Molecular Characterization of Borrelia burgdorferi from Case of Autochthonous Lyme Arthritis

LETTERS and the EMIDA ERA-NET and INIA grant APHAEA. Miguel Delibes-Mateos, Catarina Ferreira, Francisco Carro, Marco A. Escudero, and Christian Gortazar Author affiliations: Instituto de Investig- acion en Recursos Cinegeticos, a collab- orative agency of the Consejo Superior de Investigaciones Cientificas, Universidad de Castilla-La Mancha, and Junta de Co- munidades de Castilla-La Mancha, Ciudad Real, Spain (M. Delibes-Mateos, C. Gor- tazar); Trent University, Peterborough, Ontario, Canada (C. Ferreira); Estacion Biologica de Donana, Seville, Spain (F. Carro); and Ebronatura, Zaragoza, Spain (M.A. Escudero) DOI: http://dx.doi.org/10.3201/eid2012.140517 References 1. Delibes-Mateos M, Ferreras P, Villafuerte R. European rabbit population trends and associated factors: a review of the situation in the Iberian Peninsula. Mammal Review. 2009;39:124–40. http:// dx.doi.org/10.1111/j.1365-2907.2009. 00140.x 2. Calvete C. Modeling the effect of population dynamics on the impact of rabbit hemorrhagic disease. Conserv Biol. 2006;20:1232–41. http://dx.doi. org/10.1111/j.1523-1739.2006.00371.x 3. Le Gall-Recule G, Zwingelstein F, Boucher S, Le NB, Plassiart G, Portejoie Y, et al. Detection of a new variant of rab- bit haemorrhagic disease virus in France. Vet Rec. 2011;168:137–8. http://dx.doi. org/10.1136/vr.d697 4. Dalton KP, Nicieza I, Balseiro A, Muguerza MA, Rosell JM, Casais R, et al. Variant rabbit hemorrhagic disease virus in young rabbits, Spain. Emerg Infect Dis. 2012;18:2009–12. http://dx. doi.org/10.3201/eid1812.120341 5. Calvete C, Calvo JH, Sarto P. Deteccion de una nueva variante del virus de la en- fermedad hemorragica en conejos silves- tres en Espana. In: Abstracts of the 37th symposium de cunicultura de ASESCU, 2012 May 24–25. Barbastro (Spain): Aso- ciacion Espanola de Cunicultura; 2012. 6. Dalton KP, Nicieza I, Abrantes J, Esteves PJ, Parra F. Spread of new variant of RHDV in domestic rabbits on the Iberian Peninsula. Vet Microbiol. 2014;169:67–73. http:// dx.doi.org/10.1016/j.vetmic.2013.12.015 7. Abrantes J, Lopes AM, Dalton KP, Melo P, Correia JJ, Ramada M, et al. New vari- ant of rabbit hemorrhagic disease vi- rus, Portugal, 2012–2013. Emerg Infect Dis. 2013;19:1900–2. http://dx.doi.org/ 10.3201/eid1911.130908 8. Williams D, Acevedo P, Gortazar C, Escudero MA, Labarta JL, Marco MA, et al. Hunting for answers: rabbit (Oryc- tolagus cuniculus) population trends in northeastern Spain. European Journal of Wildlife Research. 2007;53:19–28. http://dx.doi.org/10.1007/s10344-006- 9. Delibes-Mateos M, Delibes M, Ferreras P, Villafuerte R. Key role of European rab- bits in the conservation of the western Mediterranean Basin hotspot. Conserv Biol. 2008;22:1106–17. http://dx.doi.org/ 10.1111/j.1523-1739.2008.00993.x 10. Garrote G. Repoblaciones del conejo de monte en gran escala para la conservacion del lince Iberico. In: Abstracts of the First International Wild Rabbit Seminar, 2013 Oct. 23–25. Beja (Portugal): Associacao IBERLINX, Instituto da Conservacao da Naturaleza e das Florestas, and Junta de Andalucia; 2013, p. 6. Address for correspondence: Miguel Delibes- Mateos. Instituto de Investgacion en Recursos Cinegeticos, IREC-CSIC-UCLM-JCCM, Ronda de Toledo s/n 13071 Ciudad Real, Spain; email: mdelibesmateos@gmail.com Molecular Characterization of Borrelia burgdorferi from Case of Autochthonous Lyme Arthritis To the Editor: The first Lyme borreliosis (LB) case reported to be acquired in California occurred in 1978 (1). During the past 10 years, 744 confirmed LB cases were reported in California; 419 (56.2%) were likely acquired in-state. The highest inci- dence of this disease occurs in north- ern coastal California, in locations such as Santa Cruz County (2), where habitat supports yearlong activity of the tick vector Ixodes pacificus (3,4). Existing data describe the genet- ic diversity of the LB agent Borrelia burgdorferi among ticks in Californa (5,6), but few instances of direct de- tection and genetic characterization of B. burgdorferi sensu stricto in samples from humans are documented in Cali- fornia. B. burgdorferi has been iso- lated from skin biopsy samples of 3 patients in California in whom LB was diagnosed (1). Seinost et al. genotyped strains isolated in the United States, including 7 isolates identified in Cali- fornia from skin, blood, or cerebrospi- nal fluid, but no documented exposure information was available (7). Girard et al. genotyped B. burgdorferi in 10- to 12-year-old stored serum samples collected from 22 northern California residents, some of whom were as- ymptomatic at time of collection. Of 22 PCR-positive specimens, 21 had the single laboratory type strain B31 genotype (3). A 12-year-old resident of Santa Cruz County, California, came to the emergency department of Dominican Hospital in September 2012 with a swollen, painful right knee and mildly painful right hip. The patient’s family reported that LB had been diagnosed by a local physician. Illness onset was in May 2010; symptoms consisted of recurrent knee swelling and pain last- ing several days every 4–5 months and positive serologic test results for B. burdorferi (not available). The pa- tient had not traveled outside of Cali- fornia during the preceding 6 years. In May 2011, an IgG Western blot of the patient’s serum that was processed at a commercial laboratory showed immunoreactive bands of 18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa. In both 2010 and 2011, the patient’s fam- ily had chosen to give the patient un- specified herbal treatments instead of antibacterial drugs. On physical examination in the emergency department, the patient’s right knee was swollen; knee flexion Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 12, December 2014

Existing data describe the genetic diversity of the LB agent Borrelia burgdorferi among ticks in Californa (5,6), but few instances of direct detection and genetic characterization of B. burgdorferi sensu stricto in samples from humans are documented in California. B. burgdorferi has been isolated from skin biopsy samples of 3 patients in California in whom LB was diagnosed (1). Seinost et al. genotyped strains isolated in the United States, including 7 isolates identified in California from skin, blood, or cerebrospinal fluid, but no documented exposure information was available (7). Girard et al. genotyped B. burgdorferi in 10to 12-year-old stored serum samples collected from 22 northern California residents, some of whom were asymptomatic at time of collection. Of 22 PCR-positive specimens, 21 had the single laboratory type strain B31 genotype (3).
A 12-year-old resident of Santa Cruz County, California, came to the emergency department of Dominican Hospital in September 2012 with a swollen, painful right knee and mildly painful right hip. The patient's family reported that LB had been diagnosed by a local physician. Illness onset was in May 2010; symptoms consisted of recurrent knee swelling and pain lasting several days every 4-5 months and positive serologic test results for B. burdorferi (not available). The patient had not traveled outside of California during the preceding 6 years. In May 2011, an IgG Western blot of the patient's serum that was processed at a commercial laboratory showed immunoreactive bands of 18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa. In both 2010 and 2011, the patient's family had chosen to give the patient unspecified herbal treatments instead of antibacterial drugs.
On physical examination in the emergency department, the patient's right knee was swollen; knee flexion was reduced to 30°. The right hip was painful on rotation. Serum laboratory values included a leukocyte count of 7,000/mL, hematocrit 33%, and erythrocyte sedimentation rate of 73mm/h. Plain radiograph images of the right hip did not show any abnormalities; the radiograph of the right knee showed suprapatellar effusion (Figure). Right knee aspiration yielded 115 mL of cloudy yellow fluid; laboratory tests showed a leukocyte count of 59,750/ mL and protein level 5 g/dL; no crystals were noted. Results of routine bacterial culture of synovial fluid were negative. Amoxicillin was prescribed for a suspected septic joint and was taken for 1 week. Nine months later, the patient was reportedly asymptomatic and had returned to normal activity.
Right knee synovial fluid was sent to ARUP Laboratories (Salt Lake City, UT, USA); results were positive for the B. burgdorferi sensu lato recA gene by use of a proprietary qualitative PCR procedure. At the University of California, Irvine, we thawed another aliquot of synovial fluid, which had been frozen without cryoprotectant, and inoculated samples into BSK II medium (8). After incubation for 2 weeks at 34°C, no spirochetes were noted. We subjected another 100 mL aliquot to DNA extraction using DNeasy Blood and Tissue Kit and the QIAcube apparatus (QIAGEN, Valencia, CA, USA). We used multiplex quantitative PCR (qPCR) and primers and specific probes for the 16S ribosomal RNA genes of LB group species and for relapsing fever group species of Borrelia in 2 replicates as described by Barbour et al. (9). By qPCR, there were 18 gene copies of an LB group species in 1 replicate and 23 copies in the other. The qPCR results for relapsing fever group species, including B. miyamotoi and B. hermsii, which are enzootic in parts of California, were negative. We genotyped the ospC allele and 16S-23S intergenic spacer (IGS) using PCR amplification of each locus and direct sequencing as described by Travinsky et al. (6). Sequencing of the targeted PCR products showed that the ospC allele was type Hb and the IGS genotype was 13.
Two years of untreated relapsing pauciarticular arthritis of the knee and hip, a B. burgdorferi-positive Western blot, and laboratory detection of B. burgdorferi from synovial fluid by PCR in 2 different laboratories leads us to conclude that the patient had Lyme arthritis. This patient likely acquired the infection locally. The prevalence of B. burgdorferi in nymphal I. pacificus ticks (range 4%-10%) in Santa Cruz County, and >10% of the geographic area of the county is categorized as being at high acarologic risk for LB (4). To our knowledge, the combination of ospC allele Hb and IGS genotype 13 has been identified only in California to date (6,8). A type "H" ospC type was reported from synovial fluid from LB patients from the eastern United States (10), but in the absence of IGS determination, this was probably type Ha, which is more typical of that region (8). The addition of the IGS locus to ospC alleles provides a precise approach to characterize genetic diversity and potential origin of B. burgdorferi in human tissue.

Acknowledgments
We thank the staff of Dominican Hospital, Santa Cruz, California, for facilitating access to case information; the staff of the County of Santa Cruz communicable disease division for their continued support; and California Epidemiologic Investigation Services for their suggestions for this manuscript and the work of their fellow, Sharon I. Brummitt.
The project described was supported by the Preventive Health Services Block Grant from the Centers for Disease Control and Prevention. Laboratory work at University of California Irvine was supported by the National Institutes of Health grant AI-065359. This work was determined exempt by The Committee for the Protection of Human Subjects, California Office of State Health and Planning.

Zoonotic Baylisascaris procyonis Roundworms in Raccoons, China
To the Editor: Baylisascaris procyonis, an intestinal roundworm that infects raccoons (Procyon lotor), causes fatal or severe neural larva migrans in animals and humans (1,2). Globally, ≈130 species of wild and domesticated animals are susceptible (2). Infections in humans typically occur in children who have the disorders pica or geophagia and ingest B. procyonis eggs in items contaminated with raccoon feces (3). Clinical manifestations include ocular disease, eosinophilic encephalitis, and eosinophilic cardiac pseudotumors; severe infection can lead to death. Since 1984, ≈24 cases of B. procyonis-related human neural larva migrans have been reported, mainly in the United States (1,3-5; K.R. Kazacos, pers. comm.). Despite few cases among humans, lack of effective treatment and widespread distribution of infected raccoons in close association with humans make B. procyonis a potentially serious public health threat (2,6). The current distribution of B. procyonis is poorly recorded in Asia (2,7), except for Japan (8). We describe B. procyonis infections among raccoons in China as part of a series of ongoing surveys of helminthic zoonoses linked to captive exotic animals in zoologic gardens (ZGs) in China.
More than 90% of raccoons in China (n >320) are raised as exotic ornamental animals in 18 ZGs. During 2011-2013, we collected 2×308 fecal samples (i.e., 1 repeat within each sampling) from 277 raccoons in 12 randomly selected ZGs (online Technical Appendix Figure 1, wwwnc. cdc.gov/EID/article/20/12/14-0970-Techapp1.pdf). Samples were stored in individual plastic bags at -20°C until use. We examined raccoons (n = 31) at the Sichuan ZGs twice, in June 2012 and May 2013. We identified B. procyonis eggs in feces using morphologic and molecular analyses (1,2,9). The nuclear first internal transcribed spacer (428 bp) and mitochondrial cytochrome c oxidase subunit 1 (cox-1, 938 bp) genes in each sample were PCR-amplified and sequenced. B. procyonis infection was confirmed by sequencing and phylogenetic analyses of both genes (7,9). We reexamined ≈60% of fecal samples to validate results. Prevalence (95% CI) was calculated for the overall population and independently for female, male, juvenile, and adult raccoons. We determined differences between the tested ZG prevalence and prevalence by sex or age of raccoons using χ 2 or Fisher exact tests in SAS (SAS Institute, Cary, NC, USA); p values <0.05 were considered significant.
Building on egg-based morphologic characterization and internal transcribed spacer 1 and cox-1 genebased phylogenies using neighborjoining trees (online Technical Appendix Figure 2), we found B. procyonis in raccoon feces from 5/12 ZGs (42%; 95% CI 14%-70%), including 2 in the most densely populated provinces, Henan and Sichuan. More infections were found in western than central and eastern ZGs (4/6 and 1/6, respectively; Table, online Technical Appendix Figure 1) (p = 0.079). Fecal samples of 35 raccoons (13%; 95% CI 9%-17%) tested positive for B. procyonis. The mean intensity of egg shedding was 5,000 eggs per gram (range 800-11,200 eggs per gram; data not shown). No significant difference was observed in the intensity of shedding by comparing sex and age of animals, and no significant differences were noted in the mean prevalence between female and male raccoons (12% versus 14%; p = 0.677) or between adult and juvenile animals (13% versus 10%; p = 0.536).
This investigation documents the presence and prevalence of B. procyonis