Bartonella henselae and B. koehlerae DNA in Birds

To the Editor: Bartonellosis, a globally emerging vector-borne zoonotic bacterial disease, is caused by hemotropic, gram-negative, aerobic, facultative intracellular Bartonella spp. (1). Of the 30 Bartonella species/subspecies, 17 have been associated with human infections (2,3). Each species has a reservoir host(s), within which the bacteria can cause intraerythrocytic bacteremia with few or no clinical signs of illness (1,3); the bacteria are transmitted by hematophagous arthropod vectors (1). Various Bartonella spp. have been identified in domestic and wild animals, including canids, deer, cattle, rodents, and marine mammals (1,4). Bartonella DNA from the blood of loggerhead sea turtles (Caretta caretta) has been PCR amplified and sequenced (5); the fact that Bartonella DNA was found suggests the possibility that persistent blood-borne infection can occur in nonmammals and that the host range for Bartonella spp. may be larger than anticipated. 
 
Growing evidence suggests that wild birds play key roles in the maintenance and movement of zoonotic pathogens such as tick-borne encephalitis virus and Borrelia and Rickettsia spp. (6–9). Bartonella grahamii DNA was amplified from a bird tick in Korea (10). The substantial mobility, broad distribution, and migrations of birds make them ideal reservoir hosts for dispersal of infectious agents. To investigate whether birds might be a reservoir for Bartonella spp., we screened 86 birds for the presence of Bartonella spp. DNA. 
 
The primary study site was a residential backyard in Morehead City, North Carolina, USA (34°43.722′N, 76°43.915′W). Of the 86 birds screened, 78 (16 species) were captured by mist net during March 2010–June 2012 and 8 (3 species) were injured birds that were to be euthanized (Table). Each bird was examined for external abnormalities and ectoparasites, weighed, measured, and tagged with a US Geological Survey–numbered band. A blood sample (0.10–0.25 mL) was collected from each bird by using a 1-mL insulin syringe with a 28-gauge × 1.27-cm needle. Blood remaining after preparation of blood smears was added to an EDTA tube and frozen (−80°C) until processed. Blood smears were examined for hemoparasites. Research was conducted under required state and federal bird banding permits and with the approval of the North Carolina State University Institutional Animal Care and Use Committee. 
 
 
 
Table 
 
Bartonella species detected in birds* 
 
 
 
Before DNA was extracted from the samples, 10 μL of blood was diluted in 190 µL of phosphate-buffered saline. DNA was automatically extracted by using a BioRobot Symphony Workstation and MagAttract DNA Blood M96 Kit (QIAGEN, Valencia, CA, USA). Bartonella DNA was amplified by using conventional Bartonella genus PCR primers targeting the 16S–23S intergenic spacer region: oligonucleotides, 425s (5′-CCG GGG AAG GTT TTC CGG TTT ATCC-3′) and 1,000as (5′-CTG AGC TAC GGC CCC TAA ATC AGG-3′). Amplification was performed in a 25-μL reaction, as described (3). All PCR reactions were analyzed by 2% agarose gel electrophoresis. Amplicons were sequenced to identify the Bartonella sp. and intergenic spacer region genotype. To compare sequences with those in GenBank, we identified bacterial species and genotypes by using Blast version 2.0 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). DNA extraction and PCR-negative controls remained negative throughout the study. 
 
Results are summarized in the Table. None of the screened birds were anemic, but 5 were PCR positive for Bartonella spp. (3 for B. henselae and 2 for B. koehlerae). B. henselae was amplified from 2 Northern Mockingbirds (Mimus polyglottos) and 1 Red-winged Blackbird (Agelaius phoeniceus) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC814161","term_id":"501412833","term_text":"KC814161"}}KC814161). The DNA sequences were identical to each other and had 99.6% (456/457 bp) sequence similarity with B. henselae San Antonio 2 intergenic spacer region genotype (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF369529","term_id":"15290601","term_text":"AF369529"}}AF369529). B. koehlerae was amplified from a Red-bellied Woodpecker (Melanerpes carolinus) and a Common Loon (Gavia immer) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC814162","term_id":"501412834","term_text":"KC814162"}}KC814162). The DNA sequences were identical to each other (404/404 bp) and to GenBank sequence {"type":"entrez-nucleotide","attrs":{"text":"AF312490","term_id":"15277539","term_text":"AF312490"}}AF312490. Lice (Mallophaga order) were found on 5 Boat-tailed Grackles (Quiscalus major), but no ectoparasites were observed on Bartonella spp.–positive birds. Hemoparasites (Haemoproteus and Plasmodium spp.) were detected in 7 of 86 birds, indicating exposure to hematophagous ectoparasites, but hemoparasites were not detected in the Bartonella spp.–positive birds. No bacteria were visualized in Bartonella PCR–positive blood smears. 
 
Bartonella spp. are increasingly associated with animal and human illnesses; thus, the identification of reservoirs and increased understanding of Bartonella spp. disease ecology are of public health importance. Our finding of 2 pathogenic species not previously reported in birds has expanded the potential sources for zoonotic infection. 
 
There is growing evidence that migratory birds serve as reservoirs and/or mechanical vectors for pathogens such as tick-borne encephalitis virus and Rickettsia spp. (6–8). Birds have been implicated as reservoirs for several Borrelia spp. (9,10) and for possible dispersion of other tick-borne pathogens (e.g., Anaplasma and Bartonella spp.) (6,10). Tick transmission of Bartonella spp. to birds should be investigated, and additional studies that investigate the reservoir host range of Bartonella spp. and the transmission of these bacteria to non–host species will improve epidemiologic understanding of bartonellosis and will identify additional risk factors for Bartonella spp. transmission to new hosts, including humans.


Bartonella henselae and B. koehlerae DNA in Birds
To the Editor: Bartonellosis, a globally emerging vector-borne zoonotic bacterial disease, is caused by hemotropic, gram-negative, aerobic, facultative intracellular Bartonella spp. (1). Of the 30 Bartonella species/subspecies, 17 have been associated with human infections (2,3). Each species has a reservoir host(s), within which the bacteria can cause intraerythrocytic bacteremia with few or no clinical signs of illness (1,3); the bacteria are transmitted by hematophagous arthropod vectors (1). Various Bartonella spp. have been identified in domestic and wild animals, including canids, deer, cattle, rodents, and marine mammals (1,4). Bartonella DNA from the blood of loggerhead sea turtles (Caretta caretta) has been PCR amplified and sequenced (5); the fact that Bartonella DNA was found suggests the possibility that persistent blood-borne infection can occur in nonmammals and that the host range for Bartonella spp. may be larger than anticipated.
Growing evidence suggests that wild birds play key roles in the maintenance and movement of zoonotic pathogens such as tick-borne encephalitis virus and Borrelia and Rickettsia spp. (6)(7)(8)(9). Bartonella grahamii DNA was amplified from a bird tick in Korea (10). The substantial mobility, broad distribution, and migrations of birds make them ideal reservoir hosts for dispersal of infectious agents.
To investigate whether birds might be a reservoir for Bartonella spp., we screened 86 birds for the presence of Bartonella spp. DNA.
The primary study site was a residential backyard in Morehead City, North Carolina, USA (34°43.722′N, 76°43.915′W). Of the 86 birds screened, 78 (16 species) were captured by mist net during March 2010-June 2012 and 8 (3 species) were injured birds that were to be euthanized (Table). Each bird was examined for external abnormalities and ectoparasites, weighed, measured, and tagged with a US Geological Survey-numbered band. A blood sample (0.10-0.25 mL) was collected from each bird by using a 1-mL insulin syringe with a 28-gauge × 1.27-cm needle. Blood remaining after preparation of blood smears was added to an EDTA tube and frozen (-80°C) until processed. Blood smears were examined for hemoparasites. Research was conducted under required state and federal bird banding permits and with the approval of the North Carolina State University Institutional Animal Care and Use Committee.
Before DNA was extracted from the samples, 10 μL of blood was diluted in 190 µL of phosphatebuffered saline. DNA was automatically extracted by using a BioRobot Symphony Workstation and MagAttract DNA Blood M96 Kit (QIAGEN, Valencia, CA, USA). Bartonella DNA was amplified by using conventional Bartonella genus PCR primers targeting the 16S-23S intergenic spacer region: oligonucleotides, 425s (5′-CCG GGG AAG GTT TTC CGG TTT ATCC-3′) and 1,000as (5′-CTG AGC TAC GGC CCC TAA ATC AGG-3′). Amplification was performed in a 25-mL reaction, as described (3). All PCR reactions were analyzed by 2% agarose gel electrophoresis. Amplicons were sequenced to identify the Bartonella sp. and intergenic spacer region genotype.
To compare sequences with those in GenBank, we identified bacterial species and genotypes by using Blast version 2.0 (http://blast.ncbi.nlm.nih. gov/Blast. cgi). DNA extraction and PCR-negative controls remained negative throughout the study.
Results are summarized in the Bartonella spp. are increasingly associated with animal and human illnesses; thus, the identification of reservoirs and increased understanding of Bartonella spp. disease ecology are of public health importance. Our finding of 2 pathogenic species not previously reported in birds has expanded the potential sources for zoonotic infection.
There is growing evidence that migratory birds serve as reservoirs and/or mechanical vectors for pathogens such as tick-borne encephalitis virus and Rickettsia spp. (6)(7)(8). Birds have been implicated as reservoirs for several Borrelia spp. (9,10) and for possible dispersion of other tickborne pathogens (e.g., Anaplasma and Bartonella spp.) (6,10). Tick transmission of Bartonella spp. to birds should be investigated, and additional studies that investigate the reservoir host range of Bartonella spp. and the transmission of these bacteria to non-host species will improve epidemiologic understanding of bartonellosis and will identify additional risk factors for Bartonella spp. transmission to new hosts, including humans.

Tick-borne Pathogens in Northwestern California, USA
To the Editor: In northwestern California, USA, the western blacklegged tick, Ixodes pacificus, is a known vector of Borrelia burgdorferi, the spirochete that causes Lyme disease. B. miyamotoi, which is more closely related to spirochetes that cause relapsing fever, has also been detected in 2 locations in California (1,2) and has recently been implicated as a human pathogen in the northeastern United States (3,4). Other studies may have unintentionally included B. miyamotoi infections among measures of B. burgdorferi if the diagnostics were for spirochetes (e.g., direct fluorescent antibody tests or dark-field microscopy) or genetically targeted for Borrelia spp. (5).
To investigate Borrelia spp. ecology in California, we collected adult I. pacificus ticks by dragging a 1-m 2 white flannel blanket along vegetation and/or leaf litter in 12 recreational areas in the San Francisco Bay area during January-May 2012 (Table). Habitat varied from chaparral and grassland to coastal live oak woodland. Ticks were pooled for examination by quantitative PCR (qPCR) for the presence of Borrelia spp. We interpreted the prevalence of Borrelia spp. from positive pools as the minimum infection prevalence (i.e., assuming 1 positive tick/positive pool). DNA was extracted from ticks by using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocols and then stored at -20°C until use. DNA was analyzed by qPCR, with use of primer and fluorescent hybridization probes previously developed to differentiate Borrelia spp. spirochetes (5). To identify the Borrelia spp. genotype, we attempted to sequence the 16S-23S (rrs-rrlA) intergenic spacer of each sample positive by qPCR (8). The nested PCR product was further purified by using the QIAquick Kit (QIAGEN) and then sequenced (Environmental Genetics and Genomics Laboratory, Northern Arizona University, Flagstaff, AZ, USA; www.enggen.nau.edu/dna.html) by using capillary Sanger sequencing on an ABI 3730 sequencer (Life Technologies, Grand Island, NY, USA). BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) was used to compare each sequence to other Borrelia spp. sequences available from GenBank.
From a total of 1,180 adult ticks, we found 43 samples positive for Borrelia spp., resulting in a minimum infection prevalence of 3.6% (Table). We obtained intergenic spacer sequence data for 27 of the positive samples; 6 samples were B. burgdorferi sensu stricto, 7 were B. burgdorferi sensu lato (both on the basis of alignments of 816 bp), and 14 were B. miyamotoi (on the basis of alignments of 503 bp). The B. miyamotoi sequences for our samples from California and those for isolates from the eastern United States (9) and Japan (8) formed a monophyletic clade that was oriented as a sister clade to the 3 Borrelia spp. that cause tick-borne relapsing fever in the United States (B. hermsii, B. turicatae, and B. parkeri).
We found borreliae-infected adult I. pacificus ticks at all 12 sites from which tick sample sizes exceeded 30. When the presence of B. burgdorferi