2.2 Molecular and serological detection of T. equi andB. caballi
Blood samples were tested for antigens of B. caballi and T.
equi using polymerase chain reaction (PCR). DNA was extracted from the
blood samples using a Maxwell RSC Whole Blood DNA kit (Promega, Madison,
WI). DNA was eluted in 50 µL volumes of elution buffer and stored at
-70°C until use. The PCR reactions included 2 µL of extracted DNA and 10
pmol of specific primer sets (Alhassan et al., 2005) (Table 1 )
and run on a C1000 TouchTM Thermal Cycler (Bio-Rad,
Pleasanton, CA) (95℃ for 10 min followed by 35 cycles at 95℃ for 1 min,
60℃ for 1 min, and 72℃ for 1 min; and finally 72℃ for 5 min).
PCR-positive samples were sequenced using Macrogen Co. (Seoul, South
Korea)
Nucleotide sequence homology searches were analyzed by the National
Center for Biotechnology Information BLAST network service and aligned
using the MegAlign software package (Windows version 7.1; DNA-STAR,
Madison, WI, USA). Phylogenetic trees were generated using
neighbor-joining algorithms and the Jukes and Cantor matrix. Support for
topology was calculated using 1,000 bootstrap replications.
Serum samples were tested for anti-B. caballi and anti-T.
equi antibodies by competitive enzyme-linked immunosorbent assay
(cELISA) using Babesia caballi and Theileria equi antibody
test kits (VMRD, Pullman, USA), respectively. cELISA tests were
performed according to the manufacturer’s recommendation. Briefly, 50 μl
of serum samples and controls were loaded into the antigen-coated plate.
After incubation, 50 μl of primary antibody was added to each well. And
50 μl of secondary antibody-peroxidase conjugate was added to each well.
Finally, substrate solution and stop solution was consecutively added to
each well and the ELISA plates were read on its optical density.
Calculation of the percent inhibition (%I) was as follows according to
the manufacturer’s manual:
%I (Inhibition rate) = 100 [1 – (sample optical density
[OD]/negative control OD)]
The one ELISA positive serum from Ulsan region was collected as a total
of three times after 4 months and 1 year later.