Human Case of Bartonella alsatica Lymphadenitis

To the Editor: Lymph node enlargement is a common medical problem that is usually caused by bacterial, viral, fungal, or protozoal agents (1). Malignancies or lymphoproliferative diseases are often found, especially in elderly patients (1). Bartonella henselae, the main causative agent of cat-scratch disease (CSD), appears to be the most common organism responsible for lymphadenopathy in adults and children (1). CSD has also been rarely associated with B. quintana (2). Recently, the epidemiology of B. quintana as an emerging source of human infection has changed because it has been isolated from the dental pulp of a domestic cat (3). Feral cats have also been found to be infected by B. quintana (4). We report a human case of B. alsatica lymphadenopathy. 
 
A 79-year-old woman came to a hospital in Agen, France, in February 2008 with a large painless axillary mass that she had noticed 10 days earlier. She reported that ≈1 month earlier she was scratched on her finger while butchering a wild rabbit. On examination, she did not have any other specific findings. Blood cell counts and levels of liver enzymes were normal. A large necrotic lymph node was surgically removed the next day. Her condition was treated with doxycycline (200 mg) for 3 weeks. 
 
Our laboratory received a fragment of the lymph node of the patient and a portion of the rabbit that had been cooked, boiled as a terrine, and stored in a freezer at –20°C in the home of the patient. DNA was extracted from these specimens by using a QIAamp Tissue Kit (QIAGEN, Hilden, Germany). The DNA was used as a template in 3 described PCRs specific for a portion of the B. alsatica 16S–23S intergenic spacer (ITS) region, ftsZ gene, and 16S rDNA (5). All results for the lymph node were positive for B. alsatica, and amplification products of the expected size were obtained from this extract. Sequences obtained shared 100% similarity with the corresponding 16S rDNA, ITS region, and ftsZ gene fragment of B. alsatica. However, the terrine specimen was negative for 16S rDNA, the ITS region, and the ftsZ gene. All negative controls showed typical results. B. alsatica have not been tested or found in our laboratory for several years. 
 
B. quintana subsp. Oklahoma, B. henselae subsp. Houston (ATCC 49882), B. vinsonii subsp. berkhoffi (URBVAIE25), B. vinsonii subsp. arupensis (ATCC 700727), and B. alsatica (CIP 105477 T) strains were used for immunofluorescence and Western blotting assays (5). A serum sample taken at admission was negative for B. alsatica by immunofluorescence assay. This result was accepted because serologic results may be negative during the onset of the disease (6). Western blotting with Bartonella spp. antigens (5) was positive for B. alsatica and after adsorption, only B. alsatica antigens retained all antibodies (Appendix Figure, panel A). 
 
Formalin-fixed, paraffin-embedded tissue specimens (3-μm thick) were stained with hematoxylin and eosin. Microscopic examination showed that the normal architecture of the lymph node was destroyed. Histologic changes were dominated by large irregular stellate or round granulomas with central neutrophil-rich necrosis (Appendix Figure, panel B). Granulomas were composed mainly of macrophages, whereas neutrophils in the necrotic areas were fragmented. These granulomas with abscess formation were similar to those described in CSD. Warthin-Starry staining showed bacteria in the necrotic center of the granulomas (Appendix Figure, panel C). 
 
Immunohistologic staining was used to demonstrate B. alsatica in the lymph node. Immunohistochemical analysis was performed by using a monoclonal antibody against B. alsatica with an immunoperoxidase kit previously described (7). Briefly, after deparaffinization, the tissue section was incubated with polyclonal-specific antibody to B. alsatica (8) diluted 1:1,000 in phosphate-buffered saline. Immunodetection was performed with biotinylated immunoglobulins, peroxidase-labeled streptavidin (HistoStain Plus Kit; Zymed, Montrouge, France), and amino-ethyl-carbazole as substrate. Slides were counterstained with Mayer hematoxylin for 10 min. Location of bacteria was superimposable on that in the Warthin-Starry–stained specimens, and clusters of microorganisms were seen in the inflammatory areas (Appendix Figure, panel D). 
 
We report lymphadenitis caused by B. alsatica. Our finding was confirmed by molecular, serologic, and staining methods. Bartonella spp. are zoonotic agents that infect erythrocytes of mammals in which they cause chronic bacteremia (9). B. alsatica was first identified in 1999 in Alsace, France, as an agent of bacteremia in healthy wild rabbits (10). However, in 2006, interest in B. alsatica increased when it was considered to be a human pathogen because it caused blood-culture–negative endocarditis in a patient who had contacts with rabbits (5). The present case confirms that B. alsatica could be a human pathogen, especially in persons who live in contact with rabbits and should be considered a cause of lymphadenopathy.


Human Case of Bartonella alsatica Lymphadenitis
To the Editor: Lymph node enlargement is a common medical problem that is usually caused by bacterial, viral, fungal, or protozoal agents (1). Malignancies or lymphoproliferative diseases are often found, especially in elderly patients (1). Bartonella henselae, the main causative agent of catscratch disease (CSD), appears to be the most common organism responsible for lymphadenopathy in adults and children (1). CSD has also been rarely associated with B. quintana (2). Recently, the epidemiology of B. quintana as an emerging source of human infection has changed because it has been isolated from the dental pulp of a domestic cat (3). Feral cats have also been found to be infected by B. quintana (4). We report a human case of B. alsatica lymphadenopathy.
A 79-year-old woman came to a hospital in Agen, France, in February 2008 with a large painless axillary mass that she had noticed 10 days earlier. She reported that ≈1 month earlier she was scratched on her fi nger while butchering a wild rabbit. On examination, she did not have any other specifi c fi ndings. Blood cell counts and levels of liver enzymes were normal. A large necrotic lymph node was surgically removed the next day. Her condition was treated with doxycycline (200 mg) for 3 weeks.
Our laboratory received a fragment of the lymph node of the patient and a portion of the rabbit that had been cooked, boiled as a terrine, and stored in a freezer at -20°C in the home of the patient. DNA was extracted from these specimens by using a QIAamp Tissue Kit (QIAGEN, Hilden, Germany). The DNA was used as a template in 3 described PCRs specifi c for a portion of the B. alsatica 16S-23S intergenic spacer (ITS) region, ftsZ gene, and 16S rDNA (5). All results LETTERS for the lymph node were positive for B. alsatica, and amplifi cation products of the expected size were obtained from this extract. Sequences obtained shared 100% similarity with the corresponding 16S rDNA, ITS region, and ftsZ gene fragment of B. alsatica. However, the terrine specimen was negative for 16S rDNA, the ITS region, and the ftsZ gene. All negative controls showed typical results. B. alsatica have not been tested or found in our laboratory for several years.
Formalin-fi xed, paraffi n-embedded tissue specimens (3-μm thick) were stained with hematoxylin and eosin. Microscopic examination showed that the normal architecture of the lymph node was destroyed. Histologic changes were dominated by large irregular stellate or round granulomas with central neutrophil-rich necrosis (online Appendix Figure, panel B). Granulomas were composed mainly of macrophages, whereas neutrophils in the necrotic areas were fragmented. These granulomas with abscess formation were similar to those described in CSD. Warthin-Starry staining showed bacteria in the necrotic center of the granulomas (online Appendix Figure, panel C).
Immunohistologic staining was used to demonstrate B. alsatica in the lymph node. Immunohistochemical analysis was performed by using a monoclonal antibody against B. alsatica with an immunoperoxidase kit previously described (7). Briefl y, after deparaffi nization, the tissue section was incubated with polyclonal-specific antibody to B. alsatica (8) diluted 1:1,000 in phosphate-buffered saline. Immunodetection was performed with biotinylated immunoglobulins, peroxidase-labeled streptavidin (HistoStain Plus Kit; Zymed, Montrouge, France), and amino-ethyl-carbazole as substrate. Slides were counterstained with Mayer hematoxylin for 10 min. Location of bacteria was superimposable on that in the Warthin-Starry-stained specimens, and clusters of microorganisms were seen in the infl ammatory areas (online Appendix Figure, panel D).
We report lymphadenitis caused by B. alsatica. Our fi nding was confi rmed by molecular, serologic, and staining methods. Bartonella spp. are zoonotic agents that infect erythrocytes of mammals in which they cause chronic bacteremia (9). B. alsatica was fi rst identifi ed in 1999 in Alsace, France, as an agent of bacteremia in healthy wild rabbits (10). However, in 2006, interest in B. alsatica increased when it was considered to be a human pathogen because it caused blood-culture-negative endocarditis in a patient who had contacts with rabbits (5). The present case confi rms that B. alsatica could be a human pathogen, especially in persons who live in contact with rabbits and should be considered a cause of lymphadenopathy.

Molecular Detection of Ehrlichia chaffeensis in Amblyomma parvum Ticks, Argentina
To the Editor: Ehrlichia chaffeensis is an obligate intracellular bacterium in the family Anaplasmataceae. It is considered an emerging pathogen in the United States because it is the causative agent of human monocytotropic ehrlichiosis (1), a fl ulike illness that can progress to severe multisystem disease and has a 2.7% case-fatality rate (2).
In Central and South America, human cases of ehrlichiosis with compatible serologic evidence have been reported in Venezuela, Brazil, Mexico, and Chile, although the bacterium has not been isolated (3). Recently, molecular evidence of E. chaffeensis infection was reported for a symptomatic 9-year-old child in Venezuela (4). In Argentina, antibodies reactive to E. chaffeensis, or an antigenically related Ehrlichia species, were detected in human serum samples during a serologic survey in Jujuy Province, where fatal cases of febrile illness were reported (5).
During November-December 2006, we collected ticks by dragging the vegetation and by examining mammal hosts, including humans, in semiarid southern Chaco, Argentina, Moreno Department, Province of Santiago del Estero. Ticks, kept in 70% alcohol, were identifi ed as Amblyomma parvum (n = 200), A. tigrinum (n = 26), and A. pseudoconcolor (n = 13). A sample of 70 A. parvum and 1 A. tigrinum ticks collected on domestic ruminants and canids were subjected to PCR and reverse line blot hybridization by using the TBD-RLB membrane (Isogen Life Science, Maarssen, the Netherlands) (6) to look for Anaplasma and Ehrlichia spp. DNA was extracted from individual ticks by using the DNeasy Blood and Tissue kit (QIAGEN Valencia, CA, USA); several negative controls (distilled water) for both DNA extraction and PCRs were run alongside the samples in random order throughout the experiments. Primers Ehr-R (5′-CGGGATCCCCA G T T T G C C G G G A C T T Y T T C t -3′) (6) and Ehr-Fint (5′-GGCTCA GAACGAACGCTG-3′; Inst. Biotecnologia, Instituto Nacional de Tecnología Agropecuaria, unpub. data) were used to amplify a 500-bp fragment of the 16S gene of Anaplasma/Ehrlichia spp. PCR products were analyzed by reverse line blot hybridization , and 11.3% (95% confi dence interval [CI] = 4.9-21.0) showed a positive signal to the specifi c E. chaffeensis probe: 8 A. parvum ticks collected from a dog (n = 1), a fox (Lycalopex gymnocercus, n = 1), goats (n = 2), and cattle (n = 4 To better characterize the positives samples, we then amplifi ed variablelength PCR target (VLPT) of E. chaffeensis (7). PCR products of variable length were detected by conventional gel electrophoresis analysis (Figure). Distilled water and R. conorii DNA were used as negative controls, and E. chaffeensis DNA as the positive control. The fi nding was confi rmed by sequence analysis (GenBank accession nos. EU826517 and EU826518) In view of these positive results, another set of 108 specimens was tested by E. Little is known about E. chaffeensis epidemiology in South America. In Brazil, wild marsh deer (Blastocerus dichotomus) are suspected to be its natural reservoir, but the tick involved in the transmission cycle is not known (8). In North America, E. chaffeensis sp. is maintained principally by the lone-star tick, A. americanum, and the white-tailed deer (Odocoileus virginianus) (2). However, the possibility of transmission by different ticks and infection among other hosts has been reported; specifi c antibodies to E. chaffeensis were detected in domestic and wild canids and goats (2), and recently experimental infection was demonstrated in cattle (9). We did fi nd E. chaffeensis organisms in ticks collected on both wild and domestic animals, but the possible role of different mammals as reservoir hosts deserves further investigation. Moreover, the fi nding of polymorphic VLPT gene fragments in our sample indicates the circulation of E. chaffeensis genetic variants in the study area. VLPT repetitive sequences vary among isolates