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Abstract 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.

Case summary A 10-year-old domestic shorthair cat presented for lethargy, anorexia and labored breathing. Significant pleural and pericardial effusions prompted thoracocentesis and pericardiocentesis. Cytologic evaluation of the pericardial effusion revealed a highly cellular hemorrhagic, eosinophilic (12%) effusion, with many markedly atypical suspected mesothelial cells, interpreted as concerning for neoplasia. Thoracoscopic subtotal pericardiectomy and histology of the pericardium revealed predominantly eosinophilic inflammation with multifocal mesothelial hypertrophy and ulceration. A peripheral eosinophilia was not present on serial complete blood counts. Initial infectious disease testing was mostly negative. Toxoplasma gondii titers were most consistent with prior exposure, although reactivation could not be excluded. The owner’s medical history included a prior diagnosis of bartonellosis. Owing to the challenges of definitive Bartonella species exclusion, the cat was treated empirically with pradofloxacin and doxycycline, and a subtotal pericardectomy. There was improvement at first but pleural effusion recurred approximately 3 months after discharge. The cat was euthanized and a necropsy was not performed. Subsequent pericardial effusion Piroplasma/Bartonella/Borrelia droplet digital PCR detected DNA of Bartonella vinsonii subspecies berkhoffii , and peripheral blood culture and sequencing revealed a rare apicomplexan organism (90% homology with Colpodella species) of unknown clinical significance. Testing for filamentous bacteria and fungal pathogens was not performed. Relevance and novel information This case offers several unique entities – eosinophilic pericardial effusion and eosinophilic pericarditis of unknown etiology – and illustrates the well-known marked atypia that may occur in reactive and hyperplastic mesothelial cells, particularly of infrequently sampled and cytologically described feline pericardial effusion, supporting a cautious interpretation of this cytology finding.

Abstract Background Ctenocephalides felis, the cat flea, is the most common ectoparasite of cats and dogs worldwide. As a cause of flea allergy dermatitis and a vector for two genera of zoonotic pathogens (Bartonella and Rickettsia spp.), the effect of the C. felis microbiome on pathogen transmission and vector survival is of substantial medical importance to both human and veterinary medicine. The aim of this study was to assay the pathogenic and commensal eubacterial microbial communities of individual C. felis from multiple geographic locations and analyze these findings by location, qPCR pathogen prevalence, and flea genetic diversity. Methods 16S Next Generation Sequencing (NGS) was utilized to sequence the microbiome of fleas collected from free-roaming cats, and the cox1 gene was used for flea phylogenetic analysis. NGS data were analyzed for 168 individual fleas from seven locations within the US and UK. Given inconsistency in the genera historically reported to constitute the C. felis microbiome, we utilized the decontam prevalence method followed by literature review to separate contaminants from true microbiome members. Results NGS identified a single dominant and cosmopolitan amplicon sequence variant (ASV) from Rickettsia and Wolbachia while identifying one dominant Bartonella clarridgeiae and one dominant Bartonella henselae/Bartonella koehlerae ASV. Multiple less common ASVs from these genera were detected within restricted geographical ranges. Co-detection of two or more genera (Bartonella, Rickettsia, and/or Wolbachia) or multiple ASVs from a single genus in a single flea was common. Achromobacter, Peptoniphilus, and Rhodococcus were identified as additional candidate members of the C. felis microbiome on the basis of decontam analysis and literature review. Ctenocephalides felis phylogenetic diversity as assessed by the cox1 gene fell within currently characterized clades while identifying seven novel haplotypes. NGS sensitivity and specificity for Bartonella and Rickettsia spp. DNA detection were compared to targeted qPCR. Conclusions Our findings confirm the widespread coinfection of fleas with multiple bacterial genera and strains, proposing three additional microbiome members. The presence of minor Bartonella, Rickettsia, and Wolbachia ASVs was found to vary by location and flea haplotype. These findings have important implications for flea-borne pathogen transmission and control. Graphical Abstract

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 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. Graphical Abstract

Surveillance of the fleas and flea-borne pathogens infecting cats is important for both human and animal health. Multiple zoonotic Bartonella and Rickettsia species are known to infect the most common flea infesting cats and dogs worldwide: Ctenocephalides felis, the cat flea. The ability of other flea species to transmit pathogens is relatively unexplored. We aimed to determine cat host and flea factors independently associated with flea Bartonella and Rickettsia infection. We also assessed flea and cat infection by flea-host pair and location. To accomplish these aims, we performed qPCR for the detection of Bartonella, hemotropic Mycoplasma, Rickettsia, and Wolbachia DNA using paired cat and flea samples obtained from free-roaming cats presenting for spay or neuter across four locations in the United States. A logistic regression model was employed to identify the effect of cat (sex, body weight, geographic location, and Bartonella, hemotropic Mycoplasma, and Rickettsia spp., infection) and flea (clade and Rickettsia and Wolbachia infection) factors on C. felis Bartonella clarridgeiae infection. From 189 free roaming cats, we collected 84 fleas: Ctenocephalides felis (78/84), Cediopsylla simplex (4/84), Orchopeas howardi (1/84), and Nosopsyllus fasciatus (1/84). Ctenocephalides felis were phylogenetically assigned to Clades 1, 4, and 6 by cox1 gene amplification. Rickettsia asembonensis (52/84) and B. clarridgeiae (16/84) were the most common pathogenic bacteria detected in fleas. Our model identified host cat sex and weight as independently associated with B. clarridgeiae infection in fleas. Rickettsia asembonensis (52/84), Rickettsia felis (7/84) and Bartonella henselae (7/84) were detected in specific clades: R. felis was detected only in Clades 1 and 6 while B. henselae and R. asembonensis were detected only in Clade 4. Wolbachia spp., also displayed clade specificity with strains other than Wolbachia wCfeT only infecting fleas from Clade 6. There was poor flea and host agreement for Bartonella spp., infection; however, there was agreement in the Bartonella species detected in cats and fleas by geographic location. These findings reinforce the importance of considering reservoir host attributes and vector phylogenetic diversity in epidemiological studies of flea-borne pathogens. Widespread sampling is necessary to identify the factors driving flea-borne pathogen presence and transmission.

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19–22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.

Lyme disease (also known as Lyme borreliosis) is the most common vector-borne disease in the United States with an estimated 476,000 cases per year. While historically, the long-term impact of Lyme disease on patients has been controversial, mounting evidence supports the idea that a substantial number of patients experience persistent symptoms following treatment. The research community has largely lacked the necessary funding to properly advance the scientific and clinical understanding of the disease, or to develop and evaluate innovative approaches for prevention, diagnosis, and treatment. Given the many outstanding questions raised into the diagnosis, clinical presentation and treatment of Lyme disease, and the underlying molecular mechanisms that trigger persistent disease, there is an urgent need for more support. This review article summarizes progress over the past 5 years in our understanding of Lyme and tick-borne diseases in the United States and highlights remaining challenges.

We describe the development, optimization, and validation of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of Babesia, Bartonella, and Borrelia spp. DNA from several sample matrices, including clinical blood samples from animals and humans, vectors, in-vitro infected human and animal cell lines, and tissues obtained from animal models (infected with Bartonella and/or B. burgdorferi). The multiplex ddPCR assay was able to detect 31 Bartonella, 13 Borrelia, and 24 Babesia species, including Theileria equi, T. cervi, and Cytauxzoon felis. No amplification of Treponema or Leptospira spp. was observed. Sensitivity of 0.2–5 genome equivalent DNA copies per microliter was achieved for different members of the Bartonella and Borrelia genus, depending on the species or matrix type (water or spiked blood DNA) tested. The ddPCR assay facilitated the simultaneous detection of co-infections with two and three vector-borne pathogens comprising four different genera (Babesia, Bartonella, Borrelia, and Theileria) from clinical and other sample sources.

Abstract 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.

Reservoir to multiple species of zoonotic pathogens, free-roaming cats (FRCs) interact with domestic and wild animals, vectors, and humans. To assess the potential for feline vector-borne pathogens to be vertically transmitted, this study surveyed ear tip and reproductive tissues of FRCs from two locations in the South Atlantic United States for Anaplasma, Bartonella, Ehrlichia, hemotropic Mycoplasma, and Rickettsia species. We collected ovary (n = 72), uterus (n = 54), testicle (n = 74), and ear tip (n = 73) tissue from 73 cats, and fetal (n = 20) and placental (n = 19) tissue from 11 queens. Pathogen DNA was amplified utilizing qPCR, confirmed by sequencing. Cats were more frequently Bartonella henselae positive on reproductive tissues (19%, 14/73) than ear tip (5%, 4/73; p = 0.02). B. henselae was amplified from fetus (20%, 4/20) and placenta samples (11%, 2/19). Bartonella spp. infection was more common in cats from North Carolina (76%, 26/34) than Virginia (13%, 5/39; p < 0.0001). Fourteen percent (10/73) of both ear tip and reproductive tissues were positive for hemotropic Mycoplasma spp. Anaplasma, Ehrlichia, and Rickettsia spp. DNA was not amplified from any cat/tissue. These findings suggest that B. henselae preferentially infected cats’ reproductive tissue and reinforces the importance of investigating the potential for B. henselae vertical transmission or induction of reproductive failure.

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.