Isolation and characterization of two virulent Aeromonads associated with haemorrhagic septicaemia and tail-rot disease in farmed climbing perch Anabas testudineus

Abstract

Diseased Anabas testudineus exhibiting signs of tail-rot and ulcerations on body were collected from a fish farm in Assam, India during the winter season (November 2018 to January 2019). Swabs from the infected body parts were streaked on sterilized nutrient agar. Two dominant bacterial colonies were obtained, which were then isolated and labelled as AM-31 and AM-05. Standard biochemical characterisation and 16S rRNA and rpoB gene sequencing identified AM-31 isolate as Aeromonas hydrophila and AM-05 as Aeromonas jandaei. Symptoms similar to that of natural infection were observed on re-infecting both bacteria to disease-free A. testudineus, which confirmed their virulence. LC₅₀ was determined at 1.3 × 10⁴ (A. hydrophila) and 2.5 × 10⁴ (A. jandaei) CFU per fish in intraperitoneal injection. Further, PCR amplification of specific genes responsible for virulence (aerolysin and enterotoxin) confirmed pathogenicity of both bacteria. Histopathology of kidney and liver in the experimentally-infected fishes revealed haemorrhage, tubular degeneration and vacuolation. Antibiotic profiles were also assessed for both bacteria. To the best of our knowledge, the present work is a first report on the mortality of farmed climbing perch naturally-infected by A. hydrophila as well as A. jandaei, with no records of pathogenicity of the latter in this fish.

Figure 1

Phylogenetic tree (UPGMA model) based on 16S rDNA (a) and rpoB (b) of two bacterial isolates (AM-31: Aeromonas hydrophila and AM-05: A. jandaei) and their closely-related species. Pseudomonas sharmana and P. pectinilytica were selected as outgroups for the 16S gene. Percentage bootstrap values (1000 replicates) are shown at each branch point.

Figure 2

Tail-rot (TR) and Ulcerations (U) on body of Anabas testudineus experimentally-challenged with bacteria (a) Aeromonas hydrophila (AM-31) and (b) A. jandaei (AM-05).

Figure 3

Haemorrhagic spots (blue arrows) on the surface of liver of Anabas testudineus experimentally-challenged with (a) Aeromonas hydrophila and (b) A. jandaei.

Figure 4

T. S. of (a) kidney and (d) liver in disease-free (as control) Anabas testudineus. T. S. of kidney of A. testudineus experimentally-challenged with (b) Aeromonas hydrophila and (c) A. jandaei [H, haemorrhage; T, vacuolar degeneration of renal tubules; V, vacuolation]. T. S. of liver experimentally-challenged with (e) A. hydrophila and (f) A. jandaei [V, vacuolation].