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Experimental Infection of Aeromonas Hydrophila in Pangasius

J Sarker
Department of Aquaculture, Chittagong Veterinary and Animal Sciences University, Khulshi, Chittagong, Bangladesh

MAR Faruk
Department of Aquaculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Experimental infections of Aeromonas hydrophila in juvenile pangasius (Pangasianodon hypophthalmus) were studied. Five different challenge routes included intraperitoneal (IP) injection, intramuscular (IM) injection, oral administration, bath and agar implantation were used with different preparations of the bacteria to infect fish. The challenge experiments were continued for 15 days. A challenge dose of 4.6×10⁶colony forming unit (cfu) fish^-1was used for IP and IM injection and oral administration method. Generally, IP route was found more effective for infecting and reproducing clinical signs in fish that caused 100% mortality at the end of challenge. IM injection, oral and bath administration routes were also found effective for infecting and reproducing the clinical signs in fish to some extent. Agar implantation with fresh colonies of bacteria also caused 100% mortality of challenged fish very quickly with no visible clinical signs in fish. The major clinical signs of challenged fish included reddening around eyes and mouth, bilateral exophthalmia, hemorrhage and ulceration at fin bases and fin erosion.

Keywords: Experimental infection, Aeromonas hydrophila, Pangasius

Location: Department of Aquaculture, Bangladesh Agricultural University (BAU), Mymensingh.

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Pangus, Diseases

Objectives:

To develop an effective experimental infection model of A. hydrophila using different challenge routes in junenile pangasius.

Methodology:

Healthy juvenile pangasius of average weight of 50 g were collected from a private fish farm in Mymensingh and stocked in 50 L rectangular glass aquariums at the laboratory of the Department of Aquaculture, Bangladesh Agricultural University (BAU), Mymensingh. Before starting the experiment, all the fish were acclimatized for 5 days providing adequate feed and better aeration by using air pump. After acclimatization the fish were used for experimental infection with laboratory stock of A. hydrophila bacteria. For challenge experiment, replicate groups of fish were placed in 15 rectangular 15-litre capacity well labeled glass aquariums. Each aquarium was aerated and one third of the water was replaced daily, dead fish were removed and debris was siphoned from the bottom of the aquarium. A. hydrophila isolate P1K was used for infecting fish. This isolate was obtained from the laboratory stock of Fish Disease Laboratory in the Department of Aquaculture, BAU. The isolate was previously isolated from the kidney of diseased pangasius from a local farm and identified as A. hydrophila by Nahar et al., (2016). It was Gram negative, motile, fermentative, oxidase positive, grown at 37ºC, resistant to O/129, TSI positive and hydrolyzed esculin. It was further confirmed using API20E microbiological kit. This isolate was found pathogenic on juvenile pangasius as determined by Nahar et al., (2016). It was cultured on Triptic soya agar (TSA) and broth at 25ºC for 48 h prior to the experiment. An amount of 5 mg of fresh culture of A. hydrophila was carefully scraped and mixed with 1 ml sterile physiological saline (0.85% NaCl) and then the desired dilution was prepared by serial decimal dilution method. In the case of broth culture, an amount of 30 µl was taken and mixed with 2.7 ml sterile physiological saline and the desired dilution was prepared by decimal dilution method. Colony forming unit (cfu ml^-1) were determined for the bacterial suspension, prepared according to the drop method described by Miles and Misra (1938) or drop count method using TSA plates. Briefly, the bacterial suspension was diluted 10 fold seven times either with fresh saline or TSA broth. Replicate drops (20 µl drop^-1) from each dilution were then placed onto a TSA plate that had been previously divided into six sections. The plates were allowed to dry before incubation at 25ºC for at least 24 h until colonies were visible and could be counted. The average number of colonies per drop was counted and cfu ml^-1 determined for the bacterial suspension using following: cfu ml^-1 = Number colonies × 20 (volume added) × dilution factor × 50. Five different challenge routes were used to administer the bacteria in fish which included intramuscular (IM) and intraperitoneal (IP) injection, oral administration, agar implantation with fresh colonies of bacteria and bath. The bacterial inoculates used in the IM and IP injection were prepared from bacteria cultured either in broth or on agar plates to examine it there was any effect on mortality between the two preparations. Two groups of 20 fish were used for each challenge route. Fish were injected either IM (below the left dorsal fin of the animal) or IP with 0.1 ml of bacterial suspension contains 4.6×10⁷ cfu ml^-1. Two corresponding group of control fish were included for each challenge route and they were given 0.1 ml of sterile broth in place of the bacteria. For oral administration two groups of 10 fish were exposed to the bacteria by dropping 0.1 ml of 4.6×10⁷ cfu ml^-1 of bacterial suspension into the pharynx of the animal using 1 ml sterile syringe. Only broth culture was used for oral administration. This was washed with a further 0.1 ml of water. For agar implantation, bacteria were implanted under the skin of the group of the animals. A small cut was made left dorsal side with a sterile scalpel blade and few colonies of A. hydrophila from 24 h TSA culture were cut from agar and placed under the skin of the animal. The control groups of fish received agar only. For bath administration, bacteria were cultured in 200 ml broth for 24 h and added to 30 litre water in an glass aquaria. Then fishes were released into it. Control fishes were released in water containing sterile broth. The experimentally infected fishes and control fishes were released in well labeled glass aquarium. The experimentally infected fish were observed for 15 days. Continuous aeration was maintained and no feed was given to the experimental fish. One-third of the water was replaced daily and debris siphoned from the bottom of the tank. Infection was recorded by observation of red mouth and fins, lesion, clinical appearance and mortality. Moribund fishes were attended, observed and waited for their death. The numbers of dead fish were recorded. Immediately after death, they were removed from the aquaria.

Source of Information: Progressive Agriculture 27 (3): 392-399, 2016

Article Link (Online Journal): DOI: https://doi.org/10.3329/pa.v27i3.30836

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This experiment established that infecting P. hypophthalmus with A. hydrophila through different routes can serve as a novel infection model that allows for the future study of host–pathogen interactions. The work presented here suggests that P. hypophthalmus represents a permissive host for A. hydrophila infections and can be used as a simple host model to assess the virulence of A. hydrophila.

Knowledge Management: Journal