Bartonella Alpha Proteobacteria Growth Medium Research Articles

Sequence typing of Bartonella henselae in small Indian mongooses (Urva auropunctata)

This study aimed to determine the sequence type (ST) of Bartonella henselae infecting small Indian mongooses from Saint Kitts via multi-locus sequence typing (MLST). This investigation used stored EDTA blood (n = 22) samples from mongooses previously identified as positive for B. henselae. Chocolate agar plates were enriched with Bartonella alpha-Proteobacteria growth medium (BAPGM) to culture and isolate Bartonella from the blood samples. To perform MLST, DNA was extracted and purified from isolates followed by amplification by conventional PCR (300–500 bp) for eight genes (16S rDNA, batR, gltA, groEL, ftsZ, nlpD, ribC, and rpoB). Bartonella henselae STs were deposited in the PubMLST repository. Out of 22 B. henselae-positive blood samples, isolates were obtained from 12 mongooses (54.5%; 12/22). Each mongoose was infected with one ST. The studied mongoose population was infected with sequence types ST2, ST3, ST8, and a novel ST represented by ST38. Bartonella henselae ST2, ST3 and ST8 infecting mongooses are known to circulate in humans and cats, with ST2 and ST8 associated with Cat Scratch Disease (bartonellosis) in humans. The results presented herein denote the circulation of B. henselae STs with zoonotic potential in mongooses with risk of B. henselae transmission to humans.

Bartonella spp. Prevalence (Serology, Culture, and PCR) in Sanitary Workers in La Rioja Spain.

Bartonella spp. are increasingly implicated in association with a spectrum of zoonotic infectious diseases. One hundred sanitary workers in La Rioja, Spain, completed a questionnaire and provided blood specimens for Bartonella spp. serology and Bartonella Alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture/PCR. Six immunofluorescence assays (IFA) were performed and aseptically obtained blood specimens were inoculated into liquid BAPGM and subcultured onto blood agar plates. Bartonella DNA was amplified using conventional and real-time PCR assays. The Bartonella spp., strain, or genotype was determined by DNA sequencing. Bartonella seroreactivity was documented in 83.1% and bloodstream infection in 21.6% of participants. Bartonella henselae, B. vinsonii subsp. berkhoffii genotypes I and III, and B. quintana were identified. IFA seroreactivity and PCR positivity were not statistically associated with self-reported symptoms. Our results suggest that exposure to and non-clinical infection with Bartonella spp. may occur more often than previously suspected in the La Rioja region.

Effectiveness of a 10% imidacloprid/4.5% flumethrin polymer matrix collar in reducing the risk of Bartonella spp. infection in privately owned cats

BackgroundBartonella henselae, Bartonella clarridgeiae and the rare Bartonella koehlerae are zoonotic pathogens, with cats being regarded as the main reservoir hosts. The spread of the infection among cats occurs mainly via fleas and specific preventive measures need to be implemented. The effectiveness of a 10% imidacloprid/4.5% flumethrin polymer matrix collar (Seresto®, Bayer Animal Health), registered to prevent flea and tick infestations, in reducing the risk of Bartonella spp. infection in privately owned cats, was assessed in a prospective longitudinal study.MethodsIn March-May 2015 [Day 0 (D0)], 204 privately-owned cats from the Aeolian Islands (Sicily) were collared (G1, n = 104) or left as controls (G2, n = 100). The bacteraemia of Bartonella spp. was assessed at enrolment (D0) and study closure (D360) by PCR and DNA sequencing both prior to and after an enrichment step, using Bartonella alpha proteobacteria growth medium (BAPGM).ResultsA total of 152 cats completed the study with 3 in G1 and 10 in G2 being positive for Bartonella spp. Bartonella henselae genotype I ZF1 (1.35%) and genotype II Fizz/Cal-1 (6.76%) as well as B. clarridgeiae (5.41%) were detected in cats of G2. Bartonella clarridgeiae was the only species detected in G1. Based on the yearly crude incidence of Bartonella spp. infection (i.e. 3.85% in G1 and 13.51% in G2; P = 0.03) the Seresto® collar achieved a preventative efficacy of 71.54%. The incidence of Bartonella spp. infection was more frequent in flea-infested cats (6/33, 18.18%) than in uninfested ones (7/112, 5.88%) (P = 0.036).ConclusionsCats living in the Aeolian Islands are exposed to B. henselae and B. clarridgeiae. The Seresto® collar provided significant risk reduction against Bartonella spp. infection in outdoor cats under field conditions. Such a preventative tool could be a key contribution for decreasing the risk of Bartonella spp. infection in cats and thus ultimately to humans.

Prevalence of Bartonella spp. by culture, PCR and serology, in veterinary personnel from Spain

BackgroundThe genus Bartonella includes fastidious, facultative intracellular bacteria mainly transmitted by arthropods and distributed among mammalian reservoirs. Bartonella spp. implicated as etiological agents of zoonoses are increasing. Apart from the classical Bartonella henselae, B. bacilliformis or B. quintana, other species (B. elizabethae, B. rochalimae, B. vinsonii arupensis and B. v. berkhoffii, B. tamiae or B. koehlerae, among others) have also been associated with human and/or animal diseases. Laboratory techniques for diagnosis (culture, PCR assays and serology) usually show lack of sensitivity. Since 2005, a method based on a liquid enrichment Bartonella alphaproteobacteria growth medium (BAPGM) followed by PCRs for the amplification of Bartonella spp. has been developed. We aimed to assess culture, molecular and serological prevalence of Bartonella infections in companion animal veterinary personnel from Spain.MethodsEach of 89 participants completed a questionnaire. Immunofluorescence assays (IFA) using B. vinsonii berkhoffii (genotypes I, II and III), B. henselae, B. quintana and B. koehlerae as antigens were performed. A cut-off of 1:64 was selected as a seroreactivity titer. Blood samples were inoculated into BAPGM and subcultured onto blood agar plates. Bartonella spp. was detected using conventional and quantitative real-time PCR assays and DNA sequencing.ResultsAmong antigens corresponding to six Bartonella spp. or genotypes, the lowest seroreactivity was found against B. quintana (11.2%) and the highest, against B. v. berkhoffii genotype III (56%). A total of 27% of 89 individuals were not seroreactive to any test antigen. Bartonella spp. IFA seroreactivity was not associated with any clinical sign or symptom. DNA from Bartonella spp., including B. henselae (n = 2), B. v. berkhoffii genotypes I (n = 1) and III (n = 2), and B. quintana (n = 2) was detected in 7/89 veterinary personnel. PCR and DNA sequencing findings were not associated with clinical signs or symptoms. No co-infections were observed. One of the two B. henselae PCR-positive individuals was IFA seronegative to all tested antigens whereas the other one was not B. henselae seroreactive. The remaining PCR-positive individuals were seroreactive to multiple Bartonella spp. antigens.ConclusionsHigh serological and molecular prevalences of exposure to, or infection with, Bartonella spp. were found in companion animal veterinary personnel from Spain. More studies using BAPGM enrichment blood culture and PCR are needed to clarify the finding of Bartonella PCR-positive individuals lacking clinical symptoms.

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.

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 5×104B. henselae (SA2 strain) or 3×104B. 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.

Infection with Panola Mountain Ehrlichia sp. in a Dog with Atypical Lymphocytes and Clonal T‐Cell Expansion

Infection with Panola Mountain <i><scp>E</scp>hrlichia</i> sp. in a Dog with Atypical Lymphocytes and Clonal T‐Cell Expansion

Phylogenetic Diversity of Bacteria Isolated from Sick Dogs Using theBAPGMEnrichment Culture Platform

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. To report non-Bartonella bacterial isolates obtained from canine samples cultured using BAPGM enrichment culture. 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. 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. 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. 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.

Bartonella henselae infection in a family experiencing neurological and neurocognitive abnormalities after woodlouse hunter spider bites

BackgroundBartonella species comprise a group of zoonotic pathogens that are usually acquired by vector transmission or by animal bites or scratches.MethodsPCR targeting the Bartonella 16S-23S intergenic spacer (ITS) region was used in conjunction with BAPGM (Bartonella alpha Proteobacteria growth medium) enrichment blood culture to determine the infection status of the family members and to amplify DNA from spiders and woodlice. Antibody titers to B. vinsonii subsp. berkhoffii (Bvb) genotypes I-III, B. henselae (Bh) and B. koehlerae (Bk) were determined using an IFA test. Management of the medical problems reported by these patients was provided by their respective physicians.ResultsIn this investigation, immediately prior to the onset of symptoms two children in a family experienced puncture-like skin lesions after exposure to and presumptive bites from woodlouse hunter spiders. Shortly thereafter, the mother and both children developed hive-like lesions. Over the ensuing months, the youngest son was diagnosed with Guillain-Barre (GBS) syndrome followed by Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP). The older son developed intermittent disorientation and irritability, and the mother experienced fatigue, headaches, joint pain and memory loss. When tested approximately three years after the woodlouse hunter spider infestation, all three family members were Bartonella henselae seroreactive and B. henselae DNA was amplified and sequenced from blood, serum or Bartonella alpha-proteobacteria (BAPGM) enrichment blood cultures from the mother and oldest son. Also, B. henselae DNA was PCR amplified and sequenced from a woodlouse and from woodlouse hunter spiders collected adjacent to the family’s home.ConclusionsAlthough it was not possible to determine whether the family’s B. henselae infections were acquired by spider bites or whether the spiders and woodlice were merely accidental hosts, physicians should consider the possibility that B. henselae represents an antecedent infection for GBS, CIDP, and non-specific neurocognitive abnormalities.

Co-infection with Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma haematoparvum in a veterinarian

BackgroundDuring a two year period, a 27-year-old female veterinarian experienced migraine headaches, seizures, including status epilepticus, and other neurological and neurocognitive abnormalities. Prior to and during her illness, she had been actively involved in hospital-based work treating domestic animals, primarily cats and dogs, in Grenada and Ireland and anatomical research requiring the dissection of wild animals (including lions, giraffe, rabbits, mongoose, and other animals), mostly in South Africa. The woman reported contact with fleas, ticks, lice, biting flies, mosquitoes, spiders and mites and had also been scratched or bitten by dogs, cats, birds, horses, reptiles, rabbits and rodents. Prior diagnostic testing resulted in findings that were inconclusive or within normal reference ranges and no etiological diagnosis had been obtained to explain the patient’s symptoms.MethodsPCR assays targeting Anaplasma spp. Bartonella spp. and hemotopic Mycoplasma spp. were used to test patient blood samples. PCR positive amplicons were sequenced directly and compared to GenBank sequences. In addition, Bartonella alpha Proteobacteria growth medium (BAPGM) enrichment blood culture was used to facilitate bacterial growth and Bartonella spp. serology was performed by indirect fluorescent antibody testing.ResultsAnaplasma platys, Bartonella henselae and Candidatus Mycoplasma haematoparvum DNA was amplified and sequenced from the woman’s blood, serum or blood culture samples. Her serum was variably seroreactive to several Bartonella sp. antigens. Despite symptomatic improvement, six months of doxycycline most likely failed to eliminate the B. henselae infection, whereas A. platys and Candidatus M. haematoparvum DNA was no longer amplified from post-treatment samples.ConclusionsAs is typical of many veterinary professionals, this individual had frequent exposure to arthropod vectors and near daily contact with persistently bacteremic reservoir hosts, including cats, the primary reservoir host for B. henselae, and dogs, the presumed primary reservoir host for A. platys and Candidatus Mycoplasma haematoparvum. Physicians caring for veterinarians should be aware of the occupational zoonotic risks associated with the daily activities of these animal health professionals.

Bartonella spp. Infection in Healthy and Sick Horses and Foals from the Southeastern United States

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. To test healthy and sick horses and sick foals from the southeastern United States for serological, microbiological, and molecular evidence of Bartonella infection. Forty-seven healthy horses, 15 sick foals, 22 horses with musculoskeletal manifestations, and 8 horses with colic were tested for Bartonella. IFA serology and PCR before and after BAPGM (Bartonella alpha-Proteobacteria Growth Medium) enrichment blood culture. 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. 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.

Experimental Infection of Horses with Bartonella henselae and Bartonella bovis

Experimental infection of horses with Bartonella species is not documented. Determine clinical signs, hematologic changes, duration of bacteremia, and pattern of seroconversion in Bartonella henselae or Bartonella bovis-inoculated horses. Twelve (2 groups of 6) randomly selected healthy adult horses seronegative and culture negative for Bartonella spp. Experimental/observational study: Group I: B. henselae or saline control was inoculated intradermally into 4 naïve and 2 sentinel horses, respectively. Group II: same design was followed by means of B. bovis. Daily physical examinations, once weekly CBC, immunofluorescent antibody assay serology, real-time polymerase chain reaction (PCR), and twice weekly blood cultures were performed for 6 weeks and at postinoculation day 80 and 139. Bartonella alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture was performed for horses that seroconverted to B. henselae antigens. Transient clinical signs consistent with bartonellosis occurred in some Bartonella-inoculated horses, but hematological alterations did not occur. Three B. henselae-inoculated horses seroconverted, whereas 1 B. bovis-inoculated horse was weakly seropositive. In Group I, B. henselae was amplified and sequenced from BAPGM blood culture as well as a subculture isolate from 1 horse, blood from a 2nd horse, and BAPGM blood culture from a 3rd horse although a subculture isolate was not obtained. All sentinels remained PCR, culture, and serology negative. Detection of Bartonella sp. in blood after experimental inoculation supports bacteremia and seroconversion. Culture with BAPGM may be required to detect Bartonella sp. Although mild clinical signs followed acute infection, no long-term effects were noted for 2 years postinoculation.

Bartonella spp. bacteremia in high-risk immunocompetent patients

Serum and blood samples from 192 patients, who reported animal exposure (100.0%) and recent animal bites or scratches (88.0%), were screened for antibodies by indirect immunofluorescence assays and for bacteremia using the BAPGM ( Bartonella alpha Proteobacteria growth medium) platform. Predominant symptoms included fatigue (79.2%), sleeplessness (64.1%), joint pain (64.1%), and muscle pain (63.0%). Bartonella spp. seroreactivity or bacteremia was documented in 49.5% ( n = 95) and 23.9% ( n = 46) of the patients, respectively; however, indirect immunofluorescence antibodies were not detected in 30.4% ( n = 14) of bacteremic patients. Regarding components of the BAPGM platform, Bartonella DNA was amplified from 7.5% of blood ( n = 21), 8.7% of serum ( n = 25), and 10.3% of enrichment culture samples ( n = 29). Polymerase chain reaction (PCR) on only extracted blood would not have detected Bartonella infection in 34.7% (16/46) of bacteremic patients. Serology, in conjunction with blood, serum, and BAPGM enrichment culture PCR, facilitates the diagnosis of Bartonella spp. bacteremia in immunocompetent patients.

Suspected Needle Stick Transmission of Bartonella vinsonii subspecies berkhoffii to a Veterinarian

Suspected Needle Stick Transmission of Bartonella vinsonii subspecies berkhoffii to a Veterinarian

Enrichment culture and molecular identification of diverse Bartonella species in stray dogs

Using pre-enrichment culture in Bartonella alpha- Proteobacteria growth medium (BAPGM) followed by PCR amplification and DNA sequence identification that targeted a fragment of the citrate synthase gene ( gltA), we provide evidence of common bartonella infections and diverse Bartonella species in the blood of stray dogs from Bangkok and Khon Kaen, Thailand. The overall prevalence of all Bartonella species was 31.3% (60/192), with 27.9% (31/111) and 35.8% (29/81) in the stray dogs from Bangkok and Khon Kaen, respectively. Phylogenetic analyzes of gltA identified eight species/genotypes of Bartonella in the blood of stray dogs, including B. vinsonii subsp. arupensis, B. elizabethae, B. grahamii, B. quintana, B. taylorii, and three novel genotypes (BK1, KK1 and KK2) possibly representing unique species with ≤90.2% similarities to any of the known Bartonella species B. vinsonii subsp. arupensis was the only species detected in dogs from both sites, B. quintana and BK1 were found in the dogs from Bangkok, B. elizabethae, B. taylorii, KK1 and KK2 were found in the dogs from Khon Kaen. We conclude that stray dogs in Thailand are frequently infected with Bartonella species that vary with geographic region. As some Bartonella species detected in the present study are considered pathogenic for humans, stray dogs in Thailand may serve as possible reservoirs for Bartonella causing human illnesses. Further work is needed to determine the role of those newly discovered Bartonella genotypes/species in human and veterinary medicine.

Bartonella vinsonii subsp. berkhoffii and Bartonella henselae bacteremia in a father and daughter with neurological disease

BackgroundBartonella vinsonii subsp. berkhoffii is an important, emerging, intravascular bacterial pathogen that has been recently isolated from immunocompetent patients with endocarditis, arthritis, neurological disease and vasoproliferative neoplasia. Vector transmission is suspected among dogs and wild canines, which are the primary reservoir hosts. This investigation was initiated to determine if pets and family members were infected with one or more Bartonella species.MethodsPCR and enrichment blood culture in Bartonella alpha Proteobacteria growth medium (BAPGM) was used to determine infection status. Antibody titers to B. vinsonii subsp. berkhoffii genotypes I-III and B. henselae were determined using a previously described indirect fluorescent antibody test. Two patients were tested sequentially for over a year to assess the response to antibiotic treatment.ResultsIntravascular infection with B. vinsonii subsp. berkhoffii genotype II and Bartonella henselae (Houston 1 strain) were confirmed in a veterinarian and his daughter by enrichment blood culture, followed by PCR and DNA sequencing. Symptoms included progressive weight loss, muscle weakness, lack of coordination (the father) and headaches, muscle pain and insomnia (the daughter). B. vinsonii subsp. berkhoffii genotype II was also sequenced from a cerebrospinal fluid BAPGM enrichment culture and from a periodontal swab sample. After repeated courses of antibiotics, post-treatment blood cultures were negative, there was a decremental decrease in antibody titers to non-detectable levels and symptoms resolved in both patients.ConclusionsB. vinsonii subsp. berkhoffii and B. henselae are zoonotic pathogens that can be isolated from the blood of immunocompetent family members with arthralgias, fatigue and neurological symptoms. Therapeutic elimination of Bartonella spp. infections can be challenging, and follow-up testing is recommended. An increasing number of arthropod vectors, including biting flies, fleas, keds, lice, sandflies and ticks have been confirmed or are suspected as the primary mode of transmission of Bartonella species among animal populations and may also pose a risk to human beings.

PCR amplification of Bartonella koehlerae from human blood and enrichment blood cultures

BackgroundCats appear to be the primary reservoir host for Bartonella koehlerae, an alpha Proteobacteria that is most likely transmitted among cat populations by fleas (Ctenocephalides felis). Bartonella koehlerae has caused endocarditis in a dog and in one human patient from Israel, but other clinically relevant reports involving this bacterium are lacking. Despite publication of numerous, worldwide epidemiological studies designed to determine the prevalence of Bartonella spp. bacteremia in cats, B. koehlerae has never been isolated using conventional blood agar plates. To date, successful isolation of B. koehlerae from cats and from the one human endocarditis patient has consistently required the use of chocolate agar plates.ResultsIn this study, Bartonella koehlerae bacteremia was documented in eight immunocompetent patients by PCR amplification and DNA sequencing, either prior to or after enrichment blood culture using Bartonella alpha Proteobacteria growth medium. Presenting symptoms most often included fatigue, insomnia, joint pain, headache, memory loss, and muscle pain. Four patients were also infected with Bartonella vinsonii subsp. berkhoffii genotype II. After molecular documentation of B. koehlerae infection in these patients, a serological test was developed and serum samples were tested retrospectively. Bartonella koehlerae antibodies were not detected (titers < 1:16) in 30 healthy human control sera, whereas five of eight patient samples had B. koehlerae antibody titers of 1:64 or greater.ConclusionsAlthough biased by a study population consisting of individuals with extensive arthropod and animal exposure, the results of this study suggest that B. koehlerae bacteremia is more common in immunocompetent people than has been previously suspected. Future studies should more thoroughly define modes of transmission and risk factors for acquiring infection with B. koehlerae. In addition, studies are needed to determine if B. koehlerae is a cause or cofactor in the development of arthritis, peripheral neuropathies or tachyarrhythmias in patients.

PCR detection of Bartonella bovis and Bartonella henselae in the blood of beef cattle

Although an organism primarily associated with non-clinical bacteremia in domestic cattle and wild ruminants, Bartonella bovis was recently defined as a cause of bovine endocarditis. The purpose of this study was to develop a B. bovis species-specific PCR assay that could be used to confirm the molecular prevalence of Bartonella spp. infection. Blood samples from 142 cattle were tested by conventional PCR targeting the Bartonella 16S–23S intergenic spacer (ITS) region. Overall, Bartonella DNA was detected in 82.4% (117/142) of the cattle using either Bartonella genus primers or B. bovis species-specific primers. Based upon size, 115 of the 117 Bartonella genus ITS PCR amplicons were consistent with B. bovis infection, which was confirmed by PCR using B. bovis species-specific primers and by sequencing three randomly selected, appropriately sized Bartonella genus PCR amplicons. By DNA sequencing, Bartonella henselae was confirmed as the two remaining amplicons, showing sequence similarity to B. henselae URBHLIE 9 ( AF312496) and B. henselae Houston 1 ( NC_005956), respectively. Following pre-enrichment blood culture of 12 samples in Bartonella alpha Proteobacteria growth medium (BAPGM) B. henselae infection was found in another three cows. Four of the five cows infected with B. henselae were co-infected with B. bovis. To our knowledge this study describes the first detection of B. henselae in any large ruminant species in the world and supports the need for further investigation of prevalence and pathogenic potential of B. henselae and B. bovis in cattle.

Identification of bacteria from clinical samples using Bartonella alpha-Proteobacteria growth medium

In an effort to overcome historical problems associated with the isolation of Bartonella species from animal and human blood samples, our laboratory developed a novel, chemically modified, insect-based, liquid culture medium ( Bartonella alpha-Proteobacteria growth medium, BAPGM). In this study, we describe the isolation of non- Bartonella bacteria from aseptically obtained human blood and tissue samples that were inoculated into BAPGM pre-enrichment culture medium, and were obtained during attempts to define each individuals Bartonella infection status. After incubation for at least 7 days in liquid BAPGM, pre-enriched inoculums were sub-cultured onto a BAPGM/blood agar plate. Bacterial DNA was extracted from pooled plated colonies and amplified using conventional PCR targeting the 16S rRNA gene. Subsequently, amplicons were cloned, sequenced and compared to GenBank database sequences using the BLAST program. Regardless of the patient's Bartonella status, seventeen samples generated only one 16S rDNA sequence, representing the following genera: Arthrobacter, Bacillus, Bartonella, Dermabacter, Methylobacterium, Propionibacterium, Pseudomonas, Staphylococcus and bacteria listed as “non-cultured” in the GenBank database. Alkalibacterium, Arthrobacter, Erwinia, Kineococcus, Methylobacterium, Propionibacterium, Sphingomonas, and Staphylococcus were isolated from nine Bartonella-infected individuals. Co-isolation of Acinetobacter, Sphingomonas, Staphylococcus spp. and bacteria listed as “non-cultured” in the GenBank database was achieved for four samples in which Bartonella spp. were not detected. Despite the phylogenetic limitations of using partial 16S rRNA gene sequencing for species and strain identification, the investigational methodology described in this study may provide a complementary approach for the isolation and identification of bacteria from patient samples.

A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: Pre-enrichment liquid culture followed by PCR and subculture onto agar plates

Historically, direct plating, lysis centrifugation, or freeze–thaw approaches have proven to be highly insensitive methods for confirming Bartonella species infection in dogs. A prospective study was designed to compare diagnostic methods for the detection of Bartonella using samples submitted to the Vector-Borne Disease Diagnostic Laboratory at North Carolina State University. Methods included indirect immunofluorescence assay, PCR, direct inoculation of a blood agar plate (trypticase soy agar with 5% rabbit blood), and inoculation into a novel pre-enrichment liquid medium, Bartonella/alpha- Proteobacteria growth medium (BAPGM). Sequential research efforts resulted in the development of a combinational approach consisting of pre-enrichment culture of Bartonella species in BAPGM, sub-inoculation of the liquid culture onto agar plates, followed by DNA amplification using PCR. The multi-faceted approach resulted in substantial improvement in the microbiological detection and isolation of Bartonella when compared to direct inoculation of a blood agar plate. Importantly, this approach facilitated the detection and subsequent isolation of both single and co-infections with two Bartonella species in the blood of naturally infected dogs. The use of a combinational approach of pre-enrichment culture and PCR may assist in the diagnostic confirmation of bartonellosis in dogs and other animals.