Thirty-nine race horses (nine were thoroughbred, 30 were cross-bred) were included in the study. Twenty-three horses were female, and 16 male. The mean age of the horses was 3.1 years (SD=3.3, range: 1-15 years). Clinical and neurological examinations of the horses were performed by a veterinarian. All horses were in a healthy condition. The study protocol was approved by the Ethics Committee of Korea University.

Forty-eight jockeys were also included. Their mean age was 31.2 years (SD=5.5, range: 22-40 years), and their mean duration of contact with horses was 11.2 years (SD=5.8, range: 1-22 years). None had any psychiatric or medical illness, as assessed by means of clinical interviews and physical examinations. Three jockeys had a current familial history of psychiatric illness (2 depression, 1 alcoholism). All the subjects in this study were included in our previous study.¹⁵ Informed consent was obtained from the jockeys and the Seoul Horse Race Association.

A sample of fasting blood (20 mL) was drawn from each subject. Preparation of peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulant(lithium heparin)-treated blood by Ficoll-hypaque gradient centrifugation. Total RNA was extracted from PBMCs using RNAzol (Gibco/BRL, Gaithersburg, MD).

Indirect immunofluorescent antibody (IFA) test was conducted according to the method previously described.¹⁸ The Madine Darby canine kidney (MDCK) cell line and MDCK cell line persistently infected with BDV (MDCK-BDV)¹⁹ cultured for 5 days with Dulbecco's Modified Eagle Medium (DMEM) (Gibco/BRL, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco/BRL, Germany), were trypsinized, suspended in phosphate buffered saline (PBS), spotted onto Teflon-coated 10-well slides, and air-dried in room temperature. Cells on the 10-well antigen slide were fixed with anhydrous acetone at -20℃ for 7 minutes and dried. Human and horse serum samples diluted in PBS as 1 : 32 were treated to MDCK cell and MDCK-BDV cell on antigen slide and the slide was incubated in humidity chamber at 37℃ for 30 minutes. After three washes with PBS, Fluorescein isothiocyanate conjugated anti-human immunoglobulin goat IgG (MP Biomedicals Inc., USA) was added to each well and the slide was incubated in humidity chamber at 37℃ for 30 minutes. The slides were mounted with glycine-buffered glycerol under cover slips and examined for characteristic cytoplasmic fluorescent pattern with a fluorescence microscope.

Real-time quantitative RT-PCR for nucleoprotein (p40) and phosphoprotein (p24) genes of BDV was performed to detect RNA genome of BDV in PBMCs. The primers and probes described in previously study were used for rRT-PCR.²⁰ For the construction of the quantitative standard, p40 and p24 genes were amplified by RT-PCR and cloned to pGEM-T Easy vector system (Promega, USA). Cloned p40 and p24 genes were transcribed to RNA using the MEGAscript T7 transcription kit (Ambion Inc., USA) in accordance with the manufacturer's instructions. The amounts of p40 and p24 RNA transcribed using by in vitro transcription system were measured by optical density. p40 and p24 RNA were used for the reverse transcription reaction template from 1×1,010 copies/µL to 1×103 copies/µL diluted by 10-fold dilution method. After the reverse transcription reaction, the Taqman Universal PCR Master Mix (applied Biosystems, USA) was used for the real-time PCR reaction as recommended by manufacturer's instruction. The real-time PCR reaction mixtures were incubated for 10 minutes at 95℃; 40 cycles of amplification were performed using ABI PRISM 7000 Sequence Detection System (Applied Biosystems, USA). Each cycle consisted of a denaturation step (15 seconds at 95℃) and an annealing-elongation step (1 minute at 60℃). Threshold cycle (Ct) values, i.e., the number of cycles for fluorescence to reach to clearly detectable levels, over 40 were regarded as negative.

To analyze demographic data, two-tailed t-test was used for continuous covariates. For discrete covariates, chi-square test was used. The null hypothesis was rejected at p<0.05. The statistical package used for the analysis was Statistical Package for the Social Sciences 11.01.