major carp Labeo rohita (Ham.)

Aquaculture Research, 2006, 37, 1215^1221

doi:10.1111/j.1365-2109.2006.01551.x

Evaluation of Bacillus subtilis as a probiotic to Indian major carp Labeo rohita (Ham.) Rajesh Kumar1, Subhas C Mukherjee1, Kurcheti Pani Prasad1 & Asim K Pal2 1

Aquatic Animal Health Management Division, Central Institute of Fisheries Education (CIFE), Mumbai, India Nutrition and Biochemistry Division, CIFE, Mumbai, India

2

Correspondence: S C Mukherjee, Aquatic Animal Health Management Division, Central Institute of Fisheries Education (CIFE), 7 Bungalows,Versova, Andheri (W), Mumbai 400061, India. E-mail: subhasmukherjee@yahoo.com

Abstract

Introduction

Bacillus subtilis, a Gram-positive, aerobic, endosporeforming bacterium, was evaluated for its probiotic potential in Indian major carp, Labeo rohita. Labeo rohita (15 2 g) were fed a feed containing B. subtilis in three concentrations for 2 weeks, e.g., 0.5 (T2), 1.0 (T3) and 1.5 (T4) 107 CFU g 1 feed. The control group (T1) was fed feed without B. subtilis for the same period. Haematological and serum parameters were monitored at weekly intervals. The response variables were total erythrocyte count, total leucocyte count (TLC), haemoglobin, total protein, albumin, globulin, albumin^globulin ratio, alkaline phosphatase activity, alanine aminotransferase activity and aspartate aminotransferase activity. Fish were challenged intraperitoneally with a virulent strain of Aeromonas hydrophila after 2 weeks of feeding to the treatment groups and positive control group, while the negative control group was challenged with phosphate-bu¡ered saline only. Clinical signs and symptoms, and mortality/survival percentage were noted in each group. The haematological and serum parameters were monitored each week and during post challenge on the third and tenth day. The B. subtilis-treated ¢sh (T4,1.5 107 CFU g 1 feed) showed maximum per cent survival (87.50%), weight gain (35.5%), TLCs (3.23 104 cells mm 3), haemoglobin content (7.4 g%), total protein (2.37 g dL 1) and globulin content (1.28 g dL 1) during the pre-challenge. Enzymes showed higher activities during post challenge (Po0.05). The result suggests that B. subtilis can be used e¡ectively as a commercial product for use in aquaculture.

The indiscriminate use of antibiotics to control diseases without knowledge of dose and pharmacokinetic data on the ¢sh poses serious environmental hazards (Baticados & Paclibare 1992). Besides this, several antibiotics including oxytetracycline often cause immunosuppression in many ¢sh (Lunden, Miettinen, Lonnstorm, Lilius & Bylund 1998). Moreover, when antibiotics are used in the lowest possible doses for economic reasons to avoid side e¡ects and to lessen environmental impact (Scotts 1993), the possibility of resistance of pathogen to the action of antibiotic increases. These pathogens are often responsible for further spreading of diseases, especially under stressful ¢sh culture conditions (Bullock & Stuckey 1975). Intensive aquaculture practices have necessitated the development of an individual’s resistance to disease rather than depending upon antibiotics and chemotherapeutics or vaccines. The role of gastrointestinal micro£ora in disease resistance has been established in human and veterinary medicine, which has led to the concept of manipulating gastrointestinal micro£ora for better health management (Fuller 1994; Kaur, Chopra & Saini 2001). In the last several decades, a number of trials have been conducted in di¡erent livestock with various indigenous and exogenous useful microorganisms called ‘probiotics’ to boost gastrointestinal micro£ora to ¢ght against infectious diseases (Salminen, Ouwehand, Benno & Lee 1999). Probiotics are bene¢cial microorganisms that protect the host from diseases. Fuller (1992) de¢ned probiotics as ‘live microbial feed supplements which bene¢cially a¡ect the host by improving its intestinal microbial balance’. Microbes play very important and critical roles in aquaculture systems, both at

Keywords: Bacillus subtilis, probiotic, Labeo rohita, haematological parameters, serum parameters, Aeromonas hydrophila r 2006 The Authors Journal Compilation r 2006 Blackwell Publishing Ltd

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E¡ect of B. subtilis on growth and survival of L. rohita R Kumar et al.

hatchery and the grow-out level, because water quality and disease control are directly a¡ected by microbial activity. Probiotic protection can be due to mechanisms such as nutritional competition and/or production of antibacterial substances. Probiotics must be considered as potentially useful for the control of ¢sh diseases (Irianto & Austin 2002). In aquaculture, persistent disease problems necessitate the use of bacteria as probiotics and as alternatives to antibiotics. There are several microbial strains used as probiotics in aquaculture systems. The common probiotics used in aquaculture, belonging to Lactobacillus sp., Bacillus sp., Bi¢dobacterium sp.,Vibrio sp., Saccharomyces sp., Enterococcus sp. Bacillus subtilis, are now being used for oral bacteriotherapy in aquaculture. Ingestion of appropriate quantities of B. subtilis is anticipated to revive the normal micro£ora after antibiotic use or critical illness (Green, Wakeley, Page, Barnes, Baccigalupi, Ricca & Cutting1999). Bacillus sp. NM 12 isolated from the intestinal content of Japanese coastal ¢sh (Anaora tentaculata) produce a wide spectrum of antibacterial substances (Sugita, Hirose, Matsuo & Deguchi 1998). The present study has been proposed to test the e⁄cacy of B. subtilis, a gastrointestinal bacterium isolated from an Indian major carp, Cirrhinus mrigala, as a candidate probiotic species for use in aquaculture.

Material and methods Fish and husbandry Labeo rohita, the most favoured ¢sh among Indian major carp, was selected for the study. Fingerlings of L. rohita, average length 12 cm and weight 15 2 g, were obtained from a carp farm at Berar, Mumbai, and were used in the experimental studies. Fish were acclimatized for 2 weeks before experimentation. Chlorine-free tap water was used throughout the course of the experiment. The physico-chemical characteristics of the test water were as follows: temperature, 27 1 1C; hardness, 82 mg L 1 (as CaCO3); alkalinity, 85 mg L 1; pH, 7.4 and dissolved oxygen concentration, 6.8 mgL 1.

Mass culture of B. subtilis Bacillus subtilis, a gastrointestinal bacterium isolated from C. mrigala, was obtained from Aquatic Animal Health Management Division, CIFE, Mumbai. A pure culture of B. subtilis was inoculated into a conical

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£ask (500 mL) containing nutrient broth and incubated at 30 1C for 24 h in a shaker incubator. The culture was centrifuged at 10 000 g for 20 min at 4 1C and the supernatant was discarded, while the pellet was re-suspended in phosphate-bu¡ered saline (PBS; pH 7.2). The suspension was similarly washed and recentrifuged three to four times and then quanti¢ed by the spread plate technique (nutrient agar, incubated at 30 1C for 24 h). Puri¢ed and quanti¢ed bacteria were kept at 4 1C in suspended form and were used for feed preparation as required. Experimental set-up Ten rectangular ¢bre tanks (100 L) were arranged in the wet lab of Central Institute of Fisheries Education with aeration. The tanks were ¢lled with 70 L of water and 12 ¢ngerlings were stocked in each tank. Four tanks were kept as controls, two positive (T1) and two negative (T5), while the other six were treatment tanks (T2, T3 and T4). Treatment groups T2, T3 and T4 were fed diets containing one of three concentrations (5, 10 and 15 mL containing 108 CFU mL 1) of B. subtilis for 15 days and the control groups received the same feed without B. subtilis. The experimental ¢sh were challenged with a virulent strain of Aeromonas hydrophila at 106 CFU mL 1 (A. hydrophila isolated from infected L. rohita and identi¢ed through biochemical tests and serial infections were developed to L. rohita with isolated A. hydrophila to check their virulency) after 15 days of feeding to the three treatment groups (T2, T3, T4) and positive control but the negative controls (T5) were injected intraperitoneally with PBS only. Five groups in duplicate were made: (1) T1 ^ positive control; (2) T2 ^ 5 mL of B. subtilis at 108 CFU mL 1/100 g feed ingredients; (3) T3 ^ 10 mL of B. subtilis at 108 CFU mL 1/100 g feed ingredients; (4) T4 ^ 15 mL of B. subtilis at 108 CFU mL 1/100 g feed ingredients and (5) T5 ^ negative control. Haematological studies Blood was drawn from the caudal peduncle region using sterile 2 mL syringes rinsed ¢rst with 2.7% EDTA solution. Blood was collected in small glass vials coated with 20 mL of 2.7% EDTA solution. The amount of haemoglobin present in the blood was measured from 20 mL of blood using the cyanmethaemoglobin method. Another 20 mL of blood was used to determine the total erythrocyte and the total leucocyte counts (TLCs).

r 2006 The Authors Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 1215^1221



vial. The mixture was shaken well to suspend the cells uniformly in the solution. Then, the cells were counted using a haemocytometer and expressed as

Table 1 E¡ect of Bacillus subtilis on weight gain (%) of Labeo rohita during pre-challenge and survival (%) after the challenge with Aeromonas hydrophila Treatments

Survival (%)

T1 T2 T3 T4 T5

18.75d 68.75c 81.25b c 87.50ab 100.00a

Number of WBC=mm3 ¼ N 500

WGw (%) 30.76c 32.87b 34.82a b 35.55a

6.25 6.25 6.25 0.00 0.00

–

where N denotes the total number of WBCs counted in four squares of the haemocytometer. The factor obtained after taking into consideration the initial dilution factors was 500. Blood haemoglobin content was analysed following the Cyanmethaemoglobin method using Darbkins Fluid (Qualigens Diagnostics Kit, Mumbai, India). Twenty microlitres of blood was mixed with 5 mL of Darbkin’s working solution. The absorbance was measured using a spectrophotometer at a wavelength of 540 nm. The ¢nal concentration was calculated after comparing with the standard. Test 251 60 Standard 100

0.24 0.37 0.42 0.80

Means in the same column with di¡erent superscripts are signi¢cantly di¡erent (Po0.05). wT1, positive control (L. rohita were fed feed without B. subtilis and challenged with A. hydrophila ); T2, T3 and T4 are the treatment groups in which L. rohita were fed feed containing B. subtilis at 0.5 107, 1 107 and 1.5 107 CFU g 1 feed respectively; T5, negative control (L. rohita were fed feed without B. subtilis and challenged with phosphate-bu¡ered saline only). Survival (%) 5 (number of ¢sh survived after challenge/initial number of ¢sh) 100. Weight gain (WG, %) 5 (¢nal weight after 15 days of B. subtilis feeding initial weight)/initial weight 100.

Haemoglobin content was expressed as g dL 1. Serum chemistry

For the total erythrocyte count (TEC, Schaperclaus, Kulow & Schreckenbach 1991), 20 mL of blood was mixed with 3980 mL of red blood cell (RBC) diluting £uid (Hayemis) in a clean glass vial. The mixture was shaken well to suspend the cells uniformly in the solution. The cells were counted using a haemocytometer (Feinoptik, Blakenburg, Germany) and expressed as:

Blood was collected from the caudal region of ¢sh without rinsing the syringe with anticoagulants and collected in an Eppendorf tube. The blood was allowed to clot for 45 min in an inclined position at room temperature, followed by 30 min incubation at 4 1C and then centrifuged at 3000 g for10 min at 4 1C. Serum was collected in a sterilized Eppendorf tube and analysed for the di¡erent serum parameters in an AR 601, semiautomatic analyzer (Qualigens Diagnostics Kit). For all of the serum parameters, Qualigen kits were used. The parameters that were analysed by a semiautomatic analyzer (AR 601, Qualigens Diagnostics Kit) were total protein (biuret method using bu¡ered dye reagent and biuret reagent, Qualigens Diagnostic Kits), albumin (bromocresol green binding method, Qualigens

Number of RBC=mm3 ¼ N 10 000 where N is the total number of counted in ¢ve squares of the haemocytometer and 10 000 is the factor obtained after taking into consideration the initial dilution factor (Tables 1^4). For the (TLC, Schaperclaus et al. 1991), 20 mL of blood was mixed with 3980 mL of white blood cell (WBC) diluting £uid (Dacies £uid) in a clean glass

Table 2 E¡ect of Bacillus subtilis on TEC, TLC and Hb content of Labeo rohita before and after challenge with Aeromonas hydrophila TEC ( 106 cells mm 3) Treatments T1 T2 T3 T4 T5

Pre-challenge bA

1.058 1.163aA 1.205aA 1.180aA

–

0.02 0.04 0.05 0.06

TLC ( 104 cells mm 3)

Post-challenge bB

0.800 1.073aA 1.175aA 1.160aA 1.092a

0.03 0.05 0.05 0.03 0.03

Pre-challenge B

3.013 3.17B 3.20B 3.236B

–

0.11 0.11 0.05 0.13

Hb3 (g%)

Post-challenge aA

3.610 3.907aA 3.970aA 3.967aA 3.080b

0.11 0.16 0.16 0.25 0.18

Pre-challenge A

7.143 7.227A 7.350A 7.4A

0.05 0.09 0.11 0.11

Post-challenge 5.802B 5.760B 6.212B 6.535B 7.037

0.41 0.28 0.32 0.37 0.06

Mean values in rows and columns containing the same superscript for individual parameters do not vary signi¢cantly (P40.05). TEC ( 106 cells mm 3), total erythrocyte count, in millions of cells per cubic millimetre; TLC ( 104 cells mm 3), total leucocyte count; Hb3 (g%), haemoglobin content of blood in gram%.


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Table 3 E¡ect of Bacillus subtilis on total protein, albumin, globulin content and A/G ratio of Labeo rohita before and after challenge with Aeromonas hydrophila Albumin (g dL 1) Globulin (g dL 1) A/G TP (g dL 1) Treatments Pre-challenge Post-challenge Pre-challenge Post-challenge Pre-challenge Post-challenge Pre-challenge T1 T2 T3 T4 T5

2.206 2.287 2.37 2.377

–

0.08 0.01 0.1 0.16

2.102 2.257 2.347a 2.287b 2.26b

0.06 0.10 0.07 0.02 0.06

1.116 1.101 1.02 1.09A

–

0.06 0.04 0.06 0.05

1.095 1.040 0.972 0.890B 1.115

0.05 1.073c 0.03 1.307A 0.12 1.043a 0.01 1.185b 0.06 1.317 0.16 0.931abA 0.05 1.215b 0.05 1.475 0.13 0.831bA 0.06 1.287a 0.11 1.397 0.08 0.863bA 0.01 – 1.157 0.03

–

0.06 0.04 .0.05 0.05

Post-challenge 0.800 0.660B 0.612B 0.647B 0.961

0.10 0.07 0.10 .0.08 0.03

Mean values in rows and columns containing the same superscript for individual parameters do not vary signi¢cantly (P40.05). TP (g dL 1), total protein content in gram/decilitre; albumin (g dL 1), albumin content in gram/decilitre; globulin (g/dL), globulin content in gram/decilitre; A/G, albumin^globulin ratio.

Table 4 E¡ect of Bacillus subtilis on ALP, AST and ALT activity of Labeo rohita before and after challenge with Aeromonas hydrophila ALP (U L 1)

AST (U L 1)

ALT (U L 1)

Treatments

Pre-challenge

Post-challenge

Pre-challenge

Post-challenge

Pre-challenge

Post-challenge

T1 T2 T3 T4 T5

65.050B 72.466 70.634 68.834

91.600A 86.625 87.400 89.525 64.875

52.067B 52.983B 54.200B 56.333B

106.450aA 77.825bA 74.025bA 70.625bA 52.800b

14.783B 16.357B 17.586B 18.195B

20.000A 25.025A 25.250A 20.475A 18.775

–

2.00 4.66 5.50 4.30

7.61 7.77 6.29 10.82 6.08

–

1.77 1.44 1.77 4.43

7.23 8.95 8.91 10.13 4.14

–

0.46 0.84 1.59 1.53

0.55 3.94 2.15 2.55 1.64

Mean values in rows and columns containing the same superscript for individual parameters do not vary signi¢cantly (P40.05). ALP, alkaline phosphatase activity, moles of substrate used up per minute of incubation per litre at 37 1C; AST, aspartate aminotransferase, moles of substrate used up per minute of incubation per litre at 37 1C; ALT, alanine aminotransferase, moles of substrate used up per minute of incubation per litre at 37 1C.

Diagnostic Kits), alkaline phosphatase (kinetic colorimetric method, Qualigens Diagnostic Kits), aspartate aminotransferase (AST, modi¢ed IFCC method, Qualigens Diagnostic Kits) and alanine aminotransferase (ALT, modi¢ed IFCC method, Qualigens Diagnostic Kits). Statistical analysis Signi¢cant di¡erences among treatment groups were tested by one-way analysis of variance (ANOVA), and the comparison of any two mean values was made by Duncan’s multiple range test. A signi¢cance level of Po0.05 was used. The mean values for pre- and post-challenge parameters were compared by a Student’s t-test. All the statistical analysis was performed by using the software program SPSS (version 11). Results

higher (Po0.05) in the treatment groups (T2, 68.75%; T3, 81.25% and T4,87.50%) fed feed containing B. subtilis compared with control (T1, 18.75%). Clinical signs observed after challenge The ¢sh were sluggish and gradually lost their equilibrium 24^48 h after challenge with A. hydrophila. The clinical signs were characterized by hyperaemic condition on the ventral side of the body, a visibly swollen abdomen and a slightly protruding reddish vent. The eyes of the infected ¢sh were opaque and during the terminal stages the animals were seen £oating dorsal side down at the water surface. They were collected before death and sacri¢ced. The abdomen was distended due to accumulation of £uid in the peritoneal cavity. The liver and the kidney were congested and pulpy. These changes were not evident in B. subtilis fed groups (T2,T3,T4).

Weight gain and survival Percentage weight gain was signi¢cantly higher (Po0.05) in (T4, 35.55%) the treatment groups, fed feed containing B. subtilis. Survival was signi¢cantly

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Haematological studies The TEC was signi¢cantly higher (Po0.05) in T4 (1.180 0.06) than in the control group of pre-




treatment. There was a signi¢cant di¡erence (Po 0.05) in the TEC between the pre and post challenge of T1. Among the post-challenge groups, T1 showed signi¢cantly less TEC than the others. TheTLCs in the post-challenge groups were higher than pre-challenge levels. There was an increase in the TLC level in the T2 (3.170 0.11), T3 (3.200 0.05) and T4 (3.236 0.13) but not at a signi¢cant level (P40.05). The haemoglobin content between pre- and postchallenge of all treatments di¡ered signi¢cantly (Po 0.05) but there was no signi¢cant di¡erence among the treatment groups of pre- or post-challenge. Serum chemistry Although total protein content in pre-challenge treatments (T2, 2.287 0.01; T3, 2.370 0.1 and T4, 2.377 0.16) had increased with respect to control (T1, 2.206 0.08), the change was not signi¢cant (P40.05). From the result it has been recorded that in post-challenge, the total protein content was signi¢cantly higher (Po0.05) inT3 (2.347 0.07). There was no signi¢cant di¡erence in albumin content among the pre- and post-challenge treatments. T4 recorded a signi¢cant change (Po0.05) in albumin content (1.090 0.05) between pre and post challenge. All the treatment groups (T2, 1.185 0.06; T3, 1.215 0.05 and T4, 1.287 0.11) fed feed containing B. subtilis showed a signi¢cantly high (Po0.05) level of globulin than the control (T1, 1.073 0.03) during the pre-challenge period. Between the preand post-challenge T1 recorded a signi¢cant di¡erence (Po0.05). The albumin^globulin ratio was signi¢cantly higher (Po0.05) in T1 (1.043 0.06) and signi¢cantly lower in T3 (0.831 0.05) during the pre-challenge. Alkaline phosphatase activity of T1 was signi¢cantly less (Po0.05) during the pre-treatment compared with the post-challenge. There was no signi¢cant change (P40.05) in alkaline phosphatase activity of the post-challenge treatment, although alkaline phosphatase activity was the lowest in T4. There was a signi¢cant increase (Po0.05) in AST activity of T1 (106.45 7.23) in post-challenge with respect to T2, T3, T4 and T5. During post-challenge, the AST activity was signi¢cantly higher (Po0.05) in all the treatments in comparison with pre-challenge. There was a signi¢cant change (Po0.05) in the ALT activity between the pre- and post-challenge; post-challenge showed a higher ALT activity.

Discussion The colonization of B. subtilis in the gut epithelium reduced the risk of pathogenic bacterial infection and hence ¢sh can develop the capacity to protect themselves from various diseases. Duncan and Klesius (1996) had reported that the percentage of erythrocytes was signi¢cantly higher in ¢sh fed diets containing S. cervisiae. Signi¢cant increases in the TEC during pre-challenge in B. subtilis fed groups are an indication of improved health of the ¢sh. The reduction in RBC count after challenge is attributed to the ability of the bacteria to haemolyse erythrocytes as explained by Oliver, Lallier and Lariviere (1981). There was a slight decrease in the mature erythrocyte count and haemoglobin content in yellowtail, Seriola quinquiradiata, infected with Nocardia kampachi (Ikeda, Ozaki, Hayama, Ikeda & Minami 1976). In our study, the reduction in total RBC count was minimal in T3 and T4 in comparison with the control T1 after challenge. This indicated that the effect of A. hydrophila was minimal in T3 and T4. Therefore, it was obvious that the growth of A. hydrophila was inhibited to a certain extent in the ¢sh fed diet containing B. subtilis. Haemoglobin content showed a trend similar to RBC. Total leucocyte count increased in ¢sh fed feed containing B. subtilis compared with ¢sh fed feed without B. subtilis. Total leucocytes count increased in yellowtail infected with N. kampachi (Ikeda et al. 1976). The result from the present experiment also revealed an increase in TLC in treatment groups (T2,T3 and T4) compared with the control (T1 and T5). This indicated the heightened immune response in the ¢sh fed feed containing B. subtilis, probably due to its the immunostimulatory e¡ect. The increase in the serum protein, and globulin levels is thought to be associated with a stronger innate response in ¢sh (Wiegertjes, Stet, Parmentier & Muiswinkel 1996). The decrease in the A:G ratio is indicative of better immunity of the animal, which may occur due to an increase in the globulin level compared with albumin. The increase in total serum protein and globulin indicates that ¢sh are immunologically strong (Nayak, Das, Kohli & Mukherjee 2004). Enzymatic activity is mainly dependent on the transport mechanism in the liver, kidney and intestinal mucosa (Vasudevan & Sreekumari 1998). The activity of ALP may increase several times during disease conditions such as infective hepatitis, cholestasis and other conditions (Jiro1989; Donald1992). In


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the present study, there was no signi¢cant change in ALP activity during pre-challenge but in post-challenge the ALP activity of T1 was high compared with other treatment groups. This may be due to pathological manifestation in the liver like hepatitis, necrotic changes in hepatic cells and constriction of the biliary ducts during the diseased condition. The decreased ALP activity in the treatment groups (T2, T3, T4) in comparison with T1 may be due to the e¡ect of B. subtilis on the immune system, which conferred su⁄cient protection to the vital organs of ¢sh. The increase in the activity of AST and ALT is mainly attributed to cortisol-induced catabolism of protein for provision of metabolic energy (Freeman & Idler 1973). During experimental A. hydrophila infection in gold¢sh, Carassius auratus, AST activity increased while the ALT activity decreased in the plasma when compared with the control during the ¢rst 36 h post infection (Brenden & Huizinga 1986). The elevated AST values in the present study may be due to the pathological manifestations leading to liver dysfunction in experimental ¢sh. In the present study, an increased growth rate was observed in L. rohita fed feed containing B. subtilis compared with control. The survival rate after challenge with A. hydrophila was signi¢cantly higher in the treatment group compared with the control. Administration of yeast glucan enhances the survival of carp infected with A. hydrophila (Selvaraj, Sampath & Sekar 2005). The high rates of establishment of bacterium in the gastro-intestinal tract of ¢sh treated with B. subtilis have suppressed the A. hydrophila infection, which ultimately resulted in the higher survival. The observed improvement in ¢sh immune parameters as well as growth and survival using probiotic bacteria may open a new chapter for screening new strains of probiotic bacteria for extensive use in aquaculture. Probiotics have an important role in disease control strategies for aquaculture, and may provide an alternative to the use of antimicrobial compounds (Verschuere, Rombaut, Sorgeloes & Verstraete 2000). The result suggests that the strain of B. subtilis used in this study can be used e¡ectively as a commercial product for use in aquaculture. Acknowledgments The senior author is grateful to the Indian Council of Agricultural Research for the ¢nancial support in the form of a junior research fellowship.

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