Introduction

Historically, studies from our research group have involved intracellular pathogens in the Genera Babesia, Ehrlichia, Rickettsia, and more recently, Bartonella. The study of these obligate intracellular, or in the case of Bartonella sp., highly fastidious pathogens, has been hampered by difficulties associated with their in vitro culture. The advent of molecular techniques that facilitate the detection of bacterial DNA in a patient blood sample has begun to revolutionize the current understanding and medical practices related to the management of infectious diseases. To a substantial degree, this molecular diagnostic revolution started with investigations related to Bartonella sp.

Although predated by a substantial number of important observations, three manuscripts, published in the New England Journal of Medicine in 1990, set the stage for a scientific revolution in our understanding of Bartonella infections. Relman and colleagues used polymerase chain reaction (PCR) to amplify bacterial DNA from lesions of AIDS patients with bacillary angiomatosis. For nearly a decade prior to the use of this approach, pathologists had observed “unculturable” silver-staining bacteria in bacillary angiomatosis lesions. When compared with known eubacterial sequences, the uncultured organisms were most closely related to Rochalimaea quintana, the cause of Trench Fever. Perkocha and colleagues reported concurrently that “unculturable” Warthin-Starry staining bacilli could be found in patients with peliosis hepatis, a rare hepatic lesion seen in people with chronic wasting diseases such as pulmonary tuberculosis, advanced cancer, or HIV infection. Independently, Slater and colleagues described the isolation of a novel, fastidious gram-negative organism in blood cultures from immunocompromised and non-immunocompromised febrile patients. The organisms described in these three manuscripts were all subsequently characterized as B. quintana or B. henselae, which was defined as a new species in 1992. During the ensuing 10 years, research efforts have begun to elucidate the importance of Bartonella species as both veterinary and human pathogens. More importantly, the potential utility of a new technique for the specific identification of bacteria in patient samples was introduced to the medical professions.

Discovery of Bartonella vinsonii (berkhoffii) in dogs.

Bartonella vinsonii (berkhoffii) was isolated from a dog with endocarditis in our laboratory in 1993. Retrospectively, long-term administration of immunosuppressive doses of corticosteroids for a presumptive diagnosis of systemic lupus erythematosus may have facilitated the isolation of the original type strain of B. vinsonii (berkhoffii) from this dog with endocarditis. Subsequent attempts to isolate B. vinsonii from immunocompetent dogs with serologic or molecular evidence of Bartonella infection have not been successful in most instances. When using the currently recommended microbiologic techniques, there appears to be considerable variation in the degree of difficulty associated with the isolation of different Bartonella spp. from the blood of different animal species. For this reason, sensitive molecular-based detection methods are proving to be diagnostically useful. For example, in our laboratory, isolation of B. henselae from the blood of naturally or experimentally infected cats is frequently successful. However, we can frequently demonstrate bartonella DNA in multiple tissues obtained from experimentally infected, specific pathogen free cats, from which we are unable to isolate the bacteria. In contrast, isolation of B. vinsonii from seroreactive dogs or dogs from which B. vinsonii (berkhoffii) DNA has been amplified from a comparable EDTA blood sample is rarely successful, even when tissue culture isolation is employed. Several lines of evidence support the conclusion that Bartonella spp. generally cause persistent infection in the susceptible host. Subclinical persistence of B. vinsonii (berkhoffii) has been documented in a dog for 14 months.

Seroprevalence

Due to the relatively recent recognition that dogs can be infected with B. vinsonii (berkhoffii) and potentially other Bartonella spp., seroprevalence data is limited. Seroprevalence was determined in 1,920 sick dogs from North Carolina or surrounding states that were evaluated at a veterinary teaching hospital. Using a reciprocal titer of > 64, 3.6% of the dogs had antibodies to B. vinsonii (berkhoffii). Risk factors that could be associated with seroreactivity included: heavy tick exposure (Odds ratio 14.2), cattle exposure (OR 9.3), rural vs. urban environment (OR 7.1), and heavy flea exposure (OR 5.6). These data were interpreted to support the possibility that exposure to B. vinsonii (berkhoffii) was more likely in dogs in rural environments, that were allowed to roam, and were likely to have a history of heavy tick infestation. Using sera from dogs experimentally infected with R. rickettsii or Ehrlichia canis, cross reactivity to bartonella antigens was not detected. However, 36% and 52% of serum samples derived from dogs naturally infected with E. canis or B. canis were reactive to B. vinsonii antigens. As both E. canis and B. canis are transmitted by Rhipicephalus sanguineous, this tick may be involved in the transmission of B. vinsonii. The possibility of tick transmission was further supported by two additional studies involving dogs infected with one or more Ehrlichia spp. from the same geographic region, in which seroreactivity to B. vinsonii (berkhoffii) antigens was 30% and 89%, respectively. Seroprevalence, using B. vinsonii (berkhoffii) antigens, was 10% (4/40 dogs) in dogs with suspected tick-borne illness from Israel and 36% in dogs with fever and thrombocytopenia from Thailand. Using an ELISA assay, 35% or 869 samples, derived from coyotes in California, contained antibodies to B. vinsonii (berkhoffii) antigens. These data suggest that B. vinsonii (berkhoffii) is distributed throughout the world. As discussed below, with the use of molecular detection techniques, we have been able to detect DNA in EDTA blood samples or tissues from dogs that were seroreactive to B. vinsonii (berkhoffii). In addition, detection of DNA has allowed us to implicate co-infection with multiple tick-transmitted pathogens in a substantially higher number of dogs than previously appreciated. Obviously, co-infection increases the difficulties associated with the diagnostic and therapeutic management of individual patients.

Disease Manifestations

The spectrum of disease associated with Bartonella infection in dogs and most other animal species is currently unknown. However, from a comparative medical perspective, dogs infected with Bartonella spp. can develop disease manifestations, including endocarditis, granulomatous lymphadenitis, granulomatous rhinitis and peliosis hepatis that are identical to disease manifestations reported in human patients. With the exception of a single case report of B. henselae associated peliosis hepatis, B. vinsonii (berkhoffii) has been implicated on a molecular basis in all other disease processes identified in dogs to date. Endocarditis, associated with B. vinsonii, occurs in large breed dogs with a potential predisposition for aortic valve involvement. Intermittent lameness or fever of unknown origin can precede the diagnosis of endocarditis for several months. Multifocal areas of severe myocardial inflammation can be found in dogs with B. vinsonii endocarditis. Potentially, myocarditis without endocarditis can result in cardiac arrhythmias, syncope or sudden death. Myocarditis was identified in a 60-year-old male from Sweden, with serologic and molecular evidence of infection with a Bartonella spp., who died suddenly during a running competition. A more recent study has implicated Bartonella sp. in the pathogenesis of both myocarditis and cardiomyopathy. Considering these preliminary and incomplete observations, derived from naturally infected dogs and human patients, the role of Bartonella spp. as a cause of myocarditis in animals and people deserves future research consideration.

Granulomatous lymphadenitis, involving the left submandibular lymph node, was diagnosed in a dog on the basis seroreactivity to B. vinsonii (berkhoffii) antigens, visualization of Warthin-Starry silver staining bacteria within the lymph node, and PCR amplification followed by Southern blot hybridization. Seven days prior to enlargement of the lymph node, the owners removed an engorged tick from the left ear. This case may be illustrative of a clinical presentation analogous to acute bartonellosis (cat scratch disease) in people, where a scratch or bite injects the inoculum, rather than the bite of a tick. Recently, B. henselae was amplified and sequenced on two independent occasions from the liver of a dog with peliosis hepatis. Although a cause of immunosuppression was not identified in this dog, extrapolation from the human scenario of peliosis hepatis in HIV infected individuals would strongly support this possibility. During a 149 day study, following experimental inoculation of SPF dogs with culture grown B. vinsonii (berkhoffii), there was sustained suppression of peripheral blood CD8+ lymphocytes, accompanied by an altered cell surface phenotype and an increase in CD4+ lymphocytes in the peripheral lymph nodes. As the clinical manifestations of natural Bartonella infection in dogs are similar to those associated with human disease, the immunopathologic consequences in dogs may prove to be similar to those ultimately detected in people.

Co-infection with multiple tick-transmitted pathogens.

Recently, simultaneous infection with more than one tick-borne pathogen has been recognized with increasing frequency in human and canine patients. The pathophysiologic consequences of co-infection in dogs with various combinations of bacteria, rickettsia, and protozoa have not been characterized clinically or experimentally. Although retrospective seroepidemiologic studies suggest that dogs may experience simultaneous infection with multiple tick-borne pathogens, microbiologic (culture), or molecular (PCR) evidence of simultaneous infection in dogs is currently limited. In nature, the risk of exposure to ticks is far greater for dogs than for humans. In addition, dogs can be infested with hundreds of ticks, and at times infestation may involve different tick species. Therefore, the unknown influences of concurrent infection with multiple tick-borne pathogens, including Ehrlichia, Rickettsia, Babesia and Bartonella species, on factors such as pathophysiology, diagnosis, prognosis or therapeutic outcome could be more readily characterized in dogs. For example, during the summer of 1997, our laboratory investigated a cluster of sick dogs with accompanying mortality, for microbiologic, serologic, and molecular evidence of tick-borne infections. Of 27 dogs, 25 were seroreactive to an Ehrlichia sp., 20 to a Bartonella sp., 17 to a Babesia sp., and 22 seroconverted to R. rickettsii antigen. Based upon PCR analysis, several dogs were co-infected with multiple Ehrlichia species, as well as a Bartonella, Babesia or Rickettsia species. Subsequently, retrospective evaluation of sick dogs, managed in our teaching hospital, has yielded molecular evidence of co-infection with multiple tick-transmitted pathogens. Our recent experience indicates that dogs with heavy tick exposure can be infected at a high rate with multiple, potentially zoonotic, tick-borne pathogens.

As recent serologic and molecular evidence indicates that coinfection in dogs with Ehrlichia, Babesia, Rickettsia, and Bartonella spp. may be more frequent than previously realized, the extent to which infection with bartonella influences the pathophysiology of ehrlichiosis, a disease of much longer historical venue, deserves critical reappraisal. For example, infection with Bartonella in dogs concurrently infected with Ehrlichia canis may contribute to the tendency to develop epistaxis. Of similar potential concern in human medicine is the finding of co-segregation of Borrelia burgdorferi, Ehrlichia equi (putative cause of human granulocytic ehrlichiosis), Babesia microti, and Bartonella vinsonii (arupensis) in Ixodes scapularis ticks in the northeastern and northcentral United States. These recent observations also serve to illustrate the potential difficulty in establishing causation in dogs or people co-infected with multiple tick-transmitted pathogens. As certain Borrelia, Ehrlichia, Babesia, and Bartonella spp. can cause chronic, insidious infection in dogs, the relative role of each of the organisms to the pathogenesis of specific disease manifestations in a sick, naturally infected dog will remain difficult to establish. Certainly, more recent evidence indicates that clinicians should screen for a panel of tick-transmitted pathogens when dealing with sick dogs with a history of tick exposure.

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