2.1 Field investigation, collection of fish and water sample Pangasius showing clinical signs of disease were collected from a private fish farm of Muktagacha, Mymensingh, Bangladesh (24.7583° N, 90.2667° E). The farm contained seven stocking ponds each with an average area and depth of 30485.55 ft2 and 2 ft respectively with well treated and a good water exchange system. The farm owner had stocked 0.5 inches sized 10-12 days old pangasius fry at a high density of about 37850 fries per decimal (~9 lac fry per hectare). The higher mortality of the reared fish was noticed at winter season. The infected fish were brought to the Fish Disease Laboratory of the Department of Aquaculture, Bangladesh Agricultural University (BAU) for bacterial and histopathological analysis. Water samples were also taken from the diseased ponds to the laboratory for the determination of water quality parameters pH, dissolved oxygen (DO), ammonia (NH3), ammonium (NH4), phosphate (PO4), nitrite (N02)? using commercially available testing kit (Sera®, GmbH, Germany).
2.2 Isolation of pathogenic bacterial isolates The collected fishes were dissected under sterile conditions and bacterial swabs were taken aseptically using a sterile loop from kidney, liver, spleen, skin, and eye lesions. For isolation of bacteria, tryptic soy agar (TSA) medium (Difco™, Becton, Dickinson and Company, NJ, USA) was used. The inoculated plate was incubated at 24 °C for 48 h. A total of ten bacterial isolates were selected from the pure cultures and were maintained on TSA plates. The glycerol stock cultures were stored at -80 °C for long term preservation.
2.3 Primary characterization and identification of isolates The experimental layout for the identification and characterization of collected isolates is depicted. Freshly recovered pure isolates were used for primary characterization. The morphological characteristics of bacterial colonies including shape, size, and color were investigated on the TSA plates. Simultaneously, physiological characteristics were studied by observing the growth of each isolated colony at various temperatures (4, 24, 37, and 40 °C) and different NaCl condition (0, 1, 2, 3 and 4%). Biochemical identification of the isolates was performed using Gram’s staining, motility, oxidase, Oxidative-Fermentative (O-F), and O/129 sensitivity tests using standard methods described earlier. Further characterizations of the isolates were conducted using the API-20E kit [6]. Esculin hydrolysis test was also performed for species determination of the Aeromonad group.
2.3.1 Gram’s staining Gram’s staining was performed on a glass slide from a single bacterial colony by crystal violet solution (1 min), iodine solution (1 min), and safranine (2 min). The slide was washed properly by running tap water before the start of the next step. The slide image analyses were performed using a light microscope (Nikon Co., Tokyo, Japan). 2.3.2 Motility test Motility test was performed using “hanging drop” method from the bacteria cultured in both agar and broth media. Briefly, a drop of bacterial suspension was taken on a glass slide. The slide was then carefully reversed to allow hanging of the culture drop. Finally, the drop was observed under light microscope (Nikon Co., Tokyo, Japan). 2.3.3 Oxidase test Oxidase test was performed using commercially available oxidase detection strips (Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA). In briefly, a few amount of bacteria was placed on the strips containing oxidase reagents (N, Ndimethyl-1,4-phenylene diammonium chloride) and the color was observed after 10 seconds.
2.4 Antibiotic sensitivity test The antibiotic susceptibility of Aeromonas sp. was determined by the disc diffusion method. Ten different discs of antibiotic were chloramphenicol (30µg/disc), oxytetracycline (30µg/disc), trimethoprim (25µg/disc), erythromycin (15µg/disc), streptomycin (25µg/disc), chlortetracycline (30µg/disc), nitrofurantoin (300µg/disc), amoxyclav (30µg/disc), doxycycline hydrochloride (30µg/disc), and azithromycine (15µg/disc) (Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA). Briefly, 200 µl of suspended culture in sterile distilled water was taken and spread over the surface of Mueller-Hinton agar (Thermo Fisher Scientific Inc., Waltham, MA, USA) plate. After drying, the antibiotic discs were placed over the plates and incubated at 24°C for 24 h. The degree of sensitivity of A. hydrophila isolates to ten antibiotics was determined by measuring the zone of clearance around the antibiotic discs.
2.5 Determination of colony forming unit (cfu ml-1) Colony forming units (cfu ml-1) were determined according to the drop count method described previously [23]. Several replicated drops (20 ?l drop-1) of 10-fold seven times diluted bacterial suspension was put on TSA plates and estimated the cfu ml-1 using following formula: cfu ml-1 = number of colonies × 20(volume added) × dilution factor × 50 2.6 Determination of pathogenicity For pathogenicity test, 8 to 10 g weighed known healthy stocks of P. hypoththalmus was acclimatized for 4 days in 15- litre capacity in a well-labelled glass aquarium. Fish were injected either intramuscularly (IM) or intraperitoneally (IP) with pre-prepared 0.1 ml of bacterial suspension contains 1.6 x108 cfu ml-1. Control groups were injected either IM or IP with 0.1 ml of sterile saline. The each group (n = 10) of fish were monitored for 14 days with daily replacement of water (one-third), removal of dead fish and debris. The mortality and morbidity were recorded on daily basis. Necroscopy was involved gross external and internal examinations of fish and inoculation of bacteria from kidney on TSA plate were done.
2.7 Histological procedure Samples (1 cm3 ) from muscle with skin, gills, liver, and kidney were collected aseptically from the infected fish and were fixed in 10% buffered formalin. The samples were then trimmed and processed in an automatic tissue processor (Shandon Citadel 1000, GMI, USA) for dehydration, clearing, and infiltration for 21 hours. Then the samples were embedded, trimmed, sectioned (5 mm thick ribbon) and stained with hematoxylin and eosin [28]. Finally observed them under a compound microscope (Olympus) and photomicrographs of the stained sections were obtained by using a photomicroscope (Olympus-CH-2, New York Microscope Company, Inc.).