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 ANTI­GEN 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 de­tects 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 (200±27g) were cultured under two feeding conditions: test diet and con­trol 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 pre­mix. Control diet contained all ingredients, except the seed. After 28d of feed­ing, 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%parafonnalde­hyde in O.lM phosphate buffer and standard procedures were followed for fur­ther 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 parti­cles (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 incorpo­rated diet, the number of gold particles was less than the control group. This in­dicated that the injected antigen was efficiently cleared in fish fed with plant in­gredient incorporated diet compared to the fish fed with control diet.

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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 re­sponses. Nature Rev. Immunol. 4:9

Joseph L.W., P. Connolly, N. Haddad, A. Le Monte. E. Brizendine, and R. Haf­ner. 2002. Antigen clearance during treatment of disseminated Histoplasmo­sis with itraconazole versus fluconazole in patients with AIDS. Antimicrobial 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

. aspera as a herbal feed ingredient. Aquaculture 238:67-73. Vasudeva R.Y. and R. Chakrabarti. 2005. Stimulation of immunity in Indian ma­

jor carp Catla catla with herbal feed ingredients. Fish & Shellfish Immunol­ogy 18:327-334.