LETTERS

(6). The sequence is distinct from a DOI: 10.3201/eid1412.080944 Human Case of small number of sequences derived from rabies viruses in Vietnam, which References alsatica suggests that China is a stronger can- Lymphadenitis 1. Smith JS, Fishbein DB, Rupprecht CE, didate for the source of the virus than Clark K. Unexplained rabies in three To the Editor: Lymph node en- her native country. immigrants in the United States: a vi- Although the case history could rologic investigation. N Engl J Med. largement is a common medical prob- not provide evidence for interaction 1991;324:205–11. lem that is usually caused by bacterial, 2. Grattan-Smith PJ, O’Regan WJ, Ellis PS, with a dog while her family was in viral, fungal, or protozoal agents (1). O’Flaherty SJ, McIntyre PB, Barnes CJ. A Malignancies or lymphoproliferative Hong Kong Special Administrative second Australian case, with a long incuba- Region, rabies was endemic within tion period. Med J Aust. 1992;156:651–4. diseases are often found, especially in the colony at the time that the pa- 3. McColl KA, Gould AR, Selleck PW, elderly patients (1). Bartonella hense- Hooper PT, Westbury HA, Smith JS. tient’s family was resident. From 1980 lae, the main causative agent of cat- Polymerase chain reaction and other labo- scratch disease (CSD), appears to be through 1984, 5 human cases were re- ratory techniques in the diagnosis of long corded (9). Only 2 case-patients had incubation rabies in Australia. Aust Vet the most common organism respon- clear evidence of a dog bite; histories J. 1993;70:84–9. DOI: 10.1111/j.1751- sible for in adults 0813.1993.tb03282.x for the remaining 3 cases provided no and children (1). CSD has also been 4. Fraser GC, Hooper PT, Lunt RA, Gould AR, rarely associated with B. quintana (2). evidence for an animal bite. From 1956 Gleeson LJ, Hyatt AD, et al. Encephalitis to 1979, Hong Kong had been free of caused by a Lyssavirus in fruit bats in Aus- Recently, the epidemiology of B. quin- rabies, but the disease had reentered tralia. Emerg Infect Dis. 1996;2:327–31. tana as an emerging source of human 5. Hanna JN, Carney IK, Smith GA, Tan- the colony shortly after its incidence infection has changed because it has nenberg AEG, Deverill JE, Botha JA, et been isolated from the dental pulp of had increased in the neighboring Chi- al. Australian bat lyssavirus infection: a nese province of Guangdong. If Hong second human case, with a long incuba- a domestic cat (3). Feral cats have also Kong was where the young girl was tion period. Med J Aust. 2000;172:597–9. been found to be infected by B. quin- 6. Zhang Y-Z, Xiong C-L, Zou Y, Wang infected, it would indicate an incuba- tana (4). We report a human case of B. D-M, Jiang R-J, Xiao Q-Y, et al. Mo- alsatica lymphadenopathy. tion period of 4.5–6 years. lecular characterisation of rabies virus Such long incubation periods are isolates in China during 2004. Virus A 79-year-old woman came to a rare for rabies virus infections. An ear- Res. 2006;121:179–88. DOI: 10.1016/j. hospital in Agen, France, in Febru- virusres.2006.05.010 lier epidemiologic study of 177 cases ary 2008 with a large painless axillary 7. Yamagata J, Ahmed K, Khawplod P, Man- mass that she had noticed 10 days ear- in Amritsar, India, demonstrated that nen K, Xuyen DK, Loi HH, et al. Molecu- rabies developed within 6 months of lar epidemiology of rabies in Vietnam. lier. She reported that ≈1 month earlier exposure in 90% of human cases (10). Microbiol Immunol. 2007;51:833–40. she was scratched on her fi nger while 8. Johnson N, Black C, Smith C, Un H, However, the thorough documentation butchering a wild rabbit. On examina- McElhinney LM, Aylan O, et al. Rabies tion, she did not have any other spe- of a small number of cases (1,2) sug- emergence among foxes in Turkey. J Wildl gests that clinicians need to be aware Dis. 2003;39:262–70. cifi c fi ndings. Blood cell counts and of the importance of including travel 9. Wong TW, Chan PK, Fung KP. Human levels of liver enzymes were normal. rabies in Hong Kong: a case review. Ann history over several years in cases of A large necrotic lymph node was sur- Acad Med Singapore. 1987;16:633–5. gically removed the next day. Her con- unexplained encephalitis. 10. Lakhanpal U, Sharma RC. An epide- miological study of 177 cases of human dition was treated with doxycycline This work was supported by the rabies. Int J Epidemiol. 1985;14:614–7. (200 mg) for 3 weeks. Department for Environment, Food and DOI: 10.1093/ije/14.4.614 Our laboratory received a frag- Rural Affairs (Defra, UK) through grant ment of the lymph node of the patient Address for correspondence: Nicholas Johnson, SE0423. and a portion of the rabbit that had been Rabies and Wildlife Zoonoses Group, WHO cooked, boiled as a terrine, and stored Collaborating Centre for Rabies and Rabies- Nicholas Johnson, in a freezer at –20°C in the home of Related Viruses, Veterinary Laboratories the patient. DNA was extracted from Anthony Fooks, Agency – Weybridge, Woodham Lane, Surrey and Kenneth McColl these specimens by using a QIAamp KT15 3NB, UK; email: [email protected]. Tissue Kit (QIAGEN, Hilden, Ger- Author affi liations: World Health Organiza- gsi.gov.uk many). The DNA was used as a tem- tion Collaborating Centre for Rabies and plate in 3 described PCRs specifi c for Rabies-Related Viruses, Weybridge, UK a portion of the B. alsatica 16S–23S (N. Johnson, A. Fooks); and Australian Ani- intergenic spacer (ITS) region, ftsZ mal Health Laboratory, Geelong, Victoria, gene, and 16S rDNA (5). All results Australia (K. McColl)

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 1951 LETTERS for the lymph node were positive for Immunohistologic staining was Author affi liations: Université de la Méditer- B. alsatica, and amplifi cation products used to demonstrate B. alsatica in ranée, Marseille, France (E. Angelakis, H. of the expected size were obtained the lymph node. Immunohistochemi- Lepidi, J.-M. Rolain, D. Raoult); and Centre from this extract. Sequences obtained cal analysis was performed by using Hospitalier d’Agen, Agen, France (A. Canel, shared 100% similarity with the cor- a monoclonal antibody against B. al- P. Rispal, F. Perraudeau, I. Barre) responding 16S rDNA, ITS region, satica with an immunoperoxidase kit DOI: 10.3201/eid1412.080757 and ftsZ gene fragment of B. alsatica. previously described (7). Briefl y, after However, the terrine specimen was deparaffi nization, the tissue section References negative for 16S rDNA, the ITS re- was incubated with polyclonal-specif- gion, and the ftsZ gene. All negative ic antibody to B. alsatica (8) diluted 1. Rolain JM, Lepidi H, Zanaret M, Triglia controls showed typical results. B. al- 1:1,000 in phosphate-buffered saline. JM, Michel G, Thomas PA, et al. Lymph satica have not been tested or found in Immunodetection was performed with node biopsy specimens and diagnosis of cat-scratch disease. Emerg Infect Dis. our laboratory for several years. biotinylated immunoglobulins, perox- 2006;12:1338–44. B. quintana subsp. Oklahoma, idase-labeled streptavidin (HistoStain 2. Raoult D, Drancourt M, Carta A, Gastaut B. henselae subsp. Houston (ATCC Plus Kit; Zymed, Montrouge, France), JA. Bartonella (Rochalimaea) quintana 49882), B. vinsonii subsp. berkhoffi and amino-ethyl-carbazole as sub- isolation in patient with chronic adenop- athy, lymphopenia, and a cat. Lancet. (URBVAIE25), B. vinsonii subsp. ar- strate. Slides were counterstained with 1994;343:977. DOI: 10.1016/S0140- upensis (ATCC 700727), and B. alsat- Mayer hematoxylin for 10 min. Loca- 6736(94)90102-3 ica (CIP 105477 T) strains were used tion of was superimposable 3. La VD, Tran-Hung L, Aboudharam G, for immunofl uorescence and Western on that in the Warthin-Starry–stained Raoult D, Drancourt M. Bartonella quin- tana in domestic cat. Emerg Infect Dis. blotting assays (5). A serum sample specimens, and clusters of microor- 2005;11:1287–9. taken at admission was negative for ganisms were seen in the infl amma- 4. Breitschwerdt EB, Maggi RG, Sigmon B, B. alsatica by immunofl uorescence tory areas (online Appendix Figure, Nicholson WL. Isolation of Bartonella assay. This result was accepted be- panel D). quintana from a woman and a cat fol- lowing putative bite transmission. J Clin cause serologic results may be nega- We report lymphadenitis caused Microbiol. 2007;45:270–2. DOI: 10.1128/ tive during the onset of the disease by B. alsatica. Our fi nding was con- JCM.01451-06 (6). Western blotting with Bartonella fi rmed by molecular, serologic, and 5. Raoult D, Roblot F, Rolain JM, Besnier spp. antigens (5) was positive for B. staining methods. Bartonella spp. are JM, Loulergue J, Bastides F, et al. First isolation of Bartonella alsatica from a alsatica and after adsorption, only B. zoonotic agents that infect erythro- valve of a patient with endocarditis. J Clin alsatica antigens retained all antibod- cytes of mammals in which they cause Microbiol. 2006;44:278–9. DOI: 10.1128/ ies (online Appendix Figure, panel chronic bacteremia (9). B. alsatica JCM.44.1.278-279.2006 A, available from www.cdc.gov/EID/ was fi rst identifi ed in 1999 in Alsace, 6. Maurin M, Rolain JM, Raoult D. Com- parison of in-house and commercial slides content/14/12/1951-appF.htm). France, as an agent of bacteremia in for detection of immunoglobulins G and Formalin-fi xed, paraffi n-embed- healthy wild rabbits (10). However, in M by immunofl uorescence against Barto- ded tissue specimens (3-μm thick) 2006, interest in B. alsatica increased nella henselae and . were stained with hematoxylin and eo- when it was considered to be a human Clin Diagn Lab Immunol. 2002;9:1004–9. DOI: 10.1128/CDLI.9.5.1004-1009.2002 sin. Microscopic examination showed pathogen because it caused blood-cul- 7. Lepidi H, Fournier PE, Raoult D. Quanti- that the normal architecture of the ture–negative endocarditis in a patient tative analysis of valvular lesions during lymph node was destroyed. Histologic who had contacts with rabbits (5). The Bartonella endocarditis. Am J Clin Pathol. changes were dominated by large ir- present case confi rms that B. alsatica 2000;114:880–9. DOI: 10.1309/R0KQ- 823A-BTC7-MUUJ regular stellate or round granulomas could be a human pathogen, espe- 8. Bonhomme CJ, Nappez C, Raoult D. Mi- with central neutrophil-rich necrosis cially in persons who live in contact croarray for serotyping of Bartonella spe- (online Appendix Figure, panel B). with rabbits and should be considered cies. BMC Microbiol. 2007;7:59. DOI: Granulomas were composed mainly a cause of lymphadenopathy. 10.1186/1471-2180-7-59 9. Breitschwerdt EB, Kordick D. Bartonella of macrophages, whereas neutrophils infection in animals: carriership, reservoir in the necrotic areas were fragmented. Emmanouil Angelakis, potential, pathogenicity, and zoonotic po- These granulomas with abscess forma- Hubert Lepidi, Atbir Canel, tential for human infection. Clin Micro- tion were similar to those described in Patrick Rispal, biol Rev. 2000;13:428–38. DOI: 10.1128/ Françoise Perraudeau, CMR.13.3.428-438.2000 CSD. Warthin-Starry staining showed 10. Heller R, Kubina M, Mariet P, Riegel P, bacteria in the necrotic center of the Isabelle Barre, Jean-Marc Rolain, Delacour G, Dehio C, et al. Bartonella al- granulomas (online Appendix Figure, and Didier Raoult satica sp. nov., a new Bartonella species panel C). isolated from the blood of wild rabbits. Int J Syst Bacteriol. 1999;49:283–8.

1952 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 LETTERS

Address for correspondence: Didier Raoult, semiarid southern Chaco, Argentina, gel electrophoresis analysis (Figure). Unité des Rickettsies, Centre National de la Moreno Department, Province of San- Distilled water and R. conorii DNA Recherche Scientifi que–Institut de Recherche tiago del Estero. Ticks, kept in 70% al- were used as negative controls, and E. pour le Développement, Unité Mixte de cohol, were identifi ed as Amblyomma chaffeensis DNA as the positive con- Recherche 6236, Faculté de Médecine, parvum (n = 200), A. tigrinum (n = trol. The fi nding was confi rmed by se- Université de la Méditerranée, 27 Bd Jean 26), and A. pseudoconcolor (n = 13). quence analysis (GenBank accession Moulin, 13385 Marseille CEDEX 05, France; A sample of 70 A. parvum and 1 A. nos. EU826517 and EU826518) email: [email protected] tigrinum ticks collected on domestic In view of these positive results, ruminants and canids were subjected another set of 108 specimens was test- to PCR and reverse line blot hybrid- ed by E. chaffeensis VLPT PCR: all ization by using the TBD-RLB mem- the ticks collected on humans (80 A. brane (Isogen Life Science, Maarssen, parvum, 1 A. pseudoconcolor, and 4 A. the Netherlands) (6) to look for Ana- tigrinum), 18 host-seeking A. parvum plasma and Ehrlichia spp. DNA was ticks, and 5 A. parvum ticks collected extracted from individual ticks by us- on armadillos of the genera Tolypeu- Molecular Detection ing the DNeasy Blood and Tissue kit tes and Chaetophractus. E. chaffeen- of Ehrlichia (QIAGEN Valencia, CA, USA); sev- sis was detected in A. parvum ticks chaffeensis in eral negative controls (distilled water) only: 5 from humans (6.2%; 95% CI for both DNA extraction and PCRs 2.1–14.0; Figure, panel A) and 3 from Amblyomma were run alongside the samples in ran- host-seeking ticks (16.7%; 95% CI parvum Ticks, dom order throughout the experiments. 3.6–41.4). In total, E. chaffeensis was Argentina Primers Ehr-R (5′-CGGGATCCCCA detected in 9.2% (95% CI 5.4–14.6) GTTTGCCGGGACTTYTTCt- of tested A. parvum ticks in the study To the Editor: Ehrlichia chaf- 3′) (6) and Ehr-Fint (5′-GGCTCA area. Of the 16 positive A. parvum, 5 feensis is an obligate intracellular GAACGAACGCTG-3′; Inst. Biotec- were infesting humans. bacterium in the family Anaplasma- nologia, Instituto Nacional de Tec- Little is known about E. chaffeen- taceae. It is considered an emerging nología Agropecuaria, unpub. data) sis epidemiology in South America. In pathogen in the United States because were used to amplify a 500-bp fragment Brazil, wild marsh deer (Blastocerus it is the causative agent of human of the 16S gene of Anaplasma/Ehrli- dichotomus) are suspected to be its monocytotropic (1), a fl u- chia spp. PCR products were analyzed natural reservoir, but the tick involved like illness that can progress to severe by reverse line blot hybridization , and in the transmission cycle is not known multisystem disease and has a 2.7% 11.3% (95% confi dence interval [CI] (8). In North America, E. chaffeensis case-fatality rate (2). = 4.9–21.0) showed a positive signal sp. is maintained principally by the In Central and South America, to the specifi c E. chaffeensis probe: 8 lone-star tick, A. americanum, and the human cases of ehrlichiosis with A. parvum ticks collected from a dog white-tailed deer (Odocoileus virgin- compatible serologic evidence have (n = 1), a fox (Lycalopex gymnocer- ianus) (2). However, the possibility been reported in Venezuela, Bra- cus, n = 1), goats (n = 2), and cattle (n of transmission by different ticks and zil, Mexico, and Chile, although the = 4). No signals to other probes pres- infection among other hosts has been bacterium has not been isolated (3). ent in the membrane were recorded reported; specifi c antibodies to E. Recently, molecular evidence of E. (A. phagocytophylum, A. marginale, chaffeensis were detected in domes- chaffeensis infection was reported A. centrale, A. ovis, E. ruminatium, tic and wild canids and goats (2), and for a symptomatic 9-year-old child in E. sp. Omatjenne, E. canis). Further recently experimental infection was Venezuela (4). In Argentina, antibod- sequence analysis of 16S fragments demonstrated in cattle (9). We did fi nd ies reactive to E. chaffeensis, or an confi rmed the result, with our se- E. chaffeensis organisms in ticks col- antigenically related Ehrlichia spe- quences showing 99.6% identity with lected on both wild and domestic ani- cies, were detected in human serum the corresponding fragment of the E. mals, but the possible role of different samples during a serologic survey in chaffeensis strain Arkansas 16S gene mammals as reservoir hosts deserves Jujuy Province, where fatal cases of (GenBank accession no. EU826516). further investigation. Moreover, the febrile illness were reported (5). To better characterize the positives fi nding of polymorphic VLPT gene During November–December samples, we then amplifi ed variable- fragments in our sample indicates the 2006, we collected ticks by drag- length PCR target (VLPT) of E. chaf- circulation of E. chaffeensis genetic ging the vegetation and by examining feensis (7). PCR products of variable variants in the study area. VLPT re- mammal hosts, including humans, in length were detected by conventional petitive sequences vary among isolates

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 1953