Persistent infection of pets within a household with three Bartonella species.

We monitored by blood culture and immunofluorescence assay (IFA) bartonella infection in one dog and eight cats in a household to determine the prevalence and persistence of the infection as well as its transmissibility to humans. Ectoparasite control was rigorously exercised. During a 3-year period, Bartonella clarridgeiae was recovered from one cat on two occasions, and B. henselae was isolated from another cat on four occasions. During a 16-month period, B. vinsonii subsp. berkhoffii was isolated from the dog on 8 of 10 culture attempts. Despite extensive household contact, the pet owner was seronegative to all three species by IFA for Bartonella-specific immunoglobulin G.


Dispatches
Bartonella species are hematotropic bacteria that have been isolated from humans, cats, dogs, wildlife species, rodents, and arthropods. The mammalian reservoir for B. clarridgeiae and B. henselae is the domestic cat, in which asymptomatic infection can be maintained for long periods (1)(2)(3). Feline infection with B. henselae is widespread throughout the world, and B. clarridgeiae has been isolated from cats in Europe and various regions of the United States (4)(5)(6). Chomel et al. have demonstrated flea transmission of B. henselae between cats (7). Prolonged bacteremia has been demonstrated in cats, but to our knowledge persistent asymptomatic B. vinsonii subsp. berkhoffii infection in dogs, as described in this report, has not been previously documented. Human exposure to B. henselae can result in cat-scratch disease (CSD) or other medical conditions (8), and B. clarridgeiae was recently implicated as the cause of CSD in a veterinarian (6).
B. vinsonii was originally isolated by Baker in 1946; splenic homogenates were cultured from voles that inhabited a former immigrant quarantine station on Grosse Isle, Canada (9). No other isolates of B. vinsonii were described until 1995 when we recovered a similar organism from a dog with valvular endocarditis (10). Subsequent genotypic evaluation of the vole and canine isolates resulted in taxonomic division of the species into subspecies. The vole and canine isolates were designated B. vinsonii subsp. vinsonii and B. vinsonii subsp. berkhoffii, respectively (11). Although the type strain of B. vinsonii subsp. berkhoffii was isolated from a dog with cardiac disease, the organism can also cause subclinical infection. The reservoir for B. vinsonii subsp. berkhoffii has not been established, but seroepidemiologic evidence supports the potential for tick transmission (12).
We describe infection of three pets within the same household with three different Bartonella species and the subclinical persistence of B. vinsonii subsp. berkhoffii infection in a dog for 14 months. This report is a component of other ongoing studies of Bartonella infection in animals. In conjunction with an earlier study, a veterinary student offered her cats as controls for an investigation into the prevalence of B. henselae associated with cases of CSD (1). Three cats and the dog were subsequently monitored for bacteremia and seroreactivity during a bartonella antimicrobial efficacy study, in which the cats received 22.7 mg enrofloxacin q12h PO for 28 days and the dog received 25 mg doxycycline q12h PO for 28 days (13).
Blood samples were collected from the dog and additional cats acquired later by the student. At each collection, all animals were assessed for general health. For the dog, packed cell volume and total plasma protein were determined from EDTA blood samples.
We monitored by blood culture and immunofluorescence assay (IFA) bartonella infection in one dog and eight cats in a household to determine the prevalence and persistence of the infection as well as its transmissibility to humans. Ectoparasite control was rigorously exercised. During a 3-year period, Bartonella clarridgeiae was recovered from one cat on two occasions, and B. henselae was isolated from another cat on four occasions. During a 16-month period, B. vinsonii subsp. berkhoffii was isolated from the dog on 8 of 10 culture attempts. Despite extensive household contact, the pet owner was seronegative to all three species by IFA for Bartonella-specific immunoglobulin G.

Dispatches
Sera were analyzed by immunofluorescence antibody assay (IFA) for reactivity against B. clarridgeiae, B. henselae, and B. vinsonii subsp. berkhoffii (1). All positive serum samples (dog, cat, and human) were reassayed at least three separate times.
Blood samples from the dog and cats were placed into 1.5-ml pediatric isolator tubes (Wampole Laboratories, Cranbury, NJ) for Bartonella culture on rabbit blood agar (1). Isolates were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene and 16S-23S intergenic spacer (ITS) region using Dde I, Mnl I, Hae III, and Alu I restriction endonuclease (6). DNA from the type strains of B. clarridgeiae (ATCC 51734), B. henselae (ATCC 49882), and B. vinsonii subsp. berkhoffii (ATCC 51672), in addition to B. vinsonii subsp. berkhoffii strains cultured from two healthy dogs, was analyzed for comparison.
The dog, cats, and pet owner remained healthy throughout the study. The packed cell volume and total plasma protein values of the dog remained within reference ranges. These values were not routinely assessed in the cats. The source, age, and order of introduction of animals into the household are depicted in Table 1. Bartonella-specific immunoglobulin G (IgG) was detected at various titers in the animals ( Table 2).
During the 3-year period (December 1993 to January 1997), B. clarridgeiae was recovered from one cat on two occasions, and B. henselae was isolated from another cat on four occasions. B. vinsonii subsp. berkhoffii was isolated from  Dispatches the dog on 8 of 10 culture attempts during 16 months of observation (Table 2). Only one species of Bartonella was identified from any individual animal. In all three animals, blood cultures performed after completion of antimicrobial therapy were negative. When compared with type strains, PCR-RFLP analysis of the 16S rRNA gene and the 16S-23S ITS region identified the feline isolates as B. clarridgeiae or B. henselae. Endonuclease digestion of the 16S gene of the isolates from the dog as well as two isolates from other subclinically bacteremic dogs produced restriction patterns identical to the type strain of B. vinsonii subsp. berkhoffii; however, digestion of the ITS region resulted in a different restriction profile for the subclinically bacteremic dogs (Figure).
We did not detect bacteremia temporally associated with exposure to known bacteremic animals in any pet newly introduced to the household. Chomel et al. (7) reported that as few as five infected fleas could transmit B. henselae infection between cats, but normal daily contact between infected and uninfected cats in ectoparasite-free surroundings was insufficient to produce bacteremia in the uninfected cats. Despite their immunologic status (seronegative) and the occasional presence of fleas, some cats in this household did not seroconvert or become bacteremic after continued exposure to infected resident animals. Although we have observed that infection with B. henselae is not crossprotective against B. clarridgeiae (Kordick and Breitschwerdt, unpub. data) and that dual infection with B. clarridgeiae and B. henselae can occur in cats (2,6), similar data are not available with regard to B. vinsonii subsp. berkhoffii. B. vinsonii subsp. berkhoffii bacteremia has not been reported in cats. Intradermal injection of B. henselae into dogs has induced seroconversion but not bacteremia (14). It is therefore not surprising that B. vinsonii subsp. berkhoffii was not recovered from any of the cats in the study or, conversely, that B. henselae or B. clarridgeiae were not recovered from the dog.
Interpretation of Bartonella IFA serologic results for species identification has been compromised because of presumed cross-reactivity. The dog in this study never seroconverted to B. clarridgeiae or B. henselae; however, the cat infected with B. henselae had a consistently elevated level of antibodies reactive against B. vinsonii subsp. berkhoffii antigen. The issue vinsonii subsp. berkhoffii isolates using Hae III. Strain differences were detected between B. vinsonii subsp. berkhoffii (T), which was the type strain cultured from a dog with endocarditis, and isolates A (described in this report), B, and C from subclinically infected dogs.

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of serologic cross-reactivity among Bartonella species deserves additional study.
Subtyping of bacteria after endonuclease digestion of the 16S-23S rRNA ITS region has been performed by other investigators because the spacer region, not appearing to encode functional proteins, demonstrates sequence variability in an operon that has been useful for taxonomic classification (15,16). In this study, different restriction profiles were obtained from isolates derived from asymptomatic dogs compared with the type strain obtained from a dog with endocarditis. Since our sample size was small, further examination of these isolates by pulsed-field gel electrophoresis or bacteriophage analysis may determine whether PCR-RFLP analysis is epidemiologically useful in predicting the pathogenicity of different strains.
The pet owner (student) remained healthy throughout the study; however, she reported that 2½ years before the study she had debilitating fatigue of 1 month duration without fever or lymphadenopathy. At the time, she lived in California and had recently acquired Cat A. A mononucleosis test was negative; her physician suspected a viral infection. Whether the fatigue was related to Bartonella exposure (CSD) or the cat was bacteremic is not possible to determine retrospectively; however, the cat was bacteremic with B. henselae when cultured 2½ years later as part of another study (1). Bartonella transmission from dogs and cats to humans appears to be relatively inefficient. It is not surprising, therefore, that despite continuous exposure to three Bartonella species, the student's serum sample was minimally reactive to Bartonella antigens.