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Investigation of a Bacterial Pathogen Isolated from Farmed Ompok Pabda

ZANNAT ARA SIMU
Department of Aquaculture Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

MD. BAZLUR RASHID CHOWDHURY
Department of Aquaculture Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

TANVIR RAHMAN
Department of Aquaculture Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

A. G. M. SOFI UDDIN MAHAMUD
Department of Aquaculture Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

KANIZ FARJANA
Department of Aquaculture Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

UMME MASUMA
Department of Aquaculture Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Studies were conducted to isolate and identify a pathogen collected from the hemorrhagic ulcer of farmed Ompok pabda and to determine the pathogenic potentiality as well as antibiotic susceptibility of the recovered isolate. Infected fish were collected from a local fish farm located at Ishwarganj under Mymensingh district, Bangladesh. The bacterial isolate was identified by using Hi Assorted™ Biochemical Test kit and further investigated to determine the antibiotic susceptibilities. To determine the virulence, a challenge model was performed using the experimental bacterium, Ar-9 by intramuscular injection of O. pabda. A total of 50 healthy experimental fish (2.3±0.44 g) were used in another challenge test by using single intramuscular injection with a pre-fixed dose (107 CFU/ fish) to find out the therapeutic effects of various available antibiotics. The results demonstrated that the collected isolate was Gram-negative rod shaped bacterium exhibiting non-wrinkled, smooth and slightly yellowish colony on TSA from 8 to 42⁰C. Biochemical identification revealed that causative bacterium (Ar-9) was Aeromonas sp. that showed variable susceptibilities against different antibiotics but marked resistance to ampicillin. Ar-9 was found virulent (LD50 = 2.2× 106.0) to O. pabda. Moreover, experiment on the therapeutic effects against the infectivity of Ar-9 revealed that CFCIN have successfully recovered 100% of challenged fish followed by O. T. C. power, Tylovet and ATIVET. Thus, the present study revealed a virulent and ampicillin resistant pathogenic bacterium which may be harmful in pabda culture in Bangladesh.

Keywords: Bacterial pathogen, Ompok pabda,

Location: Ishwargonj, Mymensingh, Bangladesh

Start Date: 00-00-2018

End Date: 00-00-2018

Program Area: Animal Health and Management

Commodity: Pabda

Objectives:

To isolate and identify a pathogen collected from the hemorrhagic ulcer of farmed Ompok pabda and to determine the pathogenic potentiality as well as antibiotic susceptibility of the recovered isolate.

Methodology:

Collection of diseased fish and isolation of bacteria: Diseased O. pabda (average weight 33.4±1.8g) were collected from a commercial fish farm, located at Ishwargonj, Mymensingh in February, 2018. Infected fish were caught using a cast net, placed in strong, clean and aseptic plastic bags then packed in column, surrounded with ice bags and immediately brought to the Fish Disease Laboratory of the Department of Aquaculture, Bangladesh Agricultural University. Upon arrival, fish were surface -disinfected with 70% ethanol and dissected. Samples for bacteriological examinations were collected by inserting sterile inoculating loop into the lesions and ulcers. The inocula were sampled from the surface as well as deeper portion of lesions and internal organs particularly from skin, fin, liver and kidneys. They were streaked on Tryptone Soya Agar (Hi-medium) plates and incubated at 30°C for 48 h. Interestingly, a common type of smooth, non-wrinkled and flat colonies were appeared from most of the specimens. A representative single colony from the body lesion sample was coded as Ar-9. After performing routine biochemical identification methods, pure culture was stored using Tryptone Soya Broth (Hi-medium) and 20% glycerol (v/v) (Merck) in 1.8 mL cryo-tube (Thermo) at -20ºC until required for further use. Biochemical identification of Ar-9: Identification of bacteria was carried out based on the morphological and biochemical tests. Morphological test of the isolate, Ar-9 was done by Gram’s staining using Grams stain kit (Hi-media) and biochemical identification was done using Hi Assorted™ Biochemical Test kit (Hi-media). The tests were based on the principle of pH change and substrate utilization. Pure culture of the isolate Ar-9 was done on the Nutrient Agar (Hi-media), freshly cultured bacterial cells were suspended in 0.85% sterile physiological saline and the suspension was adjusted to slightly more than 0.1 OD at 620 nm. The kit was opened aseptically and 50 μl of the above inoculum was inoculated in each well by surface inoculation method. The kit containing the bacterial sample was incubated at 37°C for 24 h. Identification was confirmed by comparing the biochemical reaction results of Ar-9 with the identification index (http://himedialabs.com/TD/KB002.pdf) provided with the kit. Experimental infection using Aeromonas sp. Ar-9: Experimental infections were conducted to test pathogenicity as well as to determine the virulence of isolated bacterium using O. pabda. Healthy O. pabda fingerlings (weight 2.3±0.44 g) were collected from a private fish hatchery, located adjacent to BAU for experimental infection and were acclimatized in aquaria with aeration for 72 h and checked for the disease before using in the challenge test. Ar-9 was cultured on TSA plates at 37°C for 24 h, harvested and suspended in sterile 0.85% physiological saline. Tenfold serial dilutions of the suspension were prepared and five fish per dilution were used for intramuscular injection with 0.1 ml of the suspension. Challenge doses employed for intramuscular injection were ranged from 2.2×105 to 2.2×108 CFU/ fish. Control fish received only sterile 0.85% physiological saline and kept in a separate aquarium. The average water temperature was determined. After injections, the challenged fish were kept in 35 L capacity glassaquaria facilitate with continuous aeration. Around 50% of water was exchanged daily. Water temperature was maintained with room temperature. Pathogenic activity of the pathogen, Ar-9 on the experimental fish was investigated daily by a routine observation of lesions on skin, fin, fin bases, body surface and head. Gross pathological changes, moribundness and mortalities were recorded and considered as the primary diagnosis of infection. Samples from the external lesions and internal organs, i.e., the skin, fins, liver and kidney of dead fish were directly streaked onto TSA plates and incubated at 37°C for 48 h to confirm that Aeromonas sp. Ar-9was the cause of mortality. The observation was continued up to 10 days without any feed support. Experimental infection: Apparently healthy O. pabda fingerlings (weight 2.3±0.44 g) were collected from a private fish hatchery situated adjacent to BAU. Before starting the challenged test, fish were acclimatized for three days. The experimental challenge study was performed using seven 35 L capacity plastic bucket with continuous aeration. Total 5 buckets were set for the fish to carry out this experiment of which each contained 10 O. pabda.During injection, fish were smoothly and carefully handled to avoid any physical injury and stress. All fish received a single IM injection of 0.1 mL of 2.5×107 CFU/mL of Ar-9 suspension. Control group of 10 fish was injected with 0.1 mL of sterile 0.85% saline solution. Gross pathological changes and moribundness were checked daily. The average water temperature was 31.5±0.9°C. No feed was given during experimental infection. Rearing water of each bucket was changed up to 50% as per needed. Just after the appearance of experimental infection, selected antibiotics were applied to observe their effects on the infected fish.

Source of Information: Bangladesh J. Fish. (2019) 31(2) : 243-252

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The results demonstrated that the collected isolate was Gram-negative rod shaped bacterium exhibiting non-wrinkled, smooth and slightly yellowish colony on TSA from 8 to 42⁰C. Biochemical identification revealed that causative bacterium (Ar-9) was Aeromonas sp. that showed variable susceptibilities against different antibiotics but marked resistance to ampicillin. Ar-9 was found virulent (LD50 = 2.2× 106.0) to O. pabda. Moreover, experiment on the therapeutic effects against the infectivity of Ar-9 revealed that CFCIN have successfully recovered 100% of challenged fish followed by O. T. C. power, Tylovet and ATIVET. Thus, the present study revealed a virulent and ampicillin resistant pathogenic bacterium which may be harmful in pabda culture in Bangladesh.

Knowledge Management: Journal