The Potential of Galangal ( Alpinia galanga Linn . ) Extract against the Pathogens that Cause White Feces Syndrome and Acute Hepatopancreatic Necrosis Disease ( AHPND ) in Pacific White Shrimp ( Litopenaeus vannamei )

White feces syndrome and acute hepatopancreatic necrosis disease (AHPND) are serious diseases that have recently been noted in Pacific white shrimp (Litopenaeus vannamei). Vibrio bacteria and 6 species of fungi (Aspergillus flavus, A. ochraceus, A. japonicus, Penicillium sp., Fusarium sp., and Cladosporium cladosporioides) were isolated from shrimp naturally infected with white feces syndrome. Antibiotics have been used to treatment the disease for many years, but these have been ineffective and have resulted in drug residue problems for the shrimp industry. In this study, an alternative method was tested for its efficacy in controlling these pathogens. The crude extract of galangal (Alpinia galanga Linn.), an herbal medicine, inhibited the growth of 8 vibrio species of the pathogen, V. parahaemolyticus (EMS/AHPND) in particular. The results also showed that 0.5 mg/ml of the galangal extract was a concentration that produced the strongest inhibition of the fungi A. ochraceus. Naturally infested shrimp L. vannamei were fed 2 and 4% (v/w) portions of the herb extract for 12 days and their progress was compared with that of a control group (no herb extract). At the end of the feeding trial, the numbers of total Vibrio spp. and the incidence of fungi infestation in the hepatopancreas and intestines of treated shrimp were significantly lower than that in the control group (P<0.05). Furthermore, the survival rates for the treatment groups, after injections with V. parahaemolyticus (EMS/AHPND), were significantly higher than that of the control group (P<0.05). Based on these results, we can report that the galangal extract has antimicrobial properties that are applicable as bio-medicinal agents against white feces syndrome and AHPND. Therefore, in the future this herb should be an alternative to chemotherapeutic agents that are being used in the shrimp industry.


Introduction
Disease outbreaks caused by viruses, bacteria and protozoa are important disease agents within the shrimp aquaculture, a major industry in Southeast Asia, because they can lead to serious economic losses for long periods of time.These bacterial disease pathogens have grown rapidly due to global warming, which has stressed the shrimp population by reducing immunity and enhancing the infection rates.In 2009, the first reports of an emerging disease in Penaeus sp.shrimp was initially named Early Mortality Syndrome (EMS) in China.This disease was later referred to as Acute Hepatopancreatic Necrosis Disease (AHPND).In 2011, EMS/AHPND occurred frequently in Vietnam, which led to the isolation of the causative agent, V. parahaemolyticus (Tran et al., 2013).In 2012, AHPND had a major impact on shrimp farmers in Thailand and Southeast Asian who sustained heavy losses in production (Flegel, 2012;NACA-FAO, 2011).Outbreaks of disease reported in Thailand during 2010-2011 called White feces syndrome were found mostly in Pacific white shrimp (L. vannamei).Infestations of Gregarine protozoa and Vibrio bacteria have been found that caused loose shells, decreased appetite, retarded growth, and finally sporadic mortality of the shrimp in grow out ponds.Typical symptoms of the disease can be noted by white feces floating on the water surface in the rearing pond (Tangtrongpiros, 2010).Several reports have shown that vibrio bacteria including V. alginolyticus, V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, and V. harveyi are the important pathogens, as these are always isolated from diseased L. vannamei (Lighter, 1993;Vandenberghe et al., 1999;Montero & Austin, 1999).Bacterial pathogens can cause problems that range from growth retardation to sporadic mortalities and mass mortalities of shrimp.From this point of view, vibrio bacteria represent the greatest bacterial threat to the shrimp industry.When shrimp are under stressful conditions, such as when they are reared in a high-density setting, their immunity is lowered.Under those conditions, vibrio bacteria easily attack, which leads to the occurrence of disease (Ruangpan et al., 1997;Sung et al., 2001;Karunasagar et al., 2004;Lee et al., 2009).Some aquatic fungi are also opportunistic pathogens.Fusaruosis or black gill disease is caused by Fusarium spp., and it may affect all developmental stages of penaeid shrimp (Karunasagar et al., 2004).Shrimp farmers have few choices to control the diseases that threaten their shrimp production.Many types of drugs and chemicals have been used to treat vibrio bacteria, and this has created a residual drug problem that has led to the spread of drug-resistant bacterial strains in the shrimp industry.To solve these problems, medicinal herbs have been introduced as alternative methods for the control and treatment of bacterial diseases, because herbs are known to have anti-parasite and antimicrobial effects.Galangal (Alpinia galanga Linn.) is one of the typical herbs that can be used for the treatment of diseases in shrimp, and it is available year-round at a low cost.This herb is rich in phenolic compounds such as flavonoids and phenolic acids (Mayachiew & Devahastin, 2008).The essential oil of the crude galangal extract is responsible for its antimicrobial activity (de Pooter et al., 1985;Turker et al., 2002;Oonmetta-areea et al., 2006;and, Rao et al., 2010).Therefore, we tested the potential efficacy of an ethanol galangal crude extract to inhibit and reduce the number of Vibrio spp.bacteria and fungi in natural infestations of white feces syndrome and AHPND in Pacific white shrimp (L.vannamei).

Preparation of Galangal-Ethanol Extract
Fresh galangal rhizomes were purchased from a local market in Chanthaburi, Thailand.The rhizomes were sliced into thin layers and dried at room temperature in a tray dryer followed by heating at 45 o C for 24 h.After drying, they were ground into powder using an electric blender (Philips, Cucina, Thailand).Ten grams of the galangal powder were suspended in 100 ml of ethanol, and then let stand at room temperature overnight.After filtration through filter paper (No. 1, Whatman International Ltd., Maidstone, UK), the extract was dried in a rotary evaporator (Rotary Evaporator BUCHI R-114, Vacuum pump BUCHI B-169 Switzerland) (Oonmetta-aree et al., 2006).

Bacterial Inoculum
The isolate was streaked on TCBS agar and incubated at 30 o C for 18-24 h.A pure colony was streaked on Trypticase Soy Agar (TSA, Difco, USA), and incubated at 30 o C for 18-24 h.Several colonies of bacteria were inoculated into a 1.5% sterile sodium solution, which was mixed well with a vortex mixer.The bacterial suspension was tested using McFarland Standard No. 0.5 (×10 8 cfu/ml) then diluted to ×10 6 cfu/ml for antibacterial activity testing.

Disc Diffusion Test
The efficacy of herbs for the growth inhibition of Vibrio was done using an agar disc diffusion assay and efficacy testing method developed by Oometta-aree et al. (2006).The Vibrio inoculums (×10 6 cfu/ml) were spread on Mueller Hinton agar (Difco, USA) supplemented with 1.5% NaCl (MHA-1.5 % NaCl), and then kept for about 5 min to allow the surface of the agar to dry.Each herb extract (80 µl) was applied to a paper disc (Φ8 mm, Advantec, Tokyo, Japan) then both sides were dried in a sterile laminar flow.The paper disc was then placed onto an MHA-1.5% NaCl plate inoculated with Vibrio, followed by incubation at 30 ºC for 18-24 h.The efficacy of herbs for the growth inhibition of Vibrio was estimated by measuring the diameter of the clear zone surrounding the herbal disc.

Minimum Inhibitory Concentration (MIC) Test
One milliliter of Vibrio (×10 6 cfu/ml) was inoculated into liquid Mueller Hinton broth containing 1.5% NaCl (MHB-1.5 % NaCl).Each herbal extract (0.25 g/ml) was diluted with ethanol using a 2-fold dilution method.In this method, 160 μl of each dilution was dropped onto a paper disc (Φ8 mm, Advantec, Tokyo, Japan), followed by drying in a sterile laminar flow.Then the paper disc was placed into a MHB-1.5% NaCl liquid medium and incubated at 30 ºC for 18-24 h.The MIC of ethanol extract was regarded as the lowest concentration of the extract in the liquid medium that would permit no turbidity of the tested microorganism (Oometta-aree et al., 2006).

Minimum Bactericidal Concentration (MBC) test
The tubes that showed no turbidity of the bacteria and the last tubes showing turbidity from the MIC test were used for further MBC testing.Then, 0.1 ml of culture medium used in the MIC test was spread onto TCBS Agar (Difco, USA), incubated at 30 ºC for 18-24 h, and the formations of colonies were counted.The MBC was the lowest concentration of herbal extract that formed less than 20 colonies, corresponding to an inhibition of the bacterial growth at 99.9%, or more (Oometta-aree et al., 2006).

Fungi Isolation and Identification
The samples of naturally infected shrimp were collected from 50 grow-out ponds.Artificial feed was simultaneously collected for use in rearing shrimp samples.Fungi were isolated from the hepatopancreas and intestines of diseased shrimp and artificial feed were cultured on Glucose -Yeast in seawater (GYS) agar then incubated at 25-27 o C for 2-5 days (Khomvilai, 2006).Several isolation samples were streaked on GYS agar until the fungus had formed a pure culture.Then the isolations were identified by the National Center for Genetic Engineering and Biotechnology, Thailand, as described by Klich (2002), Raper & Fennell (1965) and by Samson et al. (1994Samson et al. ( & 1995)).

Fungi growth Inhibition Test
The galangal crude extract was dissolved with sterilized seawater in 8 dilutions: 0.0005, 0.005, 0.05, 0.5, 1, 2, 4 and 8 mg/ml.Agar plates of pure fungus were cultured for normal growth.Each fungus colony was cut into a 1 cm diameter using a cork border and was immersed in each galangal extract dilution for 3 replications.The treatment group was exposed to each extract dilution for 30 minutes, then incubated at 25 o C, and the control group was immersed in sterilized seawater.After the fungi had been completely immersed, the fungi plugs were cleaned twice in sterilized seawater and placed on GYS agar followed by incubation at 25-27 o C.After incubation for 2 days, the fungi colony diameters from each plate were measured.The efficacy of the crude extract against growth inhibition was established by comparing the diameters of the colony radii of the fungal mycelia of the treatment and control groups, which was calculated as follows: Colony radius growth rate % colony radius of the treated fungus colony radius of the control 100 The growth inhibition of fungi (%) = 100 -Colony radius growth rate (%)

Test Diets
The galangal extract was dissolved in ethanol to a concentration of 0.25 g/ml, then was mixed with commercial pellet feed (CP feed, Thailand) at 2 (5 g/kg diet) and 4% (10 g/kg diet).For the control group, no herbal extract was added.The feed was kept at room temperature for 30 min to allow the absorption of the extract and the evaporation of the ethanol.The control diet was also absorbed with ethanol, and then evaporated.Next, the pellets were coated with squid fish oil (Agrithai And Development Co., Ltd., Thailand) at 10 g/kg feed to prevent the dispersion of the galangal extract in water and to reduce the smell of the extract.The feed was then dried at room temperature.These tested diets were prepared for shrimp each day.The feeding rate was 3.0% of the shrimp body weight.

Treatment of Pathogenic Vibrio Bacteria and Fungi
About nine hundred white feces syndrome diseased shrimp, L. vannamei, with average body weight 10.7 ± 1.6 g were obtained from a shrimp farm in Chanthaburi Province, Thailand.The samples were acclimatized for 3 days in 3 plastic tanks, each with a capacity of 500 L.Then, the number of Vibrio spp.bacteria and fungi in the hepatopancreas and intestines of 15 shrimp were examined and recorded.Experimental aquariums (90 L capacity) were filled with 70 L of chlorine-treated 29 ppt seawater.Flow-through water equipped with an aeration system was arranged.Each group of 25 shrimp was sampled from the stocked tanks and was transferred into the individual experimental aquariums for 5 replications of treatment and the control group.The shrimp were fed the test diet 3 times a day for 12 days.Excess feed and waste was removed before each feeding.The parameters of the water were maintained for optimal quality.Three shrimp were sampled at 1, 3, 5, 7, 10, and 12 days from each aquarium after the feeding trials.The hepatopancreas and intestines of the shrimp were examined for Vibrio spp.and fungi via the spread-plate method.
Incidence of fungi infestation % The number of infested shrimp Total number of observed shrimp 100 2.9 Effect of the Extract on Disease Resistance Caused by Vibrio spp.

Preparation of the Bacterial Samples
The pathogenic V. parahaemolyticus (EMS/AHPND) was isolated from infected shrimp.This bacterium was identified based on a scheme established by Colwell (1984) using API 20E kits (ATB System, BioMérieux, France).Identification of V. parahaemolyticus (EMS/AHPND) was accomplished via the PCR technique (Joshi et al. 2014).The isolate was grown on Thiosulfate Citrate Bile Sucrose Agar (TCBS Agar, Difco USA) incubated at 30 o C for 18-24 h.Then a pure colony was streaked on Trypticase Soy Agar (TSA Difco, USA), and incubated at 30 o C for 18-24 h.Heavy streaks of isolated bacteria were inoculated into a 0.85% sterile sodium solution, which was then mixed well via a vortex mixer.The bacterial suspensions were tested using McFarland Standard No. 0.5 (10 8 cfu/ml) then diluted to 10 5 cfu/ml for the antibacterial activity test.

Resistance Test
The water in the 90 L aquariums was managed according to the method mentioned above.Triplicate aquariums were prepared for each group.Ten shrimp (10.7 ± 0.8 g/shrimp) were transferred to each aquarium for 2 treatment groups (2 and 4% of the extract diet) and 1 control group (0% of the extract diet).The shrimp were fed test diets 3 times a day for 12 days.After the shrimp were fed a treated diet for 12 days, disease resistance tests were conducted via the injection of 100 μl of bacterial suspension, V. parahaemolyticus (2.85 ± 0.63 X 10 5 cfu/ml), into the abdominal segment.After injection, shrimp were fed a commercial diet twice a day for 15 days.Disease symptoms and mortality rates were observed and recorded daily; the surviving shrimp were counted, and statistical analysis was calculated after the end of the experiment.

Statistical Analysis
A multiple comparison (Pair wise Comparison Test: Fisher's LSD) test was used to examine the significant differences (P<0.05)among treatments and control groups using SYSTAT VERSION 5.0.As reported by Canillac & Mourey (2001), if the MBC/MIC ratio is found to be less than or equal to 4, the strain is considered to be susceptible; on the other hand, if this ratio is greater than 4, the strain is considered to be tolerant.

Antibacterial Activities of Galangal Extract
The results showed a growth inhibition zone surrounding the galangal extract disc of all species' bacteria.The MBC and MIC of 2 bacterial species, V. cholerae and V. vulnificus, showed the highest degree of sensitivity.The other 5 species, V. parahaemolyticus, V. parahaemolyticus (EMS/AHPND), V. fluvialis, V. mimicus, and V. harveyi, showed intermediate sensitivity, and V. alginolyticus showed low sensitivity among the bacteria tested.
The results of the antibacterial activity of the galangal extract in this study showed most of the species of Vibrio spp. to be sensitive (Table 1).

Fungi Isolation and Identification
Pathogenic fungi were isolated from L. vannamei, feces, and artificial feed.All fungi were identified by the 6 species shown in Table 2. Hepatopancreas and intestine Fusarium sp.

Efficacy of the Extract against Pathogenic Fungi
Table 3 shows the efficacy of the galangal extract on 6 species of fungi.The galangal extract at a concentration of 0.5 mg/ml represented the strongest inhibition of A. ochraceus.A concentration of 1 mg/ml inhibited the growth of Penicillium sp.; Fusarium sp., and C. cladosporioides.Two species, A. flavus and A. japonicus, were least susceptible to the galangal extract (8 mg/ml).

Health Status of the Naturally infected Shrimp
The hepatopancreas and intestines of shrimp from a grow-out pond that were infected with white feces syndrome were observed, and 4 species of fungi were found including C. cladosporioides, Penicillium sp., A. japonicus, and Fusarium spp.In addition, a rather high incidence of Vibrio spp.were also isolated from the same samples (100.0 ± 0.0 %).The total counts from the hepatopancreas and intestines were as high as 386.73 ± 323.73 X 10 3 cfu/g and 426.46 ± 168.21 X 10 5 cfu/g, respectively.The dominant numbers of Vibrio spp.isolated in the feces were green and yellow colonies.The totals for the number of Vibrio spp. in the hepatopancreas and intestines of infested L. vannamei shrimp fed the galangal extract (treated diet) were significantly less than that for the control group (P < 0.05).Figure 1 lists the numbers after 3 days in the hepatopancreas (A) and 5 days in the intestines (B) after feeding with the treated diet.In contrast, the total number of Vibrio spp. in the hepatopancreas (A) and intestines (B) of the control group had increased through 12 days (Figure 1).No green colonies of Vibrio spp.were found in either of the groups fed 2 or 4% treated diets.
(A) Hepatopancreas (B) Intestines Figure 1.The total number of Vibrio spp. in (A) hepatopancreas and (B) intestines of infested L. vannamei fed the 2 and 4% galangal extract, as well as the control (0%), for 12 days.Data are the means ± SD; different letters for the time interval indicates a significant difference (P<0.05)

Effect of the Galangal Extract on Pathogenic Fungi
The incidence of fungi infestation in the hepatopancreas and intestines of infested L. vannamei shrimp fed the crude galangal extract (treated diet) was significantly lower than that in the control group (P<0.05).Also, no fungi could be found in the hepatopancreas and intestines of white shrimp after feeding with the treated diet for 10 to 12 days (Figure 2 (A) and (B)).
(A) Hepatopancreas (B) Intestines Figure 2. Incidence of fungi infestation (%) in the hepatopancreas (A) and intestines (B) of infested L. vannamei fed galangal extract at 2 and 4%, along with that of the controls (0%), for 12 days.Data are the means ± SD; different letters for the time interval indicates a significant difference (P<0.05)

Effect of the Galangal Extract on Disease Resistance Challenged by V. parahaemolyticus (EMS/AHPND)
The survival rates for shrimp fed 2 and 4% galangal extract were 73.3 ± 5.8% and 83.3 ± 5.8%, respectively.Only 16.7 ± 5.8% of the survival shrimp were from the control group (0% galangal extract).The results differed significantly between the treated groups and the control group (P<0.05).Obviously, shrimp fed the galangal extract at 4% had the highest survival rate (Figure 3).remarkable that the galangal extract could be used to inhibit growth and reduce the number of Vibrio spp.and fungi that cause white feces syndrome and AHPND in shrimp.In the present study, after shrimp were fed a diet mixed with 2 and 4% of the extract with ethanol, from day 1 the numbers of Vibrio spp. in the hepatopancreas and intestines were lower than in the control group.Remarkably, galangal extract can be used to inhibit the growth and reduce the number of Vibrio spp.and fungi to prevent white feces syndrome.Moreover, resistance in L. vannamei against V. parahaemolyticus causing AHPND occurred when the shrimp were fed a diet mixed with galangal extract at 2 or 4% for 12 days.Our results also showed a higher survival rate (83.3%) of the treatment group (fed with 4% galangal extract) than the control group (feeding with 0% extract).The results revealed the efficiency of the extract, which has ability to inhibit V. parahaemolyticus (EMS/AHPND), and it may stimulate the immune system in the tested shrimp (Chaweepack et al., 2015).Therefore, shrimp fed galangal extract diet are healthier than the control group, and shrimp fed a galangal extract diet could resist infection well.For several years, antibiotics and some hazardous chemicals have been used to treat pathogenic agents in the aquaculture industry, particularly in intensive shrimp-farming systems.The development of resistant bacteria and residue in shrimp caused a major obstacle to trade and had a negative impact on the environment as well as on consumer health.The use of herbal medicines is a viable alternative to the overuse of antibacterial agents.The main advantage of herbal agents is that the crude extract contains a mixture of compounds such as phenols, acids, esters, and aldehydes, to which bacteria are unlikely to develop resistance.A synthetic agent that contains a single compound, however, is easier for bacterial stains to resist (Abraham et al., 1997).Based on the results of the present study, we recommend alternative methods for control of the disease that causes white feces syndrome and AHPND in the shrimp industry by using galangal crude extract, which has proven to be effective and safe for the environment as well as for consumers.

Conclusion
This investigation was focused on the efficacy of galangal extract for treatment of the pathogenic organisms that cause white feces syndrome and AHPND in Pacific white shrimp, L. vannamei.Ethanol galangal extract exhibited the highest potential for Vibrio spp.and fungi reduction.The highest percentage (4%) of ethanol extract was most effective in reducing all species of the pathogens of V. parahaemolyticus (EMS/AHPND).Therefore, galangal extract should be used as an antimicrobial for white feces syndrome and AHPND therapeutics in Pacific white shrimp L. vannamei and in shrimp cultures.This alternative method could assist in reducing the impact of antibiotic or chemical residue in shrimp products as well as helping to reduce the presence of resistant bacterial strains in the environment.

3. 4 . 2
Effect of the Galangal Extract on the Total Number of Vibrio spp.
Figure 3. T and 0

Table 1 .
Growth Inhibition zones, Minimum Inhibitory Concentration (MIC) and Minimum bactericidal Concentration (MBC) by the galangal extract to 8 species of Vibrio spp.

Table 2 .
The 6 species of fungi isolated from the hepatopancreas and intestines of shrimp naturally infected with white feces syndrome L. vannamei, and from artificial feed