Volume 15, Issue 1 (4-2024)
Res Anim Prod 2024, 15(1): 95-104 |
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Nazari M, Roshanfekr H, Saalabi F, Fayazi J, Mohammadian A, Kavosh F. (2024). Production of the First Effective Immune Equine Serum Antivenom against Iranian Honey Bees (Apis mellifera meda). Res Anim Prod. 15(1), 95-104. doi:10.61186/rap.15.43.86
URL: http://rap.sanru.ac.ir/article-1-1343-en.html
URL: http://rap.sanru.ac.ir/article-1-1343-en.html
Mahmoud Nazari *1 
, Hedayatolah Roshanfekr1, Fatemeh Saalabi2, Jamal Fayazi2, Ali Mohammadian2, Fariba Kavosh3
, Hedayatolah Roshanfekr1, Fatemeh Saalabi2, Jamal Fayazi2, Ali Mohammadian2, Fariba Kavosh3
1- Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Mollasani, Iran
2- Department of Venomous Animals and Anti-venom Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Ahvaz, Iran
3- Department of Venomous Animals and Anti-venom Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Ahvaz-Iran
2- Department of Venomous Animals and Anti-venom Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Ahvaz, Iran
3- Department of Venomous Animals and Anti-venom Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Ahvaz-Iran
Abstract: (1357 Views)
Extended Abstract
Background: Bee venom is a colorless, acidic liquid (pH 4.5-5.5) that contains 18 different compounds, including enzymes, peptides, and biological amino acids. Some of these compounds have anti-inflammatory properties, while others possess toxic and allergenic properties. Bee venom is produced by female worker bees, with the most important peptides being melittin, apamin, adolapin, and mast cell degranulating (MCD) peptide. The key enzymes in bee venom include hyaluronidase and phospholipase A2 (PLA2). Historically, bee venom has been used in traditional medicine to treat various diseases, such as rheumatoid arthritis, and to alleviate muscle pain. Massive bee attacks are considered a public health problem in several countries, including Iran. Currently, no specific therapy exists for bee stings; treatment typically involves chemical drugs. This has led us to explore the development of Fab-based antivenom as a potential new treatment. To date, no efforts have been made in Iran to produce Fab-based antivenom for individuals sensitive to bee stings. Therefore, this research aimed to produce an effective and safe horse serum antidote against the Iranian honey bee (Apis mellifera meda).
Methods: Crude bee venom (BV) was collected from Iranian bees (Apis mellifera meda) using an electric bee venom-collecting machine. The venom dries quickly in the air; therefore, it was collected daily from glass plates using special spatulas and stored in dark glass containers in a freezer at -20 ºC for future experiments. To remove mucus and extraneous substances, the venom was dissolved in a 1:1 ratio with saline (physiological serum) and centrifuged at high speed. After centrifugation, the upper clear solution was separated and filtered using a 0.2-micron filter. The protein concentration in the crude venom solution was determined using the Bradford protein assay method, based on a standard curve obtained from different concentrations of bovine serum albumin (BSA) solution.
The median lethal dose (LD50) of BV was determined using 24 male mice, which were divided into four groups. Different doses of venom (60, 70, 80, 90, 100, and 110 μg/ml) dissolved in sterile normal saline were injected intravenously. The control group received 0.5 ml of saline solution. Mortality was recorded after 24 hours, and LD50 was calculated using probit analysis. Three horses were used to produce antivenom against honey bee venom. The horses were immunized with Iranian bee venom seven times at 7-day intervals, and immunogenicity was evaluated under laboratory conditions using the ELISA test. The precipitated antivenom with saturated ammonium sulfate was used to perform the neutralizing test in mice. To assess the quality of the antivenom, the neutralization of phospholipase A2 activity was tested with different concentrations of antivenom. Finally, the median effective dose (ED50) was calculated using probit analysis. Three adult mares (three years old, weighing 400 to 450 kg) were used for immunization. Bee venom was injected with complete and incomplete Freund's adjuvant; complete Freund's adjuvant was used in the first two inoculations, while incomplete Freund's adjuvant was used in subsequent ones. Venom doses were incrementally increased from 50, 100, 250, 500, 1000, 2000, to 4000 micrograms over seven inoculations at 7-day intervals, administered subcutaneously (in the neck). Horse antibody isolation was performed using the saturated ammonium sulfate method. Immunogenicity evaluation was conducted via the ELISA test. The serum from immunized horses and the precipitated antivenom were also used for neutralization tests in mice. The neutralization of phospholipase A2 activity was assessed with various concentrations of antivenom. The effectiveness of the prepared antivenom was determined by calculating the ED50 value through probit analysis.
Results: The total protein concentration in the crude BV solution was calculated to be 56 mg/ml, and the LD50 of the crude BV was determined to be 4.84 μg/g. ELISA test results indicated an immune response that increased throughout the immunization schedule. Neutralization of phospholipase A2 activity with different concentrations of antivenom demonstrated that Iranian bee venom possesses phospholipase activity, and 120 μg/ml of this antidote can completely neutralize the activity of phospholipase A2. Furthermore, 55 micrograms of antivenom are required to neutralize 50% of phospholipase A2 activity in 100 micrograms of venom. The ED50 value is approximately 54 times the LD50, indicating that the antivenom can neutralize up to 54 times the LD50. Therefore, the death caused by 4 mg of bee venom can be neutralized by 1 ml of antivenom, resulting in an average effective dose (ED50) of 4 mg/ml. If 100 micrograms of venom enters the body with each bee sting, then 1 ml of the produced antivenom can neutralize 40 bee stings.
Conclusion: The results indicate that horse antivenom against honey bee venom is capable of neutralizing crude venom in a mouse model.
Methods: Crude bee venom (BV) was collected from Iranian bees (Apis mellifera meda) using an electric bee venom-collecting machine. The venom dries quickly in the air; therefore, it was collected daily from glass plates using special spatulas and stored in dark glass containers in a freezer at -20 ºC for future experiments. To remove mucus and extraneous substances, the venom was dissolved in a 1:1 ratio with saline (physiological serum) and centrifuged at high speed. After centrifugation, the upper clear solution was separated and filtered using a 0.2-micron filter. The protein concentration in the crude venom solution was determined using the Bradford protein assay method, based on a standard curve obtained from different concentrations of bovine serum albumin (BSA) solution.
The median lethal dose (LD50) of BV was determined using 24 male mice, which were divided into four groups. Different doses of venom (60, 70, 80, 90, 100, and 110 μg/ml) dissolved in sterile normal saline were injected intravenously. The control group received 0.5 ml of saline solution. Mortality was recorded after 24 hours, and LD50 was calculated using probit analysis. Three horses were used to produce antivenom against honey bee venom. The horses were immunized with Iranian bee venom seven times at 7-day intervals, and immunogenicity was evaluated under laboratory conditions using the ELISA test. The precipitated antivenom with saturated ammonium sulfate was used to perform the neutralizing test in mice. To assess the quality of the antivenom, the neutralization of phospholipase A2 activity was tested with different concentrations of antivenom. Finally, the median effective dose (ED50) was calculated using probit analysis. Three adult mares (three years old, weighing 400 to 450 kg) were used for immunization. Bee venom was injected with complete and incomplete Freund's adjuvant; complete Freund's adjuvant was used in the first two inoculations, while incomplete Freund's adjuvant was used in subsequent ones. Venom doses were incrementally increased from 50, 100, 250, 500, 1000, 2000, to 4000 micrograms over seven inoculations at 7-day intervals, administered subcutaneously (in the neck). Horse antibody isolation was performed using the saturated ammonium sulfate method. Immunogenicity evaluation was conducted via the ELISA test. The serum from immunized horses and the precipitated antivenom were also used for neutralization tests in mice. The neutralization of phospholipase A2 activity was assessed with various concentrations of antivenom. The effectiveness of the prepared antivenom was determined by calculating the ED50 value through probit analysis.
Results: The total protein concentration in the crude BV solution was calculated to be 56 mg/ml, and the LD50 of the crude BV was determined to be 4.84 μg/g. ELISA test results indicated an immune response that increased throughout the immunization schedule. Neutralization of phospholipase A2 activity with different concentrations of antivenom demonstrated that Iranian bee venom possesses phospholipase activity, and 120 μg/ml of this antidote can completely neutralize the activity of phospholipase A2. Furthermore, 55 micrograms of antivenom are required to neutralize 50% of phospholipase A2 activity in 100 micrograms of venom. The ED50 value is approximately 54 times the LD50, indicating that the antivenom can neutralize up to 54 times the LD50. Therefore, the death caused by 4 mg of bee venom can be neutralized by 1 ml of antivenom, resulting in an average effective dose (ED50) of 4 mg/ml. If 100 micrograms of venom enters the body with each bee sting, then 1 ml of the produced antivenom can neutralize 40 bee stings.
Conclusion: The results indicate that horse antivenom against honey bee venom is capable of neutralizing crude venom in a mouse model.
Type of Study: Research |
Subject:
مدیریت دامپروری و تولید
Received: 2022/12/6 | Accepted: 2023/09/13
Received: 2022/12/6 | Accepted: 2023/09/13
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