Cutting Edge: Redundant Roles for MHC Class II-, CD1d-, and MR1-restricted T Cells in Clearing Bartonella Infection.

The importance of unconventional T cells for mucosal immunity is firmly established but for systemic bacterial infection remains less well defined. In this study, we explored the role of various T cell subsets in murine Bartonella infection, which establishes persistent bacteremia unless controlled by antibacterial Abs. We found that αß T cells are essential for Ab production against and clearance of B. taylorii, whereas MHC class I (MHC-I)- or MHC class II (MHC-II)-deficient mice eliminated B. taylorii infection with normal kinetics. Similarly, animals lacking either CD1d or MR1 suppressed bacteremia with normal kinetics. Interestingly, mice with a combined deficiency of either MHC-II and CD1d or MHC-II and MR1 failed to clear the infection, indicating that the combination of CD1d- and MR1-restricted T cells can compensate for the lack of MHC-II in this model. Our data document a previously underappreciated contribution of unconventional T cells to the control of systemic bacterial infection, supposedly as helper cells for antibacterial Ab production.

Identification of the Bartonella autotransporter CFA as a protective antigen and hypervariable target of neutralizing antibodies in mice.

The bacterial genus Bartonella comprises numerous emerging pathogens that cause a broad spectrum of disease manifestations in humans. The targets and mechanisms of the anti-Bartonella immune defense are ill-defined and bacterial immune evasion strategies remain elusive. We found that experimentally infected mice resolved Bartonella infection by mounting antibody responses that neutralized the bacteria, preventing their attachment to erythrocytes and suppressing bacteremia independent of complement or Fc receptors. Bartonella-neutralizing antibody responses were rapidly induced and depended on CD40 signaling but not on affinity maturation. We cloned neutralizing monoclonal antibodies (mAbs) and by mass spectrometry identified the bacterial autotransporter CFA (CAMP-like factor autotransporter) as a neutralizing antibody target. Vaccination against CFA suppressed Bartonella bacteremia, validating CFA as a protective antigen. We mapped Bartonella-neutralizing mAb binding to a domain in CFA that we found is hypervariable in both human and mouse pathogenic strains, indicating mutational antibody evasion at the Bartonella subspecies level. These insights into Bartonella immunity and immune evasion provide a conceptual framework for vaccine development, identifying important challenges in this endeavor.

Prospective serological and molecular cross-sectional study focusing on Bartonella and other blood-borne organisms in cats from Catalonia (Spain).

BACKGROUND: There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors. METHODS: We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.). RESULTS: From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp. CONCLUSIONS: Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats.

Bartonella spp. seroprevalence in tick-exposed Swedish patients with persistent symptoms.

BACKGROUND: Bartonella spp. are emerging pathogens transmitted by arthropod vectors, possibly including ticks. We have investigated signs of bartonellosis in Swedish patients with presumed tick-bite exposure and symptom duration of at least 6 months. METHODS: Serological testing for Bartonella henselae and Bartonella quintana was performed in 224 patients. Symptoms, tick exposure, evidence of co-infection and previous treatments were evaluated. Seropositive patients were compared to a matched group (twofold larger and negative serology) from the same study cohort. RESULTS: Seroprevalence was 7% for B. henselae and 1% for B. quintana, with one patient testing positive to both agents. Tick bites were reported by 63% of the patients in the seropositive group and 88% in the seronegative group and presumed tick exposure was more common in the seronegative group. Animal contact was equally common in both groups, along with reported symptoms. The most common symptoms were fatigue, muscular symptoms, arthralgia and cognitive symptoms. Exposure to co-infections was evenly distributed in the seropositive and seronegative groups. CONCLUSIONS: Antibodies to Bartonella were more common in this cohort of patients than in cohorts of healthy Swedish blood donors in previous studies but lower than those in blood donors from southern Europe. Positive Bartonella serology was not linked to any specific symptom, nor to (suspected) tick-bite exposure.

Molecular and Serological Survey of the Cat-Scratch Disease Agent (Bartonella henselae) in Free-Ranging Leopardus geoffroyi and Leopardus wiedii (Carnivora: Felidae) From Pampa Biome, Brazil.

The genus Bartonella comprises emerging bacteria that affect humans and other mammals worldwide. Felids represent an important reservoir for several Bartonella species. Domestic cats are the main reservoir of Bartonella henselae, the agent of cat scratch disease (CSD). It can be transmitted directly by scratches and bites from infected cats and via cat fleas. This study aims to investigate the circulation of Bartonella spp. in free-ranging Neotropical wild felids from Southern Brazil using serological and molecular methods. In this study, 53 live-trapped free-ranging wild felids were sampled, 39 Leopardus geoffroyi and 14 Leopardus wiedii, from five municipalities in the Rio Grande, do Sul state, southern Brazil. All captured animals were clinically healthy. Two blood samples of L. geoffroyi were positive, by PCR, for the presence of B. henselae DNA. Conversely, none of L. wiedii blood samples were positive when tested using PCR. Indirect immunofluorescence assay (IFA) showed that 28% of serum samples of wild felids were reactive (seropositive) for B. henselae by immunofluorescence, with titers ranging from 64 to 256. The results presented here provide the first evidence of a Bartonella-enzootic cycle involving L. geoffroyi and L. wiedii, which may account for the spillover of the emerging zoonotic pathogen B. henselae for the indigenous fauna in Southern Brazil.

Molecular detection of vector-borne agents in cats in Southern Brazil / Detecção molecular de agentes transmitidos por vetores em felinos domésticos no Sul do Brasil

Abstract This study used serological and molecular methods to investigate the occurrence of vector-borne pathogens (VBP) with zoonotic potential in cats neutered at the University Veterinary Hospital in Canoinhas, Santa Catarina. The combined PCR and serological results revealed that 17 (56.6%) cats were positive for one or more pathogens. The sampled cats had antibodies to Ehrlichia spp. (7/30), Anaplasma phagocytophilum (3/30) and Leishmania infantum (2/30). The PCR assay detected DNA closely related to Ehrlichia canis in 6/30 cats, Mycoplasma haemofelis in 2/30 cats, A. phagocytophilum and Cytauxzoon sp. in one cat each. While Bartonella clarridgeiae and B. henselae were detected in two cats each, and B. koehlerae was detected in one cat.

Resumo Como os felinos podem ser parasitados por diversos patógenos transmitidos por vetores (PTV), alguns com caráter zoonótico, este estudo objetivou detectar por métodos sorológicos e moleculares, patógenos transmitidos por vetores hematófagos, em gatos atendidos em um Hospital Veterinário Universitário em Santa Catarina. Os resultados da PCR e da sorologia combinados, revelaram que 17 (56,6%) gatos foram positivos para um ou mais patógenos. Na sorologia, foram positivos 7/30 gatos para Ehrlichia, 3/30 para Anaplasma phagocytophilum e 2/30 para Leishmania infantum. Na PCR foi detectado DNA filogeneticamente associado a: Ehrlichia canis em 6/30 gatos; Mycoplasma haemofelis, em 2/30 gatos; A. phagocytophilum e Cytauxzoon sp. em 1/30 gatos cada. Enquanto Bartonella clarridgeiae e B. henselae foram detectadas, cada uma, em dois gatos, B. koehlerae foi detectada em um gato.

Molecular detection of vector-borne agents in cats in Southern Brazil.

This study used serological and molecular methods to investigate the occurrence of vector-borne pathogens (VBP) with zoonotic potential in cats neutered at the University Veterinary Hospital in Canoinhas, Santa Catarina. The combined PCR and serological results revealed that 17 (56.6%) cats were positive for one or more pathogens. The sampled cats had antibodies to Ehrlichia spp. (7/30), Anaplasma phagocytophilum (3/30) and Leishmania infantum (2/30). The PCR assay detected DNA closely related to Ehrlichia canis in 6/30 cats, Mycoplasma haemofelis in 2/30 cats, A. phagocytophilum and Cytauxzoon sp. in one cat each. While Bartonella clarridgeiae and B. henselae were detected in two cats each, and B. koehlerae was detected in one cat.

High Prevalence of Bartonella sp. in Dogs from Hamadan, Iran.

Bartonellae are emerging vector-borne pathogens infecting various domestic and wild mammals. Blood samples were collected from 66 dogs at two locations near Hamedan, Iran. Twenty dogs were rescued stray dogs and 46 dogs were from a breeding colony, with many of them infested with fleas, ticks, or lice. Serology was performed using an indirect immunofluorescent antibody test for Bartonella henselae, Bartonella clarridgeiae, and Bartonella vinsonii subsp. berkhoffii. Seroprevalence was 74.2% (range: 65.2-95%). Bartonella DNA amplification and sequencing identified B. vinsonii subsp. berkhoffii type III in seven dogs, including five rescued dogs. Two dogs were infected with Bartonella rochalimae and three dogs with Candidatus B. merieuxii, including two of the stray dogs coinfected with Bartonella vinsonii subsp. berkhoffii. Rescued stray dogs were 10 times (odds ratio (OR) = 10.13, 95% CI: 1.24-82.7; P = 0.03) more likely to be seropositive and eight times (OR = 8.82, 95% CI: 2.68-29.11; P = 0.0004) more likely to be flea-infested than breeding dogs, confirming that arthropod infestation is a major risk factor for these infections.

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.

Does co-infection with vector-borne pathogens play a role in clinical canine leishmaniosis?

BACKGROUND: The severity of canine leishmaniosis (CanL) due to Leishmania infantum might be affected by other vector-borne organisms that mimic its clinical signs and clinicopathological abnormalities. The aim of this study was to determine co-infections with other vector-borne pathogens based on serological and molecular techniques in dogs with clinical leishmaniosis living in Spain and to associate them with clinical signs and clinicopathological abnormalities as well as disease severity. METHODS: Sixty-one dogs with clinical leishmaniosis and 16 apparently healthy dogs were tested for Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum and Bartonella henselae antigens by the immunofluorescence antibody test (IFAT) and for E. canis, Anaplasma spp., Hepatozoon spp., Babesia spp. and filarioid DNA by polymerase chain reaction (PCR). RESULTS: Among the dogs examined by IFAT, the seroprevalences were: 69% for R. conorii, 57% for E. canis, 44% for A. phagocytophilum and 37% for B. henselae; while the prevalences found by PCR were: 8% for Ehrlichia/Anaplasma, 3% for Anaplasma platys and 1% for H. canis. No other pathogen DNA was detected. Statistical association was found between dogs with clinical leishmaniosis and seroreactivity to R. conorii antigen (Fisher's exact test: P = 0.025, OR = 4.1, 95% CI = 1-17) and A. phagocytophilum antigen (Fisher's exact test: P = 0.002, OR = 14.3, 95% CI = 2-626) and being positive to more than one serological or molecular tests (co-infections) (Mann-Whitney test: U = 243, Z = -2.6, n 1 = 14, n 2 = 61, P = 0.01) when compared with healthy dogs. Interestingly, a statistical association was found between the presence of R. conorii, E. canis, A. phagocytophilum and B. henselae antibodies in sick dogs and some clinicopathological abnormalities such as albumin and albumin/globulin ratio decrease and increase in serum globulins. Furthermore, seroreactivity with A. phagocytophilum antigens was statistically associated with CanL clinical stages III and IV. CONCLUSIONS: This study demonstrates that dogs with clinical leishmaniosis from Catalonia (Spain) have a higher rate of co-infections with other vector-borne pathogens when compared with healthy controls. Furthermore, positivity to some vector-borne pathogens was associated with more marked clinicopathological abnormalities as well as disease severity with CanL.

Clinical evaluation of outdoor cats exposed to ectoparasites and associated risk for vector-borne infections in southern Italy.

BACKGROUND: Cats can be carriers of infected arthropods and be infected with several vector-borne pathogens (VBP) but there is limited knowledge about their pathogenic role in cats. RESULTS: A cross-sectional controlled study investigated the clinical status and antibody (Bartonella henselae, Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum, Babesia microti and Leishmania infantum) and/or blood PCR (Mycoplasma spp., Bartonella spp., Rickettsia spp., Ehrlichia/Anaplasma spp., piroplasmids, L. infantum, Hepatozoon felis) prevalence in 197 cats. Outdoor cats lacking ectoparasiticide treatment or hosting ectoparasites (study group [SG], n = 134) and indoor cats treated against ectoparasites (control group [CG], n = 63) were enrolled. Clinical data and retroviral co-infections were compared between the two groups. Multivariable analysis tested associations between variables and VBP exposure. Lymphadenia, stomatitis, and various haematological abnormalities were statistically more frequent in SG. Antibodies against R. conorii, B. henselae, A. phagocytophylum, B. microti, E. canis and L. infantum were detected. Bartonella henselae, Bartonella clarridgeiae, Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum" and "Candidatus Mycoplasma turicensis" DNA were identified. Very high antibody (87.8%) and PCR (40.1%) positivity to at least one pathogen were detected and were significantly higher in SG. Co-infections were confirmed in about one-third of the cats and were more frequent in SG cats. Molecular and overall (antibody and PCR) positivity to Bartonella and antibody positivity to R. conorii were higher in SG. Multivariable analysis found significant associations of Bartonella spp. infection with Feline Immunodeficiency Virus (FIV) infection and increased globulins, and of Mycoplasma spp. infection with adult age, FIV infection, anaemia, and increased creatinine. CONCLUSIONS: A very high prevalence of exposure to zoonotic VBP was found in cats, with Rickettsia and Bartonella infections being most prevalent. Some risk factors were documented namely for Mycoplasma spp. and Bartonella spp. The lifestyle of cats is clinically relevant and requires specific preventative measures to protect their health.

Livestock abundance predicts vampire bat demography, immune profiles and bacterial infection risk.

Human activities create novel food resources that can alter wildlife-pathogen interactions. If resources amplify or dampen, pathogen transmission probably depends on both host ecology and pathogen biology, but studies that measure responses to provisioning across both scales are rare. We tested these relationships with a 4-year study of 369 common vampire bats across 10 sites in Peru and Belize that differ in the abundance of livestock, an important anthropogenic food source. We quantified innate and adaptive immunity from bats and assessed infection with two common bacteria. We predicted that abundant livestock could reduce starvation and foraging effort, allowing for greater investments in immunity. Bats from high-livestock sites had higher microbicidal activity and proportions of neutrophils but lower immunoglobulin G and proportions of lymphocytes, suggesting more investment in innate relative to adaptive immunity and either greater chronic stress or pathogen exposure. This relationship was most pronounced in reproductive bats, which were also more common in high-livestock sites, suggesting feedbacks between demographic correlates of provisioning and immunity. Infection with both Bartonella and haemoplasmas were correlated with similar immune profiles, and both pathogens tended to be less prevalent in high-livestock sites, although effects were weaker for haemoplasmas. These differing responses to provisioning might therefore reflect distinct transmission processes. Predicting how provisioning alters host-pathogen interactions requires considering how both within-host processes and transmission modes respond to resource shifts.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'.

Exposure to Rats and Rat-Associated Leptospira and Bartonella Species Among People Who Use Drugs in an Impoverished, Inner-City Neighborhood of Vancouver, Canada.

Rat infestations are common, particularly in impoverished, inner-city neighborhoods. However, there has been little research into the nature and consequences of rat exposure in these neighborhoods, particularly in Canada. In this study, we sought to characterize exposure to rats and rat-associated Leptospira interrogans and Bartonella tribocorum, as well as risk factors associated with exposure, in residents (n = 202) of the Downtown Eastside (DTES) neighborhood of Vancouver, Canada. There was no evidence of exposure to rat-associated L. interrogans but 6/202 (3.0%) of participants were exposed to B. tribocorum, which is known to be circulating among DTES rats. We also found that frequent and close rat exposure was common among DTES residents, and that this exposure was particularly associated with injection drug use and outdoor income-generating activities (e.g., drug dealing). These risk factors may be good targets for interventions geared toward effectively reducing rat exposure.

Chlamydiales, Anaplasma and Bartonella: persistence and immune escape of intracellular bacteria.

Intracellular bacteria, such as Chlamydiales, Anaplasma or Bartonella, need to persist inside their host in order to complete their developmental cycle and to infect new hosts. In order to escape from the host immune system, intracellular bacteria have developed diverse mechanisms of persistence, which can directly impact the health of their host.

Molecular Mechanisms of Bartonella and Mammalian Erythrocyte Interactions: A Review.

Bartonellosis is an infectious disease caused by Bartonella species that are distributed worldwide with animal and public health impact varying according to Bartonella species, infection phase, immunological characteristics, and geographical region. Bartonella is widely present in various mammals including cats, rodents, ruminants, and humans. At least 13 Bartonella species or subspecies are zoonotic. Each species has few reservoir animals in which it is often asymptomatic. Bartonella infection may lead to various clinical symptoms in humans. As described in the B.tribocorum-rat model, when Bartonella was seeded into the blood stream, they could escape immunity, adhered to and invaded host erythrocytes. They then replicated and persisted in the infected erythrocytes for several weeks. This review summarizes the current knowledge of how Bartonella prevent phagocytosis and complement activation, what pathogenesis factors are involved in erythrocyte adhesion and invasion, and how Bartonella could replicate and persist in mammalian erythrocytes. Current advances in research will help us to decipher molecular mechanisms of interactions between Bartonella and mammalian erythrocytes and may help in the development of biological strategies for the prevention and control of bartonellosis.

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

BACKGROUND: The 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. METHODS: Each 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. RESULTS: Among 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. CONCLUSIONS: High 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.

First description of Bartonella koehlerae infection in a Spanish dog with infective endocarditis.

BACKGROUND: Bartonella koehlerae has been recently described as a new cat- and cat fleas-associated agent of culture-negative human endocarditis. It has been also encountered in one dog from Israel and six dogs from the USA, but other clinically relevant reports involving this bacterium are lacking. RESULTS: A 7-year-old intact male mixed dog presented with clinico-pathological signs consistent with mitral endocarditis and cutaneous hemangiosarcoma. Molecular studies revealed the presence of Bartonella koehlerae DNA in samples from blood and mitral valve tissue. CONCLUSIONS: This is the first description of B. koehlerae in Spain, corroborating that it can also be detected in dogs. Bartonella koehlerae infection should also be considered in Spain in humans and dogs presenting with clinical disease suggestive of it, such as culture-negative endocarditis.