Recent observations have begun to support a role for Bartonella spp. as animal as well as human pathogens. Bartonella spp. are vector-transmitted, blood-borne, intracellular, gram-negative bacteria that can induce prolonged infection in the host. Persistent infections in domestic and wild animals result in a substantial reservoir of Bartonella organisms in nature that can serve as a source for inadvertent human infection. The prevalence of bacteremia can range from 50 to 95% in selected rodent, cat, deer, and cattle populations. Dogs infected with Bartonella spp. can develop lameness, endocarditis, granulomatous lymphadenitis, and peliosis hepatis, lesions that have also been reported in association with human infection. Understanding the role of Bartonella spp. as pathogens in cats and other wild or domestic animals awaits the results of additional studies. Considering the extensive animal reservoirs and the large number of insects that have been implicated in the transmission of Bartonella spp., both animal and human exposure to these organisms may be more substantial than is currently believed.

Bartonella Infection in Animals: Carriership, Reservoir Potential, Pathogenicity, and Zoonotic Potential for Human Infection

Class I. Alphaproteobacteria class. nov.

PCR targeting the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region has been proposed as a rapid and reliable method for the detection of Bartonella species DNA in clinical samples. Because of variation in ITS sequences among Bartonella species, a single PCR amplification can be used to detect different species within this genus. Therefore, by targeting the ITS region, multiple PCRs or additional sample-processing steps beyond the primary amplification can be avoided when attempting to achieve molecular diagnostic detection of Bartonella species. Although PCR amplification targeting this region is considered highly sensitive, amplification specificity obviously depends on primer design. We report evidence of nonspecific PCR amplification of Mesorhizobium species with previously published primers that were designed to amplify the Bartonella consensus ITS region. Use of these or other, less species-specific, primers could lead to a false-positive diagnostic test result when evaluating clinical samples. We also report the presence of Mesorhizobium species DNA as a contaminant in molecular-grade water, a series of homologous sequences in the ITS region that are common to Bartonella and Mesorhizobium species, the amplification of Mesorhizobium DNA with unpublished primers designed in our laboratory targeting the ITS region, and the subsequent design of unambiguous ITS primers that avoid nonspecific amplification of Mesorhizobium species. Our results define some potential limitations associated with the molecular detection of Bartonella species in patient samples and indicate that primer specificity is of critical importance if the ITS region is used as a diagnostic target for detection of Bartonella species.

Potential Limitations of the 16S-23S rRNA Intergenic Region for Molecular Detection of Bartonella Species

Cat scratch disease (CSD) is a common cause of subacute regional lymphadenopathy, not only in children but also in adults. Serological and molecular studies demonstrated that Bartonella henselae is the etiologic agent in most cases of CSD. Amplification of B. henselae DNA in affected tissue and detection of antibodies to B. henselae are the two mainstays in the laboratory diagnosis of CSD. We designed a retrospective study and investigated formalin-fixed, paraffin-embedded lymph nodes from 60 patients (25 female, 35 male) with histologically suspected CSD by PCR amplification. The sensitivities of two different PCR assays were compared. The first primer pair amplified a 296-bp fragment of the 16S rRNA gene in 36 of the 60 samples, corresponding to a sensitivity of 60%. The second primer pair amplified a 414-bp fragment of the htrA gene in 26 of the 60 lymph nodes, corresponding to a sensitivity of 43.3%. Bartonella DNA could be detected in a total of 39 (65%) of the 60 lymph nodes investigated. However, histopathologic findings are typical but not specific for CSD and cannot be considered as a “gold standard” for diagnosis of CSD. The sensitivity of the PCR assays increased from 65 to 87% if two criteria (histology and serology) were used in combination for diagnosis of CSD. Two genotypes (I and II) of B. henselae are described as being involved in CSD. Genotype I was found in 23 (59%) and genotype II was found in 9 (23%) of the 39 PCR-positive lymph nodes. Seven (18%) lymph nodes were negative in both type-specific PCR assays. Thirty (50%) of our 60 patients were younger than 20 years old (15 were younger than 10 years), 20 (33%) were between 21 and 40 years old, and 10 (17%) patients were between 41 and 84 years old. Our data suggest that detection of Bartonella DNA in patients’ samples might confirm the histologically suspected diagnosis of CSD.

Detection of Bartonella henselae DNA by Two Different PCR Assays and Determination of the Genotypes of Strains Involved in Histologically Defined Cat Scratch Disease

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp. berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp. berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp. berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31 Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, three Bartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.

Coyotes (Canis latrans) as the reservoir for a human pathogenic Bartonella sp.: molecular epidemiology of Bartonella vinsonii subsp. berkhoffii infection in coyotes from central coastal California.

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.

Comparison of different DNA fingerprinting techniques for molecular typing of Bartonella henselae isolates.

Group B streptococci (GBS) are the major cause of neonatal sepsis and meningitis. GBS infection in neonates is usually treated with a combination of penicillin and gentamicin. According to consensus guidelines, pregnant women at risk receive intrapartum prophylaxis with either ampicillin or penicillin or, in case of allergy, with erythromycin or clindamycin. We investigated the susceptibility patterns of 190 GBS strains from neonates isolated from 1993-1999 and 150 GBS strains collected from adult women in 1997 and 1999. All isolates were susceptible to penicillin, ampicillin and cefotaxime. Erythromycin resistance among all isolates from neonates and from adult women in 1997 was 4.7% and 6%, respectively. In contrast, 12% of the isolates from adult women in 1999 were resistant to erythromycin and 7% were resistant to clindamycin. These findings show an increasing macrolide resistance in recent GBS strains and indicate the need for further surveillance.

Michael Ruess

Papers

Comparison of different DNA fingerprinting techniques for molecular typing of Bartonella henselae isolates.

Antimicrobial susceptibility patterns of Streptococcus agalactiae in a German university hospital.