Bartonella DNA in dog saliva.

Bartonella species, transmitted by arthropods or animal bites and scratches, are emerging pathogens in human and veterinary medicine. PCR and DNA sequencing were used to test oral swabs collected from dogs. Results indicated the presence of 4 Bartonella species: B. bovis, B. henselae, B. quintana, and B. vinsonii subspecies berkhoffii.

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Bartonella species are being recognized as increasingly important bacterial pathogens in veterinary and human medicine. These organisms can be transmitted by an arthropod vector or alternatively by animal scratches or bites (1). Among the 11 species or subspecies known or suspected to be pathogenic in humans, 8 have been detected in or isolated from pet dogs or cats, thereby highlighting the zoonotic potential of these bacteria (2). In general, cats are implicated in the transmission of Bartonella henselae, typically resulting in cat-scratch disease; however, there have also been sporadic reports of Bartonella transmission by dogs (3-5). When B. henselae prevalence was evaluated in a population of 52 dogs, 4 dogs were seroreactive at reciprocal titers of 64 or 128, and Bartonella-positive PCR results were found in 3 of 52 blood samples, 5 of 9 oral swabs, and 5 of 9 nail clippings (5). Based on these reports and the recent recognition of B. henselae and B. vinsonii subspecies berkhoffii bacteremia in veterinarians and veterinary technicians who experience frequent cat and dog scratches and bites (6), we speculated that Bartonella species may be present in the saliva of dogs. The purpose of this study was to determine whether Bartonella DNA could be detected in oral swabs collected from dogs.

The Study

As part of an ongoing study from November 2004 to December 2006 to investigate the prevalence of Anaplasma, Bartonella, and Ehrlichia infections in healthy golden retrievers and golden retrievers with lymphoma, a buccal swab was collected using a sterile cotton applicator. The swab was placed against the inside surface of the dog's cheek. Saliva and tissue were collected by rolling the swab firmly against the cheek. Subsequently, the swab was placed into a sterile, no additive, Vacutainer (Becton Dickinson, Franklin Lakes, NJ, USA) serum tube and allowed to air dry for 10 to 15 minutes at room temperature before the tube was recapped.

Cells on the air-dried swab were resuspended in 500 [micro]L of QuickExtract DNA Extraction Solution (EPICENTRE Biotechnologies, Madison, WI, USA), according to the manufacturer's instructions. Total DNA was isolated using 200 [micro]L of the QuickExtract resuspension, which was extracted through a QIAamp DNA Blood Mini-Kit (QIAGEN, Inc., Valencia, CA, USA) according to the manufacturer's instructions. Similarly, total DNA was extracted from 200 [micro]L of EDTA-anticoagulated whole blood using the QIAamp DNA Blood Mini-Kit.

Oral swabs and blood samples (n = 44 each) were screened for the presence of Bartonella by 2 previously described PCR methods (7). The first PCR targeted a fragment of the 16S-23 S intergenic transcribed spacer (ITS) region; samples that were PCR positive for Bartonella DNA by the ITS primers were subsequently analyzed by a second PCR targeting the heme-binding protein gene, Pap31. Positive and negative controls were used in all processing steps, including DNA extraction. PCR amplicons were sequenced to identify species (Davis Sequencing, Davis, CA, USA). Sequence analysis and alignment with GenBank sequences were performed (AlignX, Vector NTI Suite 6.0, InforMax, Inc., Frederick, MD, USA). Additionally, serum samples were analyzed for IgG antibodies to B. henselae and B. vinsonii (berkhoffii) using an indirect immunofluorescence assay (IFA), as described previously (8). Reciprocal titers [greater than or equal to] 64 were considered seroreactive.

Of the 44 dogs surveyed, oral swabs collected from 5 (11.4%) dogs were PCR-positive for Bartonella DNA. Sequencing indicated that 5 different Bartonella species or subtypes were present: B. bovis, B. henselae, B. quintana, and B. vinsonii subsp, berkhoffii types I and II (Table). PCR amplification and sequencing of blood samples from these 5 dogs showed B. henselae and B. vinsonii (berkhoffii) DNA in 2 dogs (Table). None of these 5 dogs was seroreactive to B. henselae or B. vinsonii (berkhoffii) antigens. Contamination was not detected in any of the negative control samples at any stage of processing or at any time during the study. As this work was part of an ongoing study of golden retrievers with and without lymphoma, dogs 1 and 2 had lymphoma; the remaining 3 dogs were clinically healthy (Table).

Conclusions

These results demonstrate the presence of Bartonella DNA in oral swabs obtained from dogs. Notably, 3 Bartonella species and 2 B. vinsonii (berkhoffii) types were found in dog saliva. B. bovis, formerly referred to as B. weissii, was initially isolated from the blood of cats (9). Subsequently, this organism was isolated from the blood of cows in the United States, Europe, and Africa (10-12). To our knowledge, this is only the second known report of the detection of B. bovis DNA in a sample obtained from a dog (13). All 5 dogs in this study lacked serologic evidence of Bartonella infection, a finding which has been previously reported in bacteremic dogs and humans (6,13,14).

Previous studies have shown that targeting multiple Bartonella genes provides molecular evidence of coinfection with more than 1 Bartonella species or strain type (6, 7, 13). In the current work, the inability to confirm the ITS PCR results with a second PCR target has been previously reported by our laboratory (6,13,14) and likely reflects differences in PCR sensitivity, interference or inhibition of the PCR reaction by oral bacteria that are present in greater numbers than the Bartonella, or the lack of a known hemebinding protein gene in various Bartonella species, such as B. boris. The limit of detection (LOD) of Bartonella ITS PCR is 2 copies/reaction, while the LOD of Pap31 assay is 10 copies/reaction. Further, although B. henselae has a detectable Pap31 protein (Table), several researchers in our laboratory have successfully isolated B. henselae strains that lack a PCR-detectable heme-binding protein (unpub. data). Upon recognition of the discordance between ITS and Pap31, additional genes such as 16S, gltA, and rpoB were targeted; however, these analyses were negative for Bartonella and resulted in nonspecific bacterial amplification. Because inhibition of ITS PCR was suspected due the presence of other oral bacteria, Bartonella-negative DNA extracts from oral swabs were spiked with B. henselae DNA at 1.5, 2.5, 5, and 10 (0.002 pg/[micro]L) copies/reaction. Inhibition was detected at up to 5 copies/reaction, while the 10 copies/reaction sample was consistently amplified by the ITS primers.

These data, in conjunction with previous case reports (3-5), suggest that potentially viable Bartonella organisms may be transmitted to humans after a dog bite. The detection of DNA by PCR does not necessarily indicate the viability of Bartonella organisms. However, due to the extremely slow growth characteristics of Bartonella spp., isolation from the oral cavity does not seem feasible, because of competition with numerous other rapidly growing oral bacterial species. Recently, Bartonella DNA has been amplified from peripheral lymph nodes of healthy dogs (14). B. henselae was also amplified from salivary gland tissues from a dog with saladenitis (15). There are several plausible routes by which a Bartonella sp. could gain entry to the oral cavity. Future studies should determine if the tonsilar lymphoid tissues, salivary glands, or periodontal, gingival, or other oral tissues can serve as sources of Bartonella spp. contamination of canine saliva. As Bartonella infection may represent an occupational risk for veterinary professionals and others with extensive animal contact (6), additional studies should address the risk of transmission from dogs to humans following bite wounds.

Acknowledgments

We acknowledge the assistance of the veterinarians who provided samples and the owners who allowed participation of their dogs in this study.

This research was funded in part by the American Kennel Club-Canine Health Foundation, Bayer Animal Health, and the State of North Carolina.

Dr Duncan recently completed her PhD in epidemiology and biotechnology in the Intracellular Pathogens Laboratory at the College of Veterinary Medicine North Carolina State University. Her primary research interests include Bartonella species in dogs and humans.

References

(1.) Boulouis HJ, Chang CC, Henn JB, Kasten RW, Chomel BB. Factors associated with the rapid emergence of zoonotic Bartonella infections. Vet Res. 2005;36:383-410.

(2.) Chomel BB, Boulouis HJ, Maruyama S, Breitschwerdt EB. Bartonella spp. in pets and effect on human health. Emerg lnfect Dis. 2006;12:389-94.

(3.) Kerkhoff FT, Ossewaarde JM, de Loos WS, Rothova A. Presumed ocular bartonellosis. Br J Ophthalmol. 1999;83:270-5.

(4.) Keret D, Giladi M, Kletter Y, Wientroub S. Cat-scratch disease osteomyelitis from a dog scratch. J Bone Joint Surg Br. 1998;80: 766-7.

(5.) Tsukahara M, Tsuneoka H, Iino H, Ohno K, Murano I. Bartonella henselae infection from a dog. Lancet. 1998;352:1682.

(6.) Breitschwerdt EB, Maggi RG, Duncan AW, Nicholson WL, Hegarty BC, Woods CW. Bartonella species in blood of immunocompetent persons with animal and arthropod contact. Emerg Infect Dis. 2007;13:938-41.

(7.) Diniz PPVR Maggi RG, Schwartz DS, Cadenas MB, Bradley JM, Hegarty BC, et al. Canine bartonellosis: serological and molecular prevalencc in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp, berkhoffii. Vet Res. 2007;38:697-710.

(8.) Solano-Gallego L, Bradley J, Hegarty B, Sigmon B, Breitschwerdt E. Bartonella henselae IgG antibodies are prevalent in dogs from southeastern USA. Vet Res. 2004;35:585-95.

(9.) Regnery R, Marano N, Jameson R Marston E, Jones D, Handley S, et al. A fourth Bartonella species, B. weissii species nova, isolated from domestic cats. In: Abstracts of the 15th Meeting of the American Society for Rickettsiology; Captiva Island, Florida; 2000 April 30-May 3; Abstract 4. American Society for Rickettsiology; 2000.

(10.) Breitschwerdt EB, Sontakke S, Cannedy A, Hancock SI, Bradley JM. Infection with Bartonella weissii and detection of Nanobacterium antigens in a North Carolina beef herd. J Clin Microbiol. 2001;39:879-82.

(11.) Bermond D, Boulouis HJ, Heller R, Van Laere G, Monteil H, Chomel BB, et al. Bartonella bovis sp. nov. and Bartonella capreoli sp. nov., isolated from European ruminants. Int J Syst Evol Microbiol. 2002;52:383-90.

(12.) Raoult D, La Scola B, Kelly PJ, Davoust B, Gomez J. Bartonella bovis in cattle in Africa. Vet Microbiol. 2005; 105:155-6.

(13.) Duncan AW, Maggi RG, Breitschwerdt EB. A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: pre-enrichment liquid culture followed by PCR and subculture onto agar plates. J Microbiol Methods. 2007;69:273-81.

(14.) Duncan AW, Birkenheuer AJ, Maggi RG, Breitschwerdt EB. Bartonella DNA detected in the blood and lymph nodes of healthy dogs. In: Abstracts of the 20th Meeting of the American Society for Rickettsiology; Pacific Grove, California; 2006 Sept 2-7; Abstract 110. American Society for Rickettsiology; 2006.

(15.) Saunders GK, Monroe WE. Systemic granulomatous disease and sialometaplasia in a dog with Bartonella infection. Vet Pathol. 2006;43:391-2.

Address for correspondence: Edward B. Breitschwerdt, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough St, Raleigh, NC 27606, USA; email: [e-mail]

Ashlee W. Duncan, * Ricardo G. Maggi, * and Edward B. Breitschwerdt *

* North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina, USA

Table. PCR, DNA sequencing, and serologic results for the 5 dogs
positive for Bartonella DNA in oral swabs *

                      B. vinsonii
Dog    B. henselae    (berkhoffi)
no.        IFA            IFA         PCR target

1          <16            <16         ITS, Pap31

2          <16            <16         ITS, Pap31

3           32            <16         ITS, Pap31
4          <16            <16         ITS, Pap31
5          <16            <16         ITS, Pap31

Dog
no.    DNA sequence in blood

1       B. henselae and B. vinsonii
       (berkhoffii) type II, B. henselae
2         B. henselae, B. henselae

3          Negative, negative
4          Negative, negative
5          Negative, negative


Dog
no.    DNA sequence in oral swab

1          B. bovis, negative

2       B. vinsonii (berkhoffii) types I and II,
       B. henselae and B. vinsonii (berkhoffii)
                      type II
3       B. vinsonii (berkhoffii) type II, negative
4       B. vinsonii (berkhoffii) type II, negative
5               B. quintana, negative

* ITS 16S-23S intergenic transcribed spacer region; Pap31,
heme-binding protein gene; IFA, indirect immunofluorescence assay.