Isolation and identification of Aeromonas hydrophila from silver carp and its culture environment from Mymensingh region

The pathogenic bacteria, Aeromonas hydrophila from naturally infected silver carp, Hypophthalmichthys molitrix were isolated and identified from a fish farm in Mymensingh. Fish showed motile Aeromonas septicemia (MAS) like reddish head and anal region, pale body colour and external haemorrhages. Intestine, liver, kidney and environmental samples such as sediment, feed and water were inoculated onto the Aeromonas isolation medium (AIM) and TSA plates. The AIM colonies were undergone specific characterization for Aeromonas hydrophila. Quantitative studies of bacterial flora of fish on TSA plates showed that the total bacterial load in intestine, liver and kidney of the sampled fish, sediment, feed and water of the environment were 1.00 × 10 to 1.50 × 10 CFU/g, 2.7 × 10 to 4.46 × 10 CFU/g, 1.00 × 10 to 2.17 × 10 CFU/g, 1.90 × 10 CFU/g, 4.10 × 10 CFU/g and 2.50 × 10 CFU/ml, respectively. After isolation, A. hydrophila was finally identified by their specific morphological, physiological and biochemical characteristics. They were gram negative, rod shaped, motile bacteria that showed positive reaction for oxidase and catalase, fermented glucose and were resistant to vibriostatic agent 0/129. They showed positive result in esculin hydrolysis test.


Introduction
With intensive aquaculture practices, the exotic silver carp, Hypophthalmichthys molitrix, has become a main culture species of Bangladeshi farmers. A specific disease condition, clinically presented by fin rot, redness on head, enlargement of lower lip and swollen abdomen resulted in high mortality of fish which has frequently been encountered in many farms of Mymensingh region (unpublished data). Aeromonas and Pseudomonas are the important bacterial pathogens frequently isolated from the diseased fishes throughout the world as well as Bangladesh. Bacterial fish diseases, especially bacterial hemorrhagic septicaemia (Roberts et al., 1989) and motile Aeromonas septicaemia in freshwater fish caused great losses (Lio-po et al., 1992). In Bangladesh, areomonads were frequently observed in farmed fishes as well as in the natural waterbodies (Iqbal et al., 1996). Sabur (2006) isolated the pathogenic bacteria Aeromonas hydrophila in indigenous and exotic carps under their polyculture conditions. Mamnur  isolated A. hydrophila from the suspected EUS-affected shing fish, Heteropneustes fossilis. Hasan et al. (2008) observed histopahotlogical changes in intestine, liver and kidney of the affected fish. Ahmed (2009) isolated and identified A. hydrophila from carps, catfishes, perches and eel and found that they were highly pathogenic. Saeed (2010) isolated and identified bacteria from naturally infected eight fish species: rui (Labeo rohita), catla (Catla catla), mrigal (Cirrhinas cirrhosus), tilapia (Tilapia mossambicus), tarabaim (Macrognathus aculeatus), koi (Anabas testudineus), shing (Heteropneustes fossilis), and magur (Clarias batracus) from different area of Mymensingh District. Recently, a silver carp fish farm in Mymensingh was reported to have diseased condition under culture. So, the present work was undertaken to identify the disease condition and the associated pathogen.

Materials and Methods
Diseased as well as apparently healthy fish were sampled. Environmental water, sediment and feed were also collected from the farm. Trypton soya agar (TSA, Oxoid) plates were used for total bacterial count and AIM, for isolation of Aeromonas like bacteria. TSA slants were used for stocking Aeromonas bacteria for further studies.

Bacteriological investigation
The external lesions were plated onto the TSA and AIM plates. Decimal dilutions of intestine, liver and kidney samples were inoculated onto duplicate TSA plates for total bacterial count and onto AIM plates for specific Aeromonas isolation. All the plates were incubated at 25°C for 48 hrs for colony appearances.

Identification of Aeromonas bacteria
All the grown colonies of Aeromonas like bacteria from the streaked and spreaded plates of AIM were sub cultured onto TSA plates to obtain fresh 24 hrs culture and then undergone morphological characterization such as shape, size, Gram character, flagellation and motility test. Biochemical characters such as oxidase, catalase, oxidative-fermentative (OF), acid and gas production from sugars (glucose, lactose, maltose, sucrose and manitol), methyl-red, H 2 S production, Voges-proskauer (VP), arginine hydrolysis, esculin hydrolysis and 0/129 test were done to confirm their generic and specific natures. Physiological characters were studied by observing the growth of each isolate at temperatures of 4, 37 and 40°C in different concentrations of NaCl as 0, 1, 2, 3.5 and 4% to confirm the identification of the A. hydrophila bacteria.

Clinical pathology
Naturally infected silver carp fish lost their normal appearance. Fish showed reddish head and anal region, pale body colour, external ulcerative lesions with haemorrhage and reddened fin bases (Fig. 1). Moribund fish were found to swim abnormally at water surface.

Characteristics of isolated Aeromonas bacteria
Spreaded and streaked plates with AIM showed growth of Aeromonas bacteria which primarily conferred that the bacteria associated with the ulcerative lesions on silver carp. The isolates gave rise to yellowish opaque colonies on TSA agar. The colonies were smooth, raised and round in shape. The results of their comparative morphological, biochemical and physiological tests with those of Popoff (1984) are shown in Table 1. On the basis of their growth in Vibriostatic agent 0/129 the present isolates were confirmed to be Aeromonas sp., and on the basis of their esculin hydrolysis characters they were confirmed to be A. hydrophila.

Total bacterial load
Total bacterial load found in intestine, liver and kidney of the sampled fish, water, sediment and feed are shown in Table 2.

Conclusion
As Aeromonas hydrophila is an ubiquitous bacterial pathogen it might cause serious losses to the intensive culture system of Bangladesh. So, the higher loads of A. hydrophila in different organs of MAS affected silver carp fish, sediment, feed and water of the environment strongly supported the ubiquitousness of the bacteria in Mymensingh region of Bangladesh. So, it may be concluded that culturists should take proper management practices to avoid such disease of culture fishes especially silver carp and to get rid of the infection by such serious bacterial pathogen like Aeromonas hydrophila by improving their culture strategy and culture ecosystem.