Naja haje arabica (Arabian cobra) is the major cause of snake-bite mortality in Kingdom of Saudi Arabia. The treatments of the snake bite envenomation occur by anti-snake venom produced in horses previously immunized with a mixture of venom. Therefore, one of the main objectives of the current study is to produce anti Naja haje arabica immunoglobulin in high titer from the yolk of chickens by immunization of two groups of white leghorn chickens (24 weeks old) with 30 μg of Naja haje arabica emulsified in Freunds Complete Adjuvant. Chickens has been immunized with booster doses of increasing concentrations of venom at two weeks time intervals to increase the antivenom titer in the egg yolk. The characteristic IgY band of 180 kDa was observed on SDS-PAGE of the final extracted product. The ELISA antibody values reached the plateau at 2nd weeks following 4th booster dose and remained significantly high up to the end of observation period. The measured antibody titers showed significant increase following the first, second, third booster doses. However, there were no differences between the third and the fourth booster doses. Western blot technique was used to evaluate the specificity of antivenom IgY antibodies. The LD50 of the Naja haje arabica venom has been found to be 0.4 mg kg1 body weight of white Swiss mice and 100% protection against 40 LD50 of Naja haje arabica venom could be obtained by15 mg mL1 anti Naja haje Arabica specific IgY. The neutralizing power of the anti Naja haje arabica venom IgY and the absence of pyrogen, bacterial and fungal contaminations or toxic products, encourage the use of egg yolk as a cheap source of anti-venom polyclonal antibodies.
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Venomous snakebite is a one of the most important problems in tropical countries and a serious medico-legal problem. Many venomous snakes of medical importance inhabit the Kingdom of Saudi Arabia and surrounding areas, where several of the most lethal snakes are found (Gasperetti, 1976, 1988). Naja haje arabica (the Arabian cobra) and Walterinnesia aegyptia (the Egyptian black cobra; the desert cobra) are representatives of the Elapidae family.
Naja is a genus of venomous elapid snakes known as cobras. Several other genera include species commonly called cobras (for example the King cobra, Ophiophagus hannah and the rinkhals or ring-necked spitting cobra, Hemachatus haemachatus) but of all the snakes known by that name, members of the genus Naja are the most widespread and the most widely recognized as cobras. Various species occur in regions throughout Africa, Southwest Asia, South Asia and Southeast Asia.
Until recently the genus Naja had 20-22 species, but it has undergone several taxonomic revisions in recent years. Conventional antivenoms are prepared by immunizing large animals, usually horses, with individual venom or a range of different venoms obtained from several snakes to eliminate intra specific variation (Theakston, 1996; Larsson and Sjoquist, 1990). The maintenance and the production of anti snake venoms antibodies from horses are laborious and expensive. The advantages offered by avian egg yolk antibodies (IgY) over the conventional mammalian antibody production are well documented (Jensenius et al., 1981; Akita and Nakai, 1993; Schade et al., 2001; Zhang et al., 2004; De Almeida et al., 2008).
Polyvalent-Bitis and anti-Naja antivenom IgY antibodies produced by immunizing chickens with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambica venoms exhibited high antivenom activity (>100,000 U-ELISA/mL) as well as efficacy in neutralizing venom lethality (1,440 μg of IgY neutralized 62.2 LD50 of venom) and were free of toxic products, pyrogens or bacterial and fungal contaminations (Meenatchisundaram et al., 2008).
Therefore, due to their advantages, it was suggested that chicken antibodies would replace their mammalian counter parts in the future. The main goal of this study is to prepare and evaluate the protective efficacy of immunoglobulins (IgY) prepared against Naja haje arabica snake venom located in the Saudi Arabian region.
MATERIALS AND METHODS
Chickens and animals: During the period of 10th February to 30th May, 2014, five-month-old white leghorn female chickens (1.1-1.5 kg body mass), Swiss outbreed (18-20 g) mice and rabbits (0.5-1.0 kg) were used to produce IgY antivenoms; Swiss outbreed mice were used to determine venom lethality, potency, the neutralizing potency of antivenom, in addition to other in vivo assay. Rabbits were used to produce anti-IgY antiserum.
Crude venoms from Saudi Arabian Naja haje arabica snakes: The immunoglobulins IgY were prepared against local Saudi Arabian snake venom, Naja haje arabica snake venom. The Lethal Dose-50 (LD50) of venom will be estimated.
Evaluation of the lethal dose 50 of the venom: The lethal dose 50 of the Naja haje arabica (LD50) were evaluated by intra-muscular injection of five groups of Swiss outbreeds mice (20-22 g) eight mice per each group using. After 48 h the mortality rates and the LD50 were calculated according to De Almeida et al. (2008).
Immunization schedule of chickens: Two groups of eight chickens were immunized intra-muscularly in the breast region at two or three sites with 30 μg of Naja haje arabica snake venom venoms, alone or mixed as indicated in Freunds Complete Adjuvant (FCA) (De Almeida et al., 2008). Blood samples and eggs were collected before immunization to be used as negative controls either in immunochemical assays or in immunoprotection tests. The eggs of the immunized hens were collected every day and kept in refrigerators at 4°C. The egg yolks were separated from the albumin and kept at -20°C.
Extraction and purification using ammonium sulphate-caprylic acid method: The yolk from the immunized hens was separated from the egg white carefully and the IgY antibodies were extracted as previously described by De Almeida et al. (2008). The total protein content was determined by Biuret test and the IgY-preparations were filtrated using 0.45 μm filter and stored at 40°C after being a liquoted in small test tubes.
Characterization of egg antivenom IgY antibodies in vivo and in vitro and estimation of its neutralizing potency
SDS-PAGE and Western blot analyses: Egg antivenom IgY antibodies were analyzed by Western blot analysis and polyacrylamide gel electrophoresis SDS-PAGE by the method explained by De Almeida et al. (2008).
Evaluation of antibody activity: The antibody titer was estimated by indirect ELISA using polystyrene ELISA plates after coating with 0.5 μg of native Naja haje arabica venom after mixing with 50 μL carbonate bicarbonate coating buffer and kept in the refrigerator at 4°C. Antibody titer was estimated according to according to Pauly et al. (2009) and Zhen et al. (2008).
Evaluation of the neutralizing potency of anti Naja haje arabica venoms
IgY-antibodies: The neutralizing potency of IgY antivenom antibodies, produced along the immunization procedure, will be evaluated according to the recommendation of Gottstein and Hemmeler (1985) using groups of eight Swiss mice (18-20 g) for anti Naja haje arabica venom antibodies.
Statistical analysis: Statistical Analysis was performed with the SPSS Statistical Package version 12.0 (SPSS Inc, Chicago, IL). The p-values were calculated using the Mann-Whitney U and A p-value<0.05 was considered statistically significant.
Serum samples from hens immunized with Naja haje arabica venom were assayed with an ELISA just before immunization and every two weeks of immunization and boostering up to 14 weeks.
|Table 1:||Comparison between ELISA titers of Naja haje arabica-specific antibodies in serum samples and Ig Y-antibody preparations from hens immunized with Naja haje arabica vaccines at different time intervals post immunization|
|**Moderately significant (p<0.01), ***Highly significant (p<0.001), *Non significant. SDn; Standard deviation, n = 3|
Antibodies specific to Naja haje arabica appear in the serum samples of the immunized hens after two weeks from the primary immunization. Significant increase in the mean log10 antibody titer of tested serum samples was recorded 2 weeks post immunization and reached to 1.52± 0.112, as compared to a pre-immunization level of 1.27±0.016 (p<0.01). With respect to IgY-preparations, a significant increase in the mean 1og10 antibody titer of the Naja haje arabica specific IgY-antibody was first observed after 4 weeks of immunization (2 week after the 15 t b20ster dose) and reached 2.6±0.00 in IgY preparations pre-immunization level of 1.09±0.00. At 8-10 weeks of immunization, the Naja haje arabica-specific antibody titers measured in the IgY- extracts were relatively similar to that in the serum, then its level exceeds that of the serum samples and till the end of the immunization period.
The maximum antibody titers of the tested serum samples were observed at 8 weeks post immunization (2 weeks after 4th booster dose) as shown in Table 1.
The protective value of Naja haje arabica local Saudi snake venom specific IgY-antibodies as measured by neutralization test revealed that 15 mg mL1 Naja haje arabica specific IgY-antibodies produce 100% protection against 40 LD50 of Naja haje arabica venom.
Snakebite is one of the most important problems in tropical countries, therefore, the current study is aimed to evaluate the immunological properties of immunoglobulins- IgY antibodies prepared by immunization against local Saudi Naja haje arabica snake venom.
The total protein content in serum samples collected from hens prior to immunization, significant increase (p<0.001) was recorded in samples collected after two weeks of primary immunization. Boostering induced both increase and maintenance of higher levels of total protein in the examined serum samples from the immunized chicken groups. This increase continued till the end of the immunization period. The immunization-dependent increase in total protein content of serum can be attributed to the increased of production of immunoglobulins and other immunoregulatory proteins by the immunocompetent cells (Davalos-Pantoja et al., 2000; Almeida et al., 1998). Similar results were reported by Polson et al. (1980), Almeida et al. (1998), Akita and Nakai (1993) and Mclaren et al. (1994).
Analysis of results obtained with ELISA revealed that serum samples collected from hens immunized with Naja haje arabica venom showed significant increase in the specific antibodies against the snake venom after two weeks from immunization (p<0.001). The anti-venom antibody reached the maximum level after 6-8 weeks by the effect of boostering and remain significantly higher till the end of immunization period (Almeida et al., 1998; Akita and Nakai, 1993; De Almeida et al., 2008; Polson et al., 1980; Mclaren et al., 1994; Sarker et al., 2001).
Evaluation of the protective efficacy of anti Naja haje arabica venom antibodies revealed that 1 mL of IgY-antibodies (15 mg mL1 anti Naja haje arabica IgY) could protect 40 LD50 (100% protection and 75% protection against 50 LD50). The neutralizing power produced by anti Naja haje arabica IgY antibodies encourage the use of such immunoglobulin as a cheaper source of anti Naja haje arabica IgY antibodies, similar to those reported by many authors (Carroll and Stollar, 1983; Almeida et al., 1998; Carroll et al., 1992; Sarker et al., 2001; De Almeida et al., 2008).
Naja haje arabica (the Arabian cobra) are the most dangerous and the most widely recognized local Saudi cobras. Therefore, this study was aiming to produce IgY-antibodies prepared against such venom. The protective value of Naja haje arabica local Saudi snake venom specific IgY-antibodies by neutralization test revealed that 15 mg mL1 Naja haje arabica specific IgY-antibodies produce 100% protection against 40 LD50 of Naja haje arabica venom. So, this study will be helpful in further immunological investigations to produce specific IgY antibodies against more other Saudi snake venoms.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding of the study through the research group project No. RGP-VPP-162.
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