Detection of mycoplasma SP. in hyperimmune sera by bacteriological culture, PCR and LAMP (methods)

Objective: This study was performed to compare culture, PCR and LAMP methods for detection of Mycoplasmas in hyperimmune bovine sera produced to use in prophylaxis and treatment of neonatal diseases. Material and Methods: Hyperimmune sera from donor cattle immunized with bacterial antigens, their processed-final products and experimentally contaminated serum samples were used in the study. Bacteriological culture, DNA extraction, PCR and LAMP methods for detection of Mycoplasmas were optimized and compared by using experimentally contaminated serum samples. Results: In experimentally contaminated hyperimmune bovine serum samples, bacteriological culture and PCR methods detected as low as 103 Mycoplasma, and LAMP method 104 Mycoplasma. Phenol-chloroform-isoamylalcohol method was found superior to other commercial kits in the DNA extraction from samples. Mycoplasmas contamination were found in %41.6 and %25 of unproccesed hyperimmune bovine sera from donor animals with PCR and LAMP, respectively. In bacteriologic culture, Mycoplasma could not be isolated from any sample. Filter-processed samples were found negative for mycoplasmas by all three methods. Conclusion: PCR and bacteriological culture were found to be the most sensitive methods for detection of Mycoplasma contamination in hyperimmune sera. Sensitivity and repeatability of LAMP was lower when comparing with other two methods. Results also showed that Mycoplasma contamination should be considered in commercial serum production and filtration should be applied to final product.