Seroprevalence of Bartonella species, Coxiella burnetii and Toxoplasma gondii among patients with hematological malignancies: A pilot study in Romania.

Patients receiving immunosuppressive cancer treatments in settings where there is a high degree of human-animal interaction may be at increased risk for opportunistic zoonotic infections or reactivation of latent infections. We sought to determine the seroprevalence of selected zoonotic pathogens among patients diagnosed with haematologic malignancies and undergoing chemotherapeutic treatments in Romania, where much of the general population lives and/or works in contact with livestock. A convenience sample of 51 patients with haematologic cancer undergoing chemotherapy at a referral clinic in Cluj-Napoca, Romania, was surveyed regarding animal exposures. Blood samples were obtained and tested for evidence of infection with Bartonella species, Coxiella burnetii and Toxoplasma gondii, which are important opportunistic zoonotic agents in immunocompromised individuals. 58.8% of participants reported living or working on a farm, and living or working on a farm was associated with contact with livestock and other animals. 37.5% of participants were IgG seroreactive against one or more of five Bartonella antigens, and seroreactivity was statistically associated with living on farms. Farm dwellers were 3.6 times more likely to test IgG seroreactive to Bartonella antibodies than non-farm dwellers. 47.1% of the participants tested T. gondii IgG positive and 13.7% tested C. burnetii IgG positive, indicating past or latent infection. C. burnetii IgM antibodies were detected in four participants (7.8%), indicating possible recent infection. These results indicate that a large proportion of patients with haematologic cancer in Romania may be at risk for zoonotic infections or for reactivation of latent zoonotic infections, particularly with respect to Bartonella species. Special attention should be paid to cancer patients' exposure to livestock and companion animals in areas where much of the population lives in rural settings.

Prevalence of Bartonella spp. in Canine Cutaneous Histiocytoma.

Canine cutaneous histiocytoma (CCH) is a common, benign neoplastic proliferation of histiocytes of Langerhans cell origin that often ulcerate, become secondarily infected and regress spontaneously. Bartonella is a fastidious genus of facultative intracellular pathogens that can be transmitted through arthropod bites and epidermal animal scratches and has been identified previously in the cytoplasm of histiocytes within granulomatous lesions and in skin biopsy samples of inflammatory pustules and papules. Based on the established inflammatory and oncogenic properties of Bartonella, we hypothesized that Bartonella spp. DNA could be amplified from CCH more often than from non-lesional skin and bacteria could be localized within skin tumours using indirect immunofluorescence (IIF). Paraffin wax-embedded surgical biopsy samples from dogs with CCH and non-neoplastic skin adjacent to osteosarcomas (control group selected due to wide surgical margins) were retrieved from the archive of the pathology service of North Carolina State University College of Veterinary Medicine. DNA was extracted and regions of the 16S-23S rRNA intergenic transcribed spacer (ITS) region and the pap31 and gltA genes were amplified by polymerase chain reaction (PCR) using Bartonella-specific primers. IIF was performed using a primary Bartonella henselae monoclonal antibody to localize B. henselae in tissues of PCR-positive dogs. Bartonella vinsonii subsp. berkhoffii was amplified from 1/17 (5.8%) control tissues and B. henselae was amplified from 4/29 (13.8%) CCH tissues. The prevalence of B. vinsonii subsp. berkhoffii (P = 0.37) or B. henselae (P = 0.28) did not vary statistically between study groups. B. henselae could be visualized in 2/4 (50.0%) CCH tissues using IIF. Based on this study, Bartonella spp. are unlikely to cause CCH.

Phylogenetic diversity of bacteria isolated from sick dogs using the BAPGM enrichment culture platform.

BACKGROUND: Bartonella alpha-Proteobacteria growth medium (BAPGM) enrichment culture has proven useful for documenting Bartonella species infection and has facilitated growth of other fastidious bacteria from human samples. PURPOSE: To report non-Bartonella bacterial isolates obtained from canine samples cultured using BAPGM enrichment culture. ANIMALS: Between 2004 and 2008, 695 specimens from 513 dogs were tested by the NCSU-IPRL using the BAPGM enrichment culture. Over the same period of time, blood samples from 270 dogs were cultured by the NCSU-CML using Bactec-Plus Aerobic/F media. METHODS: BAPGM isolates were characterized using Bartonella genus primers and 16S rDNA primers followed by DNA sequencing. NCSU medical records were retrospectively reviewed. Blood culture results from the NCSU-CML were compared with BAPGM blood culture results. RESULTS: Seventy-nine non-Bartonella isolates were obtained from 69/513 dogs. The most commonly isolated phylum was Proteobacteria (48.1%) with alpha-Proteobacteria being the most commonly isolated class. Staphylococcus and Sphingomonas were the most commonly isolated genera. The majority of the remaining isolates were bacteria that are rarely isolated from canine samples. Comparison of NCSU-CML and IPRL (BAPGM) blood culture isolates showed alpha-Proteobacteria were isolated more often from BAPGM. CONCLUSIONS AND CLINICAL IMPORTANCE: Use of insect cell culture enrichment medium, such as BAPGM, appears to enhance the growth of alpha-Proteobacteria, but also results in isolation of non-alpha-Proteobacteria from sick dogs. Future studies are needed to elucidate the utility of BAPGM and other "nonconventional" growth media and methods for isolation of fastidious organisms and to determine if these organisms play a causal role in disease development.

Koch's postulates and the pathogenesis of comparative infectious disease causation associated with Bartonella species.

In his homage to Lucretius ('Georgica'), Vergil is credited with stating: 'Felix qui potuit rerum cognoscere causas' ('Fortunate is he who knows the causes of things'). Based on numerous commentaries and publications it is obvious that clinicians, diagnosticians and biomedical research scientists continue to struggle with disease causation, particularly in the assessment of the pathogenic role of 'stealth pathogens' that produce persistent infections in the host. Bartonella species, because of their evolutionary ability to induce persistent intravascular infections, present substantial challenges for researchers attempting to clarify the ability of these stealth bacteria to cause disease. By studying the comparative biological and pathological behaviour of microbes across mammalian genera, researchers might be able more rapidly to advance medical science and, subsequently, patient care by undertaking focused research efforts involving a single mammalian species or by attempting to recapitulate a complex disease in an rodent model. Therefore, in an effort to further assist in the establishment of disease causation by stealth pathogens, we use recent research observations involving the genus Bartonella to propose an additional postulate of comparative infectious disease causation to Koch's postulates.

Bartonella spp. infection in healthy and sick horses and foals from the southeastern United States.

BACKGROUND: Bartonella species bacteremia has been identified in numerous animal species. These bacteria cause, or have been associated with, a spectrum of clinical manifestations in dogs and human patients. The frequency of exposure to or infection with Bartonella spp. among healthy and sick horses has not been reported. OBJECTIVE: To test healthy and sick horses and sick foals from the southeastern United States for serological, microbiological, and molecular evidence of Bartonella infection. ANIMALS: Forty-seven healthy horses, 15 sick foals, 22 horses with musculoskeletal manifestations, and 8 horses with colic were tested for Bartonella. METHODS: IFA serology and PCR before and after BAPGM (Bartonella alpha-Proteobacteria Growth Medium) enrichment blood culture. RESULTS: Bartonella antibodies were not detected in foals or horses. Three Bartonella species, B. henselae, B. vinsonii subsp. berkhoffii (genotypes I and III), and a Bartonella species with closest homology to Candidatus Bartonella volans, were PCR-amplified and sequenced from blood or BAPGM enrichment blood culture samples from 1/47 healthy horses, 3/15 sick foals, 5/22 horses with musculoskeletal disease, and 0/8 horses with colic. CONCLUSIONS AND CLINICAL IMPORTANCE: Horses in the southeastern United States are naturally infected with B. henselae, B. vinsonii subsp. berkhofii genotypes I and III, and a bacteria most similar to Candidatus Bartonella volans. Antibodies were not detectable by indirect fluorescent antibody assay (IFA) testing in bacteremic foals or horses, and prolonged enrichment culture for periods up to 21 days were necessary to document bacteremia in most horses. Further investigation into the pathogenic potential of Bartonella spp. infection in horses is warranted.

Candidatus Mycoplasma haematoparvum and Mycoplasma haemocanis infections in dogs from the United States.

Mycoplasma haemocanis (Mhc) and Candidatus Mycoplasma haematoparvum (CMhp) have been described in dogs. Historically, microscopic visualization of hemotropic Mycoplasma spp. has occurred most often in immunocompromised or splenectomized dogs. The aim of this study was to determine the Mhc and CMhp prevalences among dogs from the United States. Novel 16S rRNA and RNAseP gene PCR assays were used to amplify hemotropic Mycoplasma species DNA for GenBank sequence alignment. Among the study population, hemoplasma prevalence was 1.3% (7 out of 506), with Mhc and CMhp prevalences of 0.6% and 0.8%, respectively. Two of six CMhp-infected dogs were co-infected with a Bartonella sp., and a third dog was seroreactive to Bartonella henselae antigens. The prevalence of Mhc and CMhp in this study was low; potential blood donors should be screened; and dogs and people can be co-infected with hemoplasma and Bartonella spp.

Identification of Bartonella henselae in 2 cats with pyogranulomatous myocarditis and diaphragmatic myositis.

Most cats infected with Bartonella henselae remain outwardly healthy carriers for years; however, self-limiting fever, transient anemia, neurologic dysfunction, lymphadenopathy, reproductive disorders, aortic valvular endocarditis, and neutrophilic myocarditis have been described in experimentally or naturally infected cats. Two cats in a North Carolina shelter died with pyogranulomatous myocarditis and diaphragmatic myositis. Bacteria were visualized in the lesions by Warthin-Starry silver impregnation and by B. henselae immunohistochemistry. B. henselae DNA was amplified and sequenced from the heart of 1 cat and from multiple tissue samples, including heart and diaphragm, from the second cat. This study supports a potential association between B. henselae and what has been historically described as "transmissible myocarditis and diaphragmitis" of undetermined cause in cats.

Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma sp. in dogs with splenic disease.

BACKGROUND: Among diseases that cause splenomegaly in dogs, lymphoid nodular hyperplasia (LNH), splenic hemangiosarcoma (HSA), and fibrohistiocytic nodules (FHN) are common diagnoses. The spleen plays an important role in the immunologic control or elimination of vector-transmitted, blood-borne pathogens, including Bartonella sp., Babesia sp., and hemotropic Mycoplasma sp. OBJECTIVE: To compare the prevalence of Bartonella sp., Babesia sp., and hemotropic Mycoplasma sp. DNA in spleens from dogs with LNH, HSA, and FHN. MATERIALS AND METHODS: Paraffin-embedded, surgically obtained biopsy tissues from LNH (N = 50), HSA (N = 50), and FHN (N = 37) were collected from the anatomic pathology archives. Spleens from specific pathogen-free (SPF) dogs (N = 8) were used as controls. Bartonella sp., Babesia sp., and Mycoplasma sp. DNA was amplified by PCR, followed by DNA sequencing. RESULTS: Bartonella sp. DNA was more prevalent in FHN (29.7%) and HSA (26%) as compared to LNH (10%) (P = .019, .0373, respectively) or control spleens (0.0%). The prevalence of Babesia sp. and hemotropic Mycoplasma sp. DNA was significantly lower than Bartonella sp. DNA in HSA (P = .0005, .006, respectively) and FHN (P = .003, .0004, respectively). There was no statistically significant difference in DNA prevalence among the 3 genera in the LNH group. CONCLUSIONS: The higher prevalence of Bartonella sp. in FHN and HSA warrants future investigations to determine if this bacterium plays a role in the development of these splenic diseases.

Gammopathy in a Spanish dog infected with Bartonella henselae.

Generalised pyogranulomatous disease and hyperviscosity syndrome associated with a presumed monoclonal gammopathy was diagnosed in a three-year-old intact female Pomeranian. The Bartonella henselae antibody titer was 1:64 and Bartonella species DNA was amplified from the splenic tissue. Monoclonal gammopathies in dogs are typically associated with plasma cell and lymphoid dyscrasias and other inflammatory or infectious diseases such as ehrlichiosis and leishmaniosis. Based on this case report, infection with Bartonella species should also be added to the differential diagnoses for gammopathy in dogs. To the authors' knowledge, this is the first report of molecular evidence of Bartonella species infection in a sick dog in Spain.

Molecular and serological diagnosis of Bartonella infection in 61 dogs from the United States.

BACKGROUND: Molecular diagnosis of canine bartonellosis can be extremely challenging and often requires the use of an enrichment culture approach followed by PCR amplification of bacterial DNA. HYPOTHESES: (1) The use of enrichment culture with PCR will increase molecular detection of bacteremia and will expand the diversity of Bartonella species detected. (2) Serological testing for Bartonella henselae and Bartonella vinsonii subsp. berkhoffii does not correlate with documentation of bacteremia. ANIMALS: Between 2003 and 2009, 924 samples from 663 dogs were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory for diagnostic testing with the Bartonella α-Proteobacteria growth medium (BAPGM) platform. Test results and medical records of those dogs were retrospectively reviewed. METHODS: PCR amplification of Bartonella sp. DNA after extraction from patient samples was compared with PCR after BAPGM enrichment culture. Indirect immunofluorescent antibody assays, used to detect B. henselae and B. vinsonii subsp. berkhoffii antibodies, were compared with PCR. RESULTS: Sixty-one of 663 dogs were culture positive or had Bartonella DNA detected by PCR, including B. henselae (30/61), B. vinsonii subsp. berkhoffii (17/61), Bartonella koehlerae (7/61), Bartonella volans-like (2/61), and Bartonella bovis (2/61). Coinfection with more than 1 Bartonella sp. was documented in 9/61 dogs. BAPGM culture was required for PCR detection in 32/61 cases. Only 7/19 and 4/10 infected dogs tested by IFA were B. henselae and B. vinsonii subsp. berkhoffii seroreactive, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs were most often infected with B. henselae or B. vinsonii subsp. berkhoffii based on PCR and enrichment culture, coinfection was documented, and various Bartonella species were identified. Most infected dogs did not have detectable Bartonella antibodies.

Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50% in young dogs: a longitudinal field study.

Canine vector-borne diseases (CVBDs) are highly prevalent and increasing in distribution worldwide. A longitudinal study was conducted in southern Italy to determine the incidence of and protection against CVBD-causing pathogens in dogs treated with a combination of imidacloprid 10% and permethrin 50% (ImPer). One hundred eleven autochthonous young dogs were divided into group A (n=63) and group B (n=48), both groups containing dogs positive and negative for one or more CVBD-causing pathogens. Additionally, 10 naïve male beagles were introduced in each group in May 2008. Group A was treated with ImPer on day 0 and every 21+/-2 days whereas group B was left untreated. Blood and skin samples were collected at baseline (March-April 2008) and at the first, second and third follow-up times (July and October 2008 and April 2009). Bone marrow was sampled at baseline and at the third follow-up. Serological, cytological and molecular tests were performed to detect Anaplasma platys, Babesia spp., Bartonella spp., Dirofilaria immitis, Ehrlichia canis, Hepatozoon canis and Leishmania infantum. Ectoparasites (fleas, ticks, and sand flies) were monitored throughout the study. The baseline prevalence of CVBDs was 39.6% with 44 dogs positive for at least one pathogen. A. platys (27.5%) and Babesia spp. (15.6%) were the most prevalent species and co-infections with up to two pathogens were detected in 16 (14.7%) individuals. At the end of the evaluation period, there was a 90.7% reduction in overall CVBD incidence density rate (IDR) in group A, as following: 100% reduction in L. infantum; 94.6% in E. canis; 94.4% in Babesia spp.; and 81.8% in A. platys. Initially positive treated dogs showed significantly lower pathogen prevalence at the third follow-up than untreated ones. At the end of the evaluation period, 8 of the 10 untreated beagles were infected with at least one pathogen whereas one of the treated beagles was A. platys positive at a single time point (second follow-up). Overall efficacy against ticks was 97.9%. In October 2009, samples were collected from the remaining 83 dogs (44 from group A and 39 from group B) to investigate the annual incidence of CVBDs in the same, at this time untreated, dog population. A high year incidence for tick-borne diseases (78.1%) and for L. infantum (13.6%) was detected in dogs from group A, seven months after the treatment had been withdrawn. The results demonstrate that ImPer preventive treatment against arthropods protects autochthonous and naïve beagle dogs against CVBD-causing pathogens.

Cross-contamination in the molecular detection of Bartonella from paraffin-embedded tissues.

The genus Bartonella comprises a group of gram-negative, fastidious bacteria. Because of diagnostic limitations of culture and serologic testing, polymerase chain reaction (PCR) has become a powerful tool for the detection of Bartonella spp. in blood and tissue samples. However, because many wild and domestic animals harbor Bartonella spp., transfer of Bartonella DNA during sample collection or histologic processing could result in false-positive PCR test results. In this study, we describe evidence of Bartonella DNA dissemination and transfer in the necropsy room and during the subsequent processing of formalin-fixed paraffin-embedded tissues. Bartonella DNA was amplified from different areas of the necropsy room, from the liquid paraffin in the tissue processor, and from different parts of the microtome. Unless stringent procedures are established and followed to avoid cross-contamination, the molecular detection of Bartonella spp. from tissue samples obtained at necropsy or processed in a multispecies histopathology laboratory will not be reliable.

Bartonella sp. bacteremia in patients with neurological and neurocognitive dysfunction.

We detected infection with a Bartonella species (B. henselae or B. vinsonii subsp. berkhoffii) in blood samples from six immunocompetent patients who presented with a chronic neurological or neurocognitive syndrome including seizures, ataxia, memory loss, and/or tremors. Each of these patients had substantial animal contact or recent arthropod exposure as a potential risk factor for Bartonella infection. Additional studies should be performed to clarify the potential role of Bartonella spp. as a cause of chronic neurological and neurocognitive dysfunction.

Bartonella DNA in the blood and lymph nodes of Golden Retrievers with lymphoma and in healthy controls.

BACKGROUND: Although lymphoma is the most common neoplastic process reported in dogs, its precise etiology is unknown. Golden Retrievers are more likely to develop lymphoma, suggesting a breed predisposition; however, other factors, including environment, immunity, and infection, are likely contributors to oncogenesis. HYPOTHESIS: We hypothesized that the development of lymphoma in Golden Retrievers may be associated with vector-borne infections, specifically Bartonella, Anaplasma, or Ehrlichia species infections. ANIMALS: Golden Retrievers with lymphoma and healthy Golden Retrievers from across the United States were recruited for study participation. METHODS: A matched, case-control study was performed to determine the association of lymphoma and the presence of Bartonella, Anaplasma, and Ehrlichia species in serum, blood, and lymph node aspirates. RESULTS: Using PCR analyses and DNA sequencing, single and coinfections with Bartonella henselae, Bartonella elizabethae, Bartonella quintana, and/or Bartonella vinsonii (berkhoffii) were detected in the blood and lymph node aspirates of Golden Retrievers with lymphoma (5/28 dogs, 18%) and in healthy Golden Retrievers (10/56 dogs, 18%); no Anaplasma or Ehrlichia DNA was detected in any dog. When compared with dogs with lymphoma, a higher (P <.001) proportion of healthy Golden Retrievers were receiving monthly acaricide treatments (2.6 times higher). CONCLUSIONS AND CLINICAL IMPORTANCE: Bartonella DNA can be detected in blood and lymph nodes; importantly, in this report, Bartonella was detected in the same proportion of clinically healthy dogs and dogs with lymphoma. Longitudinal studies should be conducted to determine the mode of transmission of Bartonella in dogs, whether lymphatic infection is persistent, or whether these bacteria may contribute to the development of lymphoma.