International Journal of Aquaculture, 2018, Vol.8, No.20, 151-155
152
district, West Bengal, India in oxygen filled polythene bags to the laboratory. The fish were acclimatized for an
hour followed by disinfection with 5 ppm potassium permanganate for 10 min and then stocked in 500 L capacity
fibreglass reinforced plastic tanks at 100 numbers/tank containing 400 L clean bore-well water. The fish were
acclimatized for about two weeks and fed
ad labitum
with commercial floating pellet feed containing 30% protein.
For the experimental challenge, four glass aquaria of size L60 ×H30 ×W30 cm were selected (two each for
treatment and control groups), disinfected, cleaned, dried for a week and filled with clean bore-well water. Prior to
experimentation, two apparently tilapia juveniles were euthanized, dissected aseptically and inocula from the
kidneys were streaked on to Rimler-Shotts agar and tryptic soya agar to check whether or not the fish are infected
as per Austin and Austin (2012). Ten each of healthy tilapia were released into the experimental tanks and
acclimatized for about 3 days. All the tanks were covered with nylon netting for adequate protection.
An α-haemolytic bacterium,
Aeromonas caviae
-T
1
K
2
used in this study was isolated from diseased
O. niloticus
with MAS and preserved as glycerol stock at -20°C in the Department of Aquatic Animal Health, West Bengal
University of Animal and Fishery Sciences, Kolkata, India. The glycerol stock was revived in tryptic soya broth
and the cell suspension of
A. caviae
was prepared as described in Adikesavalu et al. (2015) and used immediately.
Our preliminary studies with
A. caviae
-T
1
K
2
determined the LD
50
value in Nile tilapia juveniles as 6.76×10
8
cells/fish (data not shown). Aliquots (0.1 mL) of the undiluted
A. caviae
cell suspension (6×10
9
cells/mL) was
injected intramuscularly, i.e., on the dorsal side of the body at a 45°angle at the base of dorsal fin, in such a way
to get 6×10
8
cells/fish. The control fish received sterile saline, 0.1 mL each. The challenged fish were maintained
in their respective tanks for the observations on mortality, external signs of infections and behavioural changes.
1.2 Histopathology
The organs such as kidney and liver of the freshly dead
O. niloticus
on day 4 post-injection were fixed in Bouin’s
solution for 24 h. The fixed organs were processed, embedded in paraffin wax and thin (5 μm) sections prepared.
The sections were then stained with haematoxylin and eosin for the detection of histopathological changes as per
Roberts (2012).
2 Results
Aeromonas caviae
challenged
O. niloticus
exhibited sluggishness and abnormal behaviour like wandering around
the corners, resting at the bottom and vertical swimming. On 4-day post-injection (dpi), 60±0% of the challenged
tilapia died. The histopathological changes noted in the kidney tissues of
A. caviae
challenged
O. niloticus
are
depicted in Figures 1a-c, and those of liver and pancreas in Figure 2a-c and Figure 3a-c, respectively. The kidney
tissues had glomerulopathy with dilated Bowman’s space, nephropathy with the loss of tubular epithelial cells,
obliterated as well as inflamed nephritic tubules, melanomacrophage aggregate, necrosis, widen lumen and
thickening of lumen lining (Figures 1a-c). The liver demonstrated melanomacrophage aggregate, infiltration of
haemocytes, loss of normal architecture of the hepatic tissue and fatty changes in the hepatic parenchyma; while
the pancreas illustrated degradation and/or inflammation of the pancreas and pancreatic acinar cells and,
vacuolation in the pancreatic tissue (Figure 2a-c; Figure 3a-c).
3 Discussion
Aeromonas caviae
strain used in this study was isolated from the kidney of septicemic
O. niloticus
along with
other motile aeromonads such as
A. hydrophila
,
A. veronii
,
A. schubertii
and
A. bestiarum
(data not shown)
.
The
challenged tilapia showed haemorrhages at the site of injection and experienced 60% mortalities. The main
visceral organs, viz., kidney, liver and pancreas were affected in the injected fish, thus revealing the fact that
A.
caviae
was able to infect many visceral organs similar to
Aeromonas caviae
-like bacterium
(Thomas et al., 2013)
and
A. hydrophila
(Azad et al., 2001). Systemic infections are generally characterized by diffused necrosis in
several internal organs, primarily the liver and kidney are the target organs of an acute septicemia. These organs
when attacked by bacterial toxins cause acute haemorrhage and necrosis of vital organs and lose their structural
integrity (Huizinga et al., 1979). In control fish, the normal structure and systematic arrangement of kidney tissues
with well-defined glomerulus were observed. While, in challenged tilapia, the structural integrity of the kidney
tissues were lost and well-defined histopathological changes observed on 4 dpi, which indicated disease
