The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand.

Therapeutic antivenom against snakes was first produced by Albert Calmette in 1894. Since then antivenoms have saved the life of countless snakebite victims. However, there are still many problems associated with antivenom production, for example variable percentage of responder horses, low neutralizing potency of antivenom, the large amount of snake venom needed for immunization and the difficulties encountered in producing potent polyvalent antivenoms. These problems have led to shortage and high cost of antivenom and, in some cases, failure of treatment.In 1997, a new immunization protocol for antivenom production was reported. It involves the injection of venom at low dose (approx. 2mg/horse) emulsified in Complete Freund's adjuvant in low volume (0.1-0.2 ml/site) in a total of 10 sites around the neck area of the horse. This immunization protocol has minimized the local reaction at the injection site thus allowing the use of the potent oil adjuvant. This, together with the increase in total surface area of the droplets, allow a more effective immune response to take place, e.g. enhancing the migration and activation of more antigen presenting cells and lymphocytes. The low dose, low volume multi-site immunization has resulted in dramatic improvements on the antivenom production in terms of amount of venom used for immunization, the time required to reach hyperimmune stage, the percent of responder horses and the potency of the antivenom. Furthermore, this protocol has made it possible to produce potent truly polyvalent antivenoms against several elapid and viperid snakes. This immunization protocol has alleviated various problems associated with antivenom production and has implications for immunization in general.     

Pesticide Exposure and Cholinesterase Levels in Migrant Farm Workers in Thailand

Objectives: In this study, we examined the effects of pesticides in migrant farm workers from Cambodia after workplace exposure on fruit plantations in eastern Thailand. Methods: We studied 891 migrant farm workers employed on pineapple, durian, and rambutan plantations in Thailand. Data were collected via a detailed questionnaire survey and measurements of serum cholinesterase level (SChE). Results: The majority of subjects was male (57.7%), with an average age of 30.3 years. Most subjects (76.8%) were moderately aware of good industrial hygiene practices. SChE level was divided into four groups based on the results. Only 4.4% had normal levels of cholinesterase activity, 20.5% had slightly reduced levels, 58.5% had markedly reduced levels and were “at risk,” and 16.6% who had highest levels of cholinesterase inhibition were deemed to be in an “unsafe” range. SChE was classified into two groups, SChE value of 87.5 was “normal” and <87.5 units/mL “abnormal.” For the multiple logistic regression analysis of the abnormal SChE levels, the variables entered in the model included gender, period of insecticide use, the total area of plantation, frequency of spraying, period of daily insecticide spraying, and insecticide spraying method. The results indicated that the aOR (adjust odds ratio) for male migrant farm workers (95% confidence interval [CI]) was 1.58 (1.14, 2.17). The OR for farm migrant workers who worked on larger plantations of more than 39.5 acres (95% CI) was 2.69 (1.51, 4.82). Finally, the OR for the migrant farm workers who used a backpack sprayer (95% CI) was 2.07 (1.28, 3.34). Conclusions: These results suggest that health screening should be provided to migrant farm workers, especially those who spray pesticides on plantations of >39 acres, use a backpack sprayer, or have a low level of compliance with accepted industrial hygiene practices. These three classes of workers are at increased risk of chemical exposures and developing acute or chronic illness from pesticide exposures.     

Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol.

The purpose of this study was to prepare a potent polyvalent antivenom against three elapids namely, the Thai cobra (Naja kaouthia, NK), the King cobra (Ophiophagus hannah, OH) and the banded krait (Bungarus fasciatus, BF). Two groups of horses were immunized. Group 1, comprising five horses, was immunized twice with a mixture of postsynaptic neurotoxins followed by an additional six immunizations with a mixture of crude venoms of the three elapids. Group 2, comprising four horses, was immunized with a mixture of crude venoms throughout the course. For the first immunization, the immunogens were emulsified in Complete Freund's adjuvant and injected using a low dose, low volume multi-site immunization protocol previously developed in this laboratory (Pratanaphon, R., Akesowan, S., Khow, O., Sriprapat, S. and Ratanabanangkoon, K. (1997) Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia). Vaccine 15, 1523-1528). The second immunization was carried out with the immunogens in Incomplete Freund's adjuvant. Blood was drawn to assay the antibody titer by ELISA. Sera at the peak of ELISA titers were pooled and assayed for the median effective dose (ED(50)). The ED(50)'s of antivenom from Group 1 horses against NK, OH and BF venoms were 1.44, 0.22 and 0.23 ml serum/mg venom, respectively, while those from Group 2 horse sera were 0.88, 0.20 and 0.49 ml serum/mg venom, respectively. The potency of sera from Group 2 against BF venom was significantly higher, while the potencies against NK and OH venoms were comparable to those of the corresponding monovalent antivenoms produced under the same protocol. This potent, truly polyvalent antivenom should be useful in saving lives of victims envenomed by these elapids and the immunization protocol should be useful in the production of potent polyvalent antivenoms against other medically important elapids.