In vitro and In vivo test were carried out to find the effect of probiotics in the elimination of the EUS (Epizootical Ulcerative Syndrome) disease caused by agents i.e Aeromonas hydrophilla (bacteria) and Aphanomyces invadans (fungi) in fish. In vitro experiment revealed that the zone of inhibition to inhibit the growth of bacteria and fungi was occurred in both the probiotics; although probiotic 2 had higher zone of inhibition than probiotic 1. In vivo experiment also revealed that the elimination of pathogenic organisms observed in the form of colony forming units (cfu)/ mL i.e 8.0x10¹¹ to 3.0x10⁵ cfu/ mL by probiotic 2 was higher as compared to probiotic 1 i.e 1.8x10⁵cfu/ml. In conclusion, the present investigation showed that the viable counts of pathogenic bacterium were the highest in the fish inoculated only with the pathogenic organisms’ i.e. 6.5x1012cells/mL after a three weeks period. Probiotic cultures that were used had considerable reduction in the viable count of Aeromonas hydrophila in fish. The numbers of viable counts was the lowest in mrigal (C. mrigala) treated with probiotic 2 followed by probiotic 1 over a period of four weeks.

Keywords: Fish, Cirrihinus mrigala, Aeromonas hydrophila, Aphanomyces invadans, Colony forming unit

Cfu, Colony Forming units; NA, Nutrient Agar; EUS, Epizootic Ulcerative Syndrome

Aeromonas hydrophilla and Apanomyces invadens is the main causing agent of EUS disease. Probiotics that are the live microorganisms may release the chemical substances that have bactericidal and bacteriostatic effect on microbial population.^1,2 A probioticis defined as a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance.³ Probiotics which have the property to colonize of the potential pathogens in the digestive tract and alteration of microbial metabolism or by the stimulation of host.^4-12 The common healthy intestinal flora of fish species include Vibrio,⁵ Lactobacillus,¹³ Acinetobacter, Achromobacter and by Bacillus and representatives from the family Enterobacteriaceae.¹⁴ Keeping these in mind, the present investigation proposed to investigate the elimination of pathogenic organisms (Aeromonas hydrophila and Aphanomyces invadans) by using two probiotics (probiotic 1 and probiotic 2).

The probiotic 1 was composed by Nitrosomonas, Azosporillium, Lcithiniformes, Bacillus subtilis, Nitrobacter, Trichoderma, Bacillus megetherium. The probiotic 2 was composed by Lactobacillus sporogenes, Lactobacillus acidophilus, Bacillus subtilis, Bacillus licheniformis, Saccharomyces cervirial, Sea weed extract, Enzyme complex contains, Amylase, Phylase, Protease, Cellulose, Beta-galactosidase, Lipase, Vitamins (Vitamin C = 20g, Vitamin B6 = 1g). Both probiotics were used to find out the In vitro and In vivo antagonism against pathogenic organism.

In vitro test for determination of the antimicrobial activity

Bacterial cultures of probiotic 1and probiotic 2were examined for inhibitory effects against the pathogenic bacteria, A. hydrophila isolated from diseased fish.¹⁵ The In vitro antimicrobial activity was assessed using agar diffusion method and the inhibition zone was determined according to.¹⁶ The two probiotic bacteria were inoculated in the center of petri dishes, containing Nutrient agar (NA agar) and incubated at 30 °C for 24h. Subsequently, fresh inoculums of the pathogenic A. hydrophila, was spread over the plates (by pour plate method), previously inoculated with probiotic bacterial culture. The plates were further incubated at 30 °C for 24h, and then checked for the appearance of inhibition zone.¹²

In vivo tests of probiotics

The healthy individuals of mrigal fish weighing 20g were used to perform In vivo pathogenicity tests following.¹⁷ Nine fish were kept in each tub and the experiment was done in triplicate. The fish was inoculated with the pathogenic organisms say Aeromonas hydrophilla (bacterium) and Aphanomyces invadans (fungus). The causative nature of these pathogens to induce EUS was earlier tested by Sharma.¹⁸ All treatments given to the fish with pathogens and probiotics is shown in table 1. One fish from each replicate was sacrificed at weekly intervals and the bacterial flora from intestine; liver and kidney were taken after maceration of tissues. The viable counts of the bacterial pathogens were worked out. The difference between treatments means (at 0.05 significant levels) was investigated by following the method described by Snedecor et al.¹⁹

In vitro antagonistic test

Probiotic cultures, viz., Probiotic 1and Probiotic 2 exhibited In vitro antagonistic activity against the pathogenic A. hydrophila. Probiotic 2 developed larger inhibition zone than probiotic 1 against A. hydrophila on NA agar plates.

The In vivo results of viable counts of A. hydrophilla and A. invadans under different treatments over a period of eight weeks are presented in table 1. In the first treatment with bacteria, the number of viable counts progressively from 5.5x10¹¹ in first week to 8.7x10¹¹ in the third week when fish died. The situation became more critical when fish injected with both the pathogenic organisms (bacteria+fungi) together. The viable counts increased from 8.0x10¹¹ in first week to 6.5x10¹² in the third week. The viable count of the pathogenic organism became so high that the fish could not tolerate and subsequently also died. However, when the treatment was given with probiotics then it indicated a progressive decrease in the viable counts. The viable counts of bacteria with probiotic 1 declined from 7.6x10⁹ in first week to 2.8x10⁵ in eighth week. The viable counts of bacteria and fungi with probiotic 1 declined from 7.2x10⁹ in first week to 5.3x10⁵ in eighth week. The viable counts of bacteria with probiotic 2 decreased from 6.4x10⁹to 1.8x10⁵ in the eighth week. The viable count of bacteria and fungi together with probiotic 2 declined from 7.0x10⁹ in first week to 3.0x10⁵ in eighth week (Table 1). The tests results therefore show that pathogenic bacteria were eliminated successfully by both the probiotics.

Probiotic bacteria on the aspect of safety

Both probiotics were found to be harmless to C. mrigala as any clinical signs and mortalities were noticed during the probiotic treatments.

In the present investigation, the viable counts or colony forming units (cfu) of pathogenic organism were high in the inoculated fish. However, these counts decreased when treated along with probiotics. The results showed that the number of viable counts decreased more when using probiotic 2 than when using probiotic 1over a period of eight weeks. Similar results were observed by Zhou et al.²⁰ in their study on the inhibition ability of probiotic, Lactococcus lactis RQ516, against A. hydrophila; In vitro with 14.77 ± 1.17 mm zones of inhibition and; immune stimulator and growth promoter, In vivo in tilapia, Oreochromis niloticus. The study of Abd El Rhman et al.²¹ showed that combination of two probiotic bacteria (Micrococcus luteus and Pseudomonas sp.) gave adverse effect against A. hydrophila in Nile tilapia, Oreochromis niloticus. Although, their study was on different fish, with different probiotics and pathogenic bacterium, the pattern of inhibition in both In vitro as well as In vivo was found to be the same. Nimrat & Vuthiphandchai²² also observed similar results in marine shrimp, where they used 12 commercial probiotic products against shrimp pathogenic bacterium Vibrio harveyi. Probiotic enhanced immune system observed in a number of earlier studies^23-25 showing a decrease in the number of colony forming units of bacteria in fish. Salini et al.²⁶ observed that the diversity and intensity of microbial flora got reduced when treated with culture of medicated diet having probiotic bacteria (Bacillus). Behera & Nayak²⁷ also found that the bacterial load present inside the culture ponds does not show any harm due to presence of probiotics which helped for suppression and maintaining of a clean and hygienic environment for sustainable shrimp culture.

In conclusion, the pathogenic organisms that cause disease in fish can be effectively eliminated by the use of probiotics.

We are thankful to the Head of the Department of Zoology and Aquaculture, CCS Haryana Agricultural University, for providing the necessary facilities. The financial assistance in the form of Merit Stipend by CCS Haryana Agricultural University.

None.

1. Watson AK, Heinrich K, Lategan MJ, et al. Probiotics in aquaculture: The need, principles and mechanisms of ac–tion and screening processes. Aquaculture. 2008;274:1–14.
2. Sihag RC, Sharma P. Probiotics: The New Ecofriendly Alternative measures of disease control for sustainable aquaculture. Journal of Fisheries and Aquatic Science. 2012;7(2):72–103.
3. Fuller R. Probiotics in man and animals. J Appl Bacteriol. 1989;66(5):365–378.
4. Gatesoupe FJ. The effect of three strains of lactic bacteria on the production rate of rotifers, Brachionus plicatilis and their dietary value for larval turbot, Scophthalmus maximus. Aquaculture. 1991;96:335–342.
5. Austin B, Stuckey LF, Robertson PAW, et al. A probiotic strain of Vibrio alginoyticuseffective in reducing diseases caused by Aeromonas salmonitida, Vibrio anguillarum & Vibrio ordalii. Journal of fish diseases. 1995;18:93–96.
6. Mohanty SN, Swain SK, Tripathi SD. Rearing of catla (Catla catla) spawn on formulated diets. Aquaculture. 1996;11:253–258.
7. Gildberg A, Mikkelson H, Sandaker E, et al. Probiotic effect of lactic acid bacteria in the feed on growth & survival of fry of Atlantic cod (Gadusmorhua). Hydrobiologia. 1997;352(1):279–285.
8. Bly JE, Quiniou S M, Lawson L A, et al. Inhibition of sprolegnia pathogenic for fish by Pseudomonas florescence. Journal of fish diseases. 1997;20(1):35–40.
9. Wang XH, Ji W S, Xu HS. Application of probiotic in aquaculture. Aiken Murray corp. 1999.
10. Park SC, Shimamura I, Fukunaga M, et al. Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglosida, as acandidate for disease control. Appl Environ Microbiol. 2000;66(4):1416–1422.
11. Chang YM, Liu W Y. An evaluation of two probiotics bacterial strains, Enterococcus faecium SF68 &Bacillus toyoi, for reducing edwardsiellosis in cultured European eel, Anguilla anguilla L. Journal of fish diseases. 2002;25(5):311–315.
12. Sharma P, Sihag RC, Gahlawat SK. Relative Efficacy of two Probiotics in controlling the Epizootical Ulcerative Syndrome Disease in Mrigal (Cirrihinus mrigala). Journal of Fisheries and Aquatic Science. 2013;8(2):305–322.
13. Kacem M, Karam N. In vitro preselection criteria for probiotics Lactobacillusplantarum strains of fermented olives origin. International Journal of Probiotics and Prebiotics. 2006;1(1):27–32.
14. Ringo E, Strom E. Microflora of arctic charr, Salvelinus alpinus L. Gastrointestinal microflora of free–living fish and effect of diet and salinity on intestinal microflora. Aquaculture Research. 1994;25(6):623–629.
15. Sharma P, Sihag RC. Pathogenicity Test of Bacterial and Fungal Fish Pathogens in Cirrihinus mrigala Infected with EUS Disease. Pak J Biol Sci. 2013;16(20):1204–1207.
16. Ruiz CM, Roman G, Sánchez JL A marine bacterial strain effective in producing antagonisms of other bacteria. Aquaculture International. 1996;4(3):289–291.
17. Keskin O, Secer S, Izgor M, et al. Edwardsiella ictaluri infection in Rainbow Trout (Oncorhynchus mykiss). J Vet Anim Sci. 2004;28(4):649–653. 
18. Sharma P. Role of probiotics in controlling epizootic ulcerative syndrome disease in carps. PhD.thesis, CCS Haryana Agricultural University, India. 2009.
19. Snedecor G, Cochran W, CoxD. Statistical Methods. (8th edn), The Iowa State University Press, USA. 1989.
20. Zhou X, Wang Y, Yao J, et al. Inhibition ability of probiotic, Lactococcuslactis, against A. hydrophilaand study of its immunostimulatory effect in tilapia (Oreochromis niloticus). Int j Engineer Sci Tech. 2010;7(2):73–80.
21. Abd El–Rhman AM, Yassir AE, et al. Micrococcus luteusand Pseudomonasspecies as probiotics for promoting the growth performance and health of Nile tilapia, Oreochromis niloticus. Fish Shellfish Immunol. 2009;27(2):175–180.
22. Nimrat S, Vuthiphandchai V. In vitro evaluation of commercial probiotic products used for marine shrimp cultivation in Thailand. African Journal of Biotechnology. 2011;10(22):4643–4650.
23. Noh H, Han KI, Won TH, et al. Effect of antibiotics, enzymes, yeast culture and probiotics on the growth performance of Israeli carp. Korean J Anim Sci. 1994;36:480–486.
24. Prabhu NM, Nazar AR, Rajagopal S, et al. Use of probiotics in water quality management during shrimp culture. J Aquacult Trop. 1999;14:227–236.
25. Selvaraj V, Sampath K,Sekar V. Administration of yeast glucan enhances survival and some non–specific and specific immune parameters in carp (Cyprinus carpio) infected with Aeromonas hydrophila. Fish Shell fish Immunol. 2005;19(4):293–306.
26. Salini MP, Thomas AA, Anand J. Alterations in the microbial load at certain Alterations in the microbial load at certain non–specific immune sites of Macrobrachium rosenbergii supplemented with Centella asiatica. The Bioscan. 2013;8(2):701–704.
27. Behera DP, Nayak L. Role of probiotics in sustainable shrimp farming along balasore coast, Orissa. The bioscan. 2011;6(1):97–102.