Disease Severity in a Murine Model of Lyme Borreliosis Is Associated with the Genotype of the Infecting Borrelia burgdorferi Sensu Stricto Strain

Disease Severity in a Murine Model of Lyme Borreliosis Is Associated with the Genotype of the Infecting Borrelia burgdorferi Sensu Stricto Strain Guiqing Wang 2 3 Caroline Ojaimi 2 3 Hongyan Wu 2 3 Victoria Saksenberg 0 2 Radha Iyer 2 3 Dionysios Liveris 2 3 Steve A. McClain 0 2 Gary P. Wormser 1 2 Ira Schwartz () 1 2 3 0 Department of Pathology, Montefiore Medical Center , Bronx , New York 1 Division of Infectious Diseases, Department of Medicine, New York Medical College , Valhalla 2 Received 14 February 2002; revised 14 May 2002; electronically published 20 August 2002. Presented in part: 101st general meeting of the American Society for Microbiology , 21-25 May 2001, Orlando , Florida (abstract D-91). Animal care followed the Public Health Service Guide for the Care and Use of Laboratory Animals and the guidelines of the National Institutes of Health for care and use of laboratory animals. Financial support: National Institutes of Health (AR-41511). and Molecular Biology, New York Medical College , Valhalla, NY 10595 3 Department of Biochemistry and Molecular Biology The pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing 2 distinct ribosomal DNA spacer restriction fragment-length polymorphism genotypes (RSTs) was assessed in a murine model of Lyme disease. B. burgdorferi was recovered from 71.5% and 26.6% of specimens from mice infected with RST1 and RST3 isolates, respectively (P ! .0001). The average ankle diameter and histologic scores for carditis and arthritis were significantly higher after 2 weeks of infection among mice infected with RST1 isolates than among those infected with RST3 isolates (P ! .001). These clinical manifestations were associated with larger numbers of spirochetes in target tissues but not with the serum sensitivity of the individual isolates. Thus, the development and severity of disease in genetically identical susceptible hosts is determined mainly by the pathogenic properties of the infecting B. burgdorferi isolate. The RST1 genotype is genetically homogeneous and thus may represent a recently evolved clonal lineage that is highly pathogenic in humans and animals. - Lyme disease, which is caused by infection with the spirochete Borrelia burgdorferi, is the most common vectorborne disease in the United States. A total of 122,970 cases was reported to the Centers for Disease Control and Prevention between 1992 and 2000 [1]. Although the majority of patients show only transient erythema migrans at the site of the tick bite, some persons may develop more serious manifestations, such as carditis, neuroborreliosis, or arthritis, if appropriate antibiotic treatment is not administered at the time the cutaneous lesion appears [2]. Disease severity in humans and experimentally infected animals may depend on a number of factors. These include virulence of the infecting strain of B. burgdorferi sensu lato [35], the number of spirochetes in target tissues [69], coinfection with other tickborne pathogens [1012], and inherent differences in individual host responses to infection [13, 14]. B. burgdorferi sensu stricto is the sole species causing Lyme disease in North America. Previously, we characterized B. burgdorferi sensu stricto isolates cultured from patients with Lyme disease in Westchester County, New York, by restriction fragmentlength polymorphism (RFLP) analysis of the 16S23S rDNA spacer. All isolates analyzed were grouped into 1 of 3 rDNA spacer RFLP genotypes (RSTs) [1517]. RSTs are significantly correlated with other genotypic characteristics and can, therefore, serve as an accurate genetic marker of B. burgdorferi genotype [18]. A significantly higher percentage of patients with Lyme disease who were infected with RST1 strains had blood culture results positive for B. burgdorferi, compared with patients infected with RST2 or RST3 isolates [5]. The frequency of multiple erythema migrans lesions was also significantly greater among patients infected with RST1 strains, providing clinical evidence of an association between specific B. burgdorferi genotype and hematogenous dissemination in patients with early Lyme disease [5]. B. burgdorferi infection in laboratory mice has many clinicopathologic features in common with Lyme disease in humans [14]. Studies that used murine models of infection with B. burgdorferi or other pathogenic Borrelia species have suggested that strain differences in the spirochetes are one of the critical determinants of disease severity [4, 1922]. Arthritis severity and spirochete burden in C3H or CB-17 SCID mice infected with the relapsing fever agent, B. turicatae, appear to be determined mainly by the serotype of the infecting strain [19]. The potential impact of genotypic variation of B. burgdorferi sensu stricto on pathogenicity was established for single RST1 and RST3 isolates in a previous study, in which spirochete dissemination and disease severity in mice varied significantly according to the specific RST [9]. The present study was intended to expand on these earlier observations by studying 10 additional B. burgdorferi clinical isolates representing 2 distinct RSTs. The aims of the present study were to assess the pathogenicity of B. burgdorferi clinical isolates in C3H/HeJ mice, to determine any potential association between severity of disease and genotype of the infecting B. burgdorferi, and to identify mechanisms that may contribute to strain variation in dissemination and pathogenicity. Materials and Methods B. burgdorferi isolates. Ten clinical isolates of B. burgdorferi at passage 25 were used in this study. These isolates were cultured from blood (n p 2) or skin (n p 8) specimens from patients who were given a diagnosis of Lyme disease in Westchester County, New York, between 1998 and 2000. On the basis of rDNA spacer RFLP analysis [15], these isolates were characterized as either RST1 (isolates BL203, BL268, B479, B491, and B515) or RST3 (isolates B331, B348, B408, B418, and B500). Mice. Specific pathogenfree male and female 4-week-old C3H/HeJ mice were obtained from Jackson Laboratory. Mice were maintained in separate cages in the Department of Comparative Medicine at New York Medical College (Valhalla). Infection of mice with B. burgdorferi. Mice were randomly divided into groups of 5. B. burgdorferi was cultured in BSK-H medium supplemented with 6% rabbit serum (Sigma Chemical) at 33 C for 57 days. Spirochetes were examined for motility by darkfield microscopy, and the number of microorganisms was determined by fluorescence microscopy by mixing 10-mL aliquots of culture material with 10 mL of an acridine orange solution (100 mg/mL). All cultures were adjusted to a final concentration of 105 spirochetes/mL in incomplete BSK-H medium (without rabbit serum). Each mouse in the experimental groups was inoculated intradermally with 0.1 mL of the culture material (104 spirochetes) in the shaven back. In addition, 5 mice were inoculated with 106 (n p 2) or 1 (...truncated)