Blood Supplementation Enhances Bartonella henselae Growth and Molecular Detection of Bacterial DNA in Liquid Culture.

Bacteria of the genus Bartonella, a member of the Alphaproteobacteria, are fastidious, Gram-negative, aerobic bacilli that comprise numerous species, subspecies, and genotypes. Bartonella henselae, with a worldwide distribution, infects cats, dogs, horses, humans, and other mammals. Diagnostically, direct detection of Bartonella henselae in patient blood specimens by culture or molecular methods is required to confirm infection with this bacterium. Enrichment blood culture combined with quantitative PCR (qPCR) or ddPCR enhances the sensitivity of direct detection. The addition of sheep blood to liquid culture media increased the Bartonella henselae DNA concentration compared to controls, additionally improving PCR direct detection sensitivity. IMPORTANCE This study aims to improve diagnostic detection of Bartonella henselae. Patient samples are combined with enriched bacterial cultures aimed at growing Bartonella henselae for the best possible chance at detection. However, current Bartonella growth methods could be improved. The DNA extraction method used by most laboratories should also be optimized. Sheep blood was added to increase the growth of Bartonella henselae and multiple DNA extraction methods were to be compared to each other.

Feeding on a Bartonella henselae Infected Host Triggers Temporary Changes in the Ctenocephalides felis Microbiome.

The effect of Bartonella henselae on the microbiome of its vector, Ctenocephalides felis (the cat flea) is largely unknown, as the majority of C. felis microbiome studies have utilized wild-caught pooled fleas. We surveyed the microbiome of laboratory-origin C. felis fed on B. henselae-infected cats for 24 h or 9 days to identify changes to microbiome diversity and microbe prevalence compared to unfed fleas, and fleas fed on uninfected cats. Utilizing Next Generation Sequencing (NGS) on the Illumina platform, we documented an increase in microbial diversity in C. felis fed on Bartonella-infected cats for 24 h. These changes returned to baseline (unfed fleas or fleas fed on uninfected cats) after 9 days on the host. Increased diversity in the C. felis microbiome when fed on B. henselae-infected cats may be related to the mammalian, flea, or endosymbiont response. Poor B. henselae acquisition was documented with only one of four infected flea pools having B. henselae detected by NGS. We hypothesize this is due to the use of adult fleas, flea genetic variation, or lack of co-feeding with B. henselae-infected fleas. Future studies are necessary to fully characterize the effect of endosymbionts and C. felis diversity on B. henselae acquisition.

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis.

In vitro and in vivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E. coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

Osteomyelitis associated with Bartonella henselae infection in a young cat.

Case summary: A 1-year-old male intact domestic shorthair cat was evaluated for acute onset non-weightbearing left forelimb lameness and generalized peripheral lymphadenopathy. CT identified a monostotic aggressive bone lesion with an incomplete fracture of the left radial metaphysis. Bone aspirates yielded osteoblasts with minimal nuclear atypia. Abdominal ultrasound revealed a nodular spleen and lymphadenopathy; cytologically, both contained lymphoid hyperplasia. A urine histoplasma antigen test was negative. Bartonella henselae and Mycoplasma haemominutum DNA was amplified by PCR from peripheral blood. Indirect immunofluorescence documented strong B henselae immunoreactivity, with lower Bartonella vinsonii subspecies berkhoffii and Bartonella koehlerae antibody titers. After the administration of doxycycline and pradofloxacin for suspected Bartonella-induced osteomyelitis, lameness resolved rapidly. Six-week post-treatment radiographs identified healing of the affected bone, and Bartonella species enrichment blood culture was negative. B henselae antibody titers decreased four-fold over a year, supporting seroreversion. Relevance and novel information: B henselae is a flea-transmitted, host-adapted species, not previously implicated as a cause of osteomyelitis in cats. B henselae subclinical bacteremia is highly prevalent among cats; however, bacteremia has been associated with lymphadenopathy and febrile illness in cats. This report describes a unique clinical presentation in association with B henselae infection in a cat.

Bartonella henselae Recombinant Pap31 for the Diagnosis of Canine and Human Bartonelloses.

Bartonella spp. comprise a genus of Gram-negative alphaproteobacteria that are slow growing, fastidious, and facultative intracellular pathogens with zoonotic potential. Immunofluorescent antibody assays (IFAs), Western blot (WB), and enzyme-linked immunosorbent assays (ELISAs), the frequently used modalities for the serological diagnosis of canine and human Bartonelloses, generate numerous false negative results. Therefore, the development of a reliable serodiagnostic assay for Bartonelloses is of clinical and epidemiological importance. Pap31, a heme binding surface protein of B. henselae, is associated with bacterial adhesion and related to bacterial colonization. To our knowledge, B. henselae Pap31 and its fragments (N-terminal (NTD), middle (MD), and C-terminal (CTD) domains) have not been investigated for the serodiagnosis of canine and human Bartonelloses. In this study, we evaluate the diagnostic utility of B. henselae recombinant whole Pap31 (rPap31) and Pap31 fragments by ELISA using sera from 70 dogs (36 Bartonella spp. IFA-positive (naturally infected), and 34 Bartonella spp. IFA- and PCR-negative (control dogs)) and 36 humans (18 Bartonella spp. IFA-positive (naturally infected) and 18 controls)). In the dogs, the area under the curve (AUC) score of recombinant whole Pap31 was 0.714 with a sensitivity of 42% and specificity of 94% at an OD cutoff value of 0.8955. Among the evaluated recombinant Pap31 proteins for the diagnosis of canine Bartonelloses, rPap31-NTD yielded the highest AUC score of 0.792 (95% CI 0.688-0.895) with a sensitivity of 44% and specificity of 100% at a cutoff value of 1.198. In concordance with this finding, rPap31-NTD also had the highest AUC score of 0.747 (95% CI 0.581-0.913) among the Pap31 recombinant proteins for the diagnosis of human Bartonelloses, with 39% sensitivity and 94% specificity at a cutoff value of 1.366. Recombinant whole Pap31 (rPap31) resulted in 72% sensitivity and 61% specificity at a cutoff value of 0.215 for human Bartonelloses. Due to either low sensitivity or questionable specificity, our findings indicate that recombinant Pap31 and the selected fragments may not be appropriate diagnostic targets in detecting anti-Bartonella antibodies in Bartonella-infected dogs and humans. The findings from this study can be used to further assess the antigenicity and immunogenicity of B. henselae Pap31 as a diagnostic target.

Using Proteomic Approaches to Unravel the Response of Ctenocephalides felis felis to Blood Feeding and Infection With Bartonella henselae.

Among the Ctenocephalides felis felis-borne pathogens, Bartonella henselae, the main aetiological agent of cat scratch disease (CSD), is of increasing comparative biomedical importance. Despite the importance of B. henselae as an emergent pathogen, prevention of the diseases caused by this agent in cats, dogs and humans mostly relies on the use of ectoparasiticides. A vaccine targeting both flea fitness and pathogen competence is an attractive choice requiring the identification of flea proteins/metabolites with a dual effect. Even though recent developments in vector and pathogen -omics have advanced the understanding of the genetic factors and molecular pathways involved at the tick-pathogen interface, leading to discovery of candidate protective antigens, only a few studies have focused on the interaction between fleas and flea-borne pathogens. Taking into account the period of time needed for B. henselae replication in flea digestive tract, the present study investigated flea-differentially abundant proteins (FDAP) in unfed fleas, fleas fed on uninfected cats, and fleas fed on B. henselae-infected cats at 24 hours and 9 days after the beginning of blood feeding. Proteomics approaches were designed and implemented to interrogate differentially expressed proteins, so as to gain a better understanding of proteomic changes associated with the initial B. henselae transmission period (24 hour timepoint) and a subsequent time point 9 days after blood ingestion and flea infection. As a result, serine proteases, ribosomal proteins, proteasome subunit α-type, juvenile hormone epoxide hydrolase 1, vitellogenin C, allantoinase, phosphoenolpyruvate carboxykinase, succinic semialdehyde dehydrogenase, glycinamide ribotide transformylase, secreted salivary acid phosphatase had high abundance in response of C. felis blood feeding and/or infection by B. henselae. In contrast, high abundance of serpin-1, arginine kinase, ribosomal proteins, peritrophin-like protein, and FS-H/FSI antigen family member 3 was strongly associated with unfed cat fleas. Findings from this study provide insights into proteomic response of cat fleas to B. henselae infected and uninfected blood meal, as well as C. felis response to invading B. henselae over an infection time course, thus helping understand the complex interactions between cat fleas and B. henselae at protein levels.

Bartonella spp. seroepidemiology and associations with clinicopathologic findings in dogs in the United States.

BACKGROUND: Improved understanding of Bartonella spp. serology in dogs may aid clinical decision making. OBJECTIVE: Describe demographic and geographic patterns of Bartonella spp. seroreactivity in dogs, and describe hematologic and serum biochemical abnormalities in Bartonella spp. seroreactive and nonseroreactive dogs. ANIMALS: Serum samples from 5957 dogs in the United States, previously submitted to IDEXX Reference Laboratories. METHODS: Serum was tested using 3 indirect ELISAs for B. henselae, B. vinsonii subsp. berkhoffii, and B. koehlerae. Complete blood count and serum biochemistry panel results were reviewed retrospectively. RESULTS: Overall, 6.1% of dogs were Bartonella spp. seroreactive. Toy breeds were less likely to be seroreactive (3.9%) than mixed breeds (7.5%; adjusted odds ratio [aOR], 0.48; 95% confidence interval [CI], 0.32-0.72), and dogs <1 year old were less likely to be seroreactive (3.4%) than dogs 1 to 5.5 years of age (7.3%; aOR, 0.42; 95% CI, 0.23-0.72). Dogs in the West South Central (9.8%) and South Atlantic (8.8%) regions were more likely than dogs elsewhere in the United States to be seroreactive (aOR, 2.22; 95% CI, 1.31-3.87; aOR, 2.44; 95% CI, 1.38-4.36). CONCLUSIONS AND CLINICAL IMPORTANCE: Demographic and geographic findings for Bartonella spp. exposure were broadly comparable to previously reported patterns.

Comparison of Serological and Molecular Assays for Bartonella Species in Dogs with Hemangiosarcoma.

Currently, a gold standard diagnostic test for Bartonella infection in dogs is lacking. This represents a critical limitation for the development and evaluation of new diagnostic tests, as well as for the diagnosis of, and research on, bartonellosis in dogs. This retrospective observational study aims to compare the results of commonly performed and newly-reported Bartonella spp. diagnostic tests in banked clinical specimens from 90 dogs with hemangiosarcoma (HSA) using composite reference standard (CRS) and random effects latent class analysis (RE-LCA) techniques. Samples from each dog were tested using six serological or molecular diagnostic assays, including indirect fluorescent antibody (IFA) and Western blot (WB) for the detection of antibodies in serum, and qPCR and droplet digital PCR (ddPCR) in blood and fresh frozen tissue biopsy samples (mainly splenic HSA tumors and histopathologically normal spleen or skin/adipose tissue). Bartonella infection prevalence was estimated to be 78% based on the CRS (parallel testing with all six assays), and 64% based on the RE-LCA model. The assay with the highest diagnostic accuracy was qPCR performed on fresh frozen tissue biopsy samples (sensitivity: 94% by RE-LCA and 80% by CRS; specificity: 100%). When comparing newly-reported to traditional Bartonella diagnostic assays, ddPCR was more sensitive for the detection of Bartonella DNA than qPCR when testing blood samples (36% vs. 0%, p < 0.0001). Dogs that were positive on serological assays alone with negative molecular assays were highly unlikely (<3%) to be classified as infected by the RE-LCA model. These data indicate that Bartonella spp. DNA can be PCR amplified from fresh frozen tissues from a majority of dogs with HSA using both qPCR and ddPCR, supporting the use of these methods for future controlled studies comparing the prevalence of Bartonella spp. DNA in the tissue of dogs with HSA to that of unaffected controls.

Validation of Bartonella henselae Western Immunoblotting for Serodiagnosis of Bartonelloses in Dogs.

Bartonella spp. are etiological agents of life-threatening zoonotic diseases in dogs worldwide. Due to the poor sensitivity of immunofluorescent-antibody assays (IFAs), a reliable serodiagnostic test for canine bartonelloses is of clinical importance. The utility of Western blotting (WB) for the serodiagnosis of canine bartonelloses has not been critically investigated. The objective of this study was to characterize WB immunodominant proteins that could be used to confirm a serodiagnosis of bartonelloses. Using agar-grown Bartonella henselae San Antonio type 2 (SA2) whole-cell proteins, sera derived from four dog groups were tested by WB to assess immunodominant protein recognition patterns: group I consisted of 92 serum samples (10 preexposure and 82 postexposure serum samples) from 10 adult beagles experimentally inoculated with Bartonella spp., group II consisted of 36 serum samples from Bartonella PCR-positive naturally infected dogs, group III consisted of 26 serum samples from Bartonella PCR-negative and IFA-negative dogs, and group IV consisted of serum samples from 8 Brucella canis IFA-positive and 10 Rickettsia rickettsii IFA-positive dogs. Following experimental inoculation, 9 (90%) group I dogs were variably seroreactive to one or more of six specific immunodominant proteins (13, 17, 29, 50, 56, and 150 kDa). There was a strong but variable recognition of these proteins among 81% of group II dogs. In contrast, 24/26 group III dogs were not reactive to any immunodominant protein. In this study, the sensitivity and diagnostic accuracy of B. henselae SA2 WB were higher than those of B. henselae SA2 IFA testing. Some B. henselae SA2 immunodominant proteins were recognized by dogs experimentally and naturally infected with Bartonella spp. other than B. henselae Additional research is necessary to more fully define the utility of WB for the serodiagnosis of canine bartonelloses.

Detection of Bartonella spp. in dogs after infection with Rickettsia rickettsii.

BACKGROUND: Dynamics of infection by Bartonella and Rickettsia species, which are epidemiologically associated in dogs, have not been explored in a controlled setting. OBJECTIVES: Describe an outbreak investigation of occult Bartonella spp. infection among a group of dogs, discovered after experimentally induced Rickettsia rickettsii (Rr) infection. ANIMALS: Six apparently healthy purpose-bred Beagles obtained from a commercial vendor. METHODS: Retrospective and prospective study. Dogs were serially tested for Bartonella spp. and Rr using serology, culture, and PCR, over 3 study phases: 3 months before inoculation with Rr (retrospective), 6 weeks after inoculation with Rr (retrospective), and 8 months of follow-up (prospective). RESULTS: Before Rr infection, 1 dog was Bartonella henselae (Bh) immunofluorescent antibody assay (IFA) seroreactive and 1 was Rickettsia spp. IFA seroreactive. After inoculation with Rr, all dogs developed mild Rocky Mountain spotted fever compatible with low-dose Rr infection, seroconverted to Rickettsia spp. within 4-11 days, and recovered within 1 week. When 1 dog developed ear tip vasculitis with intra-lesional Bh, an investigation of Bartonella spp. infection was undertaken. All dogs had seroconverted to 1-3 Bartonella spp. between 7 and 18 days after Rr inoculation. Between 4 and 8 months after Rr inoculation, Bh DNA was amplified from multiple tissues from 2 dogs, and Bartonella vinsonii subsp. berkhoffii (Bvb) DNA was amplified from 4 of 5 dogs' oral swabs. CONCLUSIONS AND CLINICAL IMPORTANCE: Vector-borne disease exposure was demonstrated in research dogs from a commercial vendor. Despite limitations, our results support the possibilities of recrudescence of chronic subclinical Bartonella spp. infection after Rr infection and horizontal direct-contact transmission between dogs.

Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States.

Hemangiosarcoma (HSA), a locally invasive and highly metastatic endothelial cell neoplasm, accounts for two-thirds of all cardiac and splenic neoplasms in dogs. Bartonella spp. infection has been reported in association with neoplastic and non-neoplastic vasoproliferative lesions in animals and humans. The objective of this study was to determine the prevalence of Bartonella spp. in conjunction with two other hemotropic pathogens, Babesia spp. and hemotropic Mycoplasma spp., in tissues and blood samples from 110 dogs with histopathologically diagnosed HSA from throughout the United States. This was a retrospective, observational study using clinical specimens from 110 dogs with HSA banked by the biospecimen repository of the Canine Comparative Oncology and Genomics Consortium. Samples provided for this study from each dog included: fresh frozen HSA tumor tissue (available from n = 100 of the 110 dogs), fresh frozen non-tumor tissue (n = 104), and whole blood and serum samples (n = 108 and 107 respectively). Blood and tissues were tested by qPCR for Bartonella, hemotropic Mycoplasma, and Babesia spp. DNA; serum was tested for Bartonella spp. antibodies. Bartonella spp. DNA was amplified and sequenced from 73% of dogs with HSA (80/110). In contrast, hemotropic Mycoplasma spp. DNA was amplified from a significantly smaller proportion (5%, p<0.0001) and Babesia spp. DNA was not amplified from any dog. Of the 100 HSA tumor samples submitted, 34% were Bartonella PCR positive (32% of splenic tumors, 57% of cardiac tumors, and 17% of other tumor locations). Of 104 non-tumor tissues, 63% were Bartonella PCR positive (56% of spleen samples, 93% of cardiac samples, and 63% of skin/subcutaneous samples). Of dogs with Bartonella positive HSA tumor, 76% were also positive in non-tumor tissue. Bartonella spp. DNA was not PCR amplified from whole blood. This study documented a high prevalence of Bartonella spp. DNA in dogs with HSA from geographically diverse regions of the United States. While 73% of all tissue samples from these dogs were PCR positive for Bartonella DNA, none of the blood samples were, indicating that whole blood samples do not reflect tissue presence of this pathogen. Future studies are needed to further investigate the role of Bartonella spp. in the development of HSA.

Ewing Sarcoma in Nepal Treated With Combined Chemotherapy and Definitive Radiotherapy.

PURPOSE: To our knowledge, we conducted the first prospective oncologic clinical trial in Nepal aimed at providing state-of-the-art chemotherapy to patients with Ewing sarcoma. The efficacy of external-beam radiotherapy (RT) as the sole local treatment modality was explored and deemed justified as a result of the lack of available advanced tumor-orthopedic services in Nepal. PATIENTS AND METHODS: Twenty patients, 11 female and 9 male patients between the ages of 6 and 37 years, with newly diagnosed Ewing sarcoma were enrolled. Neoadjuvant combination chemotherapy, comprising well-established drug combinations, was administered in five courses before external-beam RT, during which one course of etoposide and ifosfamide was given. After RT, six additional chemotherapy courses were scheduled. RESULTS: RT was tolerated well, providing rapid symptom relief and local tumor control, with no pathologic fractures observed among the 15 patients who received such treatment. Eleven patients completed the entire treatment protocol; seven patients were under continued follow-up, with no evidence of disease in six patients at a median follow-up time of 2.3 years (range, 1.3 to 3.1 years) and one patient alive but with a regional recurrence. Four patients experienced metastatic relapse and died as a result of their disease. Three treatment-related deaths linked to toxicity from chemotherapy occurred. Four of the six patients who refused to complete the treatment protocol and were lost to follow-up experienced progressive disease and were assumed dead. CONCLUSION: This study was feasible with RT as the sole local treatment modality in combination with chemotherapy. As a result of the high number of patients lost to follow-up, no firm conclusions can be drawn, but the majority of the patients who completed treatment obtained durable long-term remissions.

Pasteurella canis infective endocarditis in a dog.

Infective endocarditis, an infrequent clinical syndrome in dogs, is typically associated with nondescript clinical signs such as fever, malaise and loss of appetite. Although an uncommonly reported infection in dogs, Pasteurella canis is an emerging pathogen with increasing relevance in the human microbiology literature. The goal of this study is to detail the clinical presentation and microbiological findings associated with a novel causative agent of infective endocarditis in the dog. Diagnostic evaluation as well as conventional, automated and molecular microbiological methods are highlighted. The recent literature regarding P. canis and infective endocarditis in companion animals and humans is reviewed. Although an unusual etiologic agent of infective endocarditis, awareness of P. canis as a diagnostic possibility is crucial to accurate microbial surveillance.

Bartonella henselae in a dog with ear tip vasculitis.

BACKGROUND: Bartonella henselae, a Gram-negative, zoonotic, alpha-proteobacteria has been previously implicated in association with cutaneous vasoproliferative lesions (bacillary angiomatosis), nodular panniculitis and multifocal erythema (erythema multiforme) in dogs. OBJECTIVE: Describe clinical, microbiological and histological lesions in a dog with ear margin vasculitis and B. henselae infection. ANIMALS: A 12-month-old, specific pathogen-free intact female beagle dog maintained in a vector-free laboratory animal resource facility. METHODS AND MATERIALS: Bartonella and Rickettsia serological evaluation, Bartonella and Rickettsia PCR, Bartonella alpha-proteobacteria growth medium (BAPGM) enrichment blood culture/PCR, histopathological investigation and confocal immunohistochemical evaluation. RESULTS: Serological investigation (seroreversion) and PCR testing of aural tissue biopsies failed to support Rickettsia rickettsii as a cause of the aural vasculitis; however, B. henselae, genotype San Antonio 2 DNA was amplified and sequenced from both ear tip margins and from normal-appearing abdominal skin. Seroconversion to B. henselae was documented retrospectively by IFA testing. Bartonella henselae organisms were visualized by confocal immunostaining within all three biopsies. Histopathology revealed small vessel necrotizing vasculitis and dermal necrosis. Bartonella henselae seroreversion and complete resolution of skin lesions occurred in conjunction with administration of oral doxycycline and enrofloxacin for six weeks. CONCLUSIONS AND CLINICAL IMPORTANCE: Bartonella henselae is an emerging zoonotic pathogen that has been associated with leucocytoclastic vasculitis in humans and may have had a contributing or causative role in the development of the cutaneous aural margin vasculitis in this beagle.

Evaluation of cell culture-grown Bartonella antigens in immunofluorescent antibody assays for the serological diagnosis of bartonellosis in dogs.

BACKGROUND: Because of poor sensitivity and questionable specificity of immunofluorescent antibody assays (IFAs), serological diagnosis of Bartonella species infections in dogs remains challenging. Despite limitations, IFA testing is the historical "gold standard" for Bartonella serodiagnosis in animals and humans. Because most diagnostic laboratories test against only 1 or 2 Bartonella spp., testing against a broader panel of Bartonella antigens may enhance diagnostic sensitivity and specificity. OBJECTIVE: To evaluate the sensitivity and specificity of Bartonella IFA using 8 cell culture-grown Bartonella spp. isolates. ANIMALS: Archived serum samples from 34 Bartonella spp. naturally exposed, polymerase chain reaction (PCR)-positive dogs and from 26 PCR-negative and IFA-negative dogs. METHODS: Bartonella IFA sensitivity and specificity were assessed using cell culture-grown whole cell antigens derived from 3 Bartonella henselae (Bh) strains (Bh Houston 1, Bh San Antonio Type 2, Bh California 1), 3 Bartonella vinsonii subsp. berkhoffii genotypes (Bvb I, II, and III), Bartonella koehlerae (Bk), and Bartonella quintana (Bq). RESULTS: Only 62% of 34 Bartonella spp. PCR-positive dogs were seroreactive to any of the 8 Bartonella IFA antigens, indicating low IFA sensitivity. PCR-positive dogs were most often IFA seroreactive to Bq (n = 15), to Bvb II (n = 13), or to both (n = 9) antigens. Of the 26 previously IFA-negative/PCR-negative dogs, 4 (15%) were seroreactive using the expanded antigen panel. CONCLUSION AND CLINICAL IMPORTANCE: Despite IFA testing of dogs against 8 different Bartonella isolates, IFA sensitivity remained poor, and specificity was only 85%. Development of a reliable serological assay is needed to facilitate the diagnosis of Bartonella infection in dogs.