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. (). Of the 30 Bartonella species/subspecies, 17 have been associated with human infections (,). Each species has a reservoir host(s), within which the bacteria can cause intraerythrocytic bacteremia with few or no clinical signs of illness (,); the bacteria are transmitted by hematophagous arthropod vectors (). Various Bartonella spp. have been identified in domestic and wild animals, including canids, deer, cattle, rodents, and marine mammals (,). Bartonella DNA from the blood of loggerhead sea turtles (Caretta caretta) has been PCR amplified and sequenced (); 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. (–). Bartonella grahamii DNA was amplified from a bird tick in Korea (). 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*

Bird common name	Bird species	No. birds positive/no. total	Bartonella sp.
House sparrow	Passer domesticus	0/28
Boat-tailed grackle	Quiscalus major	0/15
Mourning dove	Zenaida macroura	0/12
Herring gull†	Larus argentatus	0/6
House finch	Carpodacus mexicanus	0/5
Blue jay	Cyanocitta cristata	0/3
Song sparrow	Melospiza melodia	0/2
Northern cardinal	Cardinalis cardinalis	0/2
Northern mockingbird	Mimus polyglottos	2/2	B. henselae SA2
European starling	Sturnus vulgaris	0/2
Red-winged blackbird	Agelaius phoeniceus	1/1	B. henselae SA2
Brown thrasher	Toxostoma rufum	0/1
Tufted titmouse	Baeolophus bicolor	0/1
Red-bellied woodpecker	Melanerpes carolinus	1/1	B. koehlerae
Common grackle	Quiscalus quiscula	0/1
Common loon†	Gavia immer	1/1	B. koehlerae
Red-headed woodpecker	Melanerpes erythrocephalus	0/1
Brown pelican†	Pelicanus occidentalis	0/1
Collared dove	Streptopelia decaocto	0/1

*SA2, San Antonio 2 intergenic spacer region genotype.
†Euthanized.

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 (). 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. 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. AF369529). B. koehlerae was amplified from a Red-bellied Woodpecker (Melanerpes carolinus) and a Common Loon (Gavia immer) (GenBank accession no. KC814162). The DNA sequences were identical to each other (404/404 bp) and to GenBank sequence 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. (–). Birds have been implicated as reservoirs for several Borrelia spp. (,) and for possible dispersion of other tick-borne pathogens (e.g., Anaplasma and Bartonella spp.) (,). 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.