52 Am. J. Trop. Med. Hyg., 66(1), 2002, pp. 52–55 Copyright  2002 by The American Society of Tropical Medicine and Hygiene IDENTIFICATION OF VIRULENCE-ASSOCIATED ANTIGENS AND PLASMIDS IN RHODOCOCCUS EQUI FROM PATIENTS WITH ACQUIRED IMMUNE DEFICIENCY SYNDROME AND PREVALENCE OF VIRULENT R. EQUI IN SOIL COLLECTED FROM DOMESTIC ANIMAL FARMS IN CHIANG MAI, THAILAND SHINJI TAKAI, PRASIT THARAVICHITKUL, CHIE SASAKI, YOKO ONISHI, SHIGEKI YAMANO, TSUTOMU KAKUDA, SHIRO TSUBAKI, CHUMNAN TRINARONG, SUVICHAI ROJANASTHIEN, ANUCHA SIRIMALAISUWAN, TED TESAPRATEEP, NIWAT MANEEKARN, THIRA SIRISANTHANA, AND TERUO KIRIKAE Department of Animal Hygiene, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori, Japan; Department of Microbiology and Department of Medicine, Faculty of Medicine, and Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand; Department of Infectious Diseases and Tropical Medicine, Research Institute, International Medical Center of Japan, Tokyo, Japan Abstract. The prevalence of virulent Rhodococcus equi in soil collected from 17 domestic animal farms (from 12 cattle, 1 pig, and 4 horse farms) and in 6 clinical specimens from patients with acquired immune deficiency syndrome (AIDS) in Chiang Mai, Thailand, was investigated. The isolates were tested for the presence of 15–17-kDa antigens (VapA) and a 20-kDa antigen (VapB) by immunoblotting and for the presence of virulence plasmid DNA. Rhodo- coccus equi was isolated from most soil samples (68 of 80) obtained from the 17 farms, with 2.0  10 2 to 6.0  10 5 colony-forming units per gram of soil. We detected VapA in none of the 537 isolates from the soil samples. In one isolate from a pig farm, both VapB and virulence plasmid DNA were detected. Of the 6 clinical isolates from patients with AIDS, however, 4 isolates contained both VapB and virulence plasmid DNA. The remaining 2 isolates were avirulent. INTRODUCTION Rhodococcus equi is a facultative, intracellular, gram-pos- itive coccobacillus that causes suppurative pneumonia and ulcerative enteritis in foals aged 1–3 months. 1,2 Rhodococcus equi has been identified as an increasingly common oppor- tunistic pathogen of immunocompromised hosts such as peo- ple infected with the human immunodeficiency virus (HIV): the reported incidence of R. equi pneumonia in patients with acquired immune deficiency syndrome (AIDS) is on the rise. 3–5 The discovery of virulence-associated antigens and viru- lence plasmids has allowed for a classification of the viru- lence of R. equi strains. 2,6,7 At least 3 virulence levels of R. equi have been identified: virulence, intermediate virulence, and avirulence. Virulent R. equi is characterized by the pres- ence of virulence-associated 15- to 17-kDa antigens (VapA), virulence plasmid DNA of 85–90 kb, and suppurative pneu- monia in foals (murine 50% lethal dose [LD 50 ]  10 6 ). Rho- dococcus equi strains of intermediate virulence are identified by a virulence-associated 20-kDa antigen (VapB) and viru- lence plasmid DNA of 79–100 kb and are found in the sub- maxillary lymph nodes of pigs (murine LD 50  10 7 ). In com- parison, avirulent R. equi shows evidence of neither viru- lence-associated antigens nor plasmid DNA (murine LD 50  10 8 ). 8,9 Inhalation of R. equi is probably the main route of ex- posure in foals. 1,10 The primary source is believed to be soil. 2 The route of infection in humans remains unknown, although contact with farm animals and manure has been reported in about a third of cases. 8,11–13 The majority of R. equi isolates from patients with AIDS are virulent (VapA or VapB posi- tive), whereas most isolates from immunocompromised pa- tients without AIDS were avirulent. 8,13 Thailand has one of the highest rates of HIV-AIDS in the world. 14 It is estimated that 1 million Thais have already been infected with HIV. Pulmonary infection is a major cause of morbidity and mortality in HIV-infected patients. In Thailand, Mycobacterium tuberculosis, Pneumocystis car- inii, and Penicillium marneffei are major etiological agents of such pulmonary infection. 14 Infection by R. equi has also been reported. 15 Little is known, however, about the patho- genicity and the source of R. equi in Thailand. The purpose of the present study was to investigate the virulence-associated antigens and plasmids in R. equi isolat- ed from patients with AIDS and the prevalence of virulent R. equi isolated from soil collected from domestic animal farms in Chiang Mai, Thailand, to substantiate a possible zoonotic route of infection of R. equi in AIDS. MATERIALS AND METHODS Bacterial strains. Strains ATCC 33701 (equine origin, virulent strain) and strain 5 (human origin, strain of inter- mediate virulence) were used as reference strains because some of the protein profiles, plasmid characteristics, and vir- ulence levels of these strains have been described. 8,13,16 In addition, 6 clinical isolates obtained from patients with AIDS were examined. These bacteria were harvested from various lesions examined in the Department of Microbiolo- gy, Faculty of Medicine, Chiang Mai University. Collection and isolation of R. equi from soil. Eighty soil samples were collected from 4–12 sites from 17 farms (12 cattle, 1 pig, and 4 horse farms) in Chiang Mai in March 2000. These farms were randomly selected and were not related in any obvious way to HIV-infected patients with R. equi infection. Soil was scraped from the ground with a small spoon and poured into sterile tubes. One gram of soil was diluted serially with a 10-fold volume of sterile saline. Each dilution was inoculated onto 2 plates of nalidixic acid- novobiocin-actidione (cycloheximide)-potassium tellurite medium, as described by Woolcock and others 17 ; the plates were incubated at 30C for 2–3 days. Rhodococcus equi col- onies were counted, and the number of viable organisms per