LARVI '05 - FISH & SHELLFISH LARVICULTURE SYMPOSIUM

C.l. Hendry~ G. Van Stappen. M. Wille and P. Sorgeloos (Eds)

European Aquaculture Society, Special Publication No. 36~ Oostende, Belgium, 2005

ACHYRANTHES ASPERA ENHANCES THE EFFICIENCY OF ANTI

GEN CLEARANCE FROM THE HOST LABEO ROHITA

R. Chakrabarti and R.Y. Vasudeva

Aqua Research Lab" Department of Zoology, University of Delhi, Delhi - 110 007, India

Introduction
As soon as any foreign particle invades, the immune system of the host de
tects it, mounts immune response against it, and eliminates it from the host
body as quickly as possible. This is called antigen clearance. Antigen clearance
is associated with a downregulation of the immune response and apoptosis of
most antigen-specific T-cells (Buckland, 2004). Though antigen clearance has
been studied in several mammals by various techniques, no such study has
been conducted with fishes. Achyranthes aspera, L. (Family: Amaranthaceae)
is an herb that stimulates the non-specific immunity of carps (Vasudeva et al.,
2004; Vasudeva and Chakrabarti, 2005). This investigation aims to study the
effects of A. aspe.ra in antigen clearance from the spleen of Indian majorcarp
Labeo rohita, rohu.

Materials and methods

Rohu (20027g) were cultured under two feeding conditions: test diet and con
trol diet. Test diet was prepared using 0.5% A. aspera seed along withother feed
ingredients like fish meal, wheat flour, cod liver oil, vitamins, and mineral pre
mix. Control diet contained all ingredients, except the seed. After 28d of feed
ing, fish were injected intraperitoneally with Bovine serum albumin, BSA
(500~1). The diffused small pieces of spleen of fish were collected on d14 of
immunization. Tissues were fixed in 1% glutaraldehide and 2%parafonnalde
hyde in O.lM phosphate buffer and standard procedures were followed for fur
ther tissue processing. Ultrathin sections (70nm) were cut using glassknives in a
ultramicrotome. The sections were slowly lifted onto nickel grids and were
blocked with 20/0 fish gelatin. Then the sections were labelled by incubating with
" rabbit anti-BSA antibodies for 12h and were washed with 1% fish gelatin in
phosphate buffer. After treating with primary antibody, the grids were labelled
with secondary antibody; goat anti-rabbit-Igd conjugated with 15-nm gold parti
cles (TAAB), washed, and stained with uranyl acetate. Stained sections were
viewed for labelled particles under transmission electron microscope.

Results and discussion

Immuno-Iabelling showed the presence of gold particles in spleen sections of L.

rohita. In control group, the particles were heavily crowded and more numerous
(Fig. 1a), whereas in the spleen (Fig. 1b) of fish fed with A . aspera seed incorpo
rated diet, the number of gold particles was less than the control group. This in
dicated that the injected antigen was efficiently cleared in fish fed with plant in
gredient incorporated diet compared to the fish fed with control diet.

it

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Fig. 1. Electron micrographs of spleen (a) control (b) Achyranthes-treated rohu .

The host's immune system recognized the invading antigen and tried to clear it
as quickly as possible to confer safety of the host and to protect the host from the
onset of infection. The treatment with itraconazole reduces the concentration of
histoplasma antigen in blood and urine, suggesting the rapid clearance of
fungemia (Joseph et al., 2002). In the present study. high amount of BSAwas
found in the spleen of control fish even 14d after immunization. The immune
system of this carp is therefore poor in clearing the invaded antigen;_ hence high
mortality is recorded in this species during disease outbreaks. The feeding of
plant-incorporated diet may boost the immune system of this fish.
~
In conclusion, treatment with A. aspera has enhanced the immunity ofL. rohita
that has efficiently eliminated the BSA from the system compared to the control.
References
Buckland J. 2004 Viral Immunity: Role for Bim in termination of immune re
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Joseph L.W., P. Connolly, N. Haddad, A. Le Monte. E. Brizendine, and R. Haf
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Agents and Chemotherapy 46:248-250.
Vasudeva R.Y., R.M. Singh, A. Singh, and R. Chakrabarti. 2004. Potentiation of
antibody production in Indian major carp Labeo rohita, rohu by Achyranthes
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Vasudeva R.Y. and R. Chakrabarti. 2005. Stimulation of immunity in Indian ma
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