use of formic acid to control vibriosis in shrimp aquaculture
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Biologia 68/6: 10171021, 2013Section Cellular and Molecular BiologyDOI: 10.2478/s11756-013-0251-x
Use of formic acid to control vibriosis in shrimp aquaculture*
Derek Adams & Raj Boopathy**
Department of Biological Sciences, Nicholls State University, Thibodaux, LA 70310 USA;e-mail: Ramaraj.Boopathy@nicholls.edu
Abstract: Luminous vibriosis is a shrimp disease that causes major economic losses in shrimp industry as a result ofmassive shrimp kills due to bacterial infection caused by Vibrio species. Use of antibiotics to control Vibrio in shrimpaquaculture is not allowed in the United States and so it is necessary to develop an alternative pathogen control method forshrimp production. Short-chain fatty acids have been used as food preservatives for a long time. Organic acids are commonlyadded in feeds in animal production, such as chicken, pig, and cattle. In this study, growth inhibition eects of formic acidon ve selected Vibrio species, namely Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio parahaemolyticus andVibrio vulnicus were studied. The Vibrio bacteria were grown on both solid and liquid media using Muller-Hinton agarand alkaline peptone water, respectively, with various concentrations of formic acid. Bacterial growth was monitored in theliquid media using optical density method. The results showed signicant inhibition of growth of all ve Vibrio species byformic acid at low concentration. The eective concentration (EC50) values were calculated for all ve Vibrio species, whichwere less than 0.039% of formic acid. The results are encouraging to supplement formic acid in the shrimp feed as a controlmechanism to reduce Vibrio outbreak in shrimp aquaculture system.
Key words: vibriosis; luminescence; EC50; shrimp aquaculture; antibiotic resistance.
Abbreviations: APW, alkaline peptone water; EC50, eective concentration 50; MHA, Muller-Hinton agar.
Global aquaculture production has been growingrapidly since the 1950s. In 1982, shrimp farming ac-counted for only 5% of the world shrimp productionachieving 25% in 1990 (Gillet 2008). Currently morethan 40% of the global shrimp production come fromaquaculture.
The major bacterial diseases in shrimp aquacultureinclude vibriosis caused by Vibrio species and necro-tizing hepatopancreatitis caused by obligate intracellu-lar rickettsia-like bacteria (Lightner 2005). Vibriosis isan infectious disease caused by Vibrio species (Light-ner 1996). Within Vibrio species, Vibrio harveyi, Vib-rio vulnicus, Vibrio parahaemolyticus, and Vibrio algi-nolyticus are most frequently reported in shrimp hatch-ery, nursery and grow out ponds (Lightner 1996). Vib-riosis in shrimp ponds occurs abruptly, and infectionspreads rapidly for several days to two weeks (Brock &LeaMaster 1992). As a result, high mortality sometimeswith luminescence in shrimp ponds occurs (Lightner1996). Luminescence, cloudiness of shrimp musculature,red discoloration of appendages, and weak swimming atthe pond surfaces or edges are the visible signs of in-fection in penaeid shrimp (Brock & LeaMaster 1992).
The large numbers of bacteria are assumed to exist inthe infected shrimp, so isolation of organisms from tis-sue and haemolymph is performed to conrm the di-agnosis (Lightner 1996). Vibrio species are ubiquitousin the marine and estuarine environment (Krieg et al.1984), thus the routes of vibrio infection are presumedto be through the pit, including puncture wounds, onthe exoskeleton and through oral ingestion (Lightner &McVey 1993).
Antibiotic resistance of bacteria impacts the choiceof therapeutic drugs humans and animals can use. An-tibiotics are often used in some shrimp farms in theworld except in USA where the use of antibiotic inshrimp aquaculture is banned (Graslund & Bengtsson2001; Le & Munekage 2004; Lyle-Fritch et al. 2006).Not only emergences of antibiotic-resistant bacteria butalso antibiotic residues in food-producing animals posehealth concerns in humans and animals. In order tocontrol infectious diseases in shrimp aquaculture, re-duction in the use of antibiotics accompanied with var-ious strategies, such as improvement of both pond waterquality and the host health, are recommended (Hernan-dez Serrano 2005).
There are several alternative strategies to controlvibriosis, which include use of probiotic bacteria in
* Based on a contribution presented at the International Conference on Industrial Biotechnology (ICIB-2012), November2123, 2012, Punjabi University, Patiala (Pb.), India** Corresponding author
c2013 Institute of Molecular Biology, Slovak Academy of Sciences
1018 D. Adams & R. Boopathy
shrimp ponds. Probiotics are basically live bacterial ad-ditives that bring benecial health eects to the hostanimals (Verschuere et al. 2000). Short-chain fatty acidsinclude formic, acetic, propionic, and butyric acid. Thegenerally known antibacterial property of these organicacids is acidication of the cytoplasm (Doyle et al.2001). Some organic acids are the natural metabolicproducts of organisms, and they have been used asfood preservatives for a long time. Organic acids arealso used as feed additives to control pathogens in an-imal husbandry (Iba & Berchieri 1995; Franco et al.2004). Several studies have proven that inclusion oforganic acids in diets suppresses pathogenic bacterialgrowth in gastrointestinal tracts of poultry and swine(Iba & Berchieri 1995; Franco et al. 2004). Other stud-ies showed both short-chain and medium-chain fattyacids are eective bactericides of pathogenic Salmonellaand Campylobacter species (Khan & Katamay 1968;Chaveerach et al. 2002). In shrimp aquaculture, organicacids are not used to control bacterial infections, andthe eects of organic acids in shrimp aquaculture havenot been studied in detail (Saori & Boopathy 2011). Asseen in human food preservatives and terrestrial animalfeeds, the use of organic acids has a potential to becomealternatives to antibiotics in aquaculture. In our previ-ous study, we tested variety of organic acids on V. har-veyi and found formic acid as a potential inhibitor of itsgrowth (Mine & Boopathy 2011). We extend the cur-rent study with a major objective of nding the eectof formic acid on various species of Vibrio, includingV. harveyi, V. parahaemolyticus, V. vulnicus, V. algi-nolyticus, and V. cholerae.
Material and methods
Organism and chemicalsVibrio harveyi (ATCC 14126), V. parahaemolyticus,(ATCC17802), V. vulnicus (ATCC 29307), V. alginolyticus (ATCC17750), and V. cholerae (ATCC 14035) were obtained fromthe American Type Culture Collection, Manassas, VA, USA.All Vibrio bacteria were maintained in alkaline peptone wa-ter (APW) supplemented with 2% NaCl, adjusted to pH8.4. The cultures were maintained at room temperature (1924C) and were sub-cultured weekly. V. harveyi used in thisstudy is known to be pathogenic to shrimp (Lightner 1996).All chemicals including formic acid, acetic acid, butyric acid,and propionic acid, reagents, and media were obtained fromFisher Scientic (St. Louis, MO, USA).
Disc diusion assayThe organic acids, formic acid, acetic acid, propionic acid,and butyric acid, were used to make 5, 10, 25, 50, and 100%organic acid solutions by diluting with de-ionized water.
The method used to inoculate bacteria on agar wasbased on the Centers for Disease Control and Preven-tions method for antimicrobial susceptibility testing of V.cholerae (Anonymous 1999). Muller-Hinton agar (MHA)supplemented with 2% NaCl and adjusted pH to 8.4 wasused for disc diusion assays run in triplicate. Twenty-ve ml of MHA was poured to 100 mL Petri plates to a4 mm depth. Inocula were prepared with one loopful ofstock culture into 10 mL APW (2% NaCl, pH 8.4) followedby overnight incubation at 26C. Prior to streaking inocula
onto MHA, turbidity of the bacterial suspension in APWwas adjusted to absorbance of 0.08-0.1 at 600 nm wavelengthby adding APW. Each culture was streaked over the entireMHA surface three times with sterile cotton swabs. Within10 minutes after streaking, a lter paper (Whatman) disc(diameter, 7.03 mm) was placed on the agar surface, and itwas saturated with 23 L of an organic acid solution usinga micropipette. Petri plates were inverted, and incubated at26C for 1618 hours. After incubation, the diameter of thezones of complete inhibition was measured and recorded inmillimeters.
Determination of eective concentration (EC50) of organicacidsBased on the results from the disc diusion assay formicacid was chosen for further study. Formic acid solutions with0.025, 0.03, 0.035, 0.04, 0.045, and 0.05% were prepared bydiluting with APW (2% NaCl, pH 8.4). Fifty mL of vari-ous concentrations of formic acid solutions were dispensedinto 100 mL culture bottles in triplicate with ingredientsof APW medium. The control contained no formic acid.Two hundreds L of overnight culture of Vibrio adjustedto 0.08 absorbance at 600 nm wavelength was inoculatedto each culture bottle. Bacterial growth was monitored byabsorbance using a Genesys 20 Visible Spectrophotometer(Thermo Fisher Scientic, Inc.) at the wavelength of 600nm, and pH was measured with a calibrated UltraBASiCpH/mV meter (Denver Instruments). Both absorbance andpH were measured for 14 days, at every 12 hours for therst 4 days and every 24 hours for the rest of 10 days.
Statistical analysisThe zone of inhibition was subjected to an analysis of vari-ance (ANOVA) test (p 0.05) followed by a Tukeys posthoc analysis (SAS/Genetics Version 9.1.3, 2003, SAS Insti-tute, Cary, NC, USA).
Results and discussion
Relative toxicity of organic acidsThe relative toxicity of formic acid, acetic acid, propi-onic acid, and butyric acid on various shrimp specieswas studied using a lter paper disc assay. Sterile l-ter paper discs were saturated with various concentra-tions of organic ac