Relapsing Fever Spirochete in Seabird Tick, Japan

To the Editor: Tick-borne relapsing fever (TBRF) is caused by infection with spirochetes belonging to the genus Borrelia. We previously reported a human case of febrile illness suspected to be TBRF on the basis of serologic examination results; the vector most likely was a genus Carios tick that had fed on a seabird colony (1). However, surveillance of ticks in the area did not identify Borrelia spp. in any of the Carios ticks sampled (2). In 2007 and 2008, a borreliosis investigation was conducted on Kutsujima Island (35.71′N, 135.44′E) because a bird-associated tick, genus Carios, inhabits this island. During the investigation, 77 Carios ticks (55 nymphs, 11 adult males, and 11 adult females) were collected from colonies of seabirds: Swinhoe's storm petrel (Oceanodroma monorhis) and streaked shearwater (Calonectris leucomelas). Identification of tick species as C. sawaii was based on tick morphology and rrs gene sequence analysis of the tick mitochondrion DNA (2). Total DNA was extracted from the ticks by using a DNeasy Tissue Kit (QIAGEN, Germantown, MD, USA). For the detection of Borrelia DNA, PCR designed was based on the flagellin gene (flaB) according to Sato et al. (3). To check for contamination and amplicon carryover, we used blank tubes as a negative control for each experiment. Of 77 C. sawaii ticks that were positive by PCR of tick genes (2), 25 (14 nymphs, 6 adult males, 5 adult females) were positive for Borrelia DNA by PCR of flaB. 
 
To characterize the Borrelia spp., we sequenced amplified fragments of the flaB gene and the 16S ribosomal RNA (16SrRNA) gene of Borrelia spp. in a tick and compared the results with those of representative Borrelia spp. The primers BflaPBU and BflaPCR (3) for flaB and the 4 PCR primers (Technical Appendix) for 16SrRNA were used for direct sequencing and/or amplification. DNA sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB491928","term_id":"225703023","term_text":"AB491928"}}AB491928) of a 294-bp amplified fragment of flaB showed the following nucleotide similarities with those of Borrelia spp.: B. turicatae (98.98%), B. parkeri (98.30%), Borrelia sp. Carios spiro-1 (98.64%), and Borrelia sp. Carios spiro-2 (98.30%). DNA sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB491930","term_id":"225703027","term_text":"AB491930"}}AB491930) of a 1,490-bp amplified fragment of 16SrRNA showed the following nucleotide similarities with those of Borrelia spp.: B. turicatae (99.60%), B. parkeri (99.53%), and Borrelia sp. Carios spiro-2 (99.45%). Borrelia sp. Carios spiro-1 and Carios spiro-2, which were recently identified in C. kelleyi in the United States, have been classified into TBRF Borrelia (4,5). The Borrelia sp. found in this study, designated as Borrelia sp. K64, was closely related to B. turicatae but was distinct from other TBRF Borrelia spp. (Technical Appendix). 
 
To observe Borrelia spp. in tick tissues, we performed an indirect fluorescence assay (IFA) according to methods described by Fisher et al. (6), with minor modifications. A tick that was negative by PCRs of flab and 16SrRNA was used as a negative control. The IFA of the tick salivary gland and midgut was conducted by using acetone for fixation, goat anti-Borrelia sp. polyclonal immunoglobulin (Ig) G (1:100; KPL, Inc., Gaithersburg, MD, USA) as the primary antibody, and Alexa fluor 488-labeled rabbit antigoat IgG (1:200, Invitrogen, Carlsbad, CA, USA) as the secondary antibody. Our analysis showed a spirochete, which was stained by anti-Borrelia spp. antibody, in salivary gland and midgut tissue (Technical Appendix). However, no spirochetes were detected by IFA in the negative control (data not shown). 
 
We also attempted to isolate Borrelia spp. from tick specimens by using Barbour-Stoenner-Kelly medium (7). The motility of Borrelia-like organisms in the medium was initially observed by using dark-field microscopy. The Borrelia-like organisms were identified as Borrelia sp. K64 by sequencing of PCR-amplified fragments of flaB and 16SrRNA genes from the cultured medium. However, these Borrelia organisms were found for only 2 weeks after inoculation (data not shown). 
 
The vertebrate reservoir hosts of TBRF Borrelia are usually rodents but can be a variety of other animals (8). Although competence as a reservoir has not been determined for birds, infection of an owl with a TBRF Borrelia sp. has been reported (9). The vertebrate host of the spirochete has not yet been determined. Given our results, it is possible that seabirds are potential vertebrate hosts for Borrelia spp. 
 
In Japan, relapsing fever is a neglected infectious disease because it was not reported during 1956–1998 (10). In this study, we detected a Borrelia sp. in C. sawaii, and the spirochete we characterized is closely related to B. turicatae. Although the human health implications of infections caused by Borrelia spp. are not yet known, the findings from this study should contribute to the epidemiologic investigation of TBRF in Japan.

It is not known whether lineage VII and I viruses continue to circulate or have been replaced by lineage V and II viruses, respectively. This study confirms the long-term maintenance of distinct phylogenetically forms of Machupo virus in a small area within Beni. Although the distribution of the Machupo virus rodent reservoir (Calomys callosus) extends beyond the geographic area of the Machupo cases described, factors that limit the endemic distribution of the virus remain unknown. However, population differences among C. callosus may account for the natural nidality of BHF (5). Studies are needed to fully identify and understand the ecology of the rodent reservoir and Machupo virus transmission.
Machupo virus continues to cause sporadic cases and focal outbreaks of BHF in Bolivia. We describe 5 confirmed human cases (

Relapsing Fever Spirochete in Seabird Tick, Japan
To the Editor: Tick-borne relapsing fever (TBRF) is caused by infection with spirochetes belonging to the genus Borrelia. We previously reported a human case of febrile illness suspected to be TBRF on the basis of serologic examination results; the vector most likely was a genus Carios tick that had fed on a seabird colony (1). However, surveillance of ticks in the area did not identify Borrelia spp. in any of the Carios ticks sampled (2). In 2007 and 2008, a borreliosis investigation was conducted on Kutsujima Island (35.71′N, 135.44′E) because a bird-associated tick, genus Carios, inhabits this island. During the investigation, 77 Carios ticks (55 nymphs, 11 adult males, and 11 adult females) were collected from colonies of seabirds: Swinhoe's storm petrel (Oceanodroma monorhis) and streaked shearwater (Calonectris leucomelas). Identification of tick species as C. sawaii was based on tick morphology and rrs gene sequence analysis of the tick mitochondrion DNA (2). Total DNA was extracted from the ticks by using a DNeasy Tissue Kit (QIA-GEN, Germantown, MD, USA). For the detection of Borrelia DNA, PCR designed was based on the flagellin gene (flaB) according to Sato et al. (3).
To check for contamination and amplicon carryover, we used blank tubes as a negative control for each experiment. Of 77 C. sawaii ticks that were positive by PCR of tick genes (2) Borrelia sp. Carios spiro-2 (99.45%). Borrelia sp. Carios spiro-1 and Carios spiro-2, which were recently identified in C. kelleyi in the United States, have been classified into TBRF Borrelia (4,5). The Borrelia sp. found in this study, designated as Borrelia sp. K64, was closely related to B. turicatae but was distinct from other TBRF Borrelia spp. (online Technical Appendix).
To observe Borrelia spp. in tick tissues, we performed an indirect fluorescence assay (IFA) according to methods described by Fisher et al. (6), with minor modifications. A tick that was negative by PCRs of flab and 16SrRNA was used as a negative control. The IFA of the tick salivary gland and midgut was conducted by using acetone for fixation, goat anti-Borrelia sp. polyclonal immunoglobulin (Ig) G (1:100; KPL, Inc., Gaithersburg, MD, USA) as the primary antibody, and Alexa fluor 488-labeled rabbit antigoat IgG (1:200, Invitrogen, Carlsbad, CA, USA) as the secondary antibody. Our analysis showed a spirochete, which was stained by anti-Borrelia spp. antibody, in salivary gland and midgut tissue (online Technical Appendix). However, no spirochetes were detected by IFA in the negative control (data not shown).
We also attempted to isolate Borrelia spp. from tick specimens by using Barbour-Stoenner-Kelly medium (7). The motility of Borrelia-like organisms in the medium was initially observed by using dark-field microscopy. The Borrelia-like organisms were identified as Borrelia sp. K64 by sequencing of PCR-amplified fragments of flaB and 16SrRNA genes from the cultured medium. However, these Borrelia organisms were found for only 2 weeks after inoculation (data not shown).
The vertebrate reservoir hosts of TBRF Borrelia are usually rodents but can be a variety of other animals (8). Although competence as a reservoir has not been determined for birds, infection of an owl with a TBRF Borrelia sp. has been reported (9). The vertebrate host of the spirochete has not yet been determined. Given our results, it is possible that seabirds are potential vertebrate hosts for Borrelia spp.
In Japan, relapsing fever is a neglected infectious disease because it was not reported during 1956-1998 (10). In this study, we detected a Borrelia sp. in C. sawaii, and the spirochete we characterized is closely related to B. turicatae. Although the human health implications of infections caused by Borrelia spp. are not yet known, the findings from this study should contribute to the epidemiologic investigation of TBRF in Japan.

Backyard Raccoon Latrines and Risk for Baylisascaris procyonis Transmission to Humans
To the Editor: Raccoons (Procyon lotor) are abundant in urban environments and carry a variety of diseases that threaten domestic animals (1) and humans (2,3). A ubiquitous parasite of raccoons, Baylisascaris procyonis causes a widely recognized emerging zoonosis, baylisascariasis (3). Although only 14 human cases of severe B. procyonis encephalitis have been reported over 30 years (4), prevention is still a priority for public health and wildlife officials because of the seriousness of the resulting neurologic disease (5).
Raccoons prefer to defecate at latrines they create. Infected animals shed ≈20,000 eggs/g of feces (3), so latrines serve as the foci of parasite transmission (6). When latrines occur in close proximity to humans, the risk for zoonotic transmission increases (2). Because B. procyonis are transmitted by the fecal-oral route, young children have the greatest risk for zoonotic infection because of their tendency to put objects into their mouths (1,2). Many human cases have occurred in environments where latrines were near children's play areas. Our objective was to determine which factors encourage raccoons to create latrines in human habitats. This information will allow public health officials and wildlife managers to develop strategies to educate the public and to ultimately prevent zoonotic transmission.
We surveyed 119 backyards for raccoon latrines in the suburbs of Chicago, Illinois, USA, near the Ned Brown Forest Preserve (n = 38; 42°01′55.05′′N, 88°00′00.62′′W, Cook County) and Lincoln Marsh (n = 81; 41°51′4.54′′N, 88°5′39.019′′W, Du-Page County). Yards were selected on the basis of proximity to forest preserves and willingness of homeowners to participate in the study. We located latrines by systematically searching yards, giving special attention to horizontal substrates, such as piles of wood and the bases of large trees (6). We removed all fecal material to test for B. procyonis and stored it in plastic bags at -20 o C until analysis. Composite samples that were at least 2 g underwent fecal flotation in Sheather solution (7) (at least 1 g of every fecal deposit at a latrine) (n =131). We identified B. procyonis eggs by microscopic examination on the basis of their size and morphologic appearance (2). Multiple slides were examined for ≈10% of the samples (randomly selected) to validate our results. Prevalence was considered the proportion of positive samples from all sampled yards.
Each yard was additionally surveyed for potential latrine substrates (8) and factors believed to attract or deter raccoons. The distance of each yard from the nearest forested habitat was calculated by using ArcGIS 9.0 (Geographic Information Systems, Redlands, CA, USA). We used homogeneity tests to identify differences in the proportion of yards with latrines present and to compare the prevalence of B. procyonis between study areas. Logistic regression and odds ratios were used to evaluate a main effect model composed of 10 yard attributes, including the presence of a pet, birdfeeders, garbage cans, and sandboxes, and to evaluate a simplified model in which attributes were combined to reflect the presence of food and latrine substrates, such as pet food, birdfeed, garbage and piles of wood or logs, respectively.