Nandhakumar Balakrishnan

Flea species infesting dogs in Florida and Bartonella spp. prevalence rates.

Several Bartonella spp. associated with fleas can induce a variety of clinical syndromes in both dogs and humans. However, few studies have investigated the prevalence of Bartonella in the blood of dogs and their fleas. The objectives of this study were to determine the genera of fleas infesting shelter dogs in Florida, the prevalence of Bartonella spp. within the fleas, and the prevalence of Bartonella spp. within the blood of healthy dogs from which the fleas were collected. Fleas, serum, and EDTA-anti-coagulated whole blood were collected from 80 healthy dogs, and total DNA was extracted for PCR amplification of Bartonella spp. The genera of fleas infesting 43 of the dogs were determined phenotypically. PCR amplicons from blood and flea pools were sequenced to confirm the Bartonella species. Amplicons for which sequencing revealed homology to Bartonella vinsonii subsp. berkhoffii (Bvb) underwent specific genotyping by targeting the 16S-23S intergenic spacer region. A total of 220 fleas were collected from 80 dogs and pooled by genus (43 dogs) and flea species. Bartonella spp. DNA was amplified from 14 of 80 dog blood samples (17.5%) and from 9 of 80 pooled fleas (11.3%). B. vinsonii subsp. berkhoffii DNA was amplified from nine dogs and five of the flea pools. Bartonella rochalimae (Br) DNA was amplified from six dogs and two flea pools. One of 14 dogs was co-infected with Bvb and Br. The dog was infested with Pulex spp. fleas containing Br DNA and a single Ctenocephalides felis flea. Of the Bvb bacteremic dogs, five and four were infected with genotypes II and I, respectively. Of the Bvb PCR positive flea pools, three were Bvb genotype II and two were Bvb genotype I. Amplification of Bvb DNA from Pulex spp. collected from domestic dogs, suggests that Pulex fleas may be a vector for dogs and a source for zoonotic transfer of this pathogen from dogs to people. The findings of this study provide evidence to support the hypothesis that flea-infested dogs may be a reservoir host for Bvb and Br and that ectoparasite control is an important component of shelter intake protocols.

Bartonella quintana and Coxiella burnetii as Causes of Endocarditis, India

To the Editor: In industrialized countries, blood culture is negative for 2.5%–31% of infectious endocarditis cases (1). In developing countries such as South Africa (2), Algeria (3), and Pakistan (4), culture is negative for 48% to 56%. Negative cultures delay diagnosis and treatment, which profoundly affects clinical outcome. Negative blood cultures commonly result from previous administration of antimicrobial drugs, right-sided endocarditis, or fastidious or noncultivable pathogens (1). Our aim was to identify fastidious agents of blood culture–negative endocarditis by serology. Because of recent attention to zoonotic microorganisms as agents of this condition in developing countries (1), we investigated the prevalence of Coxiella burnetii, Bartonella spp., and Brucella melitensis among endocarditis patients in India. We cultured blood from 111 patients admitted to the Government General Hospital, Chennai, India, from August 2005 through December 2006, with a diagnosis of infectious endocarditis according to the Duke criteria (5). Informed consent was obtained from all patients. Three blood samples from each patient, collected at hourly intervals, were inoculated into brain–heart infusion broth supplemented with 0.04% sodium polyanethol sulfonate (HiMedia, Mumbai, India). Cultures were incubated at 37°C in a 5% CO2 atmosphere for 14 days and checked each day for turbidity. Subcultures were made on 5% sheep blood and MacConkey agar at 37°C at 24 hours, 48 hours, and when culture broth appeared turbid. Blood cultures were negative for 80 (72%) of the 111 patients. Serum from 63 patients was available for serologic testing. Of these patients, 30 were male and 33 were female; age range was 5–65 years and mean age was 25.5 years. Endocarditis involved the native valve for 60 (95.23%) and a prosthetic valve for 3 (4.76%). The most frequent predisposing factor was rheumatic heart disease, found in 38 (60.31%). Of the 60 with native valve endocarditis, the involved valve was mitral for most (36, 60.0%), followed by aortic (8, 13.33%), tricuspid (7, 11.66%), and pulmonary (1, 1.66%); 8 (13.33%) had both valvular and nonvalvular endocarditis. Of the 3 patients with prosthetic valve endocarditis, the involved valve was mitral for 2 and aortic for 1. Serum samples were screened for Bartonella spp. and C. burnetii by microimmunofluorescence (6,7). A diagnosis of endocarditis was based on an immunoglobulin (Ig) G titer >800 to phase I C. burnetii; this titer has a positive predictive value of 98% (6). A diagnosis of Bartonella infection was based on the combination of a positive microimmunofluorescence titer (IgG to B. quintana or B. henselae >200) and a Western blot profile consistent with endocarditis (8). Identification of the causative species was obtained by Western blot after cross-adsorption with either B. henselae or B. quintana antigens (8). Antibodies to B. melitensis were detected by agglutination by using the Rose Bengal and Brucella Wright tests (both from BioRad, Hercules, CA, USA). Of the 63 patients, 9 had a positive antibody response against a tested antigen (Table): 1 to phase I C. burnetii and 8 to Bartonella spp. (IgG >200). Of these, 7 had a 1-fold dilution higher titer to B. quintana than to B. henselae, including 1 with a low-level cross-reaction with C. burnetii, and 1 had identical titers to both. For all 8 patients, Western blot results were consistent with Bartonella endocarditis. For 7, cross-adsorption identified B. quintana as the causative species; for the other, the infecting Bartonella species remained undetermined because adsorption with B. quintana and B. henselae antigens removed all antibodies. Serologic results for B. melitensis were negative for all patients. Table Clinical findings and causative agent for 9 patients with blood culture–negative endocarditis, India, August 2005–December 2006* B. quintana is mostly associated with human body lice but has also been found in fleas (9). The predisposing factors for B. quintana endocarditis are homelessness, alcoholism, and exposure to body lice (10). For our patients, the common predisposing factors were poor hygiene and low socioeconomic status, which may expose them to ectoparasites including lice and fleas. In contrast with previous study findings, B. quintana infectious endocarditis developed on preexisting valvular lesions in all patients (10). This finding may reflect a different clinical evolution than in Europe, where studies have suggested that B. quintana infectious endocarditis followed chronic bacteremia in patients who did not have previous valvular defects (10). In summary, prevalence of negative blood culture among patients with infectious endocarditis was high (72%). The most commonly associated risk factor was rheumatic heart disease (Table). C. burnetii and Bartonella spp. were responsible for 8% of all infectious endocarditis cases and 14% of blood culture–negative cases. No case of infectious endocarditis caused by B. melitensis was identified. Our preliminary study suggests that zoonotic agents, especially Bartonella spp., are prevalent causative organisms of blood culture–negative endocarditis in India. We recommend serologic screening for antibodies to zoonotic microorganisms as diagnostic tools for this disease in India.

Analysis of seroreactivity against cell culture-derived Bartonella spp. antigens in dogs.

Background Little is known about the specificity of Bartonella spp. immunofluorescent antibody (IFA) assays in dogs. Bacteremia in sick dogs most often has been associated with Bartonella henselae (Bh), Bartonella vinsonii subspecies berkhoffii (Bvb), and Bartonella koehlerae (Bk). Clarification of the diagnostic utility of IFA serology when testing against these organisms is needed. Objective To evaluate the specificity of Bartonella IFA assays utilizing 6 cell culture–grown antigen preparations. Animals Archived sera from SPF dogs (n = 29) and from dogs experimentally infected with Bvb (n = 10) and Bh (n = 3). Methods Antibodies (Abs) to Bvb genotypes I, II, and III, Bh serotype I, strains H‐1 and SA2, and to Bk were determined by IFA testing. Results Serum from naïve SPF dogs shown to be negative for Bartonella bacteremia did not react with any of the 6 Bartonella antigens by IFA testing. Dogs experimentally infected with Bvb genotype I developed Abs against homologous antigens, with no cross‐reactivity to heterologous Bvb genotypes, Bh H‐1, SA2 strains, or to Bk. Dogs experimentally infected with Bh serotype I developed Abs against Bh H‐1, but not to Bh SA2 strain with no cross‐reactive Abs to Bvb genotypes I–III or to Bk. Conclusions and Clinical Importance Bartonella spp. Ab responses during acute experimental infections are species and type specific.

Co-infection with Anaplasma platys, Bartonella henselae, Bartonella koehlerae and 'Candidatus Mycoplasma haemominutum' in a cat diagnosed with splenic plasmacytosis and multiple myeloma.

Anaplasma platys (Apl), ‘Candidatus Mycoplasma haemominutum’ (CMh), Bartonella henselae (Bh) and Bartonella koehlerae (Bk) were confirmed by polymerase chain reaction (PCR) amplification and DNA sequencing in a cat diagnosed with multiple myeloma. Other inconsistently documented hematologic abnormalities included anemia, thrombocytopenia, eosinophilia and hypoglycemia. Persistent Apl infection was confirmed for the first time in a North American cat by sequencing three bacterial genes (16S rRNA, p44 and GroEL) in peripheral blood samples collected 100 days apart. Following doxycycline treatment for Apl, multiple myeloma was diagnosed based upon a monoclonal gammopathy and splenic plasmacytosis, and the cat was treated with melphalan, chlorambucil and prednisolone. Apl DNA was not amplified from post-treatment blood samples and the hyperglobulinemia resolved temporarily following chemotherapy. Retrospective PCR analysis of stored DNA extracts identified CMh, Bk and Bh infections. Retrospective PCR for antigen receptor rearrangements (PARR) of splenic aspirates did not confirm B- or T-cell clonality. Co-infection with multiple vector-borne pathogens should be a diagnostic consideration in cats with chronic hypergammaglobulinemia, monoclonal gammopathy and splenic plasmacytosis.

Experimental infection of dogs with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii

The lack of a suitable infection model remains an important obstacle for the pathophysiological understanding of Bartonella spp. The following pilot study was designed to determine whether cell culture-grown Bartonella henselae SA2 and Bartonella vinsonii subsp. berkhoffii genotype III would cause persistent bacteremia in dogs. Pre-inoculation screening established that two laboratory-raised Golden retrievers were naturally-infected with Bartonella koehlerae. Despite prior infection, one dog each was inoculated subcutaneously with 5 × 10(4)B. henselae (SA2 strain) or 3 × 10(4)B. vinsonii subsp. berkhoffii genotype III. Dogs were bled weekly for serological testing and culture using Bartonella alpha proteobacteria growth medium (BAPGM) diagnostic platform. Dog 1 seroconverted to B. henselae and Dog 2 seroconverted to B. vinsonii subsp. berkhoffii genotype III. Throughout the study period, Bartonella spp. DNA was neither amplified nor isolated in ante-mortem BAPGM enrichment blood cultures. B. henselae SA2 was isolated from a postmortem bone marrow from Dog 1 and B. koehlerae DNA was amplified from postmortem lung from Dog 2 following BAPGM enrichment culture. Limitations include lack of uninfected controls, a potentially suboptimal B. vinsonii subsp. berkhoffii inoculum and a relatively short duration of study. We conclude that following intradermal infection, sequestration of Bartonella spp. in tissues may limit diagnostic detection of these bacteria in dog blood samples.

“Candidatus Mycoplasma haemomacaque” and Bartonella quintana Bacteremia in Cynomolgus Monkeys

ABSTRACT Here, we report latent infections with Bartonella quintana and a hemotropic Mycoplasma sp. in a research colony of cynomolgus monkeys (Macaca fascicularis). Sequence alignments, evolutionary analysis, and signature nucleotide sequence motifs of the hemotropic Mycoplasma 16S rRNA and RNase P genes indicate the presence of a novel organism.

Serological and molecular prevalence of selected canine vector borne pathogens in blood donor candidates, clinically healthy volunteers, and stray dogs in North Carolina

BackgroundCanine vector borne diseases (CVBDs) comprise illnesses caused by a spectrum of pathogens that are transmitted by arthropod vectors. Some dogs have persistent infections without apparent clinical, hematological or biochemical abnormalities, whereas other dogs develop acute illnesses, persistent subclinical infections, or chronic debilitating diseases. The primary objective of this study was to screen healthy dogs for serological and molecular evidence of regionally important CVBDs.MethodsClinically healthy dogs (n = 118), comprising three different groups: Gp I blood donor candidates (n = 47), Gp II healthy dog volunteers (n = 50), and Gp III stray dogs (n = 21) were included in the study. Serum and ethylenediamine tetraacetic acid (EDTA) anti-coagulated blood specimens collected from each dog were tested for CVBD pathogens.ResultsOf the 118 dogs tested, 97 (82%) dogs had been exposed to or were infected with one or more CVBD pathogens. By IFA testing, 9% of Gp I, 42% of Gp II and 19% of Gp III dogs were seroreactive to one or more CVBD pathogens. Using the SNAP 4DX® assay, Gp I dogs were seronegative for Anaplasma spp., Ehrlichia spp., and B. burgdorferi (Lyme disease) antibodies and D. immitis antigen. In Gp II, 8 dogs were Ehrlichia spp. seroreactive, 2 were infected with D. immitis and 1 was B. burgdorferi (Lyme disease) seroreactive. In Gp III, 6 dogs were infected with D. immitis and 4 were Ehrlichia spp. seroreactive. Using the BAPGM diagnostic platform, Bartonella DNA was PCR amplified and sequenced from 19% of Gp I, 20% of Gp II and 10% of Gp III dogs. Using PCR and DNA sequencing, 6% of Gps I and II and 19% of Gp III dogs were infected with other CVBD pathogens.ConclusionThe development and validation of specific diagnostic testing modalities has facilitated more accurate detection of CVBDs. Once identified, exposure to vectors should be limited and flea and tick prevention enforced.

Molecular identification of Bartonella species in dogs with leishmaniosis (Leishmania infantum) with or without cytological evidence of arthritis

Recent evidence suggest that Bartonella species may cause polyarthritis and lameness in dogs. Canine leishmaniosis (CanL) due to Leishmania infantum is a multi-systemic disease often occurring in association with arthritis. We hypothesized that concurrent Bartonella infection may be a contributing factor for the development of arthritis in dogs with CanL. Hence the primary objective of this study was to investigate the molecular prevalence of Bartonella spp. in dogs with naturally occurring CanL, with or without cytologically documented arthritis. Thirty-eight dogs with CanL (31 with neutrophilic arthritis and 7 without arthritis) were retrospectively studied. Seventy-four archived clinical specimens from these 38 dogs, including 33 blood samples, 19 bone marrow (BM) samples and synovial fluid (SF) aspirates from 22 dogs were tested for Bartonella spp. DNA using the Bartonella alpha proteobacteria growth medium (BAPGM) diagnostic platform. Overall, eight (21.1%) dogs were infected with one or two Bartonella species; however, Bartonella spp. infection was not associated with arthritis in dogs with CanL. Further prospective studies are warranted to determine if there is a correlation between Bartonella spp. infection and the development of arthritis in dogs with CanL.

Isolation of a Novel Strain of Mycobacterium iranicum from a Woman in the United States

ABSTRACT A novel strain of Mycobacterium iranicum, a recently described nontuberculous Mycobacterium species, was isolated from the sputum of a woman. The source of infection was not determined; however, fomite transmission of inhaled aerosolized secretions from her husbands sleep apnea equipment was historically possible.

Bartonella henselae infection in a man with hypergammaglobulinaemia, splenomegaly and polyclonal plasmacytosis

Bartonella henselae is an infrequently reported cause of polyclonal plasmacytosis and hypergammaglobulinaemia. We herein document B. henselae infection in a 66-year-old patient who presented with hypergammaglobulinaemia, splenomegaly with polyclonal plasmacytosis, stroke, and suspected prosthetic aortic arch infection. Clinicians should remain cognizant of the heterogeneous clinical presentations associated with bartonellosis.