Reverse Line Blot Hybridization for detection and identification of Babesia spp.

RUYBAL PAULA

Centro de Investigaciones Veterinarias y Agronómicas, CICVyA, INTA, Castelar, Buenos Aires, Argentina

Otro; Babesia World Summit; 2005

Reverse Line Blot hybridization (RLB) assay allows the simultaneous detection of multiple tick-transmitted protozoan and rickettsial cattle pathogens. This technique consists of the hybridization of specie-specific PCR amplification products (16S or 18S rRNA genes from Anaplasma/Erhirichia sp. or Babesia/Theileria sp. isolates respectively) with a pathogen-specific probes attachted to a nylon membrane. RLB  technique was  applied to study tick borne diseases not only for epidemiological surveys but also as an start point for inferring phylogenetic relationship. The essence of the techniques is the hybridization of PCR products to specific probes immobilized on a membrane in order to identify differences in the amplified sequences. The conserved domains of the 18S rRNA genes of  Babesia species were used to amplify the hypervariable V4 region by PCR. Within this region, oligonucleotides were deduced for species-specific detection. A catchall Theileria and Babesia species control oligonucleotide is also included allowing the detection of  species with new sequence polymorphisms in the hypervarriable region as well as new and yet uncharacterized pathogens. This was the case for the argentinean B. bigemina isolates. Only a few percentange of these isolates did hybridized with bigemina specific probe designed using the reported mexican isolate gene sequence (GenBank accession number X59604, X59605, X59607). After sequencing the argentinian isolates PCR fragments, several sequence variations within the 20 bp probe region were detected. Furthermore, each isolate showed more that one sequence variation reflecting a mixed population composition. Based on these observations new probes were designed for detecting the new variants. In addition we search for a new specific probe based on a region inside the V4, trying to find a common region to all the bigemina variants but still different to the B. bovis ones. The aligment of the new variants allows for the identification of a specie -specific region 10 bp downstream to the former one. A critical question in RLB technique is the sensitivity, particularly for detecting carrier animals. For this purpose, we  developed an heminested PCR strategy in order to enhance detection sensitivity. For this strategy we used previously described primers such us RLB-F, RLB-Fint and RLB-R2 for Babesia/Theileria sp. 18S where the reverse primer is the biotinilated one. We performed a second step PCR reaction using an forward internal biotinilated primer. The PCR protocol was evaluated using reference and field samples. At the moment 300 serum and whole blood samples of cattle collected from Northeast (NE) and Northwest (NW) Argentina are being analyzed by serologic, PCR and RLB test for epidemiological studies. A new new RLB membranes is being designed and assembled for a better detection of argentinian B. bigemina isolates.