World Journal of Fish and Marine Sciences 4 (4): 360-368, 2012ISSN 2078-4589© IDOSI Publications, 2012DOI: 10.5829/idosi.wjfms.2012.04.04.6389

Corresponding Author: B. Rohini, CAS in Marine Biology, Annamalai University, Parangipettai-608502, Tamil Nadu, India.

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Immunostimulatory Effect of VariousFrequencies of the Marine Vibrio alginolyticus on Juvenile

Penaeus monodon Against WSSV and its Biochemical Properties

B. Rohini, W. Sylvester Fredrick, N. Sandhya and D. Subhashini

Sathyabama University, Chennai, India

Abstract: In the present study, formalin killed Vibrio alginolyticus based immunostimulants were tested indifferent frequencies by mixing in feed. These immunostimulants mixed with feed were tested on juveniles ofPenaeus monodon. The PCR analysed animals that showed negative for White Spot Syndrome Virus (WSSV)were taken for the study. The growth, survival and biochemical properties of the immunostimulated shrimpsshowed better results than the control. The prophenoloxidase activity was checked in the shrimps and itshowed high values based on the frequency of the stimulant. The WSSV challenge test of shrimps confirmsthat the immunity improved with immunostimulant feeding. However the frequency of feeding immunostimulantto the shrimp has a vital role in the immunity development and health status of the shrimp. The study delineatesthat immunostimulant, if given in the highest frequency (Daily), gives higher immunity and improves the growthand survival. However, if the immunostimulant, at the next higher frequency of weekly thrice and twicerespectively does improve the growth and survival but was not as effective as the other frequencies.

Key words: Immunostimulants % PCR % Challenge Test % Proximate Composition % WSSV

INTRODUCTION certain parasites, while those of non-infectious etiology

Shrimps form an important component in the fauna of Among the infectious diseases white spot disease hasestuarine and coastal waters and nearly 2500 species are posed a serious challenge to the shrimp cultureknown, of which slightly less than 300 are of economic operations. Traditional treatment of shrimp diseases isinterest. The genus Penaeus occurs in tropical and usually done by the use of chemical and antibioticsubtropical waters, between 40°N and to 40°S and is the products. In chemical treatment, huge amount ofmost important commercial group. Among shrimps, chemicals are required to produce the desired therapeuticpenaeids constitute the major aquaculture species effect and after effects to the ecosystem hitherto arebecause of its faster growth rate as a result of high unknown [3]. The use of antibiotics and development ofambient temperature tolerance. Availability of the plasmid mediated resistant strains of bacteria andjuveniles in inshore water is subjected to strong accumulation in the shrimp restricts continuous use of theenvironmental driven variability in recruitment and stock antibiotics [4]. However, this indiscriminate usage ofsize [1]. Economically, shrimps are more viable and cost chemicals and antibiotics and its after effects toeffective as compared to fishes. However, the life cycle of ecosystem has led to complete ban of these products [5].many penaeid shrimps occurs between the marine and the Shrimp and other decapods crustaceans have a wellestuarine environment for reproduction while post larvae defined mechanism of clearing bacteria and other invadingand juvenile stages are more adapted to tolerate the rapid foreign particles from their hemolymph. Modulatorenvironmental variations of the estuaries and hence proteins in crustaceans can recognize various cell wallspend most part of the growing period in these areas. components of bacteria and fungi resulting in enhanced

Cultured shrimps suffer from various diseases due to fighting capabilities against the invading pathogen [6].infectious and non infectious causes. Infectious diseases This variety of defense responses mostly originates fromof viral etiology are caused by viruses, bacteria, fungi and the hemocytes [7]. The shrimp hemocytes are involved in

are caused by unhealthy environmental conditions [2].

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defense processes such as phagocytosis, encapsulation, changes in shrimp in response to the immunostimulantmelanization and coagulation. Many humoral defense administration and biochemical changes in shrimp aftermolecules such as prophenoloxidase (proPO), stimulation.antimicrobial peptides and clotting factors are present inthe hemocyte granules, which are released when MATERIALS AND METHODSstimulated by pathogen invasion.

The use of probiotics, immunostimulants, Experimental Design: The juveniles of Penaeus monodonimmunomodulants, reduction in stocking density, reduced from single broodstock was obtained from a commercialwater exchange etc., are just some strategies that have hatchery. The shrimps were subjected to PCR test beforelead to a decrease in the incidence of diseases as well as the immunostimulant administration to rule out thereduction in environmental impact [8]. The mechanism of presence of WSSV and to test the efficacy of therecognizing the cell wall components of bacteria is being immunostimulant derived from formalin killed Vibrioused in stimulating the immune response in shrimp. alginolyticus. Only negative PCR samples were used forVibrios are one of the most important groups of gram the experimental trials.negative bacteria present in the brackish water and sea The experiment was conducted for 30 days in fourwater environments [9]. They possess an important cell distinct experimental groups based on the frequencywall component, the lipopolysaccharides (LPS) which (Table 1) namely, daily (T1), weekly twice (T2), Weeklyhave the capabilities of triggering and activating the thrice (T3) feed and control (T4) with each group havingshrimp elicitors. These components can both activate the triplicates. Uniform size fiber class reinforcedtransglutamase and phenoloxidase pathways of shrimp polypropylene (FRP) tanks of 100L capacity were used.immune response [10]. Hence, immunostimulation, Each were cleaned and disinfected with bleaching powdersupported by judicious water quality management has before starting the experimental trial and were filled withimmense potentialities in maximizing production from 70L filtered sea water and continuous aeration wasshrimp aquaculture systems. Thus, the concept of provided throughout the experimental period. The juvenilebiological disease control, particularly using non- shrimp weighed approximately 1g and stocked at the ratepathogenic bacterial strains for disease prevention has of 17 juveniles/tank.received wide spread attention during the last decade. The experimental animals were fed with commercial

Several commercial products branded as shrimp feed of size 1mm with known nutritionalimmunostimulants are widely used in shrimp culture [11]. composition. The commercial feed was mixed with 1.5 ml/Various Vibrio sp. used as immunostimulants have been day’s feed of the formalin killed Vibrio alginolyticusstudied in fishes and other organisms and the results bacterial suspension and dried 1 h before feeding. Theshowed the efficacy of these microbes as an control feed T4 was devoid of bacterial suspension.immunostimulant in experimental trials [12]. The immunostimulant mixed feed was given to the

So, the objective of the present study was conducted test shrimps depending on the mode of frequency ofto elucidate the use of Vibrio alginolyticus as an feeding. Rest of the days, they were fed with normal feed.immunostimulant in different frequencies mixed with the The animals were fed four times a day at 6 AM, 2 PM,feed. With these considerations, the present study was 6 PM and 10 PM in accordance of 10% of the bodyundertaken with specific focus to evaluate the weight.performance of immunostimulant in different frequency Complete (100%) water exchange was carried out inmixed with the feed and fed to tiger shrimp to enable all the tanks everyday. Daily before water exchange, waterevaluation of disease resistance in immunostimulated parameters like salinity, pH and temperature wereshrimp against WSSV and to analyze the immunological recorded. Every two days, before water exchange, water

Table 1: Experimental design

S. No. Tanks No. of animals initially stocked Immunostimulant Frequency

1 T1 17 Daily

2 T2 17 Twice/week

3 T3 17 Thrice/week

4 T4 17 Control

nd st rd nd th rd th th2 1 3 2 4 3 5 4Change in absorbance =

1 1 1 1− − − −

+ + +

Change in absorbanceProphenoloxidase =

Protein Value of Plasma

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samples were collected and estimated for bacterial load Prophenoloxidase Activity: Two replicates of 50 µl ofand the amount of ammonia and nitrite in each tank using hemolymph sample were taken and to which 50 µl ofstandard methods. trypsin was added and incubated at room temperature.

Water Quality Analysis: Before water exchange, the and the optical density was measured at 490nm on a UVabiotic parameters including water, temperature, salinity spectrophotometer every one minute interval for 5 min.and pH were recorded every day. Temperature wasrecorded using a mercury thermometer, salinity was Estimation of Protein Level of Hemolymph: Proteinrecorded using a refractometer after standardizing with concentrations of shrimp hemolymph were determined bydistilled water and pH was recorded using portable pH Lowry method [14]. To 1 ml of diluted protein standardmeter. Every two days, water samples were collected from and hemolymph sample, 5 ml of alkaline copper sulphateeach tank and analysed for ammonia and nitrite. was added and mixed thoroughly. The solution was

Ammonia Analysis: 100 ml of water sample from each tank blue color protein complex developed was measured atwas analysed immediately using the method of Xiaoyun 640 nm using UV Spectrophotometer. Using the standardLi [13] and expressed in ppm. The sample from each tank curve, protein concentration of unknown samples waswas taken and treated in an alkaline citrate medium with determined by plotting concentration (mg/ml) of BSASodium hypochloride and phenol in the presence of against standard OD at 640nm. One enzyme activity unitsodium nitroprusside, which acts as a catalyst. The blue was equivalent to an increase of 0.001 in absorbance.indophenol color formed with ammonia was measured Prophenoloxidase (unit/min/mg) of protein was calculatedspectrophotometrically. using the formula,

Nitrite Analysis: The water sample was allowed to reactwith sulphanilamide in an acid solution. The resultingdiazo compound was reacted with NED and forms a highlycolored azo dye measured spectrophotometrically.

Pro-Phenoloxidase Activity Assay: ProphenoloxidaseAcivity (proPO) in hemocytes of shrimp’s hemolymph Parameters for evaluating the efficacy ofwas measured by the formation of dopachrome from L- immunostimulant.Dihydroxyphenylalanine (DOPA). The growth and survival of shrimp in all treatment

Hemolymph Extraction: Prophenoloxidase activity in the analysed.hemolymph of P. monodon fed with or withoutimmunostimulant was estimated from 0.5 ml of Growth: The growth performance of shrimp was evaluatedhemolymph, drawn from the ventral sinus (5 peripod) of by taking the difference in final weight (after 30 days) andth

shrimp in a 1.0ml syringe pre-chilled with 0.5ml of cold the initial weight of the shrimps and the growthanticoagulant solution. The samples were thawed and percentage was calculated.immediately analysed for proPO activity.

Separation of Hemolymph: The hemolymph was taking difference in initial numbers and final numbers andhomogenized and centrifuged at 5000 rpm for 5 min. and the survival percentage was found out.the supernatant fluid i.e., Hemocyte Lysate Supernatant(HLS) was separated from plasma protein for Proximate Compositiondetermination. The cell suspension was incubated with Moisture: Live shrimps were collected from each tank andCacodylate buffer for 30 min. at room temperature. At the kept over filter paper to absorb the external moisture andend of incubation, the mixture was centrifuged for 3 min at weighed on a pre-weighed aluminium foil. The sample was6000rpm. The supernatant was collected and estimated for dried in a thermostatically controlled hot air oven atproPO activity. 80°C±5°C for 48 hrs. Then the dried sample was cooled in

After 20 min of incubation, 50µl of L-DOPA was added

incubated, added and mixed immediately. After 30min,

groups both initial and at the end of the experiment were

Survival: The survival rate of shrimps was estimated by

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a dessicator and the weight was recorded. The process Ash: A known weight of the sample in a crucible waswas repeated until difference in concurrent weighing was ignited over a Bunsen burner for charring. The charredless than 0.001g. The differences in weight were used to sample was kept in a muffle furnace at 550-600°C for 8hrscalculate the moisture percentage in the samples. until white grey ash was obtained. Then the silica crucible

Lipid: The lipid from the tissue samples were extracted by weighing was repeated till the weight was constant andchloroform-methanol procedure [15]. A known weight of the value was expressed in percentage.the sample was taken in a test tube to which chloroform-methanol (2:1) mixture was added. The test tube was Challenge Test: To test the efficacy of immunostimulant,covered with aluminium foil to prevent evaporation of challenge test of the treated shrimps is one of the bestorganic solvents and allowed to stand at 4°C for tool in identifying the time period between risk of beingovernight extraction. The mixtures were filtered and infected and the event time of death-a methodology nowrewashed two times with chloroform-methanol, an equal widely in vogue specifically in genetic selection studiesquantity of water to that of methanol was added to the in shrimps. In this infection methodology of the challengemixture. The upper milky layer contained methanol and test, the underlying assumption is that all the animals arewater mixture and the lower layer contained the equally at risk of being infected at the same time. In wellchloroform with lipid. Upper layer was aspirated using designed challenge tests criteria like mimicking a naturalglass filter and the lower layer was transferred into pre- outbreak as in the pond, control of infection dose,weighed test tubes. This test tube was dried in oven at exposure to infection at the same time, evaluation of all40°C until the whole of the chloroform evaporated leaving defense mechanism of the shrimp and identification of thebehind the lipid. The dried test tube was reweighed till state of each animal as susceptible, infected, dead orconstant weights were obtained. The percentage of lipid recovered are usually considered and followed strictlyin the tissue was calculated based on weight of the lipid [16]. Here, at the end of the experimental period, all theand sample. shrimp tanks including the control were fed with White

Protein: Protein in the tissue was estimated by Bradford survival rate was closely observed.method. Known weight of the dried sample was taken in1N NaOH and kept overnight at 37°C, so that the protein Polymerase Chain Reaction (PCR) screening for WSSV:breaks down into amino acids. Five standard solutions Different parts of the shrimp body such as pleopods,were prepared with 0.5, 1, 2, 4 and 8 µl/ml BSA. From this gills and tail were cut using a sterilized scissor. Thestock, 1.6 ml was collected separately for further assay parts were immediately digested using the solubleprocedure. To this, 400µl dye stock was added and the buffer. The contents were thoroughly homogenized withabsorbance was checked in a UV spectrophotometer at a Teflon homogenizer and the same was placed in water595nm. The procedure was immediately repeated with bath at 100°C for 5 min and then centrifuged at 6000rpmsample solutions against the blank and the protein for 5 min. 5 µl of the collected supernatant wereabsorbance enables to calculate the protein concentration transferred to PCR tubes. PCR cocktail reagents werein percentage in that sample. added and the contents of the PCR vials were mixed

Carbohydrates: The total glucose content in the sample was performed using the PCR program specific to the kitwas estimated by Phenol-sulphuric acid method. To a (Genei, Bangalore).known weight of dried tissue powder, 10% of coldTri-Chloro Acetic acid was added and incubated Amplification of DNA Using Thermocycler:overnight at 4°C. The samples were then filtered. To theknown volume of the filtrate, cold TCA, 5% phenol Initial Denaturation 93°C 5 min. followed bysolution and concentrated sulphuric acid (98%) were Denaturation (5 cycles) 93°C * 20secadded successively and the sample was incubated for 70°C * 30sec30 min at room temperature. The optical density (OD) was 72°C * 30 secmeasured against the blank reading on a UV Annealing (18 cycles) 93°C * 20 secspectrophotometer at 490nm and the percentage 58°C * 30 seccarbohydrates in the samples was obtained. 72°C * 30 sec

was cooled in a desiccators and weighed. Cooling and

Spot Syndrome Virus (WSSV) infected meat and their

gently by tapping. The amplification of DNA molecules

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Extension (25 cycles) 93°C * 20 sec ppm respectively. In the treatment groups it ranges from55°C * 20sec 0.05-0.30ppm and 0.007-0.025ppm, T2 ranges from72°C * 20 sec 0.06-0.29 and 0.008-0.032ppm and in T3 it was 0.05-

Final Extension (last cycle) 72°C * 5 min 0.25ppm and 0.004-0.031ppm of ammonia and nitrite

Preparation of Agarose Gel for Electrophoresis: About1.5% of Agarose was dissolved in 40 ml of 1X TAE Proximate Composition: The biochemical analysis of the(Tris acetate ethylene diamine tetra acetate) buffer samples from the four treatments at the end of thesolution and boiled using a microwave oven till the experimental study in terms of protein, lipid, carbohydrate,agarose melts. To the melted agarose solution, 2µl of moisture and ash content were analysed using standardethidium Bromide was added and mixed gently. The methods (Table 4).solution was poured into the gel moulder with the The mean percentage of carbohydrate in whole bodychannels and it was allowed to solidify. The amplified of shrimp was found to be the highest in T1 (daily) withproducts mixed with the required amount of gel loading the percentage of 20.25 followed by T3 (weekly thrice)dye were slowly loaded into the channels. Four channels with the percentage 17.32. The control treatment had acontained the amplified samples. Other wells contained a percentage of 12.100bp molecular marker, positive control and negative The mean percentage of protein in whole body wascontrol respectively. The samples were run in an found to be the highest in T1(daily) with the percentageelectrophoresis unit at 115 volts for half an hour. The gel of 52.23% followed by T3 (weekly thrice) with thewas documented using a gel documentation system and percentage 50.48%. The control treatment had aphotographed. percentage of 48.35%.

RESULTS treatment groups was found to be the highest in T1 (daily)

Growth: The 30 days experimental trial with different lowest content of lipid was found in T4 (control) withfrequencies of immunostimulant mixed in commercial 11.15%.shrimp feed and when fed to test shrimps, showed higher The moisture content did not vary significantlygrowth than the control (without immunostimulant) between the treatment and control. The highest moisture(Table 2). The highest growth increment (69.40%) was content was in T1 (daily) with 75.25%.observed in the shrimp fed daily with immunostimulant There was not so much variation in the ash contentmixed feed (T1), followed by T3 (thrice) and T2 (twice). also. Total ash content was observed in T1 (daily) with aThe lowest growth increment was recorded in T4 (control) percentage of 17.05%.with a percentage of 21.60%.

Survival: The survival during the experimental study (proPO) (Table 5) in shrimps in treatment and controlshowed high in all the treatments and control (Table 3). shrimps showed significant variation between the two andThe highest survival was recorded in T1 with 82.23% in the control shrimps there was not much change in thefollowed by T3 with 75.47% and T2 with 73.3% and the proPO activity. The highest proPO (39.47 units/ mg/least was recorded in control (T4) with 68.3%. protein) was recorded in T1 followed by T3 and T2 andWater Quality Parameters: During the experimental trial, the lowest in control (T4) with 17.48 units/mg/protein).the daily monitoring of a biotic factors in all theexperimental tanks did not show any variations in pH, Challenge Test: The challenge test was conducted aftertemperature and salinity but showed significant variation 30 days of experimental trial on shrimps fed with WSSVin ammonia and nitrite concentration. The daily water infected shrimp meat (one time feeding) (Table 6). Thequality parameters were monitored and the pH ranged percentage survival of shrimp after 7 days showed higherfrom 7.15-8.55, temperature ranged from 28.5-30°C and survival of shrimps in treatment as compared to thesalinity ranged from 25.5-29.0 ppt in all treatment and control. In the control, 100% mortality was observed oncontrol tanks. The ammonia and nitrite levels in the the 4 day, whereas in T1, T2 and T3, the percentagecontrol (T4) ranged from 0.03-0.26 ppm and 0.007 to 0.032 survival was 60, 20, 40 respectively even after the 7 day.

respectively.

The mean percentage of lipid in whole body of the

with 16.20%, followed by T3 (thrice) with 13.36%. The

Prophenoloxidase Activity: The prophenoloxidase activity

th

th

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Table 2: Mean percentage weight gain (g) of different concentrations of immunostimulant fed shrimp Penaeus monodon

Weeks

----------------------------------------------------------------------------------------------------------------------------------------------------------------------

Treatment 1 2 3 Mean%st nd rd

T1 1.02±0.016 1.30±0.015 1.72±0.013 69.40%

T2 1.14±0.018 1.41±0.012 1.85±0.023 65.50%

T3 1.06±0.001 1.45±0.001 2.06±0.003 66.40%

T4 1.00±0.013 1.06±0.014 1.27±0.011 21.60%

Table 3: Mean survival rate (%) of different frequencies of immunostimulants fed shrimp Penaeus monodon

Treatment Immunostimulant Frequency Survival Survival Rate (%)

T1 Daily 13±0.002 82.23

T2 Twice 15±0.001 73.30

T3 Thrice 16±0.001 75.47

T4 0 8±0.001 68.30

Table 4: Percentage of Proximate composition of whole tissue of different frequencies of immunostimulant fed shrimp Penaeus monodon

Treatments

----------------------------------------------------------------------------------------------------------------------------------------------

Biochemical Components T1 T2 T3 T4

Protein 52.23±0.21 49.48±0.18 50.48±0.74 48.35±0.38

Carbohydrates 20.25±0.26 20.6±0.270 17.32±0.33 12.00±0.41

Lipid 16.20±0.19 12.36±0.34 13.36±0.71 11.15±0.25

Ash 16.65±0.01 17.01±0.25 17.24±0.18 16.21±0.46

Moisture 77.24±0.54 72.82±0.59 75.65±0.56 75.25±0.38

Table 5: Mean Prophenoloxidase Activity (unit/min/mg/protein) in the hemolymph of different frequencies of immunostimulant fed Penaeus monodon.

Prophenoloxidase activity (unit/min/mg/protein)

-------------------------------------------------------------------------------------------------------------------------------------------------------------

Treatment 1 Day 30 Dayst th

T1 17.81±0.117 39.47±0.010

T2 17.81±0.117 31.77±0.010

T3 17.81±0.117 38.37±0.015

T4 17.81±0.117 17.48±0.015

Table 6: Mean percentage survival of post WSSV challenged at different frequencies to immunostimulant fed shrimp Penaeus monodon

Post Challenge Days PCR

------------------------------------------------------------------------------------------------------------------------------------------------- ------------------

Treatment 1 2 3 4 5 6 7 Dead Alivest nd rd th th th th

T1 100.00±0.00 100.00±0.00 100.00±0.000 100.00±0.000 80.00±10.00 80.00±10.00 60.00±0.00 + +

T2 100.00±0.00 80.00±10.00 80.00±10.00 60.00±5.770 60.00±5.770 40.00±5.770 20.00±5.77 + +

T3 100.00±0.00 100.00±0.00 80.00±10.00 80.00±10.00 60.00±5.770 60.00±5.770 40.00±5.77 + +

T4 100.00±0.00 80.00±10.00 60.00±5.770 0.00±0.000 0.00±0.000 0.00±0.000 0.00±0.00 + +

Polymerase Chain Reaction Test: The post challenged Fig. 1 and 2 depicts PCR agarose (1.5%) gellive and dead shrimps were tested for WSSV infection electrophoresis of chromosomal DNA isolatedusing PCR kit. The test showed that all shrimps showed from the shrimp Penaeus monodon withpositive post infection suggesting the presence of immunostimulant at various concentrations for theinfection in all the shrimp tissues. detection of WSSV.

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Fig. 1: Agarose gel of the PCR product of Penaeus monodon before Challenging with WSSVLANE M: DNA molecular weight marker (Genei, Bangalore); LANE 1: Tank 1(Daily dose); LANE 2: Tank 2(Weekly twicedose); LANE 3: Tank 3 (Weekly thrice dose); LANE 4: Tank 4(Control tank- feed without immunostimulant); LANE P:Positive control; LANE N: Negative Control.

Fig. 2: Agarose gel of the PCR product of Penaeus monodon after challenging with WSSVLANE M: DNA molecular weight marker (Genei, Bangalore); LANE 1: Tank 1(Daily); LANE 2: Tank 2(Weekly twice);LANE 3: Tank 3 (Weekly thrice); LANE 4: Tank 4(Control tank- feed without immunostimulant); LANE P: Positivecontrol; LANE N: Negative Control.

DISCUSSION stressful to the animal, labour intensive and costly.

Immunostimulants are substances which elicit component has proved to be the preferred option forimmune response in shrimps which has poor immunity commercial shrimp farms.status compared to higher organism. In addition to these As reported by Austin et al. [19], a strain of Vibrioimmune responses enhancements, these immunostimulant alginolyticus was found effective in reducing diseaseshave been reported to improve growth, survival and caused by Aeromonas salmonicida, Vibrio anguillariumphysiological status of shrimps [17]. Application of the and Vibrio ordalli. Laurent et al. [20], studied thatimmunostimulants is either through feed (orally) or bacteria would be found very useful in both food andthrough immersion in shrimps and hence the modulation biological control agents of fish disease and activators ofof humoral factors takes place easily in the hemolymph. the rate of nutrient regeneration in aquaculture. VibrioSubstances claimed to be ‘immunostimulatory’ have alginolyticus has been employed as an immunostimulantusually been administered by immersion or as a dietary in many Ecuadoran shrimp hatcheries since late 1992. Assupplement or by injection. The route of choice may be an a result, hatchery down time was reduced fromimportant factor in determining the success of the approximately 7days per month to less than 21 daystreatment and enhancing protection against disease, annually, while production volumes increased by 35%.regardless of the diversity of agents used and species The overall antibiotic use was decreased by 94% betweentested. Most authors have reported that administration by 1991 and 1994. The addition of immunostimulant is nowinjection is the most efficacious [18], even though it is also a common practice in commercial shrimp hatcheries.

However, administration by immersion or as dietary

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It is believed that in nature a very small percentage of using a single initial exposure to the specific virulentVibrio sp. are truly pathogenic. The addition of pathogens. The virulent strains are given through variouspotentially pathogenic bacteria to aquaculture system routes, orally through feed or by immersion or by injectionthrough water, algae and artemia was recognized. In this into the muscle. Number of views has been expressed ofstudy, the addition of the bacteria V. alginolyticus as a the viability of each mode of challenging. Amongst theimmunostimulant to mass culture tanks resulted in methods adopted, oral feeding mixed with the feed is theincreased survival rates and growth over the controls and most preferred. The other problems in challenge test areantibiotic prophylaxis. size or the age of the test animal, dosage, frequency of

Amongst abiotic factors, the water temperature is application of pathogen which is some of the issues thatprobably the most important environmental variable that are still unsolved.directly affects the metabolism. Oxygen consumption,growth, moulting and survival [21]. Temperature has a REFERENCESdirect effect on other environmental parameters such assalinity and oxygenation of water. A slow rise or seasonal 1. Oliver De Camp and Moriarty, 2007. Aquaculturechanges of temperature significantly increases the blood species profit from probiotics. Feed Mix, 15: 20-23.cell numbers with time, clotting times, total hemocyte 2. Mathieu Castex, Liet Chim, Dominique Pham, Pierrettenumber and levels of plasma protein, antibacterial activity Lemaire, Nelly Wabetea, Jean-louis Nicholas, Phillipeand phagocytosis by haemocytes, low hemocytic ProPO Schmidely and Catherine Mariojouls, 2008. Probioticat higher temperature, increase of plasmatic protein [22]. P.acidilactici application in shrimp Litopenaeus

Nwanna [23] did the proximate composition of the stylirostris culture subject to Vibriosis in New shrimp head waste silage meal, the experimental fishes and Caledonia. Aquaculture, 275: 182-193.the diets were analysed according to the methods of 3. Regunathan, C. and S.G. Wesly, 2007. Control ofAOAC [24]. A factor of 6.25 was used to convert nitrogen Vibrio spp. in shrimp hatcheries using the greento protein. Using thin-layer chromatography (TLC) algae Tetreselmis suecica. Asian fisheries Science,method described by Williams [25], carcass minerals were 17: 147-158.determined by atomic absorption spectrophotometer, 4. Javier Robalino, Jonas S. Almeida, David McKillen,while Phosphorus was determined by the calorimetric Joan Colglazier, Harold F. Trent, Yian Ann Chen,method and found that the proximate composition of the Megan E.T. Peck, Craig L. Browdy, Robert W.diets are marginally (P>0.05) different. Crude protein levels Chapman, Gregory W. Warr and S. Paul Gross, 2007.of the diets varied from 40.12 to 40.42 and the lipid Insights into the immune transcriptome of the shrimpcontent from 13.89 to 14.49, while nitrogen free extract Litopenaeus vannamei: tissue-specific expressionand crude fiber ranged between 26.17 and 27.39 and 4.37 profiles and transcriptomic responses to immuneand 4.72 respectively. Water quality parameters challenge. Physiology and Genomics, 29: 44-56.analyzed were similar in all treatments and the values 5. Franklin Pereza, B., Filip A.M. Volckaertb and Jorgetemperature (27.03-32.13°C), pH (6.2-6.62), dissolved Calderon, 2005. Pathogenecity of white spotoxygen (6.2-7.0mg/l). syndrome virus on postlarvae and juveniles of

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