Background:  Chloroform has a potential value as a substitute for thiomersal as a preservative due to its high antibacterial and antifungal activity. 
Objective: Comparative analysis of the preservative efficacy of chloroform and thiomersal in ISA206 trivalent foot and mouth disease vaccine concerning the antimicrobial activity and vaccine potency.
Method: This study was conducted on 5 prepared ISA206 trivalent foot and mouth disease vaccines, one vaccine prepared with 0.01% v/v thiomersal and four vaccines prepared with different concentrations of chloroform 0.1%,0.25%,0.5% and 0.75% v/v. Each vaccine was monthly evaluated by safety and sterility tests for 12 months. Three cattle were vaccinated intramuscularly (I/M) by each vaccine. Serum samples were collected monthly for 12 months.The humeral immune responses were monitored by Serum Neutralization Test (SNT) and Enzyme Linked Immunosorbent Assay (ELISA).The antimicrobial activity of chloroform and thiomersal in the five vaccines were determined 12 months post preparation against nine different gram negative and gram positive bacterial strains and three fungal stains. The bacterial strains were Bacillus subtilis, Staphylococcus aureus , Micrococcus luteus, Pseudo-monas aeruginosa, Escherichia coli, Salmonella typhi, Shig-ella flexneri, Salmonella para typhi A and Proteus mirabilis and fungal strains were Aspergillus flavus, Aspergillus nigar and Aspergillus pterus. Agar well diffusion method was followed in this study. The 12 monthes comparative analysis of antibacterial activity reflects that among these five vaccines. shows thiomersal as well
Results: Our results show that the incorporation of as 0.5% and 0.75% chloroform into ISA206/FMDV vaccine  are  as effective as thiomersal as a preservative.
Conclusion: Finally we recommended using 0.5% chloroform as a substitute for thiomersal as a preservative in foot and mouth disease vaccine.