Background and sample collections
There was a report of fish mortality in a backyard fish farm in Meghalaya and after that, a field investigation was carried out in 2021. It was observed that fishes were reared in cemented tanks of size 0.05 ha with a water level of 2 meters depth; mix cultured of fishes was reared Gonius (
Labeo gonius) and Common carp (
Cyprinus carpio) according to the standard ratio. The disease was reported during the December month of the winter season and mortality was observed in all the fish types. The gross pathological lesions recorded were ulceration of skins, eyes were enlarged, fins were necrosed and hemorrhages were noted on the ventral side. On postmortem, most of the organs like the liver, kidney, spleen and intestines were enlarged and hemorrhagic. To ascertain the plausible causes of death, a total of 5 dead fishes and 2 water samples from different tanks were collected aseptically and transported at 4°C for further laboratory investigation.
Bacterial isolation and identification
All the samples, namely 5 dead fishes and 2 water samples, were subjected to bacterial isolation and identification targeting the following bacterial spp.
Aeromonas spp.,
Listeria spp.,
Salmonella spp. and
Vibrio spp. Accordingly, the dead fishes were opened aseptically and liver, skin, intestine and other tissue samples were collected for inoculations. The study was conducted at the Division of Animal and Fisheries Science laboratory, ICAR RC NEHR, Umiam.
Accordingly, for
Aeromonas spp., the samples (tissues and water) were first enriched in Alkaline peptone water (APW, HiMedia) overnight to 18 hrs at 37°C. A loopful of cultures was inoculated on Ampicilin Dextrose agar plates (ADA, HiMedia) and incubated at 37°C for 24 hours. Smooth and opaque yellow colonies were further subjected to various biochemical tests after visualizing Gram’s negative coccobacillary morphology. Subjected biochemical tests were oxidase test, haemolysis test, Lecithinase test, esculin test, TSI, Citrate, Urease, Lysine, Arginine, Ornithine, Nitrate, Voges-Proskauer, Methyl red and Carbohydrate tests (Mannitol, Sucrose, Inositol, Cellobiose, Arabinose, salicin and Lactose) following already described protocols (
West and Colwell, 1984;
Shome et al., 2005).
Targeting for
Vibrio spp., water and ground tissue samples were pre-enriched in alkaline peptone water containing 3% NaCl at pH 8.5 and incubated at 37°C for 18 hrs. Further, a loopful of overnight culture were streaked on thiosulphate citrate bile salts sucrose agar (TCBS, HiMedia) and incubated at 37°C for 24 hrs. However, there was no growth of any typical colonies specific to
Vibrio spp. (green colonies with blue center), it was not investigated further.
To isolate
Listeria spp., water and minced tissue samples were inoculated in University of Vermonth (UVM)-1 (HiMedia) broth enriched with acriflavin and Nalidixic acid (inhibitor of gram-positive and negative organisms) at 30°C for 24 hrs and further on UVM-II. However, further growth was not detected, which arbitrarily ruled out the presence of Listeria spp.
Lastly for
Salmonella spp. the samples were inoculated in test tubes containing 5 ml of Rappaport-Vassiliades broth as an enrichment medium. The tubes were incubated aerobically at 42°C for 24 to 48 hours. A loopful of broth culture was then streaked onto Mac Conkey Lactose agar. After that, the non-lactose fermented colonies were streaked on Brilliant Green Agar (BGA) and incubated for 24 hours at 37°C. Salmonella suspected pink or red colonies were smeared and stained by Gram’s staining method and examined microscopically. Characterization and preliminary identification of suspected Salmonella cultures were made based on morphology, colony characteristics and biochemical tests
(Quinn et al., 2002).
Samples were also inoculated on Mac Conkey Lactose agar, discerning lactose fermenter and non-lactose fermenter, lactose fermenter typical of
E.
coli were streaked on Eosin methylene blue agar for confirming its metallic sheen nature.
Molecular confirmation
Based on the cultural, morphological and biochemical tests the isolates were designated as
Aeromonas hydrophila and
Salmonella spp. The pure colonies of these isolates were subjected to bacterial DNA isolation using a kit (Qiagen). Thereafter, a Polymerase chain reaction was conducted in a thermal cycler (Biorad) for detecting the virulence-determining genes of
Aeromonas hydrophila viz ahh-1 (haemolysin gene) and
aer-A (aerolysin gene) following the described PCR protocols of Wang
et al., 2003. The primer sets corresponding to ahh1 and aerA will yield an amplified product of 309bp and 130bp respectively. The PCR was set up for the final volume of 25 ul reaction mixture comprising 12.5 ml of 2x PCR master mix (Fermentas, Life Sciences, Germany), 10 pm of each forward and reverse primers, nuclease-free water and 1 ml of respective template DNA. The cycling parameters were an initial denaturation for 5 min at 95°C, followed by 35 cycles of 50 sec at 95°C, annealing at 59°C for 50 sec and extension at 72°C for 50 sec, followed by a final extension at 72°C for 10 min and hold at 4°C.
The suspected Salmonella isolates were confirmed as
Salmonella by detecting the
Salmonella-specific gene by simplex PCR using a specific primer reported by
Elder et al., (1997) corresponding to the invA gene. The PCR reaction mixture was set up for a reaction volume of 25µl and the reaction mixture composition was similar as above except for respective primers. The cycle parameters were 1 cycle of 5 min at 94°C; 30 cycles of 45 sec at 94°C, 40 sec at 60°C, 1 minute at 72°C; 1 cycle of 5 min at 72°C and hold at 4°C. The positive reaction will yield an amplified product of 457 bp.
DNA extraction for the suspected
E.
coli colonies was done using the snap-chilled method. The crude DNA extracted was subjected to detection of the stx1 and stx2 genes using already reported primers and protocols. (All primers used in the study are detailed in Table 1).
The amplified products of all the genes were analyzed by electrophoresis in 1.5% agarose gel in Tris-borate buffer containing 2 μl ethidium bromide (10 mg/ml), visualized with a UV transilluminator and photographed by gel documentation system.
MPN determination
To assess the microbial load of the water from the fish tank, the most probable count (MPN) of coliform was done per standard microbiological methods
(Quinn et al., 2002). The aseptically collected water samples were processed within the same day of collection per the standard protocol using an inverted Durham’s tube in 9 Mac Conkey broth test tubes (HiMedia). The tubes were arranged in three rows of 3 tubes per row. The first row contains 10 ml of double-strength broth plus 10 ml of water samples. Subsequent rows contain 10 ml single-strength broth inoculated with 1 ml and 0.1 ml water respectively giving up to 1:1000 dilutions. All the tubes for separate samples were incubated at 37°C for 24 to 48 hr. The MPN index determined the MPN.
Water analysis- Temperature, pH, dissolved oxygen, alkalinity, chloride, nitrite, conductivity
The temperatures of the fish tanks were recorded and the pH of the water was also determined. Limnological water analysis was performed in the laboratory following standard techniques (APHA, 2005).
