The study was conducted for isolation and identification of various bacterial species from skin affection cases of freshwater fish of Punjab. Total 210 cases were examined during the period July 2009 to March 2010. Bacterial isolation was done from forty seven cases of skin affections. The various organs of fish showing gross lesions were collected aseptically. The isolation and identification of bacteria was done depending upon morphology, staining characteristics and biochemical testing using conventional methods as per the standard protocol. The bacterial species isolated were Aeromonas spp, Bacillus spp., Citrobacter spp., E. coli, Enterobacter spp., Flavobacter spp., Klebsiella spp., Lactobacillus spp., Micrococcus spp., Proteus spp., Pseudomonas spp., Salmonella spp., Staphylococcus spp., Streptococcus spp., Vibrio spp. and six gram negative bacterial isolates. Out of these Aeromonas was most predominantly isolated species followed by Vibrio. Gram negative bacteria were the most dominant species isolated from skin affection cases.
Bacteria are ubiquitous in nature. Their role in causation of different diseases in aquaculture is well known. Fish are potentially more vulnerable to water quality and environmental factors when injured, stressed or suffering from weaked immune systems with concurrent finding of skin problems. Etiology of the skin affections in fish is complicated as many causative agents had been isolated from the skin lesions viz. parasites fungus, viruses and bacteria (Kinnune, et al., 1997; Srivastava, et al., 1994 and Hedrick, et al., 1990). Out of these bacterial infections most commonly associated with skin lesions (Wiklund, et al., 1999). Presence of bacteria in internal organs indicate the break in immunological tolerance of fish (Cahill, 1990), in such conditions of decreased immune competence the bacteria can flare up to produce the disease (McVicar, 1997). Occurrence of the bacterial diseases in fish poses great threat to sustainable aquaculture also some of these bacteria are zoonotic too. Therefore it is imperative to have the knowledge of the bacteria that are prevalent in the skin affection cases of fish, to determine and implement an adequate prophylactic measures
Materials and Methods
Bacterial isolation was done from forty seven cases of skin affections of freshwater out of 210 fish examined during the period July 2009 to March 2010. Skin was taken from all the cases while gills, kidney, liver and other organs were also collected whenever gross lesions were observed.
The relevant organ(s) viz. skin, kidney, liver, and gills were aseptically collected in sterilized plastic bags and placed on ice. The various cultural media used for isolation of micro-organism(s) included: Peptone water, Tryptone soya agar (TSA), Nutrient agar, Blood agar, MacConkey’ lactose agar (MLA) and Eosin methylene blue agar (EMB) (Hi-Media). The media were prepared as per the directions given by the manufacturer.
Identification of Bacteria
The organ(s) viz. skin, kidney, liver and gills were collected aseptically and minced by sterile scissor and inoculated into peptone water for 18-20 hrs, followed by inoculation on TSA and MLA plates and subsequently incubated at 370C. The petri dishes were examined for growth of micro organisms after incubation at 370C for 24-48 hrs. MLA was used to differentiate between lactose and non lactose fermenters. Lactose fermenters produce pink colored colonies on MLA and then these colonies were streaked on EMB agar for confirmation of E. coli, which produced colonies with greenish metallic sheen.
The colony characteristics were observed and Gram staining was carried out. The biochemical tests like oxidase, catalase, indole, methyl red, Voges-Proskauer, urease, citrate and growth on triple sugar iron agar (TSI) besides sugar fermentation tests were also carried out for the identification of isolated organisms as per the Quinn et al (1994) and Roberts and Smail (2001). The pure culture colony of individual isolate was stored on tryptone soya agar slant.
Results and Discussion
Bacterial isolation was done from forty seven cases of skin affection(s). Skin was taken from all the cases while gills, kidney, liver and other organs were also collected whenever necessary. Total fifteen bacterial species (127 isolates) were obtained. The bacterial species isolated from different organs are given in Table 1.
Table 1: Bacterial species isolated from different organs of skin affections cases of fish.
|Other gram negative bacteria||3||1||0||0||1||1||6||04.72|
Out of one hundred and twenty seven isolates of bacteria ninety six (75.6%) bacterial isolates were Gram negative while thirty one (24.4%) bacterial isolates were Gram positive. Gram negative bacteria dominate over Gram positive bacteria these findings are in accordance to those of Mastan and Rao (2007). Pure bacterial colonies were obtained from 57.4% (27/47) of skin samples while 75% each form gill (15/20) and intestine (3/4), 70 % from kidney (7/10) and 100% from liver (7/7) and 0% from fin (0/5). These findings are comparable with the observations of Austin and Austin (2007).
Most of the bacteria isolated during the study were known as potential zoonotic pathogens indicating public health significance. While isolation of bacterial species viz. Klebsiella spp. and Proteus spp. was suggestive of secondary invasion of the lesions.
The prevalence of Aeromonas spp. was highest with 22.83 % overall prevalence. This result was comparable with findings of Patil (2009) who reported the prevalence of Aeromonas spp. in fish from various source in Ludhiana to be 37.50%. Incidence of Aeromonas spp. in fish ranges from 22-69% (Agarwal 1997; Radu et al 2003). High prevalence of Aeromonas could be attributed to its presence as apart of the intestinal flora of the healthy freshwater and its ability to its rapid growth rate in the living fish body, Newman and Natnarich, (1982). Vibrio spp. was the second most prevalent in skin affections with total 15.74% prevalence. These findings were comparable to the observations of Al-Sunaiher et al. (2010). The overall prevalence of the other bacterial species (Micrococcus spp., Streptococcus spp., Bacillus spp., Enterobacter spp. and Salmonella spp.) was in the range 6 to 8 % while prevalence of rest of isolated bacterial species was even lower (0.5 to 5%).
Thus the bacterial isolation studies revealed that Aeromonas spp. was the most common bacteria isolated from the freshwater fish exhibiting skin affections followed by Vibrio spp. Gram negative bacteria were the dominant bacterial species in skin affection cases of fish. Internal organs viz liver, kidney were most suitable for isolation of pure bacterial culture from the diseased fish.
Agarwal RK.1997. Characterization of virulence factors of aeromonads isolated from foods of animal origin. Ph.D. Thesis, Deemed University, IVRI, Izatnagar, India.
Al-Sunaiher AE, Ibrahim ASS and Al-Salamah AA. 2010. Association of Vibrio Species with Disease Incidence in Some Cultured Fishes in the Kingdom of Saudi Arabia. World Applied Sciences Journal. 8 (5): 653-660.
Austin B and Austin DA. 2007. Bacterial fish pathogens, diseases of farmed and wild fish. 2nd Edn., Chichester, UK, Springer Praxis Publishing.
Cahill MM. 1990. Bacterial flora of fishes: a review. Microbiology Ecology. 19: 21-41.
Hedrick RP, Groff JM, Okihiro MS and McDowell TS. 1990. Herpesviruses detected in papillomatous skin growths of koi carp (Cyprinus carpio). Journal of Wildlife Diseases. 26(4):578-81.
Mastan SA and Rao MLVG. 2007. Isolation and characterization of bacteria associated with ulcerative disease fish, Clarias batrachus. Biosciences Biotechnology Research Asia. 4(2): 665-670.
Mcvicar AH. 1997. Interactions of pathogens in aquaculture with wild fish populations. Bulletin- European Association of Fish Pathologists. 17(6): 197-200.
Newman SG and Natnarich JJ. 1982. Direct immersion vaccination of juvenile Rain Bow trout, salmo gairdnen, and juvenile Coho salmon, Oncorhymchus kisutch, with a Yarisina ruckeri bacterin. Journal of Fish Diseases. 5:338-341.
Patil SS. 2009. Isolation and molecular characterization of Aeromonas species from food and its public health significance. Thesis done in Department of Veterinary Public Health. Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab.
Quinn PJ, Carter ME, Markey B and Carter GR. 1994. Clinical Veterinary Microbiology. Published by Wolfe Publishing, an imprint of Mosby-Year Book Europe Limited.
Radu S, Ahmad N, Ling FH and Reezal A. 2003. Prevalence and resistance to antibiotic for Aeromonas species from retail fish in Malaysia. International Journal of Food Microbiology. 81: 261-266.
Roberts RJ and Smail DA. 2001. Laboratory methods. In: Roberts R.J. (ed). Fish Pathology. 3rd Edn. pp 380- 412.W B Saunders, London.
Srivastava AK. 1979. Fungal infection of the young hatchlings of Labeo rohita (Ham.). Mykosen. 22(2):48.
Wiklund T, Tabolina I and Bezgachina TV. 1999. Short communication: Recovery of atypical Aeromonas salmonicida from ulcerated fish from the Baltic Sea. ICES Journal of Marine Science. 56:175-179.