The free-dried snake venom powder of Echis carinatus venom was obtained from Irula’s Snake Catchers Industrial Co-operative Society Limited, Chennai and was stored at 4ºC. Twenty four week old, single comb white leghorn chickens obtained from the Abinaya Poultry farm, Namakkal were maintained in our animal facility. They were used in the study for the production of antivenom (IgY). Male inbreed Swiss albino mice 18-20 gm were used for the studies of venom toxicity and in the experiments of venom neutralization. Institutional Animal Ethics Committee clearance at Institute of vector control and Zoonooses, Hosur, was obtained to conduct the experiment. All the animals were conditioned in standard cages.

The lyophilized snake venom powder of Echis carinatus venom was dissolved in 0.9% Phosphate Buffered Saline (PBS) in the concentration of 1mg/ml. The diluted venom samples were then centrifuged and filter sterilized to remove the impurities. The protein concentration of Echis carinatus venom was estimated by the method of Lowry (1951). To prepare the Freund's adjuvant/venom antigen mixture, 50µlof venom mixed and emulsified with Freunds Complete Adjuvant (FCA) in the ratio of 1:1 using the technique of Herbert (1973). To prepare the bentonite adjuvant/venom antigen mixture, one volume of native venom was mixed with one volume of a sterile, 2% (w/v) bentonite (Sigma) suspension to adsorb the venom proteins to the particulate.Then the solution was injected subcutaneously into multiple sites of the breast muscles into six white leghorn hens on Day zero. Three birds received the received the antigen with CFA. The remaining three birds received the antigen absorbed to bentonite. Test bleeding was made frequently to check the presence of antivenom antibodies in the serum. Eggs were collected from day 0 until the end of the experiment and stored at 4°C until testing by the indirect ELISA.

The antibodies were extracted from egg yolk by the method of Polson et al. (1980) using Polyethylene and Ammonium sulphate precipitate method. Then the content was desalted by dialysis process. The crude fraction of IgY thus obtained was further purified by DEAE cellulose ion exchange column chromatography. The IgY fraction was then concentrated with Poly Vinyl Pyrolidone (PVP) at room temperature. The protein content of the purified IgY fraction was determined by the method described by Lowry et al (1951). The purity of the generated antivenom was observed by Sodium Dodecyl Sulphate Polyacryamide gel electrophoresis using 12% seperating gel and 5% stacking gel by the method of Laemmli (1980). Coomassie Brilliant Blue R 250 was used for visualizing the protein bands.

The antibody titer of the antibodies generated against Echis carinatus venom was determined by indirect ELISA described by Voller et al., 1976. Nunc polysorp ELISA plates were coated with venom at a concentration of 1µg/100µl/well using coating buffer (0.05M carbonate-bicarbonate buffer, pH 9.6) and incubated at 4ºC overnight. After coating plates were washed with PBST for 3 times. The empty sites blocked by 1% BSA (200µl/well) and incubated at 37ºC for 1hour. Plates were subsequently washed and incubated with antivenom antibodies (100µl/well) at appropriate dilutions. PBST and preimmune sera served as controls. Wells were washed thrice with PBST and 100µl of diluted (1:1000) rabbit antichicken immunoglobulin coupled to Horse Radish Peroxidase (Genei pvt Ltd, Bangalore) was added and incubated. Then the plates were washed and 100l of freshly prepared substrate solution (4mg of O-phenylene diamine dissolved in 10ml of 50mM citrate buffer, pH 5.0 containing 10l of 30% hydrogen peroxide) was added. The plates were allowed to stand at room temperature in dark for 20 minutes. The reaction was stopped by adding 50l of 4N H₂SO₄ and plates were read at 490nm in an ELISA reader. All samples were tested in triplicates.

The western blot analysis to determine the specificity of IgY antibodies was carried out according to the method described by Towbin et al., 1979. The Echis carinatus venom separated on SDS PAGE was transferred onto nitrocellulose membrane in an electroblot apparatus for 1 hour at 350mA. After transblotting the membrane was blocked with 5% BSA in PBS and incubated. The membrane was washed with PBST, and incubated with chicken egg yolk derived IgY antibodies for 2 hours at room temperature. After thorough washing, the membrane was incubated with rabbit antichicken immunoglobulin coupled to horseradish peroxidase enzyme (1:1000) for 1 hour at room temperature. After incubation and washing, the substrate solution containing TMB/H₂O₂ (Genei Pvt. Ltd, Bangalore) was added onto the membrane and incubated at room temperature for 20 minutes in dark with constant shaking. The Nitrocellulose membrane was washed with distilled water, air dried and the color development was observed.

Lethal toxicity

Edema- forming Activity

Haemorrhagic activity

Phospholipase activity

Procoagulant activity

Statistical evaluation was performed using XL stat 2007 and SPSS 10 Softwares. P< 0.005 was considered statistically significant.

White leghorn Chickens were immunized intramuscularly with sublethal dose of Echis carinatus venom emulsified with Freund’s Complete Adjuvant for the generation of antivenom antibodies. The booster doses of increasing concentrations of venom was given at regular time intervals to raise the antivenom level in the egg yolk. In another sets of chickens bentonite coated Echis carinatus venom antigens were immunized intramuscularly at the multiple sites of breast muscles in chicken. Booster doses also given in combination with bentonite specific time interval. The preimmune sera and hyperimmune sera were collected at specified time intervals during and after the various immunization schedules. Then specific antibodies were detected in egg yolk after a week. The antibodies were extracted by ammonium sulphate and Polyethylene Glycol (PEG) precipitation methods and further purified by DEAE Cellulose Ion Exchange Column Chromatography. The protein content egg yolk antibodies generated against adjuvant (FCA) coated Echis carinatus venom varied from 5 - 6.65mg/ml and antibodies raised against bentonite coated Echis carinatus venom varied from 0.4 - 5.96mg/ml. The electrophoretic analysis of column purified IgY fraction showed 180KDa-200KDa molecular weight protein band confirming the purity of IgY.

The ELISA there was a gradual increase in the antibody titer in both egg yolk antibodies (FCA and Bentonite) and reached a pleatue and remained stable till 180th day of observation. The booster doses administered at regular time intervals increased and maintained the antivenom level in yolk. The birds immunized with freunds adjuvant coated venoms showed very high titer at dilutions of more than 1:10000 detecting even less than 0.080µg of specific antivenom (Fig 1).

Figure 1

The birds immunized with bentonite venoms also showed good titre value but lesser than FCA. All the birds show a significant titer as compared with the unimmunized control. The chickens immunized with FCA coated venoms did not produce any side effects in birds but Bentonite caused a decrease in the laying frequency of the bird. These side affects caused by bentonite may lead to decrease in antivenom titres in egg yolk of immunized hens.

The antivenom potential of chicken egg yolk antibodies generated against bentonite and Adjuvant(FCA) coated Echis carinatus venom were tested against Echis carinatus venom by in vivo and in vitro methods. The lethal toxicity (LD₅₀) of Echis carinatus venom was assessed using 18g, Balb/c strain mice. About 12μg of Echis carinatus venom was found to be LD₅₀ for 18g of mice. The neutralization of lethality was done by mixing constant amount of venom with various dilutions of chicken egg yolk antibodies and incubated at 37°C for 30 minutes prior to injection. We found that 1.27mg of IgY (FCA coated venom) and 1.33mg of IgY (Bentonite coated venom) were able to completely neutralize the lethal activity of 2LD₅₀ of Echis carinatus venom (Table 1).

Figure 2

When comparing to IgY generated against bentonite coated venom, IgY generated against F CA coated Echis carinatus venom was effectively neutralize the lethal toxicity (Fig 2).

Figure 3

In edema forming activity, the mice immunized with Echis carinatus venoms showed increase in footpad thickness. About 7μg of Echis carinatus venom induced edema formation within 3h which is considered as 100% activity. The edema was reduced up to 30% when 3500μl of IgY/mg venom was given. There was no further reduction in the percentage of edema even when there was an increase in antivenom dose (Fig 3).

Figure 4

In the case of hemorrhagic activity, Echis carinatus venom produced visible hemorrhagic spot and both chicken egg yolk antibodies were effectively neutralized Echis carinatus venom induced hemorrhagic activity. In phospholipase activity (PLA2), Echis carinatus venom able to produce hemolytic haloes in agarose-sheep erythrocytes gels. About 10μg of Echis carinatus venom produced 11mm diameter hemolytic halo, which is considered to be 1U (U/10μg). This shows that Echis carinatus venoms have the enzymes (PLA2) that has the ability to lyse sheep RBC's. Both chicken egg yolk antibodies were capable of inhibiting PLA2 dependent hemolysis of sheep RBC's induced by Echis carinatus venom in a dose dependent manner. The minimum coagulant dose (MCD) was determined as the venom concentration inducing clotting of plasma in 60s. About 120μg of Echis carinatus venom clotted human citrated plasma within 60s. In the neutralization assay, the absence of clot formation shows the neutralizing ability of both chicken egg yolk antibodies.