groups fed on diet contain 0.1 gm./kg feed respectively (Table 6) and(Fig.5).

Table 2:      Effects of different treatments on differential leucocytic counts of sea bass during experimental period:

Means in the same column with the different small litter at the same week are significantly different at (P>0.05)

Fig. 1: Differential leucocytic counts of seabass during experimental period

Fig. 2: Cotrisol level, phagocytic activity and phagocytic index of seabass during experimental period.

Table 3: Effects of different treatments on cortisol level, phagocytic activity and phagocytic index of seabass during experimental period:

Means in the same column with the different small litter at the same week are significantly different at (P>0.05)

Table 4: Effects of different treatments on total serum protein, albumin, globulin and A/G ratio of seabass during experimental period.

Means in the same column with the different small litter at the same week are significantly different at (P>0.05)

Fig. 3: Total serum protein, albumin, globulin, and A/G ratio of sea bass during experimental period.

Fig. 4: Serum lysozyme and bactericidal activity of seabass during experimental period

Table 5: Effects of different treatments on serum lysozyme and bactericidal activity of seabass during   experimental period:

 Means in the same column with the different small litter at the same week are significantly different at (P>0.05).

Table 6: Effects of different treatments on mortality percent of seabass after challenge with Vibrio alginolyticus

Fig. 5: Mortality percent of seabass after challenge with Vibrio alginolyticus

DISCUSSION

Ammonia exposure may act as a stressor and induce some adaptive endocrine responses and complex metabolic adaptations (Mommsen et al., 1999). Fish experiencing stress show a number of physiological changes that are expressed through a number of particular indicators.

In general stress affects aquatics through weakening immune system (Mahshid and Leila, 2013).

A lot of bacterial cultures containing nitrifying bacteria to control the ammonia level in culture water are available commercially. Nitrifies are responsible for the oxidation of ammonia to nitrite and subsequently to nitrate (Asaad et al., 2013).

Some Lactic acid bacteria (LAB) have proven to be very useful probiotics in different animals including fish / shellfish. The promising probiotic strains of LAB include the members of genera Lactobacillus, Lactococcus and Enterococcus from the representative species Enterococcus faecium (E.faecium). Similarly LAB can improve haematological indices such as hemoglobin concentration, haematocrit percentage, total erythrocytes counts, total leucocytes counts, total serum protein, globulin, glucose, and cholesterol level in fish. Several immunological studies have confirmed the ability of some LAB to stimulate fish immune systems and have been found to elevate several immunological parameters like phagocytic activity, lysozyme level, peroxidase/anti-peroxidase level, complement activity and respiratory burst activity in teleosts (Sukanta, 2013).

Haematological and biochemical parameters have been acknowledged as valuable tools for monitoring fish health.

The results indicated that although increasing ammonia concentration had a significant negative impact on the differential leucocytic count but protexin significantly improve lymphocytes and incontrary neutrophils significantly decreased in all treated groups comparing to the control. These findings were nearly obtained by Manal A. A. Essa   et al. (2012) after supplementation of E.faecium in the diet of Oreochromis niloticus but without using ammonia as stress.

The present study showed, significant decrease in phagocytic activity and phagocytic index; similar results were obained by Winton et al. (2004) where ammonia caused a depression in immune Parameters as phagocytic activity of H. diversicolor supertexta but Protexin improve the previous picture to the favor of nearly maintain phagocytic activity and phagocytic index as the control group these results nearly supported by (Sukanta, 2013).

It is well known that cortisol plays an adaptive function against stressors. However, chronically elevated cortisol levels may become damaging for several physiological functions e.g. suppression of the immune system (Person et al., 2003). In the present study, serum cortisol was measured as an indicator of primary stress response. The analysis of serum cortisol levels carried out in this study gave the evidence that the groups fed with protexin showed a better tolerance to ammonia as stress rearing conditions. This was evidenced by the lower cortisol levels detected in the treated experimental groups with comparing to control nearly these results  were supported by (Carnevali and Sulpizio 2006) where cortisol levels obtained in the probiotic treated European seabass juveniles (Dicentrarchus labrax,L.), fishes were significantly lower than those in the control that untreated with probiotic.

With increasing concentration of Total Ammonia Nitrogen (TAN), haemoglobin and serum protein content were reduced in mrigal (Cirrhinus mrigala) (Hamilton) (Das et al., 2004) similar results were observed in the present study but with increasing protexin concentration in seabass feed the serum total proteins as well as total globulin levels were significantly improved in all treated groups than the control. Similar results were also obtained after E.faecium supplementation in the diet of O.niloticus by Manal A. A. Essa et al. (2012) but without using ammonia as stress.

Lysozyme, being an enzyme with antibacterial activity, can split peptidoglycan in bacterial cell walls especially of the gram positive species and can cause lysis of the cells. The Lysozyme increased with activation of immune system.

The present study revealed decreasing lysozyme activity as well as Bactericidal activity during the experimental period in all treated groups by increasing ammonia concentrations but using Enterococcus faecium relatively improve this picture to be better than the control, These results supported by Kim et al. (2012) in  investigation of the effect of a probiotic, Enterococcus faecium, on olive flounder (Paralichthys olivaceus) moreover also (Taoka et al., 2006) showed that viable probiotics administered to Japanese flounder (Paralichthys olivaceus), increased nonspecific immune response, determined by parameters such as lysozyme activity, neutrophil migration and bactericidal activity, which improved the resistance of fish to infection. On contrary, dietary supplementation of probiotics like Enterococcus faecium in O.niloticus failed to elevate lysozyme level (Wang et al., 2008) and (Zhou et al., 2009). This is may be due to different fish species and water.

Concerning mortalities of ammonia stressed seabass challenged with Vibrio alginolyticus were significantly lower in all treated groups than the control but mortalities increased by increasing the ammonia concentrations moreover the lowest mortality percent was recorded in groups fed on diet contain 0.3 gm./kg feed of protexin. These results supported by Liu and Chen (2004) as well as Cheng et al. (2004) and (Winton et al., 2004) where ammonia in water caused a depression in the immune response and an increase in mortality of Litopenaeus vannamei from the V. alginolyticus infection and mortality of H. diversicolor supertexta from V. parahaemolyticus respectively. The obtained results in the present study may be attributed to that ammonia caused suppression of the cellular or the non-specific defense mechanisms or to the effect of ammonia on other physiological systems (vascular, respiratory, etc.) Hurvitz et al. (1997) where studying  the effect of sublethal concentrations of ammonia on rainbow trout (Oncorhynchus mykiss Walbaum), vaccinated against and challenged with Streptococcus iniae survival and the antibody response referred  decreasing of protection against S. iniae in ammonia-exposed trout.  Inclusion of protexin in seabass feeding have positively impacted the resistance of fish to Vibrio alginolyticus infection as was indicated by significantly lower mortality rates of the ammonia treated fish challenged by Vibrio alginolyticus in comparison to the control. These results were supported by Krummenauer et al. (2009) after dietary application of E.faecium to shrimp challenged by Vibrio parahaemolyticus as well as Gopalakannan and Arul (2011) after challenging E.faecium MC13 supplemented Cyprinus carpio by Aeromonas hydrophila.

CONCLUSION

It could be concluded that supplementation of protexin (Enterococcus faecium) as probiotic in seabass feeding have positively impacted the resistance of fish to stress (sub-lethal concentrations of ammonia) as was indicated by  significant increasing of lymphocytes, total protein, globulin, phagocytic activity, index of phagocytes, lysozyme activity and bactericidal activity which enhanced the resistance of challenged fish to Vibrio alginolyticus as was indicated by significant decrease in mortalities rates in protexin treated groups than the control. The highest dietary level 0.3 gm. /kg feed of protexin showed best results than 0.2 gm./kg feed then 0.1 gm./kg feed. For seabass ammonia stress tolerance Enterococcus faecium supplement should be recommended.

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