USE OF INDIGENOUS BENEFICIAL BACTERIA ( Lactobacillus spp . ) AS PROBIOTICS IN SHRIMP ( Penaeus monodon ) AQUACULTURE

Probiotic Shrimp Lactobacillus Antagonistic effect Aquaculture The present study was conducted to study in-vitro the antagonistic effect of Lactobacillus spp. against the pathogenic bacterial vibrio harveyi on shrimp. For this purpose, shrimp samples were collected from three different Ghers at Batiaghata upazilla, Khulna. Gills and intestines were taken out from the samples to identify the load of Lactobacillus spp. and Vibrio spp. The results revealed that the load of Lactobacillus spp. was found more than Vibrio spp. both in gills and intestines; the gills also contained higher load of Vibrio spp. than in the intestines. V. harveyi was separated from the isolated Vibrio spp. with different types of biochemical tests: Gram stain, Motility test, Indole test, VP test, MR test, Arginine dihydrolase, Salt tolerance test, growth at different temperature ranges and colony color on TCBS agar media. The isolated V. harveyi was subjected for in-vitro test. In in-vitro challenge test, the potential antagonistic effect of Lactobacillus spp. against V. harveyi was gradually obtained at 0, 4, 8, 12 hour of treatments. Interesting finding was that, with the time, the load of V. harveyi was reduced gradually and the lowest load was obtained after 12 hours of probiotic inoculation. The present study revealed an excellent in-vitro antagonistic probiotic effect of Lactobacillus spp. on V. harveyi. Therefore the result suggested that probiotic treatment might be an effective alternative to the use of antibiotics in treatments of bacterial diseases in shrimp aquaculture.


INTRODUCTION
Shrimp aquaculture has been recognized as a profitable business in Bangladesh.In spite of having great potentiality, this sector is affected with a wide range of microbial disease.It is one of the limiting factors to shrimp production.A dozen of Vibrio species are responsible for bacterial disease.Specially V. harveyi, V. logei,, V. alginolyticus, V. pelagicus, V. splendidus, V. alginolyticus, V. vulnificus, V. parahaemolyticus and V. damsella, are commonly found as causative agent of vibriosis (Lightner, 1983).Gram-negative bacterium V. harveyi as the causative agent of luminous bacterial disease and it is considered a serious pathogen of larval shrimp in hatcheries (Lavilla-Pitogo et al., 1990; Karunasagar et al., 1994).Both juvenile and adult shrimp can be attacked by this bacterium causing mass mortalities.From over the years, a verity of antibiotics has been used to control various pathogenic bacteria (Baticados et al., 1990).Disinfectants and antimicrobial drugs (antibiotics) are not easy to buy and most of time it is applied in semi-intensive and intensive aquaculture run by the rich farmers.But the situation is different in our country where most of the people farmer live below the poverty line during the starting of the culture and practice traditional or improved traditional culture systems.It is really difficult for them to afford such extra expenses.Furthermore, antibiotics is not encouraged to use nowadays as there is a growing concern about the abuse of antimicrobial drugs not only in human medicine and agriculture but also in aquaculture (Alberman, 1988).Frequent use of chemotherapeutic agents, especially antibiotics, leads to the emergence of resistant strains bacteria (Akoki, 1975) pathogenic to the animals.If antibiotics or disinfectants are used to kill bacteria, some bacteria will survive, because they carry genes for resistance.These will then grow rapidly because their competitors are removed (Moriarty, 1999).Vaccinations to prevent infections have been successful in laboratory scale but yet to be proved under field conditions.
However, it is an urgent issue to find an alternative of antibiotics that would be easy to get and will no harm the nature in anyway.The promising alternative approach for using antibiotics for controlling fish diseases is the use of probiotics or beneficial bacteria.Probiotics bacteria could prevent the establishment of pathogenic bacteria by out-competing them for adhesion and colonization sites in the intestines and other tissues of the animal (Vine et al., 2004a).They could also produce inhibitory substances actively preventing pathogen establishment (Verschuere et al., 2000).When added to rearing water, they may act as bioremediation agents improving water and sediment quality, augment nutrient cycling in the system and initiate colonization of other beneficial micro-flora affecting an overall positive impact on growth rates and productivity (Prabhu et al., 1999).Based on above background the study was undertaken in-vitro to find out the beneficial bacteria (Lactobacillus spp.) from shrimp for possible use as probiotics on V. harveyi infected shrimp.

Sampling and sample size
The present study was conducted on three ghers of Batiaghata upazilla, Khulna.The ghers were randomly selected for collecting shrimp samples.Nine (9) specimens of shrimps, Paeneus monodon (8.10cm-12.4cm in length and 13.12g-22.6g in weight) were collected from three ghers of the study area.

Preparation of stock solution of the target organs
The target organs (gill and intestine) of the samples were separated aseptically and weighed in electric balance.Organs (gills weight: 0.22g-0.34gand weights of intestinal tracts: 0.13g-0.25g.) were taken into eppendorf tubes with peptone water (James and Hirsch, 1960) for isolating Lactobacillus spp.and alkaline saline peptone water was used for isolating Vibrio spp.Then they were homogenized using tissue homogenizer.Then homogenized solutions were centrifuged at 3000 rpm for 3 minutes (James and Hirsch, 1960).After centrifugation the supernatant liquid portion was collected with a micropipette and taken into eppendorf tube and preserved in deep freeze.

Experimental design
Isolation of probiotic bacteria (Lactobacillus spp.) Lactobacillus spp. was isolated from gills and intestinal tracts of the collected shrimp samples.The stock solution was diluted (tenfold dilution) with peptone water (James and Hirsch, 1960).0.1 ml suitable dilution of each stock solution was inoculated in MRS Lactobacillus agar media and incubated at 37 0 C temperature for 24-48 hours.After incubation at 37 0 C temperature for 24-48 h, cream-colored colonies with yellow halos were collected and preserved for further experiment.

Isolation of Vibrio spp.
Vibrio spp. was isolated from the gills and intestinal tracts of the collected shrimp samples from study area.ISO method was followed for isolating Vibrio spp.0.1ml stock solution of each gill and intestinal tract was taken and mixed with 0.9ml alkaline saline peptone water (ASPW) in sterilized test tubes.Then the mixture was incubated at 37 °C for 6 ±1 hr.After that, whole culture of each test tube obtained in first selective enrichment was taken and transferred into other test tubes each containing 10 ml ASPW.Then the solution was incubated at 41.5 °C for 18 ± 1 hr.This was the second selective enrichment.Then serial dilution (tenfold) was done and 0.1ml suitable dilution of each culture was inoculated in thiosulfate citrate bile and sucrose (TCBS) agar plates.Then the inoculated TCBS agar plates were incubated at 37 °C.After 24 h ± 3 h of incubation, the plates were examined for the presence of typical colonies of presumptive Vibrio spp.(ISO/TS 21872-1, 2007).

Biochemical tests
The isolates were identified at the species level with the use of biochemical key.

Gram Staining
Gram stain was done to identify the gram positive and gram negative bacteria (Cowan and Steel's, 1993).

Indole test
The Indole test was performed in a 48h culture in peptone water adding about 1ml ether; shake; run 0.5 ml Ehrlich's reagent down the side of the tube.A red color in the solvent indicates positive reaction (Cowan and Steel's, 1993).

Voges-Proskauer (VP) test
The VP test was performed after completion of the methyl red test adding 0.6ml 5% α-naphthol solution and 0.2 ml 40% KOH aqueous solution; shake well, slope the tube, and examine after 15 min & 1 h.A positive reaction is indicated by a strong red color (Cowan and Steel's, 1993).

Arginine hydrolysis
Arginine hydrolysis inoculated 5ml Arginine broth & after incubation for 24h adding 0.25ml Nessler's reagent.Arginine hydrolysis is indicated by the development of a brown color (Cowan and Steel's, 1993).

MR test
The MR test inoculated the isolated bacteria with buffered glucose broth & incubated at 37°C for 48h.After incubation adding a few drops of methyl red solution to the culture, read immediately.A red color represents a positive test (Cowan and Steel's, 1993).

Motility test
The motility test stabbed inoculates tubes of motility medium to a depth of about 5mm.Incubate at or below the optimum growth temperature.Motile organisms migrate throughout the medium, which becomes turbid; growth of non-motile organisms is confined to the stab inoculum (Cowan and Steel's, 1993).

Salt Tolerance Test
This test was done on nutrient agar media supplemented with varying amounts of NaCl (0%, 1%, 3%, 6%, 8%, and 10%).This was performed to study the salt tolerance range of the isolated species and the optimum concentration, once determined was supplemented in various media required to test their biochemical properties (Cowan and Steel's, 1993).

Growth at different temperature range
The isolated Vibrio spp. was kept in incubator after inoculation at subsequent interval at temperature 4 0 C, 28 0 C, 37 0 C, 55 0 C and checked for their survivability and colony formation obtained at 28 0 C and 37 0 C (Cowan and Steel's, 1993).

In-vitro challenge test and determination of antagonistic activity of the probiotics
After identification of Vibrio harveyi by biochemical test one colony of V. harveyi was taken into eppendorf tube by isolating loop into 0.9ml peptone water.Prepared stock solution of V. harveyi (ISO/TS 21872-1, 2007).The stock solution was diluted (tenfold dilution) with peptone water (James and Hirsch, 1960) and prepared test solution of V. harveyi.Then 0.5 ml isolated probiotic solution was separately mixed with 0.5ml of test solution (V.harveyi).0.1ml suitable dilution of the mixer solution was inoculated in TCBS agar media after at subsequent intervals of 4 hour up to 12hour.This procedure was done for 2 times.Test solution of V. harveyi ; without probiotic was also inoculated in TCBS agar media at 0 hour, 4 th hour, 8 th hour and 12 th hour subsequently.All the inoculated TCBS agar plates were incubated at 37 0 C for 24± 3 hours.Standard plate count was done after incubation.

Data collection and analysis
Collected data were stored, explored and analyzed using Microsoft Excel (Microsoft office, 2007) and Statistical Package for the Social Sciences (SPSS version 16.0; SPSS, Inc., Chicago, IL).Independent sample t-test was applied to address the differences between the treatment and control at 1% significance level using SPSS (version 16.0).

Figure 2. In-vitro challenge test with isolated probiotics on vibrio harveyi
The average load of Lactobacillus spp. in the gills and intestinal tracts of the shrimp samples collected from 3 experimental ghers were 1.65× 10 5 , 1.65× 10 5 , 1.18× 10 5 CFU/g and 4.14× 10 4 , 2.74× 10 4 , 3.10× 10 4 CFU/g respectively (Table-1).The average load of Vibrio spp. in the gills and intestinal tracts of the shrimp samples collected from 3 experimental ghers were 1.29 × 10 4 , 1.90 × 10 4 , 1.40 × 10 4 CFU/g and 5.44 × 10 3 , 4.00 × 10 3 , 8.49× 10 3 CFU/g respectively (Table 1).Nine biochemical tests were performed (Table 2) to identify V. harveyi from the isolated colonies of Vibrio spp.The isolated colonies were inoculated in different biochemical media, supplemented with 1% NaCl to provide the optimum condition for growth, and incubated overnight at 30 o C. The Gram stain result was observed under total magnification by using a light microscope and showed all V. harveyi isolates to be Gram negative short rods.All V. harveyi isolates showed positive results to the Motility test, Indole ring, MR test.Majority of V. harveyi isolates (96.7%) were able to grow in salt tolerance test at 0% to 5%.These bacterial isolates showed inhibition at temperatures of 4°C and 55°C but were able to grow well at temperatures of 28°C and 37°C.The green and yellow colonies were observed on TCBS agar plates.
The biochemical test was done with 10 colonies of Vibrio spp.From the conformation test it was found that 80% was V. harveyi among 10 Vibrio spp.After that with the isolated V. harveyi colonies were further invitro tested to determine the antagonistic effect of Lactobacillus spp. on V. harveyi.
The result of the in-vitro challenge test with and without probiotics had been presented in Figure 2. In-vitro challenge tests were performed to investigate the antibacterial effect of the isolated probiotics (Lactobacillus spp.) on the Vibrio harveyi of infected shrimp.When in-vitro challenge tests were run it was found that the average load of V. harveyi. in zero (0) hour was 4.69× 10 3 CFU/g and it was 2.30× 10 4 , 2.36× 10 5 and 4.24× 10 5 at 4 th , 8 th and 12 th hour respectively.However, after treatment with the isolated probiotics, the average load of V. harveyi at 4 th , 8 th and 12 th hour were reduced to 1.16× 10 4 , 7.41× 10 3 and 1.13× 10 3 respectively.The present study showed a slight reduction of V. harveyi load at the 4 th hour of probiotic application where as a drastic and significant reduction in the load of V. harveyi had been obtained at the 8 th and 12 th hour of probiotic application.

DISCUSSION
The present study showed that the load of Lactobacillus spp.and Vibrio spp. was more in gills than in the intestinal tracts of shrimp samples.Gill is an essential organ for the respiration of fish and always remains in contact with the aquatic environment.Water is the major source of various types of microorganisms.So, gill possesses the greater possibility of association with different types of bacteria.The present study was also similar with some other research work.Such as, potential pathogens were able to maintain themselves in the external environment (water) of the animal and proliferate independently of the host animal (Hansen and Olafsen, 1999;Verschuere et al., 2000).
Various potential pathogens are taken up constantly by the animal through the processes of respiration, osmoregulation and feeding.This might be the main reason of such better occurrence of Lactobacillus spp.and Vibrio spp. in gills than intestinal tracts of the experimental samples.This result also agreed well with the findings of Ringo and Gatesoupe (1998).They reported that, Lactobacillus spp. is less abundant in intestinal tract.It is also well known that the population level of lactic acid bacteria associated with the digestive tract is affected by nutritional and environmental factors like dietary polyunsaturated fatty acids, chromic oxide, stress etc.These affecting factors might be another reason why there was less abundance of Lactobacillus spp. in intestinal tracts than in the gills.Moriarty (1990) reported that, aquatic farmed animals are surrounded by an environmental milieu that supports opportunistic pathogens independently of the host animal, and so the pathogens can reach high abundance on the external organs of the animal.In aquaculture ponds, where animal and algal population densities are very high, Vibrio spp.numbers can also become high compared to the open sea.This might be another reason of such better occurrence of Vibrio spp. in gills than the intestinal tracts of the experimental samples, which was found in the present study.Verschuere et al., (2000) stated that although lactic acid bacteria are not dominant in the normal intestinal microbiota of larval or growing fish, several trials have been undertaken to induce an artificial dominance of lactic acid bacteria in aquatic animals.This statement supports the findings of the present study.
The present study revealed that Lactobacillus spp.loads were higher than Vibrio spp.both in gill and intestine.Lactobacillus spp. is a beneficial bacteria and the presence of higher amount of Lactobacillus spp. in gill and intestine is a good sign.Because this Lactobacillus spp.; act as natural probiotics in infected shrimp.These results also agreed with the findings of two other separate experiments (L ee et al., 2000) and (Vine et al., 2004) who stated that successful probiotics bacteria are usually able to colonize the intestine, at least temporarily, by adhering to the intestinal mucosa.The adhesive probiotics bacteria could prevent the attachment of pathogens, such as coliform bacteria and clostridia, and stimulate their removal from the infected intestinal tract.Both of these experiments revealed the temporary colonization and adherence of probiotic bacteria.The gills may appear susceptible to bacterial penetration because they are covered by a thin exoskeleton (Taylor and Taylor, 1992), but their surfaces are cleaned by the setobranchs.
From the biochemical test it was found that most of the Vibrio spp.were Vibrio harveyi .From the present it has been proved that there is a significant antagonistic effect of Lactobacillus spp.against V. harveyi.In invitro challenge test, the potential antagonistic effects of Lactobacillus spp.against V. harveyi was gradually obtained at 4 th , 8 th and 12 th hour of probiotics treatment.This finding is very much similar to Balca'zar, (2003) also works on effect of probiotics on shrimp, his findings also relate with the findings of the present study.He found that the administration of a mixture of bacterial strains (Bacillus spp.and Vibrio spp.) positively influenced the growth and survival of juveniles of white shrimp and presented a protective effect against the pathogens Vibrio harveyi and white spot syndrome virus.
Sumon et al., (2018) also found the antagonistic effect of probiotic bacteria on Vibrio harveyi that also supports the present study.Austin and Brunt, (2008) also mentioned that the probiotics actively interfere with the colonization of potential pathogens in the digestive tract by antibiosis or by competition for nutrients, oxygen and/or space, by alteration of microbial metabolism, and/or by the stimulation of the innate immune response, including enhancement of phagocytic and respiratory burst activities and lysozyme reduction.Jiravanichpaisal and Chuaychuwong et al., (1997) used successfully Lactobacillus spp. as the probiotic bacteria in the tiger shrimp (P.monodon Fabricius).They designed to investigate an effective treatment of Lactobacillus spp.against vibriosis and white spot diseases in P. monodon.Results of the present study indicated that probiotics treatment offers a promising alternative to the use of antibiotics in shrimp aquaculture.These works strongly suggest the effective control of microflora in fish and shellfish in culture environments by antibiotic-producing bacteria.

CONCLUSION
From this in-vitro challenge test with Lactobacillus spp. it was found that it significantly reduced the V. harveyi load of the selected shrimp samples.From this in-vitro test it was proved that Lactobacillus spp., had the inhibitory property of a biocontrol agent for use in control of shrimp pathogens and might be useful for replacing the commercial antibiotics.The present investigation also clearly demonstrated that, putative probionts isolated from infected shrimp possesses an excellent antibacterial effect and could be applied in aquaculture operation as an effective tool of treatment to prevent and cure shrimp diseases caused by V. harveyi.On the basis of the results obtained in this work, it could be concluded that Probiotics, as 'bio-friendly agents' such as Lactobacillus spp., could be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms.

Figure 1 .
Figure 1.Average Load of Vibrio spp.and Lactobacillus spp. in Gill and intestine

Table 1 .
Load of Lactobacillus spp. in gills and intestinal tracts of collected shrimp

Table 2 .
Biochemical test to identify Vibrio harveyi