Molecular diagnostic tools for simultaneous detection of Babesia bovis, Babesia bigemina and Anaplasma marginale

DE LA FOURNIERE S; PAOLETTA M; GUILLEMI E; LOPEZ ARIAS L; WILKOWSKY S. E; FARBER M

Cape Town

Congreso; Joint 8th International Ticks and Tick-borne Pathogens (TTP8) and 12th Biennial Society for Tropical Veterinary Medicine (STVM) Conference; 2014

Parasitology Society of Southern Africa

Bovine Babesiosis and Anaplasmosis are tick-borne diseases that impose important constraints for livestock industry in tropical and subtropical areas. In endemic regions, Babesia bovis, B. bigemina and Anaplasma marginale are often found together within a single bovine host. Given the importance of simultaneous detection for epidemiological evaluation and the detection and quantification during outbreaks, we developed a multiplex PCR (mPCR) and a quantitative Real Time PCR (qPCR) assays. The mPCR was rationally designed targeting specific multi-copy genes for each microorganism (ves1a, rap1 and msp1b); the products generated were easily detected in a 2% agarose gel due to their different sizes. The assay sensitivity curve tested on artificial mixtures of reference DNA samples was equivalent to the single PCR for each pathogen. Nevertheless a reduced sensitivity was observed when the mPCR was tested on field samples. In addition, a qPCR involving the use of SYBR Green/ROX was developed to easily quantify field samples and compared with previous protocols for the three hemoparasites. We demonstrated for both B. bovis and B. bigemina that the already reported qPCR based on mitochondrial cytochrome B (cytb) was not enough reproducible, as cytb copy number varies between samples and also in the same sample within a period of time. Thus, we set up a new qPCR targeting single copy genes with equivalent sensitivity to the described cytb qPCR. The ongoing development of these methodologies would assist to the rapid and specific detection of a large number of coinfected field samples from endemic areas where these parasites are prevalent.