FRACTIONATION AND DETOXIFICATION OF NAJA NAJA VENOM, PREPARATION OF ANTI TOXOID - IGY ANTIBODIES AND IN VITRO NEUTRALIZATION STUDIES

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1 Journal of Cell and Tissue Research Vol. 8(2) (28) ISSN: (Available online at Original Article FRACTIONATION AND DETOXIFICATION OF NAJA NAJA VENOM, PREPARATION OF ANTI TOXOID - IGY ANTIBODIES AND IN VITRO NEUTRALIZATION STUDIES? MANJULA, J., PAUL, K. AND SUBBA RAO, P. V. 1 Vittal Mallya Scientific Research Foundation, P O Box 46, K.R. Road, Bangalore 56 4, 1 Bigtec Labs., SID Entrepreneurship Center, Indian Institute of Science, Bangalore: E. mail: manjula751@rediffmail.com Received: April 17, 28; Accepted: May 24, 28 Abstract: In order to separate lethal toxins from less toxic or non-toxic proteins, Naja naja venom was fractionated into low molecular weight and high molecular weight fractions by centrifugal concentration using 1kDa Centricon. The LD5 studies in mice revealed LMW venom fraction to be lethal than HMW fraction. Further the lethality of the crude whole venom as well as LMW venom fraction was abolished by detoxification using formalin treatment. Antitoxoid antibodies were produced in chickens by immunizing chickens with increased concentrations of whole or LMW toxoids. ELISA and immunoblots showed the specific binding of anti-toxoid antibodies to the crude venom proteins. Finally, ELISA inhibition and in vitro neutralization studies in murine model proved venom neutralization efficiency of anti-toxoid antibodies. Key words: Naja naja; Anti-toxoid antibodies INTRODUCTION Venomous snakebite is a worldwide problem, especially in tropical regions [1]. Throughout the world, more than 5 million snakebite cases occur annually, while in India alone the incidences are about 3,, of which 1 percent are fatal [2]. The majority of snakebites in the Indian subcontinent are caused by members belonging to the families Elapidae and Viperidae [3,4]. Among Viperidae, Echis carinatus (saw-scaled viper) is responsible for almost two-thirds of the bites [5]. Currently in India, snakebite envenomation is treated by the administration of anti-snake venom (ASV) produced in horses immunized with a mixture of venoms from the four poisonous species of snakes viz. Naja naja, Bungarus caeruleus, Vipera russelli and E. carinatus. Such polyvalent, partially purified antivenom contains large quantities of nonspecific serum proteins that could elicit clinical side effects some times severe enough to be fatal [6,7]. In addition, maintenance and production of ASVs from horses is laborious and expensive. The above drawbacks prompted us to search for an alternative animal model for the production of a pharmaceutically safe and cost effective monovalent antidote for snakebite. Of late, chicken egg as an antibody source has attracted several investigators throughout the world for the noninvasive production of antibodies with applications in research, diagnosis and immunotherapy [8,9]. The predominant class of immunoglobulin in chicken is called IgY, which is transferred from serum to the yolk to confer passive immunity to the developing embryo [1]. Although the avian IgY and mammalian IgG are functionally equivalent, they differ with respect to certain physicochemical properties [11]. Production of avian antibodies offers many advantages over mammalian antibodies [12] and has several applications in immunotherapy. IgY raised against common gastrointestinal infections might be used for fortification of infant food products [13-15]. 1463

2 J. Cell Tissue Research Experimental studies have shown that passive immunization of IgY produced against Helicobacter pylori urease is effective against tissue injury and ulcer formation caused by the production of ammonia by H. pylori [16]. IgY antibodies raised against Streptococcus mutans glucan binding protein B has been shown to induce protection against experimental dental caries [17]. In other reports, chicken antibodies raised against venoms of Brazilian snakes [18] and scorpion and rattlesnakes [19] have shown venom neutralization activity. Also in the field of genomics, development of gene or protein specific IgY antibodies may prove helpful in gene expression studies as well as screening drug targets [2]. We have already shown the isolation, purification and neutralization efficacy of anti-echis carinatus venom antibodies from chicken egg yolk using whole venom [21]. Extensive toxicological studies have also been completed in order to establish the biosafety of these venom-specific IgY antibodies for parenteral administration. The details of this study have been published by Manjula et al. [22]. Anti-Naja naja venom antibodies were also generated in chickens but because of the high toxicity of elapid venoms (Naja naja and B. caeruleus) only small amounts of venom could be injected and the efficiency of these antibodies in neutralizing the crude venom was poor. Hence, in order to over come this an attempt was made to make venom toxoids and produce anti-toxoid antibodies. Toxoids are inactivated protein toxins used in vaccines. The venoms can be detoxified by exposing to various forms of radiations, including both ionizing and nonionizing types or subjecting to chemical treatment [23]. Photooxidation was used as a method of choice for detoxification and toxoiding of C. atrox, V. russelli, A. piscivorus and Micrurus fulvius fulvius venom [24,25]. These investigations have shown that photooxidation in the presence of methylene blue leads to rapid detoxification of a variety of snakevenoms with a concomittant reduction of their necrogenic, hemorrhagic and neurotoxic properties. Immunization of rabbits with these toxoids resulted in the production of antibodies which protected mice against the toxic effects of their respective unaltered venoms. Huang et al. [26,27] investigated the effects of photooxidation on the detoxification and immunogenicity of two Formosan snake venoms, those of Bungarus multicinctus and Trimeresurus mucrosquamatus. Both venoms were detoxified, and the loss of toxicity was found to be proportional to the rate of O 2 uptake. Antiserums produced against the detoxified venoms were similar to that produced against the untreated venoms. Photo oxidation of proteins causes destruction of specific amino acid chains, in particular, histidine and tryptophan. These primary changes lead to alterations in the specificity, enzymatic activity, antigenicity, and other properties of protein molecules. The dye methylene blue sensitizes proteins to photo oxidation by visible light. Thus, since snake venom is composed of many proteins and polypeptides, some of which are responsible for the toxicity of the venom, it is reasonable to expect photo oxidation to detoxify these components. Flowers [28] devised a method for preparation of Agkistrodon piscivorus venom toxoid by x-ray irradiation. The antivenom produced against this toxoid proved to be very effective in neutralizing the lethality of the whole venom. Venom protease and hemorrhagic activities can be removed by adding EDTA, a powerful chelating agent, to venoms. The EDTA treated venom of Agkistrodon piscivorus was capable of stimulating the production of antibodies which reacted with and neutralized the toxins of untreated venom [29]. Modern molecular biological techniques have refocused efforts to develop toxoids through inactivation of toxin proteins via site-directed mutagenesis [3-34]. Formalin can be used to produce toxoids of venoms. Formalin has been used to produce a toxoid of habu venom for use in active immunization as well as in antivenin production [35,36]. Toxoidation involves detoxification of the venom to a major extent while still retaining immunogenicity of the toxin [37]. These toxoids can be further used to produce venomspecific antibodies. The present investigation describes fractionation of Naja naja venom through centrifugal concentration using 1 kda centricon. The fractionated as well as whole venoms were converted to toxoids by detoxification using formalin. LD 5 dose was determined for both fractionated venoms and toxoids in mice. Antibodies were generated in chickens by immunizing White leghorn chickens with fractionated LMW and whole toxoids. The efficiency of fractionated LMW as well as whole anti-toxoid antibodies against the lethal effects of venom was determined by in vitro neutralization studies in mice. 1464

3 Manjula et al. MATERIALS AND METHODS Materials: Naja naja venom, Freund s complete and incomplete adjuvant, bovine serum albumin (BSA), rabbit anti-chicken IgY, orthophenylenediamine (OPD), 3,3 -Diaminobenzidine (DAB) (SIGMA Chemicals Co., St. Louis, MO), avidin-horse radish peroxidase (avidin-hrp) (vector labs), casein acid hydrolysate, Tween-2 (Himedia), polystyrene microtiter plates (Costar, Cambridge, MA), nitrocellulose membrane (Schleicher & Schuell Inc., Keene, NH),.22µ millipore filter (Millipore corpn, Bedford, MA), formaldehyde solution (37-41% w/ v), sodium sulphate, hydrogen peroxide (H 2 2 ) (Merck), coomassie brilliant blue R-25, acryl-amide, bis-acrylamide, sodium dodecylsulphate (SDS), N,N,N,N -tetramethyl ethylenediamine (TEMED), ammonium persulphate (Bio-rad laboratories). Fractionation of Naja naja venom: Naja naja whole venom (1 mg/ml) was fractionated on 1 kda molecular weight cut-off centricon by centrifugation at 5 rpm for 3 min at 1 C. The high molecular weight and low molecular weight venom fractions were collected separately and subjected to SDS-PAGE on a 1 % gel. Briefly, 2µg of each sample taken in SDS-PAGE sample buffer was boiled for 5 min, and loaded into adjacent wells. After the run, gel was stained with Coomassie Brilliant blue R-25. Toxoid preparation: Naja naja whole venom as well as fractionated Naja naja LMW venom were converted into toxoids by subjecting 1 mg/ml of each of these venoms to formalin treatment at ph 7.4, 37 C in the presence of.1 M lysine. Initially formalin was added at a concentration of.2% and then at increasing concentrations of.2% on 3 rd, 5 th, and 7 th days at 37 C. After removing excess formalin by extensive dialysis both LMW toxoid as well as Whole toxoid were used for immunization. LD 5 determination: LD 5 dose was determined by injecting 7, 8 and 1 µg Naja naja LMW and 1, 12, 2, 25, 5 and 1 µg of Naja naja HMW venom fractions to individual group of BALB/c mice containing 2 animals each. Similarly LD 5 dose was determined for toxoids by injecting 5 and 1 µg of Naja naja whole and 25 µg of LMW toxoid, to BALB/c mice containing 2 animals each. The animals were kept under observation for 24h. Immunization: 18 weeks old White Leghorn chickens were intramuscularly injected with 1 mg/kg b.w. of Naja naja whole or LMW toxoid in Complete Freund s adjuvant (CFA). Subsequent boosters were given in Freund s incomplete adjuvant on days 14 and 28. Eggs were collected and antibodies were purified. Antibody extraction and purification: Anti-toxoid antibodies were extracted from the egg yolks by acid water dilution method. described by Akita and Nakai [38]. Briefly, yolks were separated from egg whites and diluted with acidified water to ph 5.5. The suspension was allowed to settle for 6 h at 4 C. The water soluble fraction (WSF) containing the antibodies was separated by centrifugation and clarified by filtration. The crude protein fraction was further purified to homogeneity by 19% sodium sulphate salt fractionation. The antibody fraction was dialyzed in.9% saline, filter sterilized and stored at 4 C. The protein content in the WSF, 19% salt pellet and 19% supernatant was estimated by Bradford s dye binding method using BSA as standard. Quantitation by ELISA: The venom-specific antibody titers in egg yolk were determined by ELISA. Briefly, polystyrene 96-well microtiter plates were coated with 1µg/ml native Naja naja venom in coating buffer (.1M sodium carbonate-bicarbonate buffer, ph 9.6) for 1 hr at 37 C. The wells were washed 6 times with rinse buffer, PBST (phosphate buffer saline ph 7.4 with.5% Tween-2), and blocked with 2% casein in rinse buffer for 1 hr at 37 C. After washing, the wells were incubated with 2.5µg/ml concentration of anti-whole or LMW toxoid antibodies for 1 hr at 37 C. The wells were then rinsed with rinse buffer and incubated with anti-rabbit IgY biotin conjugated (1: 2) for 3 min at 37 C. The wells were washed again and incubated with avidin-hrp conjugate (1:2) at 37 C for 5 min. After rinsing the wells thoroughly, the enzyme activity was quantitated by addition of HRP substrate solution (.5mg/ml o-phenylenediamine and.6% H 2 O 2 in.2m potassium phosphate buffer, ph 7.) and colour was developed in dark for 2 min. The enzyme reaction was terminated by the addition of 5µl of 2N HCl and the absorbance was read at 49 nm by automated ELISA reader. Immunoblot: Immunodetection of the Naja naja venom proteins by anti-toxoid antibodies was performed by immunoblot. Briefly, fifteen micrograms 1465

4 J. Cell Tissue Research of native Naja naja venom was separated electrophoretically on a 1% SDS-polyacrylamide gel and electroblotted on to a nitrocellulose membrane using semi dry Western blotting apparatus (LKB, Uppsala, Sweden). The proper transfer of venom proteins on to the membrane was determined by staining the blot with Ponceau-S. The stain was removed by washing excessively with PBST (phosphate buffer saline, ph 7.4 with.5% Tween-2). The unoccupied sites on the membrane were blocked with blocking buffer (2% casein in rinse buffer) for 1 hr at room temperature or overnight at 4 C. The blots were then incubated with 1µg/ml concentration of anti-whole or LMW toxoid antibodies at 37 C for 1 h. The blots were then washed with rinse buffer and incubated with antirabbit IgY-biotin conjugate (1:2) for 1 hr at room temperature. The blots again washed with rinse buffer and incubated with avidin-hrp (1:2) for 3 min at room temperature. Finally the blots were washed and developed with developing solution (1mg/ml 3,3'- diaminobenzidine and.3% H 2 O 2 in 5 mm sodium acetate buffer, ph 5). Inhibition ELISA: Polystyrene 96 well microtiter plates were coated with 1 µg/ml concentration of venom protein in coating buffer (.1M sodium carbonate-bicarbonate buffer, ph 9.6) for 1 hr at 37 C and subsequently blocked with blocking buffer (2% casein in rinse buffer). The anti-whole or LMW toxoid antibodies at fixed dilution were preincubated at 37 C for 3 min with log-fold dilutions of native venom before adding to the venom coated wells. After 1 hr of incubation, the wells were washed with rinse buffer and incubated with anti-rabbit IgY-biotin conjugate (1: 2) for 3 min. Remaining steps of the assay procedure were performed as described in the ELISA protocol. The percent inhibition was calculated by the difference in absorption at 49 nm between inhibited and uninhibited control wells. In vitro neutralization: The ability of anti- whole or LMW toxoid antibodies in protecting mice against lethal effects of the venom was determined by in vitro neutralization studies. BALB/c mice weighing 18-2 g were divided into two groups, test and control groups each group consisting of 6 animals. Test group was injected with 2 LD 5 (24 µg) of native Naja naja venom pre-incubated with 5 mg of commercial horse ASV or anti- whole or LMW toxoid antibodies for 3 RESULTS Naja Naja venom Fractionation: Fractionation of Naja naja whole venom proteins was carried out using 1kDa centricon to separate low molecular weight proteins from the higher molecular weight proteins. The SDS-PAGE profile of fractionated venom showed the separation of HMW proteins from the LMW proteins (Fig.1). LD 5 studies: The LD 5 studies in mice revealed that mice injected with 8 and 1 mg of Naja naja LMW venom showed 1% mortality while mice injected with 7 mg showed 5% mortality and 5% survival. Hence 7 mg was taken as LD 5 dose for Naja naja LMW venom. Similarly mice injected with 1, 12, 2, 25, 5 and 1 mg of Naja naja HMW venom or 5 and 1 mg of Naja naja whole toxoid or 25 mg of LMW toxoid did not show Fig. 1 mortality after 24 h (Fig.2). Purification of anti-toxoid antibodies: Antitoxoid antibodies were extracted from the egg yolks by a combination of acid water dilution method followed by salt fractionation. The total protein in the WSF, 19% salt pellet, and 19% supernatant was found to be 365, 8, and 28 mg/egg as determined by Bradford s dye binding method. Specific activity of anti-toxoid antibodies: Activity for the presence of anti-toxoid antibodies was screened by ELISA. The activity obtained for anti- whole or LMW toxoid antibodies were 95,625 and 96, ELISA units/egg respectively (Fig.3). Immunoblot: Immunoblot was performed by incubating the blots separately with anti- whole or LMW toxoid antibodies. Results obtained revealed the specific binding of anti- whole or LMW toxoid antibodies to the native venom (Fig.4). Inhibition ELISA: Inhibition ELISA experiments were performed to demonstrate the specific binding of anti-whole or LMW toxoid antibodies to native Naja naja venom. At an inhibitor concentration of 1µg/ml, Naja naja venom inhibited the binding of anti-whole or LMW toxoid antibodies to the native venom immobilized to microtiter wells by 81.4% and 81.68% respectively (Fig.5). min at 37 C. Control group was injected with 2 LD 5 (24 µg) of Naja naja venom alone. The animals were kept under observation and the protective ability was measured in terms of % survival after 24 h Fig. 3 In vitro neutralization studies: The potency of anti- whole or LMW toxoid antibodies in protecting mice against the lethal effects of the venom was

5 Manjula et al. 97kDa fi 66kDa fi 45kDa fi Fig. 1: SDS-PAGE profile of Naja naja venom fractionated on 1 kda Centricon. Twenty micrograms of samples were applied to 1% SDS-PAGE and the resolved venom proteins were visualized by staining with Coomassie brilliant blue R-25. Lanes: (1) Molecular weight markers; (2) Naja naja whole venom; (3) Naja naja HMW venom fraction; (4) Naja naja LMW venom fraction. 29kDa fi 14kDa fi Percentage Survivial 12% 1% 8% 6% 4% 2% % Fig. 2: Determination LD 5 dose for fractionated venoms and toxoids in BALB/ c mice. LD 5 dose was determined by injecting different concentrations of fractionated venom and toxoids to BALB/ c mice. The animals were kept under observation for 24h and time of death was recorded after 24 h. (1) Naja naja LMW fraction; (2) Naja naja HMW fraction; (3) Naja naja whole toxoid; (4) Naja naja LMW toxoid Whole toxoid antibodies LMW toxoid antibodies Fig. 3: Activity of anti-whole and LMW toxoid antibodies in WSF. Polystyrene microtiter wells were coated with 1µg/ml of native Naja naja, venom and incubated with anti-whole or LMW toxoid antibodies. Enzyme reaction was developed as described in materials & methods. (1)Antiwhole toxoid antibodies (2)Anti-LMW toxoid antibodies. 1467

6 J. Cell Tissue Research Fig. 4: Immunoblot for binding of anti- whole and LMW toxoid antibodies to Naja naja venom. Native venom (15µg) was transferred on to nitro cellulose membrane and probed with anti-whole or LMW toxoid antibodies. Lanes: (1) Control; (2) antiwhole toxoid antibodies; (3) anti-lmw toxoid antibodies. % I N H I B I T I O N LMW toxoid abs 2 Whole toxoid abs Venom Conc. in µg/ml Fig. 5: Inhibition of antibodies to whole and LMW toxoids with Naja naja venom. Polystyrene microtiter wells were coated with 1µg/ml of Naja naja venom and incubated with anti- whole or LMW toxoid antibodies after pre-incubation with 1µg/ml of native Naja naja venom. 12% 1% 8% 6% 4% 2% % Control Horse ASV Whole toxoid Abs LMW toxoid Abs Fig. 6: In vitro venom neutralizing efficacy of antiwhole or LMW toxoid antibodies against 2LD5 doses of Naja naja venoms in mice and comparison with commercial horse ASV. The antivenin and venom were preincubated at 37 C for 3 min before administration to mice. Control mice received venom alone. Time of survival was recorded after 24 hr. Lanes: (1) Control (2) Horse ASV (3) Whole toxoid antibodies (4) LMW toxoid antibodies. 1468

7 determined by in vitro neutralization studies. The mice injected with a pre-incubated mixture of a 2 LD 5 dose (24 µg) of Naja naja venom with 5 mg of commercial horse ASV or antibodies to whole or LMW toxoid showed 1% survival after 24 h, while the control group animals did not show any survival (Fig.6). DISCUSSION Snake venom has a very complex heterogeneous composition, containing enzymes, lethal peptides, nonenzymatic proteins, metals, carbohydrates, lipids, biogenic amines, free amino acids and direct hemolytic factors. Snake venoms have complex and varied composition, evolved as specialized secretory products of exocrine glands. They have high efficiency in immobilizing, killing and initiating the digestion of the prey. In addition to its offensive role as in the capture and digestion of the food, it may also serve as a defensive armament protecting the reptile against predators and aggressors. Earlier all the deleterious and pharmacological effects of snake venoms were attributed to the enzymes of the venom. Recently, short chain and long chain neurotoxins and cardiotoxins from elapid venoms which are nonenzymatic have been thoroughly characterized. These investigations clearly suggested that the nonenzymatic peptides contribute signifiantly towards snake venom s lethal potency and pharmacological activities. In present study Naja naja venom was subjected to fractionation by centrifugal concentration on a 1kDa centricon to separate LMW venom fraction from the HMW fraction. This was confirmed by the separation pattern obtained by SDS-PAGE profile. LD 5 studies conducted for these fractions in mice. The results obtained revealed that LMW venom fraction produced mortality in mice at a concentration of 7 µg, while the HMW fraction did not produce mortality even at a concentration of 1µg. The LD 5 dose for native venom was found to be 12 µg. This was much higher than LD 5 dose obtained for the LMW fraction. This may be due to separation of LMW venom fraction from the HMW fraction. In order to produce antibodies with high venom neutralizing capabilities the amount of venom that could be injected in chickens became a limitation as snake venoms are highly toxic. Hence lethal effects of the venom were abolished by converting them into toxoids by formalin treatment. It is a known fact that Manjula et al. when snake venoms are treated with formalin most of the venom toxins lose their toxicity. Bizzini and Raynaud [39] have discussed the possible mechanisms by which protein toxins are detoxified by formaldehyde and reported that tyrosyl and histidyl residues participate in the toxicity but lysyl residues do not. Similarly, Linggood et al.. [4] and Scheibel and Christensen [41] have demonstrated that when purified diphtheria toxins were detoxified with formalin, reversion to toxin occurred when such toxoids were diluted and stored for 2 months at room temperature or at 32 C. But when lysine-formalin mixtures were added to toxin a stable non-reversing toxoid was formed. The concentration of lysine and the ph of detoxification have been shown to be critical. Both Naja naja whole and Naja naja LMW venoms were detoxified by treating with.2% formalin in the presence of.1m lysine at ph 7.4, 37 C. LD 5 studies were conducted for these toxoids by injecting different concentrations in mice. The results obtained revealed that the animals injected with 5 and 1 µg of Naja naja whole or 25 µg of Naja naja LMW toxoid, did not show any signs of mortality. Thus detoxification using formaldehyde might have abolished toxicity of the venom. White leghorn chickens were immunized individually with Naja naja whole or LMW toxoids. The concentration of toxoid used for immunization was 15 times more than the concentration that can be used for the native venom. Antibodies of both whole and LMW toxoids were purified from immunized hen eggs and venom-specific activity was screened by ELISA and Immunoblot techniques. Both preparations of the anti-toxoid antibodies recognized native venom proteins in a similar manner as native venom antibodies. The efficiency of anti-whole or LMW toxoid antibodies in neutralizing the venom was determined by in vitro neutralization studies in BALB/c mice. Mice were injected with a pre-incubated mixture of 2 LD 5 (24 µg) of native Naja naja venom with 5mg of commercial horse ASV or anti-whole or LMW toxoid antibodies and protectivity was measured in terms of percentage survival after 24 h. The mice injected with a pre-incubated mixture of 2 LD 5 (24 µg) of native N. naja venom with commercial horse ASV or anti-whole or LMW toxoid antibodies showed 1% protection. By looking at the results of in vitro neutralization studies, it can be said that conversion of venom to toxoid and administration of higher doses of the resultant toxoid for immunization could produce 1469

8 J. Cell Tissue Research antibodies probably with high venom neutralization capability. Thus detoxification using.2% formalin in presence of.1m lysine could be useful for preparing an effective venom toxoid with good immunogenicity. ACKNOWLEDGEMENTS This work was funded by a grant from the Department of Biotechnology, Government of India. REFERENCES [1] Warrell, D.A.: Med. J. Aust. 159 (11-12): (1993). [2] Thachil, R.T., Tony, J.C., Jude, E., Ross, C., Vincent, N.D.J. and Sridhar, C.B.: Tropical Doctor 22(3): (1992). [3] Ananthapadmanaban, J.: Snake bite. In: Medicine update. Association of Physicians of India (Dalal, P. M. ed.), Bombay, pp (1991). [4] Jena, I. and Sarangi, A.: Snakes of Medical Importance and Snake Bite Treatment. Ashish Pub. House, New Delhi, India (1993). [5] Bhat, R. N.: J. Indian Med. Assoc. 12: (1974). [6] Sutherland, S. K. and Lovering, K. E.: Med. J. Aust. 2 (13): (1979). [7] Malasit, P., Warrell, D. A., Chanthavanich, P., Viravan, C., Mongkolsapaya, J., Singhthong, B. and Supich, C.: Br. Med. J. 292: 17-2 (1986). [8] Schade, R., Schniering, A. and Hlinak, A.: ALTEX 9 (2): (1992). [9] Gross, M. and Speck, J.: Dtsch Tierarztl Wochenschr. 13 (1): (196). [1] Larsson, A., Balow, R. M., Lindahl, T. L. and Forsberg, P. O.: Poult. Sci., 72(1): (1993). [11] Leslie, G. A. and Clem, L.W.: J. Exp. Med., 13 (6): (1969). [12] Schade, R., Pfister, C., Halatsch, R. and Henklein, P.: ATLA 19, (1991). [13] Marquardt, R.R., Jin, L.Z., Kim, J.W., Fang, L., Frohlich, A. A., Baidoo, S.K.: FEMS Immunol. Med. Microbiol. 23 (4): (1999). [14] Sunwoo, H.H., Nakano, T., Dixon, W.T., Sim, J.S.: Poult. Sci., 75 (3): (1996). [15] Sarker, S.A., Casswall, T.H., Juneja, L.R., Hoq, E., Hossain, I., Fuchs, G..J., Hammarstrom, L.: J. Pediatr. Gastroenterol. Nutr., 32 (1): (21). [16] Shin, J-H., Yang, M., Nam, S.W., Kim, J.T., Myung, N. H., Bang, W.G.. and Roe, I.H.: Clin. Diagn. Lab. Immunol. 9 (5): (22). [17] Smith D.J., King, W.F., Godiska, R.: Infect. Immun. 69 (5), (21). [18] Almeida, C. M., Kanashiro, M. M., Rangel Filho, F. B., Mata, M. F., Kipnis, T. L. and Dias da Silva, W.: Vet. Rec. 143 (21), (1998). [19] Thallay, B.S., Carroll, S.B.: Biotech. (NY). 8: (199). [2] Zhang, W.W.: Drug Discov. Today, 8(8): (23). [21] Paul, K., Manjula, J., Deepa, E.P., Selvanayagam, Z.E., Ganesh, K.A., and Subba Rao, P.V.: Toxicon., 5: (27). [22] Manjula, J., Kusum, P., Sairam, A.K., Murthy P.B. and Subba Rao P.V.: J. Cell Tissue Res. 6(2): (26). [23] Anthony, T.T.U.: Venoms: Chemistry & Molecular Biology. A Wiley Intersci. Pub., New York (1977). [24] Kocholaty, W.: Toxicon. 3: (1966). [25] Kocholaty, W.F.,Ashley, B.D. and Billings, T.A.: Toxicon. 5: (1967). [26] Huang, C.T., Huang, J.S., Ling, K.H., and Hsieh, J.T.: J. Formosan Med. Assoc., 71: (1972). [27] Huang, C.T., Huang, J.S., Ling, K.H., and Lin, S.Y.: J. Formosan Med. Assoc., 72: (1973). [28] Flowers, H.H.: Toxicon., 1: (1963). [29] Goucher, C.R. and Flowers, H.H.: Toxicon., 2: (1964). [3] Killeen, K.P., Escuyer, V., Mekalanos, J.J., and Collier, R.J.: Proc. Natl. Acad. Sci. U.S.A. 89: (1992). [31] Pizza, M., Bartoloni, A., Prugnola, A., Silvestri, S. and Rappuoli, R.: Proc. Natl. Acad. Sci. U.S.A. 85, (1988). [32] Pizza, M., Fontana, M.R., Giuliani, M.M., Domenighini, M., Magagnoli, C., Giannell, V., Nucci, D., Hol, W., Manetti, R. and Rappuoli, R.: J. Exp. Med. 18: (1994). [33] Barbieri, J.T., Armellini, D., Molkentin, J. and Rappuoli, R.: Infect. Immun., 6: (1992). [34] Marsili, I., Pizza, M., Giovannoni, F., Volpini, G., Bartalini, M., Olivieri, R., Rappuoli, R. and Nencioni, L.: Infect. Immun., 6, (1992). [35] Kondo, S., Sadahiro, S., Yamuchi, K., Kondo, H. and Murata, R.: Jap. J. Med. Sci. Biol. 24: (1971) [36] Someya, S., Murata, R., Sawai, Y., Kondo, H. and Ishii, A.: Jap. J. Med. Sci. Biol. 25, (1972). [37] Das Gupta, S.C., Gomes, A., Aparna Gomes, Basu, A. and Lahiri, S.C.: Indian. J. Exp. Biol., 27: (1989). [38] Akita, E.M. and Nakai, S.: J. Food Sci., 57(3): (1992). [39] Bizzini, B. and Raynaud, M. : Biochimie. 56, (1974). [4] Linggood, F.V., Stevens, M., Fulthrope, A.J., Woiwod, A J. and Pope, C.G.: Brit. J. Exptl. Pathol. 44: (1963). [41] Scheibel, I. and Christensen, P.E.: Acta Pathol. Microbiol. Scand. 65, (1965). 147