Evaluation of Bacillus spp. as Potent Probiotics with Reduction in AHPND-Related Mortality and Facilitating Growth Performance of Pacific White Shrimp (Litopenaeus vannamei) Farms

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease affecting shrimp aquaculture worldwide. In this study, natural microbes were used in disease prevention and control. Probiotics derived from Bacillus spp. were isolated from the stomachs of AHPND-surviving Pacific white shrimp Litopenaeus vannamei (22 isolates) and mangrove forest soil near the shrimp farms (10 isolates). Bacillus spp. were genetically identified and characterized based on the availability of antimicrobial peptide (AMP)-related genes. The phenotypic characterization of all Bacillus spp. was determined based on their capability to inhibit AHPND-causing strains of Vibrio parahaemolyticus (VPAHPND). The results showed that Bacillus spp. without AMP-related genes were incapable of inhibiting VPAHPND in vitro, while other Bacillus spp. harboring at least two AMP-related genes exhibited diverse inhibition activities. Interestingly, K3 [B. subtilis (srfAA+ and bacA+)], isolated from shrimp, exerted remarkable inhibition against VPAHPND (80% survival) in Pacific white shrimp and maintained a reduction in shrimp mortality within different ranges of salinity (75–95% survival). Moreover, with different strains of VPAHPND, B. subtilis (K3) showed outstanding protection, and the survival rate of shrimp remained stable among the tested groups (80–95% survival). Thus, B. subtilis (K3) was further used to determine its efficiency in shrimp farms in different locations of Vietnam. Lower disease occurrences (2 ponds out of 30 ponds) and greater production efficiency were noticeable in the B. subtilis (K3)-treated farms. Taking the results of this study together, the heat-shock isolation and genotypic-phenotypic characterization of Bacillus spp. enable the selection of probiotics that control AHPND in Pacific white shrimp. Consequently, greater disease prevention and growth performance were affirmed to be beneficial in the use of these probiotics in shrimp cultivation, which will sustain shrimp aquaculture and be environmentally friendly.


Introduction
Acute hepatopancreatic necrosis disease (AHPND) is one of the most serious threats to shrimp farming [1].AHPND is caused mainly by Vibrio parahaemolyticus (VP AHPND ), which carries plasmid encoding-specific toxin genes [2].This pathogen affects penaeid shrimp, including the Pacific white shrimp Litopenaeus vannamei and black tiger shrimp Penaeus monodon, with mortality up to 100% within 20 to 30 days of cultivation, resulting in significant economic losses in shrimp aquaculture [1,3].Although the use of antibiotics is effective against this bacterial disease, the risk of resistance against drugs among bacteria in the environment requires alternative strategies to address this disease.
In general, bacteria, which are promising contributors to shrimp health, are widely applied in shrimp farms for health management as an alternative strategy to reduce the risk of diseases [4].Probiotics, 'live microorganisms which when administered in adequate amounts confer a health benefit on the host' [5], and biological control agents [6] have been studied in the research field of aquaculture.Previous studies suggest that oral administration of exogenous bacteria stimulates shrimp immune reactions [7,8], and bacterial antagonism occurs in the environment, including the shrimp gut and pond water [9,10].However, the exact mechanism of the probiotic effects of bacteria on shrimp and the appropriate application are uncertain.
Bacillus spp. is one of the most studied and used bacteria as a probiotic or biocontrol agent in aquaculture [11].In shrimp, oral administration of spores or vegetative cells of specific strains of Bacillus spp.reduces the mortality of shrimp caused by bacterial infections, with induction of the host immune system and/or antagonism between the bacteria as possible mechanisms [7,8,[12][13][14][15].Antimicrobial peptides (AMPs) secreted by Bacillus spp., such as bacillomycin, fengycin, iturin, surfactin, bacilysin, and subtilin, inhibit the growth of other microorganisms, especially bacteria and fungi [16,17].The risk of resistance among bacteria against those AMPs is thought to be small [18]; thus, the application of AMP-producing Bacillus to aquaculture fields is promising.
The stomach microbiota of penaeid shrimp may play a crucial role in protecting against bacterial infections.Vibrio bacteria, such as VP AHPND and Vibrio penaeicida, colonize the shrimp's stomach during the initial stages of infection [19,20].Moreover, in contrast to mammalian animals, shrimp stomachs host a diverse range of bacteria [21,22], with variations observed in the presence or absence of AHPND development [1,23].This knowledge suggests that the bacterial community in the stomach microbiota of shrimp, similar to the intestinal microbiota of mammals, might confer beneficial effects on shrimp.
Southeast Asian countries have been heavily impacted by AHPND [24].However, interestingly, not all shrimp farms or shrimp ponds were affected by the disease.Thus, we expected that shrimp may have specific factors that reduce the risk of AHPND.In this study, Bacillus spp.were isolated from the shrimp stomach and environment, and their potential as beneficial bacteria for shrimp was evaluated.

Bacillus spp. Isolation
Bacillus spp. in this study were isolated from two sources: (1) from the stomachs of surviving shrimp from ponds that were positive for AHPND outbreaks on local farms in Samut Songkhram Province and (2) from soils in mangrove forests in Thailand.The isolation of Bacillus spp.followed the heat-cold shock method [25].Briefly, stomachs of AHPND-surviving shrimp and soils from mangroves were ground and diluted in 0.85% Normal saline solution (NSS) and then heated at 80 • C for 20 min before rapidly chilling on ice for 1 to 2 min.Suspensions were serially diluted in 0.85% NSS and spread on tryptic soy agar (TSA).After incubation at 30 • C for 20 h, Bacillus-like colonies were selected on the basis of their morphology and kept at −80 • C until use.

VP AHPND Isolation and Identification
To obtain the V. parahaemolyticus (VP) AHPND strain (VP AHPND ), the stomachs of diseased shrimp from different areas in the eastern and southern areas of Thailand were collected, including Chanthaburi (CT), Rayong (RY), Trat (TR), Nakhon Si Thammarat (NK), Surat Thani (SR), Chumphon (CP), and Songkhla (SK).The shrimp stomachs were aseptically dissected, minced, and serially diluted 10-fold in 0.85% NaCl (normal saline; NSS), spread on thiosulfate citrate bile salt (TCBS) agar, and incubated at 30 • C overnight [26].Individual green colonies were chosen and identified using specific primers for VP [27] and VP AHPND [28].One VP isolate from each province that was positive with both primer sets was examined for pathogenicity in a shrimp challenge assay.VP isolates that caused AHPND were designated VP AHPND hereafter.VP AHPND isolates were collected in glycerol stocks and stored at −80 The soft agar overlay technique was used to determine inhibition on a solid medium [29].Soft agar was prepared by mixing melted TSA and TSB at a ratio of 1:2.Then, 3 mL of soft agar was mixed with 50 µL of overnight cultured VP AHPND .The mixture was poured onto the surface of solidified TSA in 87 mm diameter Petri dishes and left for 20 to 30 min to solidify.Then, blank antimicrobial susceptibility disks (Oxoid) were placed on the surface of the agar, and 10 µL of 1 × 10 8 CFU/mL of each Bacillus culture was dropped on the blank disks.After incubation at 30 • C for 16 h, the diameters of the clear zones surrounding the disks were measured.

In Vitro Inhibition Assay: Liquid Medium
Overnight cultures of different Bacillus spp.were diluted to 1 × 10 5 CFU•mL −1 in 10 mL of TSB.The overnight culture of VP AHPND was prepared as a 100-fold dilution with Bacillus culture and shaken at 200 rpm 30 • C for 20 h.TSB was used as the control (without Bacillus).For the negative control, TSB was not inoculated with either Bacillus or VP AHPND .Samples with different Bacillus isolates were 10-fold serially diluted in 0.85% NSS and spread on TCBS to count VP AHPND and TSB to count Bacillus spp.All assays were performed in triplicate for each Bacillus isolate.

Genotypic Characterization of Bacillus spp. 2.4.1. Species Identification
The species identification of isolated Bacillus spp. was based on the entire sequence of 16S rRNA gene (1500 bp) using universal primer 8f and 1490r as described below.The isolated Bacillus-like colonies were cultured in tryptic soy broth (TSB) at 30 • C for 20 h.Bacterial DNA was extracted for species identification following the standard phenolchloroform extraction method.Primers 8f (3 GAGTTTGATCCTGTGCTCAG5 ) and 1490r (5 GACTTACCAGGGTATCTAATCC-3 ) were used as 16S rRNA universal primers for the bacteria.PCR products were purified with a GeneJET PCR Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced using Sanger sequencing using a 3730XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA).Bacterial identification was performed using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi,20 April 2020).

Preparation of Bacillus spp. and VP AHPND
Bacillus isolates and VP AHPND in glycerol stocks were streaked on TSA and TCBS agar, respectively.After incubation at 30 • C overnight, a single colony was inoculated in TSB and shaken at 250 rpm and 30 • C for 20 h.The bacterial amount was quantified using the plate count method on TSA and TCBS agar.

Pathogenicity Analysis of Isolated VP AHPND in Shrimp
Three hundred sixty shrimp, 0.5 ± 0.03 g, were acclimated in aerated experimental 200-L tanks for 3 days.The shrimp were separated into two groups: VP AHPND challenge and control group, 60 shrimp in each group with triplicate.In the challenge groups, shrimp were inoculated with isolated VP AHPND at 10 4 CFU•mL −1 using the immersion method [37].In the control group, shrimp were inoculated with 100 mL of TSB.The hepatopancreas of moribund shrimps were examined for the presence of VP AHPND using PCR [28], and histomorphology was determined using H&E staining.The number of dead shrimp was observed every 24 h after the challenge until 10 days after the challenge.All assays were performed in triplicate for each VP AHPND isolate.

Efficiency of Isolated Bacillus in Controlling AHPND: Laboratory Level
Three hundred sixty shrimp, 0.5 g ± 0.03 g, were kept in a 400-L container with aeration, and the diseased contaminant was determined prior to testing.To test the disease control, 60 shrimps in each group were directly immersed in different Bacillus isolates (including K3, K5, K6, K11, K12, K13, K19, P4, and P6) at 1 × 10 5 CFU•mL −1 for 10 h.After that, VP AHPND at 1 × 10 4 CFU•mL −1 was immediately added to the aquarium.For the control group, 60 shrimp were treated with TSB instead of Bacillus spp.All experiments were performed in triplication.Moribund shrimp were examined for the presence of VP AHPND using PCR.The number of dead shrimp was observed every 24 h.All assays were performed in triplicate for each Bacillus isolate.

Evaluation of AHPND Control Efficiency at Different Salinities
Shrimp were kept in tanks with salinities of 5, 20, and 40 ppt.Shrimp were immersed in Bacillus spp. at 1 × 10 5 CFU•mL −1 for 10 h and then challenged with VP AHPND at 1 × 10 4 CFU•mL −1 using the immersion method.The other methodology of this experiment was the same as that mentioned above.All assays were performed in triplicate for each salinity.

Validation of Disease Control Efficiency against Different Strains of VP AHPND
The efficacy of Bacillus sp.against various strains of VP AHPND was evaluated.Shrimp were immersed in 1 × 10 5 CFU•mL −1 Bacillus sp. for 10 h and then challenged with 1 × 10 4 CFU•mL −1 of different VP AHPND strains (strains CT, RY, TR NK, SR, CP, and SK) in a salinity of 20 ppt.The other methodology of this experiment was the same as that mentioned above.All assays were performed in triplicate for each VP AHPND isolate.

Efficiency of Isolated Bacillus in Controlling AHPND: Field Level
Field trials were conducted in nursery farms in Quang Binh, Binh Dinh, Ninh Thuan, Ben Tre, Bac Lieu, and Kien Giang at local farms in Vietnam (Supplementary date S1).Healthy postlarvae were reared in 250 m 3 aerated aquaculture tanks at a density of 1600 (pcs/m 3 ) for 35 days.Shrimp were initially randomly screened for the presence of AHPND, EHP, WSSV, IHHNV, TSV, and YHV using PCR based on previously described methods.Culture conditions for shrimp were maintained during the whole experiment as follows: pH at 7.8-8.2,temperature at 28-32 • C, salinity at 20 ppt, alkalinity at 170-190 mg, TAN less than 1 ppm and NO 2 − less than 1 ppm.In the treatment group (30 tanks), Bacillus spp. was added directly to the water at a dose of 10 5 CFU•mL −1 every 3 days.In the control group (30 tanks), postlarvae did not receive any probiotics.The number of final shrimp (pcs), total weight (kg), final size (pcs•kg −1 ), total feed (kg), FCR, % survival, and VP AHPND infection were recorded at the end of the experiment.

Statistical Analysis
In bacterial inhibition analysis, Dunnett's multiple comparisons test was performed to compare the bacterial number in the test groups and the control.In challenge tests, the survival of shrimp was analyzed using the log-rank test.These statistical analyses and figure preparations were conducted using GraphPad Prism v.6 (GraphPad).

Bacillus spp. Isolation
To collect the Bacillus spp., the stomachs of AHPND-surviving shrimp and the soil from the AHPND outbreak area were targeted for Bacillus spp.isolation.After heat and cold shock treatment, Bacillus spp.were entered into the sporulation stage, while other bacteria were killed.Thus, the spore suspension was spread on solid agar to allow the Bacillus spp. to re-enter the vegetative stage.The bacteria showing different morphological characteristics of the colonies were collected, and their species were determined based on 16S rRNA analysis.A total of 22 isolates, designated K1-K22, were isolated from AHPND-surviving shrimp stomachs, whereas 10 isolates, named P1-P10, were isolated from the soil in mangrove forests (Table 1).

VP AHPND Isolation, Identification, and Pathogenicity of Isolated VP AHPND
VP AHPND was isolated from the stomachs of naturally AHPND-infected shrimp in commercial shrimp farms.Seven strains were collected from different provinces in Thailand: Chanthaburi (CT), Rayong (RY), Trat (TR), Nakhon Si Thammarat (NK), Surat Thani (SR), Chumphon (CP), and Songkhla (SK).Those strains were confirmed using PCR using specific primers for V. parahaemolyticus and VP AHPND .The pathogenicity of these isolates demonstrated acute mortality, with survival rates ranging from 40.56-67.78%(Supplementary date S2).The mortality of AHPND-challenged shrimp ceased at 6-8 days after infection.The presence of VP AHPND in the hepatopancreas of moribund shrimp was confirmed using PCR, and the AHPND histomorphology was confirmed using H&E staining (Supplement data S1 and S2).Pathogenic bacteria were reisolated from dead shrimp, demonstrating authentic infection using the tested VP AHPND.

In Vitro Inhibition Assay: Solid Medium and Liquid Medium
To assess the inhibition activity of Bacillus isolates against VP AHPND in vitro, the width of the inhibition zone on agar plates and the growth of bacteria in liquid media were evaluated.The twenty-five Bacillus isolates exhibited inhibition zones ranging from 14.33-34.67mm against VP AHPND (strain RY, topic 3.4).Isolates K3, K4, K14, K15, P1, P2, and P3 displayed outstanding inhibition with clear zone diameters ranging from 27.00-34.67mm (Figure 1A).
In liquid media, Bacillus isolates showed significant (Dunnett's multiple comparisons test, p < 0.001) inhibition against the growth of VP AHPND , except isolates K13, P9, and P10 (Figure 1B).These results concurred with the inhibition assay on a solid medium.The order corresponds to Table 2, which shows the presence of AMP-related genes.
In liquid media, Bacillus isolates showed significant (Dunnett's multiple comparisons test, p < 0.001) inhibition against the growth of VPAHPND, except isolates K13, P9, and P10 (Figure 1B).These results concurred with the inhibition assay on a solid medium.

Species Identification
Upon 16S rRNA identification, the nucleotide sequences (1500 bp) from the isolated Bacillus spp.were compared with references sequenced in the NCBI database to identify their species.In conclusion, the populations of isolated Bacillus species were B. subtilis (11 isolates), B. amyloliquefaciens (5 isolates), B. velezensis (4 isolates), and B. licheniformis (4 isolates) (Table 3).

Antimicrobial Peptide (AMP)-Related Gene Determination
To determine the inhibition activity of isolated Bacillus spp.against VP AHPND , six different AMP genes were tested.Analysis based on gene-specific PCR detection showed diverse distributions of AMP genes in different Bacillus isolates (Table 2).From a total of 32 isolates, the srfAA gene was most frequently found (25 isolates), followed by bacA (19 isolates), bmyB (11 isolates), fenD (11 isolates), and ituC and spaS (1 isolate).More than half of the isolates (21 isolates) harbored at least two of the tested AMP genes, while seven isolates had none of the tested genes.The most frequent patterns of AMP genes were srfAA + -bacA + (7 isolates) and srfAA + -bacA + -bmyB + -fenD + (7 isolates).Bacillus isolates carrying AMP genes contained at least srfAA + .

Evaluation of AHPND Control Efficiency at Different Salinities
To ensure that B. subtilis (K3) had the highest AHPND control effectiveness, whether it remained effective at different salinities was evaluated.The challenge test was monitored after treatment with B. subtilis (K3).Survival rates of shrimp 76.67% (5 ppt), 42.78% (20 ppt), and 0% (40 ppt) were observed in control shrimp after immersion with VPAHPND (strain RY) without pretreatment with B. subtilis (K3) (Figure 3).This result indicated that the virulence of VPAHPND (strain RY) was salinity-dependent.However, after treatment with B. subtilis (K3), a significantly higher survival rate than the control groups for each salinity (p < 0.0001) was observed (Figure 3).

Evaluation of AHPND Control Efficiency at Different Salinities
To ensure that B. subtilis (K3) had the highest AHPND control effectiveness, whether it remained effective at different salinities was evaluated.The challenge test was monitored after treatment with B. subtilis (K3).Survival rates of shrimp 76.67% (5 ppt), 42.78% (20 ppt), and 0% (40 ppt) were observed in control shrimp after immersion with VP AHPND (strain RY) without pretreatment with B. subtilis (K3) (Figure 3).This result indicated that the virulence of VP AHPND (strain RY) was salinity-dependent.However, after treatment with B. subtilis (K3), a significantly higher survival rate than the control groups for each salinity (p < 0.0001) was observed (Figure 3).
by the immersion method.Shrimp were reared at different salinities, including 5 ppt, 20 ppt, and 40 ppt.**** p < 0.0001 compared with the control for each salinity.

Discussion
Probiotics in aquaculture are a well-known method to improve the health status of aquatic animals; thus, the use of probiotics to control disease has been widely discussed.However, the validity of their use should be confirmed using rational probiotic screening and selection, as well as the elucidation of their efficiency in laboratory and field trials.
Upon AHPND devastation, we noticed that some shrimp survived in the AHPNDpositive ponds.Hence, the ecosystem of the microbiome in the shrimp digestive system might reflect the regulation of the population of pathogenic bacteria by healthy bacteria.Therefore, probiotics, Bacillus spp., were isolated from two different sources: (1) from the surviving shrimp from the AHPND outbreak pond and (2) from the soils in mangrove forests in Thailand.
By using the heat-cold shock method, Bacillus spp.can be isolated from other bacterial species [25].Based on genotypic and phenotypic identification, AMP identification was used to screen and group all Bacillus spp.AMP synthesis-related genes tested in this study have long been known to be responsible for the inhibitory activity of Bacillus spp.against other microorganisms [31], and it is not surprising that various Bacillus isolates possess these genes.However, a diverse pattern of AMPs containing Bacillus spp. was found, and it is not appropriate to classify them regarding the presentation of AMPs.In addition, the phenotypic determination of their capability to inhibit and control VP AHPND growth and pathogenesis in vitro and in vivo facilitated the selection of candidate Bacillus spp.for further analysis.
B. subtilis (K3) markedly reduced shrimp mortality both at the laboratory level and at the farm level.The bacteria harbor srfAA and bacA genes.The BacA gene is known to contribute to the synthesis of bacilysin, but insufficient reports on controlling or inhibiting Gram-negative bacteria, including Vibrio spp., have been noted.Conversely, many inhibitory effects of srfAA-mediated surfactin against Vibrio were reported [38-40].Therefore, it is expected that surfactin secreted by B. subtilis (K3) would inhibit the growth of VP AHPND in vitro, and a similar mechanism would be proposed in vivo.However, the effect of secreted AMPs or bacterial components on the activation of the shrimp immune system, which contributes to retained survival, should be further elucidated.
Despite the apparent differences in environmental conditions between shrimp stomachs and mangrove forest soil, there were no clear patterns in the distributions of the tested AMP-related genes among Bacillus isolates from each origin.It is expected that the Bacillus present in the shrimp stomach is not specifically adapted to the shrimp stomach environment but rather that Bacillus that could be present in the external environment is ingested orally and colonizes the shrimp stomach.While some previous studies claimed that bacteria administered by feeding colonized shrimp [15,41], it is not clear whether supplied Bacillus in water can stably colonize the digestive tract of shrimp.A previous report showed very low colonization rates of Bacillus bacteria in the digestive tracts of shrimp, especially in earthen ponds [42].For this reason, the field trial in this study was conducted with continuous administration of the test bacterium, but the optimization of dosing methods is needed.
In general, strains of VP AHPND are halophilic, and the salinity in the water affects their virulence [43,44].Experimental infections in this study also showed different mortality rates of shrimp in a salinity-dependent manner.However, in shrimp hatcheries, it may be difficult to reduce the salinity [45].In contrast, inland water aquaculture of Pacific white shrimp uses low-salinity water for cultivation [46,47].In this study, at each salinity, mortality during VP AHPND challenge was lower in the experimental group using B. subtilis (K3).This indicates that the effects can be expected under a variety of environmental salinity conditions.
Given the definition of the term probiotic [5], it may not be appropriate to use the designation probiotic for the use of Bacillus in this study (exposure via immersion).However, shrimp are expected to take up bacteria in the water, as evidenced by the fact that oral infection of Vibrio is experimentally established using immersion [26].Further research is needed on the dynamics of the supplied bacteria during immersion, but it might become possible to use the term probiotics for this strategy of the use of beneficial bacteria for shrimp in this method.
Although Bacillus bacteria have been isolated from the shrimp gut [14,15,48], previous analyses of the 16S rDNA-based microbiome show that the genus Bacillus is rarely the dominant genus in the shrimp gut [49], despite its strong inhibitory activity against other bacteria.In the environment of the shrimp digestive tract, it is likely that the balance of microbes is maintained among many bacterial species via bacterial competition or interrelationships with the host.Human studies and subsequent mouse model studies have shown that rare Bacillus in the gastrointestinal tract reduces the risk of infectious disease outbreaks [50].Surely, the findings in mammals cannot be easily applied to shrimp, but there are phenomena that cannot be fully elucidated by sequencing microbiome analysis alone, and isolation of bacterial strains and subsequent in vitro and in vivo analysis remain useful.
This study was based on selected AMP-related genes for genetic analysis rather than whole genome analysis.We cannot deny that the isolates might harbor novel or overlooked AMP genes that contribute to bacterial inhibition.In addition, it remains unclear whether the differences in inhibitory activity between isolates in vitro are dependent on the amount of AMP secreted or the activity of each peptide molecule.Because of the limitations of this study, it is not certain that similar results can be obtained with other Bacillus isolates.
Importantly, selected B. subtilis (K3) isolate do not possess antibiotic resistance properties.It is suggested that the use of B. subtilis (K3) as a probiotic is safe for aquatic animal cultivation and suitable for producing fishery products for human consumption.In the future, the replacement of antibiotics with probiotics not only reduces the use of chemicals in agriculture, which leave residues in the environment, but also reduces bacteria harboring AMR, which reduces the transfer of antimicrobial-resistant bacteria to humans.
In conclusion, an isolate of Bacillus spp., which was obtained in this study, decreased the mortality of shrimp challenged with VP AHPND .The criterion to screen potential beneficial Bacillus spp. is useful for the search for probiotics for shrimp.This study also shows one of the possible mechanisms of the beneficial effects of probiotics on shrimp.

Microorganisms 2023 , 15 Figure 1 .
Figure 1.Growth inhibition analysis of Bacillus spp.against VPAHPND.(A) Solid agar; isolated Bacillus spp.were grown on the lawn of VPAHPND (strain RY) on solid agar plates.The inhibition zone was measured and demonstrated as the diameter of the clear zone (mm).(B) Liquid medium; Bacillus spp.were cocultured overnight with VPAHPND (strain RY).The control is the culture without Bacillus.The order corresponds to Table2, which shows the presence of AMP-related genes.

Figure 1 .
Figure 1.Growth inhibition analysis of Bacillus spp.against VP AHPND .(A) Solid agar; isolated Bacillus spp.were grown on the lawn of VP AHPND (strain RY) on solid agar plates.The inhibition zone was measured and demonstrated as the diameter of the clear zone (mm).(B) Liquid medium; Bacillus spp.were cocultured overnight with VP AHPND (strain RY).The control is the culture without Bacillus.The order corresponds to Table2, which shows the presence of AMP-related genes.

Figure 3 .
Figure 3.Effect of water salinity on B. subtilis (K3) efficiency in controlling AHPND.Shrimp were treated with B. subtilis (K3) for 10 h following the challenge with VPAHPND (strain RY) at 10 4 CFU•mL −1 by the immersion method.Shrimp were reared at different salinities, including 5 ppt, 20 ppt, and 40 ppt.**** p < 0.0001 compared with the control for each salinity.

Figure 3 .
Figure 3.Effect of water salinity on B. subtilis (K3) efficiency in controlling AHPND.Shrimp were treated with B. subtilis (K3) for 10 h following the challenge with VP AHPND (strain RY) at 10 4 CFU•mL −1 by the immersion method.Shrimp were reared at different salinities, including 5 ppt, 20 ppt, and 40 ppt.**** p < 0.0001 compared with the control for each salinity.

3. 5 . 4 .
Efficiency of Isolated Bacillus in Controlling AHPND: Field Level Field trials were performed in a local nursery farm in Vietnam to evaluate the protection efficacy of B. subtilis (K3).Shrimp ponds that were treated with B. subtilis (K3) showed a low number of AHPND occurrences, and if eventually positive for AHPND, those ponds can be continually cultivated until harvested.Moreover, with B. subtilis (K3) threat, higher weight with lower FCR compared to the control was observed, which led to greater shrimp production (Table
2.5.In Vivo AHPND Challenge Test and Efficacy Analysis 2.5.1.Experimental Shrimp Healthy Pacific white shrimp were kindly provided by Aquatic Animal Research Mae Klong, Charoen Pokphand Foods Public Co., Ltd.(CP), Bangkok, Thailand.Shrimp were maintained in aerated aquaculture tanks at Kasetsart University until the challenge test.

Table 3 .
Presence of AMP genes in Bacillus isolates.

Table 4 .
Results of the field trial.Number of AHPND infection ponds, number of drain ponds, % average survival, total shrimp weight (kg), total feed (kg), and FCR of shrimp receiving B. subtilis (K3) and the control group.