Characterization of probiotics bacillus spp isolated from striped catfist pangasianodon hypophthalmus (sauvage, 1878) and the application as feed supplement

pumilus 47B CFU/g gut in striped catfish Appendix Table 1 Antimicrobial activity of 36 Bacillus isolates against pathogenic bacteria and their protease screening 129 3 Mortality of

CHARACTERIZATION OF PROBIOTICS Bacillus

ISOLATED FROM STRIPED CATFISH [Pangasianodon

(Sauvage, 1878)] AND THE APPLICATION

AS FEED SUPPLEMENT

HO THI TRUONG THY

GRADUATE SCHOOL, KASETSART UNIVERSITY

2016

Bacillus spp Pangasianodon

AND THE APPLICATION

GRADUATE SCHOOL, KASETSART UNIVERSITY

THESIS

CHARACTERIZATION OF PROBIOTICS Bacillus spp ISOLATED FROM STRIPED CATFISH [Pangasianodon hypophthalmus (Sauvage,

1878)] AND THE APPLICATION AS FEED SUPPLEMENT

HO THI TRUONG THY

A Thesis Submitted in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy (Aquaculture) Graduate School, Kasetsart University

2016

Ho Thi Truong Thy 2016: Characterization of Probiotics Bacillus spp Isolated from Striped Catfish [Pangasianodon hypophthalmus (Sauvage, 1878)] and

the Application as Feed Supplement Doctor of Philosophy (Aquaculture), Major Field: Aquaculture, Department of Aquaculture Thesis Advisor:

Associate Professor Nontawith Areechon, Ph.D 132 pages

Identification of 6 species of Bacillus (B subtilis, B amyloliquefaciens, B

cereus , B megaterium, B licheniformis and B pumilus) by PCR technique was

developed to identify 120 isolates collected from striped catfish farms in 3 provinces

in the southern part of Vietnam: An Giang, Dong Thap, and Can Tho The highest

number of Bacillus population collected from the three provinces was B megaterium and there was no isolate identified as B licheniformes The two Bacillus spp chosen for in vivo experiments were 54A-B amyloliquefaciens (BA) with high protease activity and 47B-B pumilus (BP) with high inhibition activity against Ewardseilla

ictaluri These two strains were also resistant to stomach acidic pH and low bile salt condition after 3 h and 24 h of exposure Evaluation of antibiotic susceptibility

showed that all six probiotics candidates were susceptible to all antibiotics except

spectinomycin, oxytetracycline, and sulfamethoxazole

Three doses of mixed probiotics spore of BA and BP were applied in striped catfish feed at 1 × 108, 3× 108, and 5×108 CFU/g feed Although there were no

significant effects on % SGR and FCR (P>0.05) of fish, AWG was significantly

higher in fish fed with 5× 108 CFU/g feed compared to the control (P<0.05) Similarly,

the significant increase of lysozyme, phagocytic activity, and respiratory burst activity

of innate immunity were recorded in fish fed with the highest dose of probiotics

(P<0.05) This might induce the more resistance of fish against Ewardsiella ictaluri

infection during 15 days of challenge All doses of probiotics stimulated significantly higher stress tolerance to ammonia concentration at 150 ppm TAN

Student’s signature Thesis advisor’s signature

First of all, I want to express sincere gratitude to my thesis advisor: Associate Professor Nontawith Areechon, Ph.D and my committee members: Assistant Professor Sasimanas Unajak, Ph.D and Assistant Professor Ruangvit Yoonpundh, Ph.D., for their suggestion and encouragement during the period of my study in Kasetsart University I greatly appreciate Associate Professor Wara Tapahudee, Ph.D

as the chairperson and Ms Varin Tanasomgwang, Ph.D as an external referee for my thesis defense examination

I also give thanks to my sponsor, Ministry of Agriculture and Rural Development, for the financial support of my Ph.D study

Next, I should not forget to thank the fish farmers at MeKong Delta who provided the necessary information and the samples for this study, as well as sincerely gratitude to Dr Nguyen Huu Thinh and Assistant Professor Nguyen Nhu Tri, Ph.D from Faculty of Fisheries, Nong Lam University for their suggestions and assistances

Finally, I am grateful to express my acknowledgment to my parent and my husband who give me support, love, encouragement, and their sacrifices to help me to finish my study

Ho Thi Truong Thy

June 2016

LIST OF TABLES

1 Timeline of striped catfish grow out development 5

2 Differences and similarities between between E ictaluri and E

4 Protease activity of six Bacillus spp and their identification 58

5 Survival of Bacillus spp in low pH after period of incubation

9 Total count of Bacillus spp identified as B amyloliquefaciens

54A and B pumilus 47B (CFU/g gut) in striped catfish

Appendix Table

1 Antimicrobial activity of 36 Bacillus isolates against

pathogenic bacteria and their protease screening 129

3 Mortality of striped cat fish in the challenge with E ictaluri

4 Mortality of fish challenge with ammonia for 6 hours (TAN

150 ppm, unionized NH3 3.8 ppm, T 30oC, pH 7.5) 131

LIST OF FIGURES

1 Distribution of striped catfish farming in Mekong Delta 8

2 Trend in export of striped catfish from 2009 to 2013 9

4 Interaction between probiotics bacteria and intestinal mucosa 23

5 Some probiotic mechanism whereby induce beneficial host

9 Spore of Bacillus after 6 days of cultured on spore media 46

11 System of challenge disease and stress experiment 48

12 Nine pure cultured colony morphology of Bacillus spp in TSA

13 Spore and long rod vegetative cell in Gram positive staining 55

14 Detection of Bacillus spp isolated from the gut of striped

catfish by Polymerase Chain Reaction using universial primer

of Bacillus spp designed on 16S rRNA gene (amplicon size:

16 Inhibition activity of Bacillus isolates (A) Percentage of

Bacillus isolates with and without inhibition activity against

E ictaluri by cross treak method (B) Percentage of Bacillus

isolates with different zone inhibition against E ictaluri 57

17 Protease screening Protease (A) and none protease clear zone

18 PCR identification of Bacillus spp by specific primers 61

LIST OF FIGURES (Continued)

19 Percentage of Bacillus spp classified by specific PCR

identification in the total of 120 isolates 62

20 Distribution of Bacillus spp in An Giang (AG), Can Tho (CT)

and Dong Thap (DT) in every 40 isolate sampling in each

22 The growth performance of striped catfish after 90 days of

feeding three different levels of the mixed probiotics of B

amyloliquefaciens 54A and B pumilus 47B and the control diet 67

23 Disease symptoms caused by E ictaluri infection on striped

24 Ewardseilla ictaluri identification by specific primer 70

25 Survival rate of striped catfish after challenge to E ictaluri 71

26 Mortality rate of striped catfish after 6 hours exposure in 150

27 Phagocytic activity of striped catfish fed control, and probiotic

28 Respiratory bust of striped catfish fed control, and probiotic

29 Lysozyme activity of striped catfish fed control, and probiotic

30 Superoxide dismutase of striped catfish fed control, and

probiotic feed (1× 108, 3×108 and 5×108 CFU/g) 75

31 Alternative complement of striped catfish fed control, and

probiotic feed (1× 108, 3×108 and 5×108 CFU/g) 75

CHARACTERIZATION OF PROBIOTICS Bacillus spp

ISOLATED FROM STRIPED CATFISH [Pangasianodon

hypophthalmus (Sauvage, 1878)] AND THE APPLICATION AS

FEED SUPPLEMENT

INTRODUCTION

In recent times, Vietnam aquaculture has made a great development in the

world of fishery market Especially, striped catfish [Pangasianodon hypophthalmus

(Sauvage, 1878)] has become the most important product for export with output estimated to reach $ 4.7 billion dollars in 2012 and in 2013, it was expected to reach between 7.45 and 8.55 billion U.S dollars (Khoi, 2010) With increasing consumption

of striped catfish, the culture system needs to enhance large scale intensive farming Therefore, environment can be affected due to water discharge and sludge from ponds, subsequently, disease outbreaks occur severely and widely

In the industry of striped catfish farming in Mekong Delta, the diseases

occurring commonly and seriously are Bacillary Necrosis caused by Edwardsiella

ictaluri (Dung et al., 2004) The E ictaluri infection can cause 50-90 % mortality (Dung et al., 2004), and it occurs in over 90 % of striped catfish farms in Mekong Delta (Phan et al., 2009)

Although the traditional treatments using antibiotics and chemical disinfectants obtain some effective results, it creates drug-resistance and increases

virulent bacteria (Huong et al., 2010) Simultaneously, antibiotic residues in fish

muscle reduce the value of products and affect human health So, today, prevention and treatment by biological approach such as vaccines, immunostimulant, and probiotics have been received more attention and interest from many researchers Vaccines may be the most effective methods for prevention and can be applied with a various number of diseases caused by bacteria and viruses However, the use of

vaccines is still limited, due to the high cost, taking long time to study and often cause stress to fish (Grisez and Tan, 2005) Therefore, the usage of beneficial microorganisms as probiotics become a tool for prevention, effective treatment of many diseases in aquaculture through the ability to improve water environment,

enhance the growth of fish and inhibition of pathogenic microorganisms (Jose et al.,

2006)

The group of beneficial microorganisms that are used in aquaculture

commonly includes Lactobacillus, Lactococcus, Leuconostoc, Enterococcus,

Carnobacterium, Shewanella, Bacillus, Aeromonas, Vibrio, Enterobacter,

Pseudomonas , Clostridium, and Saccharomyces species (Nayak, 2010) Bacillus is an

important candidate for the production of probiotics This species has the characteristics such as: the ability to sporulate, and produce secondary products such

as antibacterial substances, enzymes that are useful for intestinal environment of

humans and animals Enzymes produced by Bacillus are efficacious to digest carbohydrate, lipid, and protein There have been many studies on the Bacillus in

many fish in the world and provide good outcomes such as Chinese grouper, Indian

carp, yellow croaker, and rainbow trout (Ai et al., 2011; Nayak et al., 2007; Fyzul et al., 2007; Yun-Zhang et al., 2010)

Newai-Recently, more than 400 brands of commercial probiotics are available in the Vietnam market, but most imported or local products of probiotics with unknown

origin The strains are used as a probiotics mainly from Bacillus, Lactobacillus,

Nitrosomonas , Saccharomyces, and Nitrobacter (Tinh, 2010) Some domestic in vitro experiments in Vietnam about Bacillus species isolated from soil and water in striped

catfish ponds have been carried out, and one research group in Auburn University

conducted in vivo testing on channel and striped catfish by using Bacillus isolated from the intestine of channel catfish (Ran et al., 2012) However, the bacteria applied

in these studies were isolated from soil, water, and channel catfish intestine, and they were only determined for the antimicrobial activity and mortality rates in the challenge test

In this research, number of Bacillus strains were isolated from intestine of striped catfish raised in farms at Mekong Delta, Vietnam The isolates were tested in

vitro on probiotics activities that are beneficial for fish, for example inhibition test

against pathogenic bacteria E ictaluri The in vivo experiments were also conducted

on Bacillus supplemented feed to observe growth, disease resistance, stress tolerance

as well as immune response of striped catfish

3 To determine the efficacy of dietary Bacillus spp supplementation diet on

growth performance, survival, non-specific immunity, disease resistance and stress tolerance in striped catfish

LITERATURE REVIEW

1 Development of striped catfish farming industry

From the 1990, striped catfish (Pangansianodon hypophthalmus) was cultured

only at household for family income and their consumption Farmers collected the seed from Mekong river near Cambodia border, the good place for their migration of spawning every year (Nguyen, 2009) Several years later, farmers changed culture model from ponds to cage and pen culture system mainly in An Giang, Can Tho, Dong Thap, and Vinh Long province In 1994, Cambodian government banned the exploitation and fishing wild stock on their river area This induced local people to find the way of artificial breeding on striped catfish, which grew faster than other catfish species and become the candidates for intensive culture (Ngor, 1999; Nguyen, 2009)

Cage and pen culture system faced the problems of water quality in the river during summer seasons, due to high sediments as well as pollution This can affect negatively to fish farming such as poor growth and disease infection On the other hand, small scale farming in the pond resulted good evidence of better production (Anonymous, 2009) After 2007, the cage and fence culture of striped catfish was

abandoned and changed to pond culture along Mekong river (Davy et al., 2011)

Table 1 Timeline of striped catfish grow out development

1940-1950

Striped catfish culture in small, family ponds using wild-collected fingerlings commenced in An Giang, and Dong Thap provinces, which are upstream of Mekong

Table 1 (Continued)

1981 – 1982: trials of pond culture First trials of striped catfish intensive culture

in small ponds conducted by a farmer in Can Tho city using wild-caught fingerlings

1996-1999: expension of pond

culture and trials of cage culture

Striped catfish intensive culture in ponds expanded gradually to other provinces First trials of striped catfish culture in cages and pens were also conducted Both production systems used wild and hatchery-reared fingerlings

2000-2004: rapid expansion of cage

and pond culture

Striped catfish intensive culture in cages and ponds expanded rapidly Hatchery-reared fingerlings met the demand for stocking Productivity was significantly improved Farmers gradually shifted from homade feeds

to commercial feeds

2005-to present There were significant improvements to pond

culture techniques and increases in productivity Introduction of sustainable production standards such as SQF-1000, AquaGAP, GlobalGAP and BMPs

Source: Davy et al (2011)

The striped catfish (P hypophthalmus), commonly known as Tra catfish, is

now transformed to industrial scale operation with millions tons of production

annually According to FAO (2006) report, Vietnam is the largest producers, with export revenue exceeding 1.4 billion USD in 2010, creating 180,000 jobs for rural poor people NACA (2011) concluded about Vietnam striped catfish farming system

Lands for striped catfish farms distribute in two main branches of Mekong River including Tien Giang, and Hau Giang (Figure 1) In 2000, the industry of striped catfish farm began to grow gradually, and after 2004, their production rose rapidly, then kept steadily from 2008 to 2010 (VASEP, 2010), and increase slightly in

2011, but reached plateau until 2013 (Figure 2) According to data of local authorities (News, 2010), there were 5,393 striped catfish farms in operation in 2009, of which there were 4,416 farms with water surface less than 1 ha (81.9 %), 812 farms from 1

to 5 ha water surface (15.1 %), and 165 farms with water surface larger than 5 ha (3.1 %)

Figure 1 Distribution of striped catfish farming in Mekong

catfish culture provinces;

nursery location,

Source: Sena and Nguyen

Recently, there has been an increase

companies, and this trend encouraged by the authorities By contrast, number of small scales farms decrease due to drop of selling

inability to make more investment

Distribution of striped catfish farming in Mekong River, Vietnam

catfish culture provinces; newly developed catfish provinces;

nursery location, main hatchery location

uyen (2011)

has been an increase in large scale farms owned by processing this trend encouraged by the authorities By contrast, number of small rms decrease due to drop of selling price because of economic regress

investment

, Vietnam main newly developed catfish provinces; main

in large scale farms owned by processing this trend encouraged by the authorities By contrast, number of small

economic regression and

Figure 2 Trend in exports of striped catfish from 2009 to 2013

Source: VASEP (2013)

2 Enteric Septicemia of catfish (ESC) caused by Edwardsiella ictaluri

The industry of channel catfish (Ictalurus punctatus) farming in USA has been

damaged by the Enteric Septicemia of Catfish (ESC) caused by Edwardsiella ictaluri

Approximately 47 % of ESC disease occurred in Mississippi areas every year, and the economic loss in channel catfish industry was estimated to millions of dollars yearly

with sign of further increase of disease outbreak (Hawke et al., 1998) In 1976, this disease was first identified from channel catfish at Southeastern Cooperative Fish Disease Laboratory, Auburn University (Hawke, 1979) The most susceptible species to E ictaluri is channel catfish (Hawke et al., 1998), and the next species is striped catfish (P hypophthalmus) that have just recognized as a new disease at Pangasius farms in Mekong Delta of Vietnam (Crumlish et al., 2002; Crumlish et al., 2010) E ictaluri can be isolated in other fish species such as brown bullhead Ameiurus nebulous (Plumb and Sanchez, 1983), danio Danio devavio (Watman et al., 1985), green knife fish Eigemmannia virescens (Kent and Lyons, 1982) , Japanese eel Angiulla japonica, puntius Puntius conchonus,

white catfish Ameiurus catus (Plumb and Sanchez, 1983) and walking catfish Clarias

batrachus (Kasornchandra et al., 1987)

According to Francis-Floyd et al (1987), the disease occurs seasonally during

spring and fall period at temperature 22-28C and they also concluded that 25 C is the

condition that caused the highest rate of mortality in experimented fish Wise et al

(1993) reported that other environment factors such as high density, poor water quality, low nutrient, poor transport method, and stress also induce disease outbreak The fish infected by E ictaluri often swim

erratically with tail down in water External clinical signs often observed in fish include discoloration of skin pigmentation, hemorrhage in fin, anus, and “hole in the

head” (Shotts et al., 1986) Internal symptoms usually occur severely in trunk kidney,

spleen, and liver with many necrotic foci (Areechon and Plumb, 1983) ESC has been found in striped catfish at all sizes, and sometimes it appears in basa catfish

(Pangasius bocourti) The highest rate of mortality was in fingerling fish, but economic loss mostly in stocking stage from size 300-500 g (Dung et al., 2004).

E ictaluri is in a family of Enterobacteriaceae with general characteristics of

short rod, gram negative cells and weak motile (Hawke et al., 1998) To isolate

bacteria, using TSA with 5 % sheep blood or brain heart infusion BHI agar would be

better for the bacterial growth The growth of E ictaluri often detect after 48 hours of incubation with small, puctates, and white colonies (Hawke et al., 1998) E ictaluri is quite similar to E tarda with 96 % genomic similarity on 16S rRNA sequence (Panangala et al., 2005) E tarda and E ictaluri can be distinguished by incubated condition such as temperature, time, salt tolerance, indol

production, and H

2

S production in TSI

Table 2 Differences and similarities between E ictaluri and E tarda

result has been accepted until now The typical symptoms were the presence of white necrotic, and pyogranulomatous foci on surface of internal organs, remarkably on spleen, kidney, and liver (Crumlish and Dung, 2006) Miyazaki and Plumb (1985)

reported that E ictaluri can invade to fish from water through olfactory organ and

migrate to the nerve before attack to the host brain The bacteria can also move through intestine, then enter to the blood stream to cause the septicemia infection

(Shotts et al., 1986)

With the rapid development of aquaculture, using an effective method control the disease out break is really big challenge for fish farm industry Many methods have been applied to control disease including immunostimulant, genetic selection for disease resistance, using drug and chemical, monitor water quality and so on

(Dunham et al., 2002; Hawke et al., 1998) Using antibiotic is the old procedure and

more effective to treat in serious cases with high mortality in fish farm However, there are some limitation when applying antibiotic to fish recently due to high cost of drug, as well as bacterial resistance, and drug residue in fish product affecting to

human health According to Dung et al (2008), the percentage of resistance of E

ictaluri isolated from striped catfish to Streptomycin was detected in 83 %, to

Oxytetracycline in 81%, and to Trimethoprim in 71% of the isolates In 2009, Dung et

al found plasmid IncK containing tetracycline resistant gene in E ictaluri strains from striped catfish farm in Vietnam Reger et al (1993) also tested the antimicrobial susceptibility of E ictaluri isolates from USA and found full susceptibility to

Enrofloxacin, Gentamicin, and Doxycycline

Some vaccine have been developed for prevention of ESC disease in channel catfish in 2002, and the live-attenuated vaccine of Intervet was supplied widely

against ESC (Shoemaker et al., 2002) The safety and efficacy of ESC vaccine to

reduce mortality in channel catfish fry and fingerlings has been demonstrated in

lab-experiments (Lawrence and Banes, 2005; Shoemaker et al., 2002; Wise, 2006)

Studies in the farms carried out on larger fingerlings, and the data on survival

improvement was quite similar as test in laboratory studies (Carrias et al., 2008;

Shoemaker et al., 2009; Wise, 2006) In Vietnam, applying primary vaccination with

inactivated E ictaluri by immersion and oral boost showed the good results of protection for disease control on striped catfish (Thinh et al., 2009) The first

vaccination (Figure 3) by injection method developed for Vietnam Pangasius by Pharmar company has been used on a commercial scale in the Mekong Delta (Thanh

et al., 2013) Attenuated vaccine using gene mutation method on gene WZZ of bacteria Edwardsiella ictaluri initially tested with striped catfish fry showed positive

result in laboratory of Biotech Center, Ho Chi Minh city (Dat Viet News, 7/2012)

Figure 3 Vaccine inject on striped catfish

Source: Magazine of Aquaculture Trading (8/2012)

3 Overview of probiotics in aquaculture

Consumer needs for seafood is increasing in the world and the supply from natural fisheries exploitation cannot meet the demand Therefore, aquaculture is an urgent issue to solve this need However, the aquaculture industry is currently face difficulties by disease problems, mainly caused by bacteria When detecting the disease, farmers often use antibacterial compounds to treat diseases Many farmers use antibiotics to prevent disease, even when they can not identify pathogen yet The solution to this problem lies in the field of ecology rather than in the field of pharmacology (Nayak, 2010) Farmers need to learn to use the complex microbial communities and control them The method using the beneficial bacteria, i.e probiotics to eliminate harmful bacteria is much better than using antibiotics Today, antibiotics have been banned to use as growth promoter and disease prevention in aquaculture (Pilar, 2005) Trends in use of probiotics in aquaculture are increasing Many studies showed that it has the ability to increase productivity and prevent disease (Aditya et al., 2008) There are two methods of using probiotics in aquaculture: 1/Supplement to the culture water, inducing probiotic bacteria localized in the water, 2/ Mix into feed and supply to fish or shrimp (Gatesoupe, 1999)

The term probiotics is derived from Greek language "probios" means "for life" Through time, along with the development of science and human, the understanding

of the term is redefined many times (Hamilton et al., 2003) Lilly and Stilwell (1965)

descried that probiotics is defined as promoting factors produced by microorganisms

In 1974, Parker described probiotics based on the relationship between the host and microorganism in intestine “Organisms and substances that have a beneficial effect on the host animal by contributing to its intestinal microbial balance” By 1989, Fuller expanded the definition of probiotics as “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance" Havenaar and Huis In't Veld (1992) defined “A viable mono- or mixed culture of microorganisms which applied to animal or man, beneficially affects the host by improving the properties of the indigenous microflora” Gatesoupe (1999) reported

"probiotics are live organisms as food supply to improve the health of the host." Reid

et al (2003) defined "probiotics are microorganisms are tightly controlled, with the

appropriate amount of benefit for the host." In 2003, Fioramonti et al defined

probiotics briefly concisely “the probiotic are microbial cells that have beneficial effects on health and well being of the host" Previously, the probiotics was often for humans and terrestrial animals, hence the term usually implies that the probiotic

bacteria is Lactobacillus mainly Gram positive (Fuller, 1989) Gradually, organisms

used as probiotics are increasing diverse, including negative bacteria, positive bacteria, bacteriophage, yeast and unicellular algae, with wider range of uses, besides to humans and animals, today probiotics are commonly applied in aquatic

Gram-animal subjects (Aditya et al., 2008)

Unlike humans and land animals, aquatic animals are surrounded by water and the environment in which the pathogens exist independently with the host The pathogenic microorganisms are constantly migrating to the host through food consumption Aquatic animals engulf large amounts of bacteria in water, which lead

to the result of the interaction between the natural microorganism in the environment and fisheries objects (Viswanath, 2012) In aquaculture, the interaction between probiotics and the host is not only in the host's intestinal tract, but also operate on the

gills, skin and the environment around the host Therefore, Veschuere et al (2000)

have proposed a new definition allows extended applications of the term probiotics in aquaculture "Probiotics are live microorganisms mixture supplied to provide beneficial effects for the host by changing the factors related to the host or microbial communities around to improve feed utilization or value nutrition, enhance disease resistance, and improve the quality environment for the host’s life”

General characteristics of probiotics including:

- Able to survive in acid environment of stomach

- Existing or forming colonies temporarily in the small intestine

- No harm to the host

- Probiotics should be taken to the correct position to impact the host's body

-Expression of benefit to the host (competitive antagonism with pathogenic organisms, or produce enzymes to help digestion)

-Does not contain toxicity genes or antibiotic resistance genes (Veschuere

et al., 2000)

The mechanism of probiotics such as adhesion ability or antagonism to harmful pathogens is important properties of probiotics to reduce toxicity during

infection The antagonism of probiotics to pathogenic bacteria in vitro and in vivo

conditions under several different mechanisms were reported by Nayak (2010) Most studies in the past decades explained the mechanism of probiotics as follows:

- Production of inhibitory compounds

Bacterial resistance is a natural phenomenon in nature Therefore, the interaction between microorganisms plays an important role to ensure equilibrium

between useful microbes and pathogenic microorganisms (Balca'zar et al., 2006) The

different species of bacteria can release a number of chemical compounds to inhibit other species These compounds include: bacteriocin, siderophore, lysozyme, protease,

hydrogen peroxide and organic acids There are many in vitro studies that have

demonstrated the ability to inhibit pathogenic bacteria of some selected bacteria supplemented on larval rearing environment (Vaseeharan and Ramasamy, 2003) Gaixa (1889) was the first to report the existence of bacteria in seawater inhibits

Vibrio sp Then, Rosenfeld and Zobell (1947) studied the production of microbial inhibitors marine pathogenic bacteria and next manuscript studies towards using microorganisms as biological control agents Nogami and Maeda (1992) and Nogami

et al (1997) demonstrated Thalasobacter utilisinhibited Vibrio anguillarum, thus the survival ability of blue crab larvae Portunus trituberculatus increased, and also reduced the number of Vibrio in the water used to culture larvae The follow-up study showed that this strain prevents the growth of bacteria V angillarum on the matured turbot Scophthalmus maximus and the common dab Limanda limanda Using V

alginolyticus as probiotics has been mentioned to enhance the survival and

development of white shrimp larvae (Litopenaeus vannamei) in Ecuador According

to Chythanya et al (2002), saltwater bacteria, Pseudomonas I2 were able to inhibit

Vibrio pathogens in shrimp This microorganism compounds were heat stable, soluble

in chloroform, and the hydrolysis resistance (Brown et al., 1996; Pan and Chiu, 2002)

- Competition nutrients or energy

Competing of beneficial microorganism to obtain the nutrients and available energy can play an important role to survive in the gut or in the aquaculture environment Typical example for this effect is the ability of producing siderophore All bacteria need iron for growth, and siderophore is low weight molecular, strong affinity to iron ions Siderophore can precipitate soluble iron in the form that is easy

to use, so it is an effective tool to collect iron molecule (Vaseeharan and Ramasamy, 2003) The beneficial strains could produce siderophore to compete iron with

pathogenic bacteria in the iron-deficient environment (Gatesoupe, 1994; Teresa et al., 2005) Vibrio strain E can improve resistance to pathogenic bacteria Vibrio splendidus

in halibut larvae (Gatesoupe, 1994) In vitro studies showed that Vibrio strain E can

survive in an environment without iron while V splendidus did not Moreover, halibut larval fed rotifers enriched siderophore deferoxamine produced from Vibrio strain E achieved higher survival rate after challenge with V splendidus, compared with the control group From relevant results in experiments in vitro and in vivo, the scientists concluded that the probiotics of Vibrio E may be partly due to the struggle to get the

iron atoms competed with pathogens (Gatesoupe, 1994)

- Compete with harmful bacteria for adhere position

Colony-forming and adhesion ability of probiotics in the host intestinal epithelium and the intestinal mucus layer is the defense mechanisms against pathogens through competition for adhesion receptors, nutrition, space, the oxygen and the production of antimicrobial substances Some mechanisms involved in microbial adhesion to intestinal epithelial cells including hydrogen bonds, electrostatic interactions, based on the position of specific structures These features are important competitive advantages to help maintain intestinal bacteria in animals

(Balca'zar et al., 2006; Bengmark, 1998), (Robertson et al., 2000)

Recent studies have demonstrated that Lactobacillus plantarum is capable of

produce colonization and adhesion by using a specific binding site which is mannose

to compete with gram-negative pathogenic strains for adhere position in the fish intestine In addition, microorganism formation and adhesion ability of probiotics plays an important role in the regulation and stimulate the immune system of the host

cell (Kelly et al., 2007; Nikoskelainen et al., 2001)

- Contributions on nutrition and digestive enzymes

Some studies have shown that probiotics can influence the process of digestion of aquatic animals: detoxify toxic compounds in food, break down indigestible compounds such as chitin, cellulose in food by the cellulase and amylase Also, probiotics provides vitamins (biotin and vitamin B12), carotenoids, proteins, short chain fatty acid and unsaturated fatty acid chains (PUFAs - polyunsaturated fatty

acids) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)

essential for growth of fish (Dalmon et al., 2007)

At the in vivo level, there were reports that the fish feed probiotics at

concentrations 103-1012 cfu/g during period of 30-90 days could increase the survival

rate, weight gain, reduced food conversion ratio (Laurent et al., 2000) In fish, there are reports that Bacteroides and Clostridium provide nutrition to host especially fatty acids and vitamins (Gildberg et al., 1995) Some micro-organisms such as

Agrobacterium sp., Pseudomonas sp., Brevibacterium sp., Microbacterium sp., and

Staphylococcus sp can contribute to digestive processes in the Arctic trout char

(Salvelinus alpinus L.) (Vendrell et al., 2008; Ringø et al., 1995) Some strains of

Bacillus (Bacillus licheniformis, B subtilis, B polymixa, B laterosporus and B

circulans ) stimulate the growth of persian sturgeon larval (Acipencer persicus):

increased length and weight, increased feed conversion ratio, increased specific

growth rate (Moein et al., 2011) In addition, some bacteria are involved in the

digestion of bivalve molluscs by producing extracellular enzymes such as protease, lipase, and provides the necessary nutritional elements (Yazawa, 1996) The same study showed that probiotics source localized in intestine of Chinese white shrimp

(Penaeus chinensis) produce enzyme digestion and assimilation compounds similar in land animals (Xu et al., 2012)

- Improve water quality

Supplementation of probiotics strains, especially strains of Bacillus sp can

contribute to improve water quality The Gram-positive strains are capable of converting CO2 into organic compounds better than Gram-negative strains It has been

reported that the use of the strain Bacillus sp improves water quality, increase the survival rate, growth rate of Penaeus monodon and reduces pathogenic Vibrio (Gómez et al., 2007) The treatment of aquaculture with probiotics could reduce the

effects of organic matter in water ponds, reduce BOD (biological oxygen demand) and the toxicity of ammonia, nitrite and hydrogen sulfide, control institutional

bacteria, and increase production (Jose et al., 2006)

- Stimulate immune response

The immune system can be nonspecifically stimulated by probiotics Colony formation and adhesion of probiotics in the intestines of fish is a necessary condition

to enhance the immune response by the bond-link receptors such as e-BAMPs bacteria (bacteria associated molecular patterns) that have high conservation value and special receptors on the surface of intestinal epithelial cells Several studies have

demonstrated Bacillus probiotics help increase resistance of shrimp to Vibrio sp and increased phagocytic activity of leukocytes Rengpipat et al (2000) suggested that using Bacillus sp (strain S11) protected Penaeus monodon from pathogens by

activating the immune system in shrimp Balca´zar (2003) demonstrated using a

mixture of Bacillus sp and Vibrio sp impacted positively on the growth and survival

of white shrimp larvae and protected shrimp against the virus WSSV and Vibrio

harveyi This protection comes from stimulation of the immune system by increasing phagocytosis and antimicrobial activity In addition, many authors reported using

lactic acid bacteria Lactobacillus rhamnosus (strain ATCC 53103) at a concentration

of 105 cfu/g, and Lactococcus lactis, Leuconostoc mesenteroides and L sakei stimulated immunity in trout Oncorhynchus mykiss by promoting phagocytic activity, superoxide production, lyzozyme, enhanced complement activity (Balca'zar et al., 2006; Balcázar et al., 2007; Kim and Austin, 2006; Nejad et al., 2006; Europe, 2008)

- Antiviral Effects

Some bacteria have been used as antiviral probiotics, although this mechanism has not been fully understood The laboratory test showed the virus inactivated by chemical and biological compounds from marine algae and

extracellular bacteria extracts Kamei et al (1988) reported that the Pseudomonas sp.,

Vibrios sp., Aeromonas sp and Coryneform sp isolated in hatchery salmon farms

have antiviral activity to IHHNV (infectious hypodermal and hematopoietic necrosis virus)

Direkbusarakom et al (1998) isolated two strains of Vibrio sp NICA 1030 and

1031 NICA, which showed antiviral activity against IHHNV and Oncorhynchus

masou virus (OMV) with a decrease in the density of these two virus by 62 % and 99 % respectively

Some benefits of using probiotics in aquaculture (Gatesoupe, 1999; Jose et al.,

2006):

When supplying probiotics into pond water, microorganisms grow rapidly in the aquatic environment The activity of beneficial microorganisms will have the following effects in aquaculture ponds: decomposition of organic matter in the water, absorbs dead organism and reduces the increase of sediment; reducing toxic gas in the environment (due to gases: NH3, H2S arising), thereby reducing bad odors in the water; helps stabilize the pH of water, water color stability due to probiotics utilizes organic matter dissolved in the water, and consequently restrict algae growth So, farmer can reduce change of water

It can enhance non-specific immunity of fish and shrimp, and inhibit development of harmful microorganisms due to competition for nutrients and adhere position with harmful microorganisms

In addition, some probiotics are used by mixing into feed to improve feed intake of the fish, reduce FCR and prevention of intestinal infections for fish Therefore, the use of probiotics will improve the economic efficiency for aquaculture such as: Increase feed efficiency (FCR), growth rate of fish in short culture time, survival rate and productivity of fish, reduce the cost of water change, using drugs, antibiotic and chemical to treat disease

Many studies on probiotics have been published in the past decades The early research focused on 80 studies using probiotics for the same object and commercial fish However, most of the research at this stage focused on nutritional effects of

probiotics on the host such as the study of Brown et al (1996), Ringø et al (1995),

and Sakata (1990) Gatesoupe (1994), Gildberg and Mikkelsen (1998) demonstrated

the effect of probiotics on disease resistance of fry and fingerlings Austin et al (1995), Nikoskelainen et al (2001), Sakai et al (1995) demonstrated the ability of probiotics on immune stimulation The research by Gildberg et al (1995), Kelly et al.,

(2007) and Nikoskelainen (2006) reported enhancement of probiotics adhesion and formation of colonies in the gut

In recent years, to minimize the disadvantages caused by the use of chemicals

in aquaculture, application of probiotics to prevent disease and improve the environment in aquaculture are on growing in Vietnam According to statistics from the Department of Protection of Aquatic Resources, over 400 brands of probiotics and vitamins currently are sold on the domestic market The majority of biological products derived from the U.S., India, and China are sold at high prices Vietnamese scientists began studying 22 probiotics sold in domestic market with lower costs to

improve profits of farmers (Tinh, 2010) Hanh et al (2004) studied BIO II on

decomposition of food waste and toxic gas at the bottom of the pond, stable pH and color of pond water, inhibition of the growth of microorganisms that cause disease in

fish and shrimp such as Vibrio sp and increase farming productivity Probiotics EM

(Effective microorganism) of Japanese Professor Teruo Higa, invented in 1980, is used in the fields of agriculture and fisheries in the world (Microbes, 2012) However, the EM in liquid form are sub-cultured from original EM from Japan with cell density

in low efficiency for aquaculture (<107cfu/ ml) To help improvement of the efficiency, by the financial support of the Department of Science and Technology, HCMC, Institute of Tropical Biology studied probiotics of VEM VEM (Vietnamese effective microorganisms) includes a collection of useful microorganisms in the EM

probiotics Also, some species of Bacillus sp were selected to improve water in

aquaculture and compete with pathogenic bacteria in shrimp and fish with the density

107 and 1010 cfu/ ml (Hanh et al., 2004)

Scientists at the Department of Chemistry and Natural Products, Institute of Science and Technology of Vietnam have successfully studied probiotics Hudavil –

HUD 5 included Bacillus subtilis, Nitrosomonas sp, Nitrobacter sp, Thiobacter

thioparus with density 108 cfu/ml for environmental treatment The probiotics Biochie

for aquaculture including some strains of Bacillus (B subtilis, B megaterium, and B

licheniformis ) and Lactobacillus acidophilus which decomposed the organic

compounds from excess feed and waste by producing enzymes protease, and amylase Beside that, this probiotics compound also has the ability to synthesize antimicrobial

agents to reduce the overgrowth of pathogenic microorganisms such as Vibrio,

Aeromonas in poor water environment Using probiotics Biochie for shrimp and fish farming is useful to reduce the amount of organic sludge, reducing water cycle change and improve environment (increase dissolved oxygen, reduce COD, BOD) In addition, it is effective to reduce mortality, stunting, and increase production Dr Nguyen Huu Phuc, Ho Chi Minh City Institute of Tropical Biology researched successfully two probiotics for shrimp Two probiotics named Probact for mixing in the shrimp feed, and Ecobact used to treat the water in the pond Two probiotics may

be useful in reducing the pathogenic microorganisms shrimp, and improve water quality in the shrimp pond These two products have been used in the trials at farms in Can Gio, Nha Be, some in Mekong Delta provinces, with the quite good results Shrimp grow faster, less sensitive to disease, and increase high production yields

4 The beneficial activity of probiotics in the host gut

Probiotics bacteria are believed to have an important role as mediators of

intestinal function and stability to relief injury in the gut (Schulzke et al., 2009; Zeissig et al., 2004) With injured intestinal wall, exposure of mucosal immune cells

to material, molecular, or antigens inside gut will lead to abnormal immune activity The positive effects on function of intestinal barriers by probiotics include the prevention of pathogen attachment to epithelia cell, production of defenses molecules,

production of proinflammaroty cytokines and prevention apoptosis (Mennigen et al.,

2009) In addition, the adjustment of probiotic bacteria to dendritic cells (DCs) in cell immunity at gut mucosa is worth to be considered, especially they induce DCs to

regulate T cells in a strain specific manner at mucosal surface (Christensen et al.,

2002) (Figure 4) Probiotics also can produce bacteriocin which is a small, heat stable

anti-microbial peptide, and hydrogen peroxide against pathogens

(Corr et al., 2007; Pridmore

Figure 4 Interaction between probiotic

Source: Corthesy et al (

, and hydrogen peroxide against pathogens in gut environment

Pridmore et al., 2008)

Interaction between probiotics bacteria and intestinal mucosa

(2007)

in gut environment

Figure 5 Some probiotic mechanism whereby induce beneficial host response

Source: Saad et al (2013

Beside that, the probiotic

microorganism can be caused

regulate gut environment condition, and

(Figure 5) Adhesion of

electrostatic interactions, hydrophobic, steric force

structures such as external appendages covered by lectins

2003) Probiotics microorganism can first

receptor and then stimulate mass of immunol

transported by cells localized in

antigens and micro-organism can be also activated by transepithelial vesicular transport in enterocytes and M cells

2005) After that, Dendritic cells (DCs) in the lamina propria (LP) can actively extend their dendrites through epithelial tight junction

Some probiotic mechanism whereby induce beneficial host response

2013)

he probiotics activity in host gut against pathogenic can be caused by competition for nutrients and site of adhersion, regulate gut environment condition, and adjustment of the immune response in gut

of probiotics in the gut can be achieved by passive force, electrostatic interactions, hydrophobic, steric forces, lipoteichoic acids and specific structures such as external appendages covered by lectins (Alain and Marie

microorganism can first attach to intestinal epithelial cells (IECs) by receptor and then stimulate mass of immunological defense mechanism or can be transported by cells localized in the follicle called Payer’s patches The uptake of

organism can be also activated by transepithelial vesicular transport in enterocytes and M cells (Neutra and Kraehenbuhl, 1993; Snoeck After that, Dendritic cells (DCs) in the lamina propria (LP) can actively extend their dendrites through epithelial tight junctions and thus, process directly the

Some probiotic mechanism whereby induce beneficial host response

in host gut against pathogenic competition for nutrients and site of adhersion, adjustment of the immune response in gut

passive force, teichoic acids and specific Alain and Marie- Helene, attach to intestinal epithelial cells (IECs) by ogical defense mechanism or can be

The uptake of organism can be also activated by transepithelial vesicular

Snoeck et al.,

After that, Dendritic cells (DCs) in the lamina propria (LP) can actively extend

s and thus, process directly the

probiotics in the gut lumen (Marco and Kleerebezem, 2006; Rescigno et al., 2001)

The innate and adaptive immune system then can be triggered after antigenic peptides transported across the intestinal barriers Nevertheless, the cell wall components of probiotic bacteria, such as lipoteichoic acid are capable to stimulate TNF-α and IL-10 generated by macrophage through an important surface receptors such as Toll-like

Receptor (TLR) (Dogi et al., 2010; Matsuguchi et al., 2003) It is known that

Lipoteichoic acid (LTA), a major constituent of cell wall of Gram positive bacteria were recognized by Toll-like receptor 2 (TLR 2), while lipopolysaccharide (LPS) on

Gram negative bacteria were recognized by TLR 4 (Ryu et al., 2009) To stimulate

adaptive immunity, many probiotics bacteria can react with IgAs produced by B cells and activation of T helper cells and macrophage by cytokine activation There were wide range of response from cytokines induced by different strains of probiotics

(Arseneau et al., 2007)

5 Overview of Bacillus

According to Bergey’s classification (1998), the name “Bacillus” is a large,

diversified genus with 51 species correctly identified, and many species have not been clearly classified belong to family of Bacillaciae This family is divided into five more

genuses including: Bacillus, Sporolactobacillus, Clostridium, Sporosarcina,

Desulfotomaculum, and the characteristic of this family is the formation of spores

Characteristics of Bacilllus are described as follows: The rod-shaped cells, or

straight from 0.5 to 2.5 x 1.2 to 10 micrometers, and the cells are usually arranged in pairs or chains, round or slightly square All of them are Gram positive, and move by filament One cell has only one internal spore with oval or cylindrical shape, and are resistant to adverse conditions such as temperature and acid Beside that, spore

formation is not hindered by exposure to air Bacillus spp cell is Gram positive in the

early stages of development, metabolic pathway by fermentation or respiration, and most species are catalase-positive This genus is aerobic or anaerobic bacteria, withstand in wide range of pH, temperature, salinity Because of survival ability in severe conditions for a long time, most of them are commonly found from many

different sources Some species are pathogenic for people such as B anthracis, and B

cereus (Holt et al., 1994)

The previous classification of Bacillus based on two characteristics: growth in

aerobic conditions and the ability to form internal spores However, due to the heterogeneity of physical characteristics, ecology and genetics, it leads to difficulty

for classification of genus Bacillus The result from application of molecular biology

in the categorization of Bacillus shows that GC composition of about 32-69 % The phylogenetic tree based on the 16S rRNA sequence shows more Bacillus species

closely related to a number of groups with non-spore forming including genus

Enterococcus, Lactobacillus, Streptococcus, Listeria and Staphylococcus.

Furthermore, some previous members of the genus Bacillus have been reclassified and they are in new family consisting of: Acyclobacillaceae, Paenibacillaceae and

Planococcaceae (Holt et al., 1994)

Most species of the genus Bacillus are heterotrophic organisms that have

capability of respiratory to use a number of simple organic compounds such as sugars, amino acids and organic acids In some cases, they have the ability to ferment

carbohydrates to produce glycerol and butanediol Most species of the genus Bacillus (except B megaterium and some other species) require amino acid, vitamin B, or both

compounds for growth (Turnbull, 1996) Most of them are thermophilic bacteria with optimal growth temperature from 30 to 45 C; moreover, there are a number of heat-resistant bacteria with optimal growth temperature up to 65 C and other species

adapted to cold, and create spores at 0 C The development of Bacillus species can be

in a wide pH range from 2-11 In the laboratory, Bacillus can grow in 16 hours under optimal nutrient condition (Holt et al., 1994)

Spore cell formation was first described by Cohn (1872) when he researched

on B subtilis and then Koch (1875) described the study about B anthracis Cohn has demonstrated the ability to resist the heat of B subtilis spores, and Koch also described the spore formation in B anthracis Due to the anatomy and physiology, the

spores are known as the most sustainable form of existence in the nature and they are able to survive in harsh conditions for millions of years

In the discolored state, the spores are black at the edges, very light and photorefractive The structure of a spore, including: exosporium, spore coat, cortex and inner membrane (Figure 6)

Figure 6 Cross section of a spore Bacillus

Source: Kitty (2002)

Non spore formed during active growth and cell division Internal spores only formed when cells begin to phase of stability and in adverse environmental conditions that often lack nutrients or produce harmful compound Normally, each cell forms a spore and spores are released from the cell after maturation In mature spores, there are not metabolic processes in dormancy state which is able to withstand harsh environmental conditions such as temperature at which species can survive in boiling for several hours, radiation, acid, and detergent So, the formation of the spores is a mechanism to ensure the survival of the bacteria in extreme conditions, and it is not a reproductive mechanism (Graumann, 2012)

In some previous studies, the process of Bacillus sporulation is similar The

whole process takes place within 6-7 hours and involves regulation of more than 50 genes It starts with the DNA helix, and a chromosome is surrounded by pristine membrane forming primitive spores, ending this process with the formation of spore coat, spores and spore capsule shell Subsequently, spore dehydrate to form mature spores and released from the mother cell (Ole and Anne-Brit, 2011)

Bacillus applications in aquaculture can increase survival, promote growth, stimulate digestion, enhance immune response, and improve water quality (Aditya

et al., 2008) Bacillus strains produce extracellular enzymes such as proteases and

other enzymes contributing to the natural enzyme activity of fish, and multiple

micronutrient supplementation (Ghosh et al., 2002) Moreover, some Bacillus strains such as B subtilis produces antimicrobial compounds, nutrient competition and living space contributed to inhibit pathogenic bacteria (Qinghui et al., 2011) B

licheniformis was proven to have antiviral activity, whereas B pumilus produce

extracellular enzymes including amylase, cellulase, a key enzyme in the digestive

activity of the fry (Wang et al., 2008) Using B licheniformis, B subtilis, B polymixa,

B laterosporus and B circulans increase feed efficiency and growth of Beluga larval

Acipenser nudiventris (Moein et al., 2011)

Besides, Bacillus reduces effects of organic sludge, water cycle changes and

improves the environment, due to the ability to remove nitrate and generate the decompose organic matter by the extracellular enzyme such as protease, amylase,

lipase Also, antibacterial activity of some Bacillus strains were found against to

Vibrio sp and Aeromonas sp and they could be used to inhibit and prevent disease in aquaculture (Hong H et al., 2005; Wang et al., 2008)

6 Using Bacillus spp for biological control in aquaculture

The genus Bacillus is antagonistic bacteria against other pathogenic bacteria

considered using in aquaculture for prevention of disease or environmental improvement (Newaj et al., 2007) Another potential benefit is to replace antibiotic

This can prevent development of the resistant gene in bacteria due to using antibiotic

overdose (Moriarty, 1998) Evelyn (1996) demonstrated some strains of Bacillus are

able to adhere to the intestinal surface and hinder the invasion of pathogens to the blood Another study of Brunt and Austin (2005) obtained good results on stimulation

of innate immunity of rainbow trout (Oncorhynchus mykiss) by some Bacillus

candidates

According to Jack et al (1996) and Robertson et al (2000), in vitro

experiment to select candidates against pathogen probably is priority due to producing inhibitory compounds, such as organic acids, and hydrogen peroxide (Gatesoupe, 1999; Ring and Gatesoupe, 1998) However, secretion of inhibitory substrate is

impacted by factors outside experiments lead to different results of in vitro and in vivo (Riquelme et al., 1997) Thus, more particular experiments should be conducted to

select acceptable candidates

Aquaculture industry these days applied successfully many antagonistic bacteria to control different disease affected seriously to farms, such as disease caused

by A salmonicida in rainbow trout and Atlantic salmon with symptom of furuncle skin (Irianto and Austin, 2000; Nikoskelainen et al., 2001), septicemia caused by E

tarda on European eel, Anguilla anguilla (Chang, 2002), Lactococcus garvieae and

Streptococcus iniae caused lactococcosis and streptococcosis in rainbow trout (Brunt

and Austin, 2005) The study of Wang et al (2008) showed that Bacillus spp with

their antimicrobial capability and forming spores can be used as a effective microorganism in the control of disease as well as improvement of growth in animal, due to their resistance in severe condition of environment and long storage time

Many previous results with Bacillus sp showed increase survival rate of aquatic animal in challenge with various disease bacteria The presence of B

amyloliquefaciens as potential probiotic at optimal level of 109 cfu/ml elevated immunity level persistently, hence considering resistance against Edwardsiellosis in

Catla (Catla catla) (Anushree et al., 2013) The research of Qinghui et al (2011) reported that supplementation of B subtilis (1.35 × 107 cfu/g) enhanced not only the growth performance and feed utilization of juvenile large yellow croaker

(Larimichthys crocea), but also the non specific immune response and disease resistance An publication of Guiherme et al (2014) reported that B subtilis

supplementation with concentration of 5× 106 cfu/g feed decreased stress due to raising in a high stocking density, and improved the innate immune system on Nile

tilapia (Oreochromis niloticus) Application of multi species of Bacillus with mixture composed of B subtilis, B licheniformis, and B pumilus provided via rotifers and

Artemia nauplia and added to water increased growth in terms of standard length and

body weight at laval and juvenile stages of sea bream (Sparus aurata) (Matteo et al.,

2010)

Furthermore, Bacillus species have been used in crustacean feed to improve digestive enzyme activities, growth and survival rate (Moein et al., 2011) Several

studies reported some positive results from using a single or two strains in shrimp

culture B subtilis and B licheniformis exist naturally in fresh and marine water

environments and live in intestinal tracts of prawns They are practical probiotics for

shrimp aquaculture (Wang et al., 2008) Bacillus group produce large amount of

exo-enzymes that effectively break large molecules of organic matters in shrimp pond (Moriarty, 1998) In 2004, Moriarty found in his experiment at Taknomin farm, India

that 25 % of black tiger shrimp Penaeus monodon reached 34 gram in 3 ponds which

were added probiotics in feed Meanwhile, in the control ponds, there was only 25

gram increase on shrimp weight during the time of trial B subtilis E20 isolated from

human health food, enhanced the digestive enzyme activity and food absorption of

white shrimp (Litopenaeus vannamei) (Liu et al., 2009), and also stimulating immunity and disease resistance of L vannamei against Vibrio alginolyticus (Tseng et

al , 2009) Besides, this strain has great potential for use as probiotics in feed of L

vannamei larval at 109 cfu/L through increasing larval survival rate, stress tolerance,

and immune status (Kuan-Fu et al., 2010) The study of Hadi et al (2013) showed that the vegetative cell suspension of two B subtilis strains, L10 and G1 at 105 and

108 cfu/ml reduced significant level of ammonia, nitrite, and nitrate ions under in vitro and in vivo conditions in experiment on juvenile white shrimp The addition of

Lactobacillus plantarum to rearing water showed positive effect to larvi-culture in

crab (Portunus pelagicus) by improving significantly higher survival rate, enzyme

activities, and reduction in bacterial load, especially Vibrio spp (Talpura et al., 2013) The probiotics B cereus in feed at concentration of 0.4 % stimulated the growth as well as immune response in shrimp P monodon cultured an outdoor system (Manohar

et al., 2013)

There were some research about the effects of probiotics on immunity and the

growth of striped catfish Son et al (2013) applied mixtures of Bacillus circulans B3,

Bacillus subtilis N26.3, and Pediococcus acidilactici LA61 to evaluate their effects on non specific immunity of striped catfish (Pangansianodon hypophthalmus), and their effects on survival of striped catfish after challenge with E ictaluri at dose of 1×107

cfu/g of feed for 4 weeks They found that the survival rates increase to 42.2% compared to control group Moreover, probiotics mixture increased significantly phagocytic activity (PA) and respiratory burst, and lysozyme activity However,

phagocytic index (PI) was not significantly different between treatments Ran et al (2012) isolated Bacillus subtilis and demonstrated the high efficacy of this probiotics

on the resistance of striped catfish against E ictaluri in Vietnam

MATERIALS AND METHODS

- Strains of Bacillus species isolated from Dong Thap, Can Tho, and An

Giang province in MeKong Delta areas of Vietnam

- Brain Heart Infusion Agar (BHIA), and Brain Heart Infusion Broth (BHIB) for bacteria culture and stocking

- Centrifuge tube of 1.5 ml, and 15 ml

- Gram stain chemical (crystal violet, safranine, iodine, and acetone)

- PCR reagents (DNA template, primer, Taq polymerase, buffer solution,

dNTP, magnesium ions Mg2+), DNA extraction reagents (phenol chloroform, protease, RNase, isopropanol), plasmid extraction Kit for sequencing

- Incubator, laminar, PCR themocycler, gel electrophoresis apparatus, centrifuge machine

1.3 Inhibition activity

- Identified Bacillus, Edwardsiella ictaluri