Álvarez‑Fernández et al. Parasites & Vectors https://doi.org/10.1186/s13071-021-05105-6 (2022) 15:6 Parasites & Vectors RESEARCH Open Access Prospective serological and molecular cross‑sectional study focusing on Bartonella and other blood‑borne organisms in cats from Catalonia (Spain) Alejandra Álvarez‑Fernández1, Ricardo Maggi2, Gerard Eduard Martín‑Valls3, Marta Baxarias1, Edward Bealmear Breitschwerdt2 and Laia Solano‑Gallego1* Abstract 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 associ‑ ated risk factors. Methods: We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quin- tana-, 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 quin- tana, 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., Hepa- tozoon, 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 *Correspondence: laia.solano@uab.cat 1 Department of Animal Medicine and Surgery, Facultat de Veterinària, Universitat Autònoma de Barcelona, Cerdanyola del Valles, Spain Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 2 of 14 detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowl‑ edge, this study is the first to document M. wenyonii is infection in cats. Keywords: Bartonellosis, Hemotropic Mycoplasma, Mycoplasma wenyonii, Ehrlichia, Anaplasma, Piroplasma, Co-infection, Cats, Spain Background result guarantees the absence of infection [3]. Under Bartonellosis, caused by Bartonella spp. parasites, is a this premise, infection can be confirmed only by positive vector-borne infectious disease that is currently consid- diagnostic test results derived from molecular modalities, ered an emerging zoonosis [1]. More than 40 Bartonella such as conventional (cPCR) or real-time PCR (qPCR), species that are adapted to infect a broad spectrum of ideally accompanied by DNA sequencing, or the isolation reservoir mammalian hosts, including cats, are described and identification of the bacteria by enrichment culture, in the literature [2, 3]. Transmission to cats is mainly rather than exposure [17, 18]. In addition to technical by flea feces, potentially ticks, and scratches and bites limitations inherent in culture and PCR diagnostic tech- between hosts. The cat has been described as the main niques, Bartonella may not be present in sufficient quan- reservoir for Bartonella henselae, Bartonella clarridgeiae tities in the blood at the time of specimen collection to be and Bartonella koehlerae [4]. However, cats can be spo- detected. As an example, Bartonella DNA was amplified radically infected with two other zoonotic Bartonella from fresh frozen tissues of dogs with hemangiosarcoma, species: Bartonella quintana [5, 6] and Bartonella vinso- where qPCR from blood failed to amplify bacterial DNA nii subsp. berkhoffii [7]. [19]. Thus, choosing the correct sample for culture or The spectrum of disease manifestations associated PCR testing could also be critical for the definitive diag- with Bartonella spp. infections in cats continues to nosis of bartonellosis [19]. expand [8], despite the fact that it is not easy to dem- Indirect immunofluorescence assays (IFA) are the onstrate an association between clinical signs, labora- most frequently used serological technique for the tory abnormalities, and Bartonella spp. infection [9, detection of antibodies against Bartonella spp. [20– 10]. This factor is primarily due to the long duration 23], but other serological assays are available, such as of relapsing bacteremia and the high percentage of enzyme-linked immunosorbent assay (ELISA) and infected healthy cats in endemic areas [3, 11]. Although western immunoblot [12, 24]. A serological negative the majority of acute infections caused by Bartonella result does not ensure that a cat is not infected with a spp. are thought to be self-limiting in cats [12], persis- Bartonella sp., and a positive result only documents the tent infections can be associated with a wide variety presence of antibodies against the pathogen, but does of clinical signs and abnormalities. These manifesta- not confirm infection. ‬ Although technically challeng- tions in cats can range from intra- or extra-erythrocytic ing and more expensive than selecting individual tests, subclinical bacteremia to fever of unknown origin, considering a combination of diagnostic techniques and lymphadenomegaly, endocarditis, myocarditis, ocu- optimal specimen types in conjunction with the correct lar disease (neuroretinitis, uveitis), skin inflammation, interpretation of the results is likely a good strategy to and other less common disease manifestations [13, increase ‬ ‬ ‬ ‬ ‬ diagnostic success in cats with suspected 14]. Various factors allow Bartonella spp. to persist in bartonellosis [3, 19].‬‬ ‬ the blood of hosts, causing a chronic intravascular and Bartonella spp. epidemiological studies involving endotheliotropic infection that can ultimately result cats, carried out in many parts of the world, have docu- in the appearance of nonspecific or specific clinical mented substantial differences in prevalence from one manifestations. Factors that influence symptomatology area to another [3, 25]. In Europe, many studies includ- include virulence differences among Bartonella spp. ing various cat populations have reported seroprevalence and strains, the mode of transmission, differences in ranging from 0 to 71.4% and molecular prevalence rates the host immune response and clinical status (comor- ranging from 0 to 83.5% [3, 26, 27]. In Europe, including bidities), concurrent infectious or noninfectious dis- Spain, clinical and epidemiological knowledge regard- eases, bacterial load, therapeutic- or infection-induced ing Bartonella infection in cats remains limited. Fur- immunosuppression, and malnutrition [15, 16]. thermore, there are no serological studies comparing Due to the abovementioned factors, establishing dis- the presence of antibodies against different Bartonella ease causation or a diagnosis of Bartonella spp. infections species in cats, and only a few molecular studies have can be clinically challenging, particularly in cats. There assayed bacteremia in cats [28, 29]. In addition, there are no available diagnostic techniques whose negative are limited feline Bartonella studies that investigated Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 3 of 14 co-infections with other vector-borne pathogens and the Teflon-coated slides (Cel-Line/Thermo Scientific), air- associated risk factors [29, 30]. The objective of this study dried, acetone-fixed, and stored frozen. Serum samples was to test cat serum samples for the presence of anti- were diluted in phosphate-buffered saline (PBS) solution bodies against B. henselae, B. quintana, and B. koehlerae containing 1% normal goat serum, 0.05% Tween 20, and antigens. To assess for co-infections, blood samples were 0.5% powdered nonfat dry milk (Bio-Rad, Hercules, CA, tested by PCR for Anaplasma spp., Bartonella spp., Ehr- USA) to block nonspecific antigen binding sites. Cat sera lichia spp., and hemotropic Mycoplasma spp. and using were further tested with twofold dilutions out to a final primers that amplified Babesia, Cytauxzoon, Hepatozoon, dilution of 1:16384, and 10 µl of each serum dilution was and Theileria spp. applied per well. Previously tested positive and negative controls were selected and added in each slide. The slides Methods were incubated for 30 min at 37 °C and then washed A prospective cross-sectional study, conducted between with PBS under moderate agitation for an additional 2017 and 2019 in cats from Barcelona province (Spain), 30 min. Once slides were dry, 10 µl of fluorescein-con- was designed to investigate Bartonella spp. seropreva- jugated goat anti-cat immunoglobulin G (IgG) (MP Bio- lence and bacteremia prevalence using qPCR and cPCR, medicals) at a dilution of 1:100 was added into each well. with all DNA amplicons sequenced to confirm species The slides were incubated for another 30 min at 37 °C identity. In addition, co-infection with other feline vec- in the dark to protect the photosensitive conjugate. The tor-borne pathogens was assessed by cPCR testing. washing procedure described above was repeated, add- ing a few drops of Tween 20 (Sigma-Aldrich). After the Cats and specimen collection last washing procedure, some drops of antifade mount- A total of 117 blood specimens from apparently healthy ing medium (Vectashield, Vector Labs, Burlingame, cats and 18 blood specimens from sick cats were col- CA, USA) were added on the cover slips. The slides lected by venipuncture. A general physical examination were evaluated using a fluorescence microscope (Leica was performed in all cats. A clinical questionnaire was DM6000 B; Leica Microsystems, Wetzlar, Germany) at completed for each cat including information about age, ×200 and ×400 magnification, and each well was com- sex, colony origin or owned pet, breed, weight, clinical pared to the fluorescence pattern seen in the positive history, travel history, health status (sick versus healthy), and negative controls. To avoid confusion with possible exposure to fleas and/or ticks or bites, and the use of aca- nonspecific binding found at low dilutions, a cutoff of ricide products. In most cases (n = 110), the blood speci- 1:64 was selected as a seroreactive antibody titer [31, 32]. mens were obtained under anesthesia during a neutering Antibody titer results were classified as low seroreactiv- procedure. A signed consent form was obtained from the ity from 1:64 to 1:512 and high seroreactivity > 1:512 for owners or by the colony origin person in charge of the comparative analyses [32]. cats. Approximately 8–10 ml of peripheral blood was col- lected by jugular or cephalic venipuncture from each cat Blood DNA purification at the time of enrollment. Two milliliters was collected EDTA tubes were centrifuged at 1300×g for 5 min. into an EDTA-anticoagulant tube for DNA extraction, Plasma was obtained for further studies and the cellu- and 4–6 ml was injected into serum separator tubes with lar pellet was used for DNA extraction. Total DNA was clot accelerator and granule serum separator for serology. extracted from blood cell pellets in 110 samples and Samples were subsequently stored at −80 °C until testing. from EDTA whole blood in 25 samples using the DNA Gene extraction kit (Sigma-Aldrich) following the Bartonella IFA serological testing manufacturer’s instructions, with slight modifications. For all 135 cats, three IFAs were used to detect serum Forty microliters of proteinase K solution was added to antibodies directed against three Bartonella species all samples. Four hundred microliters of whole blood (B. henselae, B. quintana, and B. koehlerae antigens) as was used for all sample DNA extractions. The other described previously [31, 32]. Briefly, to obtain antigens steps were performed as described previously [33]. for IFA testing, B. henselae SA2+ (feline origin Missy S Distilled water was used as a negative control for all 95 FO-099), B. quintana cynomolgus monkey origin DNA extractions. 11-MO-01 (China/Pfizer), and B. koehlerae (originally from blood of Trillium B, feline) were passed from agar- grown cultures of each organism into DH82 (a con- Conventional and quantitative real‑time PCR analysis tinuous canine histiocytic cell line) cultures. For each DNA extracted from each blood sample was screened for antigen, infected cell cultures were spotted onto 30-well the presence of Bartonella spp. using cPCR and qPCR, Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 4 of 14 and for Ehrlichia spp., Anaplasma spp., piroplasmids, 0.2 µl of 100 µM of each primer (Roche Diagnostics). and hemotropic Mycoplasma spp. DNA using only cPCR. PCR testing was carried out using 5 µl of DNA. PCR neg- The primers used for cPCR and qPCR to establish species ative and positive controls were prepared as described for strain identification by amplicon product size or melting cPCR. Real-time PCR conditions were as follows: a single temperature, respectively, are summarized in Table 1. hot-start cycle at 95 °C for 3 min followed by 44 cycles of denaturing at 94 °C for 10 s, 10 s of annealing at 66 °C, Conventional PCR analysis and extension at 72 °C for 10 s. When present, Bartonella reaction containing 12.5 µl of Taq-Ex® Premix (Fisher Amplification was performed in a 25-µl final volume spp. amplicons were detected by fluorescence readings at the appropriate wavelength [19]. Scientific), 0.2 µl of 100 µM of each forward and reverse primer (Roche Diagnostics, Basel, Switzerland) 7.1 µl of Sequencing of positive PCR amplicons distilled ­H2O, and 5 µl of DNA from each sample tested. The molecular characterization of Bartonella and hemo- PCR negative controls were prepared by adding distilled tropic Mycoplasma spp., as well as the confirmation ­H2O. Positive controls were used in each reaction and of cPCR and qPCR positive results, was performed by were prepared by serial dilution (using cat blood DNA) Sanger sequencing of cPCR and qPCR amplicons, fol- of genomic DNA from each pathogen down to 0.001 pg/ lowed by chromatographic evaluation and sequence μl (equivalent to 0.5 bacteria/μl). Conventional PCR was alignment. For bacterial species identification, DNA performed in a VeritiPro Thermal Cycler (Applied Bio- sequences were analyzed by comparing similarity with systems, Waltham, MA, USA) under the following condi- other sequenced bacteria deposited in the GenBank tions: a single hot-start cycle at 95 °C for 2 min followed database using the Basic Local Alignment Search Tool by 55 cycles of denaturing at 94 °C for 15 s, annealing at (BLAST). Only PCR results confirmed by sequencing 66 °C for 15 s, and extension at 72 °C for 18 s. Amplifi- were considered positive in this study. cation was completed by an additional cycle at 72 °C for 1 min, and products were analyzed by 1.5% agarose gel Statistical analysis electrophoresis with detection using ethidium bromide A descriptive analysis was carried out for the data from under ultraviolet light. each cat along with a comparative analysis according to the IFA and PCR results. Frequency analysis was per- Real‑time PCR analysis formed for age, sex, colony/owned, ectoparasites, health status, and three Bartonella spp. IFA, cPCR, and qPCR Dx Real-Time PCR instrument (Applied Biosystems™) in Bartonella spp. qPCR was performed using a 7500 Fast results. Comparative analysis of categorical data was per- 25-µl final volume reactions. Master mix was prepared formed using the Chi-square test or Fisher’s exact test. The kappa (κ) statistic was used to assess the degree of ID™ One-Step RT-PCR Reagents, Applied Biosystems™), using 12.5 µl of buffer and 1 µl of enzyme mix (AgPath- (inter-rater) agreement between IFAs performed against 5.9 µl of H2O, 0.2 µl of 100 µM of fluorescent probe, and three Bartonella species. The Shapiro–Wilk test was Table 1 Target gene employed, size in base pairs of amplicons and cPCR and qPCR primer sequences used Target gene Size in base Primer names and sequences References pairs (bp) Conventional PCR Bartonella spp. 500–680 bp BsppITS325s: 5′ CTT​CAG​ATG​ATG​ATC​CCA​AGC​C TT​TGGCG 3′ [19] 16S-23S ITS BsppITS1100as: 5′ GAA​CCG​ACG​ACC​CCC​TGC​T TG​CAA​AGCA 3′ Mycoplasma spp. (hemotropic group) 588 bp Myco16S-322 s 5′ GCC​CAT​ATT​CCT​ACG​GGA​AGC​AGC​AGT 3′ [19] 16S rRNA Myco16S-938as 5′ CTC​CAC​CAC​T TG​T TC​AGG​TCC​CCG​TC 3′ Anaplasma or Ehrlichia 420 bp GEPs 5′ CTG​GCG​GCA​AGC​Y TA​ACA​CAT​GCA​AGT​CGA​ACGGA 3′ [33] 16S rRNA GEPr 5′ CTT​C TT​C TR​TRG​GTA​CCG​TCA​T TA​TCT​TCC​CYA​Y TG​ 3′ Babesia, Cytauxzoon, Hepatozoon, and 663 bp Piro18S-144 s 5′ ACC​GTG​C TA​ATT​GTA​GGG​C TA​ATA​CA 3′ [19] Theileria spp. Piroplasma18S-722as 5′ GAA​TGC​CCC​CAA​CCG​T TC​C TA​T TA​AC 3′ 18S rRNA Real-time PCR Bartonella spp. 200–231 bp BsppITS325s: 5′ CTT​CAG​ATG​ATG​ATC​CCA​AGC​C TT​Y TG​GCG​ 3′ [34] 16S-23S ITS BsppITS543as: 5′ TAA​AYT​GGT​GGG​CCT​GGG​AGG​ACT​TG 3′ Probe BsppITS500: 5′ FAM-GTT​AGA​GCG​CGC​GC TTG​ATA​AG—IABkFQ 3′ Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 5 of 14 performed to detect the normality of the distribution of against one Bartonella species. Interestingly, all cats that the samples. A P-value < 0.05 was considered statistically were seroreactive against B. koehlerae or B. quintana significant. Statistical analyses were performed using the antigens were also seroreactive against B. henselae IFA R i386 version 3.3.1 (R Development Core Team) and the antigens. Five cats had antibodies against B. henselae but DeduceR version 1.7-16 (DeduceR: A data Analysis GUI were not seroreactive to the other two Bartonella spp. for R) software programs for Windows. Kappa agreement analysis between Bartonella spp. IFA is summarized in Table 2. Cats seroreactive against B. quin- Results tana and B. koehlerae antigens were more likely to be B. Cat description henselae-seroreactive (100%) than seronegative (Fisher’s All cats sampled were European domestic shorthairs. exact test: P < 0.001, OR = 262.9, 95% CI = 15.3–4515.9 Seventy-five of 135 cats (55.5%) were female and 60 and OR = 1081.6, 95% CI = 57.3–20,432.6). The serum (44.5%) were male. Only one female and two males IFA geometric mean antibody titer for B. henselae, B. were intact. All remaining cats were neutered. Cat age quintana, and B. koehlerae antigens was 1:2050, 1:1028, ranged from 4 months to 7 years, with a median age of and 1:1460, respectively. The maximum titer was 1:16384 1.2 years. Cats ≤ 2 years old were considered young cats for B. henselae and 1:8192 for B. quintana and B. koe- and > 2 years old were considered old cats. Fifty-eight hlerae. The frequency of seronegative, low seroreactive, percent (79/135) were colony cats and 41.5% (56/135) and high seroreactive antibody titers for the three Bar- were owned pet cats. A recent history of tick exposure or tonella antigens studied is graphically displayed in Fig. 1. flea infestation was reported in 81.5% (110/135), whereas When IFA results were compared with data collected 18.5% (25/135) were flea- or tick-infested at the time of from each cat, we found that colony cats were more likely blood collection, including 23 colony cats and two owned to be Bartonella spp. (100%)-seroreactive than owned pet cats. Colony cats were more likely to have a recent pet cats (53%) (Fisher’s exact test: P < 0.001, OR = 136.9, history of ectoparasites (100%) compared to owned cats 95% CI = 8–2319.8). Cats with a recent history of tick (3.6%) (Fisher’s exact test: P < 0.001, OR = 3466.2, 95% and flea infestation were more likely to be Bartonella confidence interval [CI] = 163.2–73,624.6). Routine spp. (99%)-seroreactive (Fisher’s exact test: P < 0.001, treatment with ectoparasiticides was reported for 18.5% OR = 5450, 95% CI = 177.8–167,014.7) when compared (25/135) of the cats. One hundred and seventeen cats with non-infested cats. Cats with ectoparasites were (86.7%) were clinically healthy, and 18 (13.3%) cats had more likely to be B. henselae-seroreactive (100%) (Fish- clinical signs at the time of sampling. Gingivitis was the er’s exact test: P = 0.001, OR = 15.5, 95% CI = 0.9–263.2), most frequent clinical sign, found in 55.6% (10 of 18, 95% B. koehlerae-seroreactive (100%) (Fisher’s exact test: CI = 32.6–78.5%) of the sick cats). P = 0.004, OR = 19.6, 95% CI = 1.2–332.5), and B. quin- tana-seroreactive (84%) (Fisher’s exact test: P = 0.05, Bartonella IFA in cat serum samples OR = 3, 95% CI = 1–9.4). Cats ≤ 2 years old were more Considering antibody titers ≥ 1:64, 80.7% (109 of 135, 95% likely to be Bartonella spp.-seroreactive (91.5%) than CI = 74.1–87.4%) of the cats were seroreactive against at cats > 2 years old (41.4%) (Chi-square test: χ2 = 36.8, least one Bartonella sp. antigen. Bartonella henselae, B. df = 1, P < 0.001). Of the 109 cats that were seroreactive quintana, and B. koehlerae seroreactivity was 80.7% (109 against any Bartonella species tested, 17 cats were sick of 135, 95% CI = 74.1–87.4%), 67.4% (91 of 135, 95% (15.6%, 95% CI = 8.8–22.4%), whereas the remaining cats CI = 59.5–75.3%), and 77% (104 of 135, 95% CI = 69.9– were clinically healthy. There was no statistical difference 84.1%), respectively. Eighty-eight of 109 cats (80.7%, associated with serology and health status. No further 95% CI = 73.3–88.1%) were seroreactive against all three significant associations were found. Bartonella spp. antigens, 32 cats (29.3%, 95% CI = 20.8– 37.9%) were seroreactive against two Bartonella species, and five cats (4.6%, 95% CI = 0.7–8.5%) were seroreactive Table 2 Comparison of agreement between IFAs performed against three Bartonella species Test pair κ ± SE κ ­interpretationa B. henselae IFA versus B. quintana IFA 0.661 ± 0.070 Substantial agreement B. henselae IFA versus B. koehlerae IFA 0.889 ± 0.048 Almost perfect agreement B. quintana IFA versus B. koehlerae IFA 0.726 ± 0.065 Substantial agreement a The interpretation for each κ value is shown in the final column according to the following scale: ≤ 0, no agreement; 0.01–0.20, none to slight; 0.21–0.40, fair; 0.41– 0.60, moderate; 0.61–0.80, substantial; and 0.81–1.00, almost perfect agreement. IFA, immunofluorescence antibody assay; κ, Cohen’s kappa value; SE, standard error Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 6 of 14 Fig. 1 Frequencies of negative, low seroreactive, and high seroreactive by means of IFA for the three Bartonella spp. antigens studied. Bartonella spp. antibody titers were classified as low seroreactive from 1:64 to 1:512 and high seroreactive > 512. The most prevalent group is boxed in pink for each Bartonella sp. PCR and sequencing and Genbank accession number are summarized in Bartonella spp. DNA was amplified from the blood of Tables 3, 4, and 5. 11.9% (16 of 135, 95% CI = 6.4–17.3%) of the cats using Based upon PCR results, 29 of 135 cats (21.5%, 95% cPCR or qPCR. Based on DNA sequence alignments, CI = 14.6–28.4%) were infected with at least one bac- cats were infected with B. henselae (n = 12), B. clar- terial organism. Seven cats (5.2%, 95%; CI = 1.4–8.9%) ridgeiae (n = 3), or B. koehlerae (n = 1). From 16 posi- were co-infected with two pathogens based on molecu- tive cats, Bartonella DNA was amplified using cPCR lar assays. Bartonella spp.-infected PCR-positive cats in 10 cases (Table 3) and using qPCR in eight cases were more likely to be infected with hemotropic Myco- (Table 4). Bartonella DNA was amplified by both cPCR plasma spp. (37.5%) than Bartonella spp. PCR-negative and qPCR in only two of 16 cats. Based on PCR test- cats (10.9%) (Fisher’s exact test: P = 0.0115, OR = 4.89, ing, 81.3% (95% CI = 62.1–100.4%) of bacteremic cats 95% CI = 1.5–15.7). originated from a colony and were ≤ 2 years of age. Of Detailed clinical, serological, and PCR data for infected the 16 Bartonella spp.-infected PCR-positive cats, 15 cats is summarized in Table 6. Two cats had gingivitis were Bartonella spp.-seroreactive. Fourteen out of 15 and one a purulent nasal discharge. There were no sta- Bartonella-seroreactive and PCR-positive cats had IFA tistical associations when hemotropic Mycoplasma PCR antibody titers ≥ 1:1024 to one or more test antigens. results were compared with categorical clinical data or Hemotropic Mycoplasma spp. DNA was amplified Bartonella spp. IFA serology results. by cPCR from 14% (19/135, 95% CI = 8.2–19.9%) of the cats. Based on DNA sequencing, Mycoplasma hae- Discussion mofelis (n = 8), Candidatus M. haemominutum (n = 6), The Bartonella spp. seroprevalence in this study was Candidatus M. turicensis (n = 4), and Mycoplasma 80.7%. Similar seroprevalence results (71.4%) were wenyonii (n = 1) were identified. Anaplasma spp., reported in a study published in 2006 involving cats from Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and the Barcelona area [31]. There has seemingly been little Theileria spp. were not detected in any of the samples progress in preventing flea exposure among colony/stray studied. and owned pet cats in this region. Despite the high Bar- For all DNA amplified by PCR, sequences were com- tonella spp. seroprevalence, 84.4% of the seroreactive cats pared with sequences available in the GenBank data- were apparently healthy. Gingivitis was the most frequent base by nucleotide sequence homology using BLAST. clinical finding among the sick cats. In addition, clinical Percentage of similarity, species, sequence description, abnormalities observed in Bartonella spp.-infected cats in this study were mild, highlighting the historically long Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 7 of 14 Table 3 Bartonella spp. 16S-23S internal transcribed spacer sequences obtained by cPCR (primers Bspp. 325–1100) compared with sequences available in the GenBank database by nucleotide sequence homology using BLAST ID GenBank accession no. Bartonella spp. Homology % (bp) Compared sequence GenBank accession no. GUAB-14 OK624784 B. henselae 100% (409/409) JQ316963.1 GUAB-27 OK624785 B. henselae 99.45% (364/366) EF209013.1 GUAB-35 OK624786 B. henselae 100% (409/409) JQ316963.1 GUAB-59 OK624787 B. clarridgeiae 99.77% (429/430) AB896695.1 GUAB-60 OK624788 B. henselae 99.96% (585/587) DQ529247.1 GUAB-75 OK624789 B. henselae 100% (458/458) KY464065.1 GUAB-77 OK624790 B. henselae 100% (393/393) HM042285.1 GUAB-78 OK624791 B. clarridgeiae 100% (525/525) AB896695.1 GUAB-79 OK624792 B. henselae 100% (365/365) DQ383228.1 GUAB-105 OK624793 B. clarridgeiae 100% (509/509) MN170544.1 ID identification, % percentage, bp base pairs Table 4 Bartonella spp. 16S-23S internal transcribed spacer sequences obtained by qPCR (primers Bspp 325–543) compared with sequences available in the GenBank database by nucleotide sequence homology using BLAST ID GenBank accession no. Species Homology % (bp) Compared sequence GenBank accession no. GUAB-14 OK624794 B. henselae 99% (148/149) AB896699.1 GUAB-35 OK624795 B. henselae 100% (140/140) MT095053.1 GUAB-39 OK624796 B. henselae 100% (148/148) AB896699.1 GUAB-41 OK624797 B. henselae 99% (167/168) MT095054.1 GUAB-45 OK624798 B. henselae 100% (151/151) MT095050.1 GUAB-46 OK624799 B. henselae 99% (148/149) MT095048.1 GUAB-49 OK624800 B. henselae 100% (144/144) MT095053.1 GC-13 OK624801 B. koehlerae 94% (120/127) KX499345.1 ID identification, % percentage, bp base pairs and apparently efficient co-evolution of fleas, cats, and against B. henselae (≥ 1:1024), compared with a previous Bartonella spp., as well as the poor relationship between study from the same area carried out 17 years earlier that Bartonella seroreactivity and clinical status. In the pre- reported 23% seroreactivity, with maximum antibody sent study, seroprevalence in owned and colony cats was titers of 1:512 [37]. Interestingly, the geometric mean 53% and 100%, respectively. However, other studies per- antibody titer in the present study was 1:2050, com- formed in the Barcelona area found seroprevalence of pared to 1:256 in the previous study, which may reflect 29.6% in owned pet cats [36] and 44% in shelter or colony age-related differences in seroreactivity [31]. The mean cats when cats in both studies were tested only with a B. age of cats in the present study and in the previous study henselae assay [28]. Seroprevalence reported in cats from was 1.2 and 4.4 years, respectively, suggesting that young other areas of Spain has ranged from 24.7% in the Madrid cats may have higher antibody titers than older cats. In area [29] to 50% described in a study that included sev- Europe, similar seroprevalence has been reported in Por- eral locations within the Mediterranean areas of Spain tugal (64.9%) against Bartonella spp. [38] and in Ger- (Canary Islands, Galicia, and Madrid) [37]. many (68.7%) against B. henselae antigen [39]. Variations To the best of our knowledge, this is the first study between studies are likely due to differences in the study measuring antibodies against three different species populations (e.g., owned versus colony) and differences of Bartonella and the first evidence of B. quintana and in Bartonella spp. prevalence among geographic areas, B. koehlerae seroreactivity in cats from Spain. In this study, 49.6% of seroreactive cats had high antibody titers Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 8 of 14 Table 5 Hemotropic Mycoplasma spp. 16S-23S internal transcribed spacer sequences obtained by cPCR compared with sequences available in the GenBank database by nucleotide sequence homology using BLAST ID GenBank accession no. Hemotropic Mycoplasma spp. Homology % Compared sequence (bp) GenBank accession no. G-UAB-27 OK624802 M. haemofelis 93% (466/499) MN240855.1 G-UAB-30 OK624803 M. haemofelis 99% (508/509) EU442638.1 G-UAB-37 OK624804 M. haemofelis 100% (526/526) KU645929.1 G-UAB-40 OK624805 Candidatus M. haemominutum 95% (533/560) KR905456.1 G-UAB-48 OK624806 M. haemofelis 95% (496/524) MG594502.1 G-UAB-49 OK624807 Candidatus M. haemominutum 100% (547/547) MT926039.1 G-UAB-58 OK624808 Candidatus M. haemominutum 100% (508/508) MN240865.1 G-UAB-60 OK624809 M. haemofelis 99% (529/530) KU645929.1 G-UAB-63 OK624810 Candidatus M. haemominutum 99% (471/472) KM275257.1 G-UAB-67 OK624811 Candidatus M. turicensis 97% (499/512) KY046312.1 G-UAB-70 OK624812 M. haemofelis 100% (515/515) KU645929.1 G-UAB-77 OK624813 M. wenyonii 99% (539/546) MT241310.1 G-UAB-78 OK624814 Candidatus M. turicensis 100% (480/480) MK632342.1 G-UAB-79 OK624815 Candidatus M. turicensis 99% (461/466) MK632342.1 G-UAB-88 OK624816 Candidatus M. haemominutum 100% (534/534) MT926039.1 G-UAB-94 OK624817 Candidatus M. haemominutum 100% (546/546) KR905457.1 G-UAB-109 OK624818 M. haemofelis 100% (31/31) MK632350.1 G-UAB-112 OK624819 M. haemofelis 99% (529/530) KU645929.1 G-UAB-115 OK624820 Candidatus M. turicensis 99% (467/468) EU789558.1 ID identification, % percentage, bp base pairs differences in flea exposure, and differences in diagnostic the three species of Bartonella studied. Furthermore, the techniques used among published studies [29, 40]. three cats infected with B. clarridgeiae had high anti- In this study, all Bartonella spp.-seroreactive cats had body titers against B. henselae (1:2048–1:8192), B. quin- previously been exposed to fleas and/or ticks, in agree- tana (1:256–1:1024), and B. koehlerae (1:512–1:2048). ment with the well-known relationship between exposure The present findings suggest serological cross-reactivity, to fleas and the transmission of Bartonella spp. infection as previously reported in cats [49, 50], or that cats were in cats [41–44]. Although Bartonella transmission by co-infected with several Bartonella spp. when infested ticks remains controversial, there is enough evidence (i.e., with numerous fleas or both fleas and ticks. In this study, artificial membrane feeding system experiments, clinical increasing the number of antigens by adding B. quintana and epidemiological studies in cats, dogs, and humans) to and B. koehlerae to B. henselae did not increase the num- consider that Ixodes ricinus ticks can act as a vector for B. ber of seroreactive cats. When experimentally infected henselae [45–48]. The presence of Bartonella spp. DNA with B. henselae or B. vinsonii subsp. berkhoffii, dogs has also been documented in Rhipicephalus sanguineus naturally infected with B. koehlerae elicited a species- ticks removed from cats and dogs [44, 49, 50]. However, specific antibody response for the first 100 days of the less information is available regarding the potential role study [57]. Differentiating cross-reactivity from co-expo- of R. sanguineus ticks or other tick species as potential sures to multiple Bartonella spp., potentially occurring vectors for Bartonella spp. transmission to cats, dogs, or at multiple vector exposure time points among naturally humans [44, 51]. exposed animals, often infested with numerous fleas and Several studies have assessed serological cross-reactiv- ticks, remains difficult to impossible with current diag- ity between Bartonella species or strains in cats [52, 53], nostic modalities. dogs [54], and humans [55, 56]. In our study, 65.2% of The majority of young cats ≤ 2 years were Bartonella the cats examined had antibodies against all three Bar- spp.-seroreactive. Furthermore, 14 of 16 PCR-seroreac- tonella spp. IFA antigens. All cats that were seroreactive tive cats were ≤ 2 years of age, although compared to cats against B. koehlerae or B. quintana antigens were also older than 2 years, there was no significant difference. seroreactive against B. henselae antigen. In addition, sub- In agreement with previous studies, young cats were stantial to almost perfect agreement was found between more likely to be bacteremic and seroreactive [58, 59]. Table 6 Bacteremic PCR-positive cats and the corresponding IFA results for each of the three Bartonella spp. IFA antigens Cat ID Lifestyle Age (months) Clinical signs Ectoparasites IFA cPCR Bartonella qPCR Bartonella cPCR hemotropic Mycoplasma Álvarez‑Fernández et al. Parasites & Vectors B. henselae B. quintana B. koehlerae GUAB-14 O 24 AH Np 1:1024 1:256 1:1024 B. henselae B. henselae Neg GUAB-27 C 8 AH Np 1:512 1:512 1:512 B. henselae Neg M. haemofelis (2022) 15:6 GUAB-35 C 6 Gingivitis Np 1:512 1:4096 1:2048 B. henselae B. henselae Neg GUAB-39 C 18 AH Otodectes cynotis 1:4096 1:1024 1:4096 Neg B. henselae Neg GUAB-41 C 6 AH Np 1:1024 1:4096 1:1024 Neg B. henselae Neg GUAB-45 C 24 Purulent nasal discharge Fleas 1:4096 1:2048 1:2048 Neg B. henselae Neg GUAB-46 C 18 AH Np 1:4096 1:1024 1:1024 Neg B. henselae Neg GUAB-49 C 8 AH Np 1:4096 1:2048 1:2048 Neg B. henselae Candidatus M. haemominutum GUAB-59 C 24 AH Fleas 1:2048 1:256 1:512 B. clarridgeiae Neg Neg GUAB-60 C 36 AH Fleas 1:2048 1:1024 1:2048 B. henselae Neg M. haemofelis GUAB-75 C 5 AH Np 1:1024 1:1024 1:8192 B. henselae Neg Neg GUAB-77 C 6 AH Np 1:2048 1:1024 1:2048 B. henselae Neg M. wenyonii GUAB-78 C 6 AH Np 1:8192 1:1024 1:2048 B. clarridgeiae Neg Candidatus M. turicensis GUAB-79 C 6 AH Np 1:8192 1:256 1:2048 B. henselae Neg Candidatus M. turicensis GUAB-105 C 10 Gingivitis Fleas 1:4096 1:512 1:512 B. clarridgeiae Neg Neg GC-13 O 38 AH Np Neg Neg Neg Neg B. koehlerae Neg O owned, C colony, AH apparently healthy, Np not present, Neg Negative, cPCR conventional polymerase chain reaction, qPCR real-time polymerase chain reaction Page 9 of 14 Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 10 of 14 Moreover, in the present study, all cats with Bartonella [53]. Therefore, we might hypothesize that this cat was spp. antibody titers ranging from 1:512 to 1:8192 were recently infected or did not seroconvert despite being bacteremic, which is consistent with a previous study chronically infected, a phenomenon commonly described [58] and suggests that antibodies do not elicit effective in bacteremic dogs [84]. Furthermore, it is important immune elimination of bacteremia. ‬ ‬ to highlight that in the present study, B. koehlerae was Bartonella spp. DNA was detected in 11.9% of the 94.49% identical to the B. koehlerae subsp. bothieri strain cats in this study. The most prevalent Bartonella species 1178 16S-23S ribosomal RNA intergenic spacer partial based on molecular analysis was B. henselae (8.9%), also sequence as described previously [85], and therefore, this consistent with worldwide PCR data [3], followed by B. microorganism might be a new B. koehlerae strain or a clarridgeiae (1.5%) and B. koehlerae (0.7%). The preva- closely related Bartonella species. For this reason, dif- lence for B. henselae bacteremia has ranged from 0.3 ferences in antigenicity among B. koehlerae strains could to 38.3% in different areas of Spain [28, 29, 36, 60, 61]. make detection of antibodies in infected cats more diffi- Bartonella clarridgeiae infections have previously been cult [85]. reported in the Barcelona area, with similar bacteremia Although B. quintana seroprevalence was documented results ranging from 0.6 to 1% [31, 60]. A higher B. clar- in this study, B. quintana has not previously been ampli- ridgeiae bacteremia prevalence (10.9%) was reported fied from the blood of any cat in Spain. To the best of in northern Spain [62]. In Europe, bacteremic preva- our knowledge, there is no previous molecular evidence lence rates for various combinations of B. clarridgeiae, documenting B. quintana infection in cats in Spain; B. henselae, and B. koehlerae have ranged from 0.7 to however, B. quintana has been previously documented 83.5% [27, 63–65]. The differences in bacteremic preva- in sick humans [86, 87] and healthy humans in Spain lence are related to different climatic conditions, the cat [22, 88]. Nevertheless, B. quintana infection was docu- population tested, and PCR techniques used, as well as mented from cat dental pulp in France [5] and from two intrinsic differences in bacteremic behavior linked to bacteremic feral cats in the USA [6]. Failure to isolate the microorganisms themselves. For example, it is possi- or PCR-amplify B. quintana DNA from cats may reflect ble that cats infected with B. koehlerae maintain a lower infrequent or low-level bacteremia, potentially requir- level of bacteremia than cats infected with B. henselae or ing enrichment blood culture/PCR or testing at multiple B. clarridgeiae, as B. koehlerae is infrequently isolated or time points to document potential B. quintana infection. detected by PCR testing [3, 66]. Despite this low bactere- Previous research has confirmed the ability of five Bar- mic prevalence, cats are considered the main reservoir of tonella species including B. quintana to persist in C. felis B. koehlerae infection worldwide [67–69]. [43], and B. quintana DNA has been amplified from cat To the best of our knowledge, this is the first descrip- fleas in France [80]. Therefore, fleas could be a potential tion of B. koehlerae infection in an apparently healthy cat alternative vector for B. quintana, although flea transmis- from Spain, as has previously been reported in Greece sion has not yet been proven. [65], France [70], Israel [71], and the USA [67, 72]. More- In the present study, the hemotropic Mycoplasma over, B. koehlerae infections have also been described infection rate was 14%, including the amplification and in dogs from Israel [73], Spain [35, 74], and the USA sequencing of M. haemofelis (5.9%), Candidatus M. [75], and infection has been diagnosed sporadically in haemominutum (4.4%), and Candidatus M. turicensis humans from Israel [76] and the USA [77, 78]. Interest- (3%) DNA. Frequency and species distribution are simi- ingly, B. koehlerae infection was not diagnosed in a large lar and in agreement with previous studies from Spain, study in veterinary personnel from Spain, despite 41.6% where hemotropic Mycoplasma prevalence of 7.8% [28] of participants being B. koehlerae-seroreactive, using the and 12% [69] was reported from the Barcelona area and same antigen as used in this study [22]. However, there 10.6% from Madrid [89]. Similar prevalence and species is more worldwide evidence of B. koehlerae DNA in cat distribution have also been documented in Italy (11.6% fleas, with prevalence exceeding 30% [79–83], suggesting and 18.3%) [90, 91] and Ireland (16.4%) [92]. However, that B. koehlerae infections in cats are potentially more higher hemotropic Mycoplasma prevalence has been common than studies have documented thus far [3]. In reported from Greece (26.4%) [30] and Portugal (27.1%) the present study, the B. koehlerae DNA-positive cat was [93]. Therefore, hemotropic Mycoplasma spp. are widely seronegative against all three test antigens. In an experi- distributed and prevalent among European cats. Further- mental study, seroconversion was detected between of 7 more, co-infection with hemotropic Mycoplasma and to 15 days after B. koehlerae inoculation in cats [53]. The Bartonella spp. appears to be highly common among cats infected in that study had serological cross-reactiv- cats in the Barcelona area. Bartonella spp. and hemotro- ity with B. henselae antigens, but antibody titers against pic Mycoplasma spp. co-infection prevalence was 4.4% B. henselae were lower than B. koehlerae antibody titers in this study, compared with other cat studies from Italy Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 11 of 14 that reported co-infection prevalence of 3% [91] and Conclusion 0.1% [90]. However, Bartonella and hemotropic Myco- In conclusion, Bartonella spp. and hemotropic Myco- plasma co-infection has not been documented in other plasma infections were found to be prevalent in cats feline [65] or canine [94] studies. Interestingly, Bartonella residing in the Barcelona area, whereas no infection spp. and hemotropic Mycoplasma spp. co-infection has with Anaplasma, Ehrlichia, or Piroplasma species was been sporadically reported in humans [95, 96]. It remains detected by PCR testing. We report, for the first time, B. unknown whether there are factors that predispose cats koehlerae infection in one apparently healthy cat in Spain. or humans to co-infection with Bartonella spp. and Co-infection with hemotropic Mycoplasma appears to be hemotropic Mycoplasma spp.; therefore, further studies common in Bartonella-infected cats. Of note, M. wenyo- are needed to elucidate these findings. nii infection in cats is documented herein for the first Surprisingly, and to the best of our knowledge, this is time. Serological testing with the three Bartonella spp. the first time that M. wenyonii DNA has been amplified antigens used in this study did not increase the overall from the blood of a cat worldwide and the first descrip- seroprevalence as compared to IFA testing using only the tion of this hemoplasma in an animal from Spain. Myco- B. henselae antigen. This and previous reports highlight plasma wenyonii has been widely identified in bovines the importance of combining serological and molecular worldwide [97–99] but also sporadically documented in diagnostic methods for the detection of Bartonella spp. other ungulates such as water buffalo and red deer from infection. Future studies should focus on risk factors for central Europe [100], France [101], and Cuba [102]. The acquiring co-infections with different Bartonella spe- mode of transmission for the majority of hemotropic cies and subspecies and whether co-infections might Mycoplasma species, including M. wenyonii, remains influence the clinical status and diagnosis of bartonello- unknown [101]. One study reported potential vertical sis in cats. In addition, investigations are needed to bet- transmission for M. wenyonii. in bovines [103]. Inter- ter characterize the humoral immune response against estingly, M. wenyonii DNA was confirmed in one wild- Bartonella spp. to facilitate a better understanding of caught mosquito pool by DNA sequencing in the USA the immunological response to these bacteria in healthy [104]. Those mosquitos were captured near feral cat and sick cats. Based upon our serology and PCR results, colonies to specifically evaluate wild-caught mosquitoes the risk of Bartonella and hemotropic Mycoplasma spp. for evidence of hemotropic Mycoplasma species DNA transmission among cats, humans, and other animals in and to determine whether the feline hemoplasmas could the Barcelona area may be substantial. be transmitted by Aedes aegypti mosquitoes in a labora- tory setting. Laboratory transmission to naive cats was Abbreviations not documented, suggesting that this mosquito is not a Bp: Base pair; DNA: Deoxyribonucleic acid; EDTA: Ethylenediaminetetraacetic biologically competent vector [104]. Further studies are acid; ELISA: Enzyme-linked immunosorbent assay; IFA: Indirect immunofluo‑ needed to evaluate whether cats are infected with M. rescence assay; ITS: Internal transcribed spacer; κ: Cohen’s kappa value; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; cPCR: Conven‑ wenyonii accidentally or more commonly in areas where tional polymerase chain reaction; qPCR: Real time polymerase chain reaction; large ungulates such cows and cats might have close rRNA: Ribosomal ribonucleic acid; SE: Standard error. contact. Acknowledgements Anaplasma and Ehrlichia spp. DNA was not detected The authors thank all veterinarians and students who contributed to this in this study. A low PCR prevalence (1%) for these two study. In particular, we are grateful to Toni Richarson and Henry Marr (Intracel‑ tick-transmitted genera was previously reported in the lular Pathogens Research Laboratory College of Veterinary Medicine, North Carolina State University, NCSU), Diana Andrea Murillo, David Prandi, Iciar Barcelona area [60]. In contrast, high molecular preva- Martínez and Lourdes Alarcón (Universitat Autònoma de Barcelona, UAB) for lence of Ehrlichia canis (9.9%) and Anaplasma phagocyt- the assistance with sampling and laboratory work. ophilum (8.4%) was described in cats from Madrid, Spain Authors’ contributions [29]. Anaplasma and Ehrlichia infections in cats are rare LS and AA designed the research study. LS, RM and GM supervised technical or sporadically documented worldwide [90, 105, 106], in work. AA, LS and MB contributed with data analysis and interpretation. AA agreement with the present study. Furthermore, Babesia, wrote the manuscript. LS and EB revised the manuscript. AA coordinated enrolment of cats, sampling and performed IFAs and PCRs. All authors read Cytauxzoon, and Hepatozoon DNA was also not detected and approved the final manuscript. in the present study. These tick-associated protozoan infections in cats appear to be rare, and have only been Funding This research was funded by Bayer Animal Health/Elanco. Publication of this sporadically documented in cats from Mediterranean manuscript has been sponsored by Elanco. regions [107–109], as was found in the present study. Álvarez‑Fernández et al. Parasites & Vectors (2022) 15:6 Page 12 of 14 Availability of data and materials 11. Pennisi MG, Marsilio F, Hartmann K, Lloret A, Addie D, Belák S, et al. The datasets supporting the conclusions of this article are included within the Bartonella species infection in cats: ABCD guidelines on prevention and article. All analyzed data are available from the corresponding author upon management. J Feline Med Surg. 2013;15:563–9. reasonable request. Representative sequences generated in this study were 12. Lappin MR, Breitschwerdt E, Brewer M, Hawley J, Hegarty B, Radecki submitted to the GenBank under accession numbers OK624784-OK624820. S. Prevalence of Bartonella species antibodies and Bartonella species DNA in the blood of cats with and without fever. J Feline Med Surg. 2009;11:141–8. Declarations 13. Chomel BB, Wey AC, Kasten RW, Stacy BA, Labelle P. Fatal case of endocarditis associated with Bartonella henselae type I infection in a Ethics approval domestic cat. J Clin Microbiol. 2003;41:5337–9. This study was conducted according to the guidelines of the Declaration of 14. Varanat M, Broadhurst J, Linder KE, Maggi RG, Breitschwerdt EB. Helsinki, but ethical review and approval were waived for the study, due to Identification of Bartonella henselae in 2 cats with pyogranulomatous non-invasive procedures and use of residual specimens. Furthermore, a signed myocarditis and diaphragmatic myositis. Vet Pathol. 2012;49:608–11. consent form was obtained from the owner or the person in charge of the 15. Cheslock MA, Embers ME, Cheslock MA, Embers ME. Human bartonel‑ cats. losis: an underappreciated public health problem? Trop Med Infect Dis. 2019;4:69. Competing interests 16. Breitschwerdt EB. Bartonellosis, one health and all creatures great and The authors declare no conflict of interest. Edward B. Breitschwerdt is a small. Vet Dermatol. 2017;28:96-e21. founder, shareholder and chief scientific officer for Galaxy Diagnostics, 17. La SB, Davoust B, Boni M, Raoult D. Lack of correlation between Research Triangle Park, North Carolina, USA. Galaxy Diagnostics was not Bartonella DNA detection within fleas, serological results, and results of involved in any aspect of this study. The funders had no role in the design of blood culture in a Bartonella-infected stray cat population. 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