A comparison of Bartonella henselae infection in immunocompetent and immunocompromised mice.

Bartonellosis refers to disease caused by the Bartonella genus of bacteria. The breadth of disease manifestations associated with Bartonella is currently expanding and includes regional lymphadenopathy, rheumatic, ocular, and neurological disorders. The dearth of knowledge regarding diagnosis, treatment and pathogenesis of this disease can be partially attributed to the lack of a reliable small animal model for the disease. For this study, Bartonella henselae, the most common species associated with human disease, was injected into Swiss Webster (SW) mice. When the outcome indicated that productive infection did not occur, SCID/Beige (immune compromised) mice were inoculated. While SW mice may potentially harbor an acute infection, less than 10 days in length, the SCID/Beige model provided a sustained infection lasting up to 30-days. These data indicate that SCID/Beige mice can provide a model to study Bartonella infection, therapeutics, and vector dynamics in the future.

Eosinophilic pericardial effusion and pericarditis in a cat.

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.

Viability and Desiccation Resistance of Bartonella henselae in Biological and Non-Biological Fluids: Evidence for Pathogen Environmental Stability.

Pathogen environmental stability is an often-neglected research priority for pathogens that are known to be vector-transmitted. Bartonella henselae, the etiologic agent of Cat Scratch Disease, has become a "pathogen of interest" in several serious human illnesses, which include neoplastic, cardiovascular, neurocognitive, and rheumatologic conditions. Survival in the flea gut and feces as well as the association with a biofilm in culture-negative endocarditis provides insight into this organism's ability to adjust to environmental extremes. The detection of B. henselae DNA in blood and tissues from marine mammals also raises questions about environmental stability and modes of pathogen transmission. We investigated the ability of B. henselae to survive in fluid matrices chosen to mimic potential environmental sources of infective materials. Feline whole blood, serum and urine, bovine milk, and physiologic saline inoculated with a laboratory strain of B. henselae San Antonio 2 were subsequently evaluated by culture and qPCR at specified time intervals. Bacterial viability was also assessed following desiccation and reconstitution of each inoculated fluid matrix. Bartonella henselae SA2 was cultured from feline urine up to 24 h after inoculation, and from blood, serum, cow's milk, and physiologic saline for up to 7 days after inoculation. Of potential medical importance, bacteria were cultured following air-desiccation of all fluid inoculates. The viability and stability of Bartonella within biological and non-biological fluids in the environment may represent a previously unrecognized source of infection for animals and human beings.

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

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

The association of host and vector characteristics with Ctenocephalides felis pathogen and endosymbiont infection.

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.

Identification of microbial taxa present in Ctenocephalides felis (cat flea) reveals widespread co-infection and associations with vector phylogeny.

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.

Vector-borne and other pathogens of potential relevance disseminated by relocated cats.

Large populations of unowned cats constitute an animal welfare, ecological, societal and public health issue worldwide. Their relocation and homing are currently carried out in many parts of the world with the intention of relieving suffering and social problems, while contributing to ethical and humane population control in these cat populations. An understanding of an individual cat's lifestyle and disease status by veterinary team professionals and those working with cat charities can help to prevent severe cat stress and the spread of feline pathogens, especially vector-borne pathogens, which can be overlooked in cats. In this article, we discuss the issue of relocation and homing of unowned cats from a global perspective. We also review zoonotic and non-zoonotic infectious agents of cats and give a list of practical recommendations for veterinary team professionals dealing with homing cats. Finally, we present a consensus statement consolidated at the 15th Symposium of the Companion Vector-Borne Diseases (CVBD) World Forum in 2020, ultimately to help veterinary team professionals understand the problem and the role they have in helping to prevent and manage vector-borne and other pathogens in relocated cats.

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

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

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

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

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.

Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species.

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.

Recent Progress in Lyme Disease and Remaining Challenges.

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.

Prevalence of Vector-Borne Pathogens in Reproductive and Non-Reproductive Tissue Samples from Free-Roaming Domestic Cats in the South Atlantic USA.

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.

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

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

Bartonella henselae Detected in Malignant Melanoma, a Preliminary Study.

Bartonella bacilliformis (B. bacilliformis), Bartonella henselae (B. henselae), and Bartonella quintana (B. quintana) are bacteria known to cause verruga peruana or bacillary angiomatosis, vascular endothelial growth factor (VEGF)-dependent cutaneous lesions in humans. Given the bacteria's association with the dermal niche and clinical suspicion of occult infection by a dermatologist, we determined if patients with melanoma had evidence of Bartonella spp. infection. Within a one-month period, eight patients previously diagnosed with melanoma volunteered to be tested for evidence of Bartonella spp. exposure/infection. Subsequently, confocal immunohistochemistry and PCR for Bartonella spp. were used to study melanoma tissues from two patients. Blood from seven of the eight patients was either seroreactive, PCR positive, or positive by both modalities for Bartonella spp. exposure. Subsequently, Bartonella organisms that co-localized with VEGFC immunoreactivity were visualized using multi-immunostaining confocal microscopy of thick skin sections from two patients. Using a co-culture model, B. henselae was observed to enter melanoma cell cytoplasm and resulted in increased vascular endothelial growth factor C (VEGFC) and interleukin 8 (IL-8) production. Findings from this small number of patients support the need for future investigations to determine the extent to which Bartonella spp. are a component of the melanoma pathobiome.

Schizophrenia and Bartonella spp. Infection: A Pilot Case-Control Study.

Recently, infections with emerging zoonotic bacteria of the genus Bartonella have been reported in association with a range of central nervous system (CNS) symptoms. Currently, it remains unknown if Bartonella spp. infection is associated with symptoms of schizophrenia/schizoaffective disorder (SCZ/SAD). The objective of this study was to determine if there is an association between Bartonella species infection and SCZ/SAD. A secondary objective was to determine if SCZ/SAD symptoms were more severe among participants with documented Bartonella spp. infection. Using a case-control study design, 17 cases and 13 controls were evaluated with a series of clinical and cognitive assessments. Blood samples were collected and tested for Bartonella spp. infection using serological, microbiological, and molecular techniques. People with SCZ/SAD were more likely than healthy volunteers to have Bartonella spp. DNA in their bloodstream, with 11 of 17 cases (65%) positive by Bartonella spp. droplet digital PCR (ddPCR). In comparison, only one healthy volunteer was Bartonella spp. ddPCR positive (8%, p = 0.0024). Based on serology, Bartonella spp. exposure was common among people with SCZ/SAD (12 of 17) as well as among healthy volunteers (12 of 13), with no significant difference between the groups (p = 0.196). Within the case group of people with SCZ/SAD, there was no significant difference in SCZ/SAD severity scores between people with and without ddPCR evidence of Bartonella spp. infection. This pilot study provides preliminary evidence in support of future investigations that should examine a potential contribution of Bartonella spp. infection to SCZ/SAD.

Bartonella Associated Cutaneous Lesions (BACL) in People with Neuropsychiatric Symptoms.

Bartonella species are globally important emerging pathogens that were not known to infect animals or humans in North America prior to the human immunodeficiency virus (HIV) epidemic. Ongoing improvements in diagnostic testing modalities have allowed for the discovery of Bartonella species (spp.) DNA in blood; cerebrospinal fluid; and the skin of patients with cutaneous lesions, fatigue, myalgia, and neurological symptoms. We describe Bartonella spp. test results for participants reporting neuropsychiatric symptoms, the majority of whom reported the concurrent development of cutaneous lesions. Study participants completed a medical history, a risk factor questionnaire, and provided cutaneous lesion photographs. Bartonella spp. serology and Bartonella alpha proteobacteria enrichment blood culture/PCR were assessed. Within a 14-month period, 33 participants enrolled; 29/33 had serological and/or PCR evidence supporting Bartonella spp. infection, of whom 24 reported concurrent cutaneous lesions since neuropsychiatric symptom onset. We conclude that cutaneous lesions were common among people reporting neuropsychiatric symptoms and Bartonella spp. infection or exposure. Additional studies, using sensitive microbiological and imaging techniques, are needed to determine if, or to what extent, Bartonella spp. might contribute to cutaneous lesions and neuropsychiatric symptoms in patients.

Parasites and vector-borne diseases disseminated by rehomed dogs.

The Companion Vector-Borne Diseases (CVBD) World Forum is a working group of leading international experts who meet annually to evaluate current scientific findings and future trends concerning the distribution, pathogenesis, clinical presentation, diagnosis and prevention of vector-borne infections of dogs and cats. At the 14th Symposium of the CVBD World Forum in Trieste, Italy (March 25-28, 2019), we identified the need to (i) bring attention to the potential spread of parasites and vectors with relocated dogs, and (ii) provide advice to the veterinary profession regarding the importance of surveillance and treatment for parasites and vector-borne infections when rehoming dogs. This letter shares a consensus statement from the CVBD World Forum as well as a summary of the problem faced, including the role of veterinary professionals in parasite surveillance, causal issues, and the importance of interdisciplinary cooperation in addressing the problem. To limit opportunities for dissemination of parasites and vectors, whenever possible, underlying problems creating the need for dog rehoming should be addressed. However, when it is necessary to rehome dogs, this should ideally take place in the country and national region of origin. When geographically distant relocation occurs, veterinary professionals have a vital role to play in public education, vigilance for detection of exotic vectors and infections, and alerting the medical community to the risk(s) for pathogen spread. With appropriate veterinary intervention, dog welfare needs can be met without inadvertently allowing global spread of parasites and their vectors.

Hopping species and borders: detection of Bartonella spp. in avian nest fleas and arctic foxes from Nunavut, Canada.

BACKGROUND: In a warmer and more globally connected Arctic, vector-borne pathogens of zoonotic importance may be increasing in prevalence in native wildlife. Recently, Bartonella henselae, the causative agent of cat scratch fever, was detected in blood collected from arctic foxes (Vulpes lagopus) that were captured and released in the large goose colony at Karrak Lake, Nunavut, Canada. This bacterium is generally associated with cats and cat fleas, which are absent from Arctic ecosystems. Arctic foxes in this region feed extensively on migratory geese, their eggs, and their goslings. Thus, we hypothesized that a nest flea, Ceratophyllus vagabundus vagabundus (Boheman, 1865), may serve as a vector for transmission of Bartonella spp. METHODS: We determined the prevalence of Bartonella spp. in (i) nest fleas collected from 5 arctic fox dens and (ii) 37 surrounding goose nests, (iii) fleas collected from 20 geese harvested during arrival at the nesting grounds and (iv) blood clots from 57 adult live-captured arctic foxes. A subsample of fleas were identified morphologically as C. v. vagabundus. Remaining fleas were pooled for each nest, den, or host. DNA was extracted from flea pools and blood clots and analyzed with conventional and real-time polymerase chain reactions targeting the 16S-23S rRNA intergenic transcribed spacer region. RESULTS: Bartonella henselae was identified in 43% of pooled flea samples from nests and 40% of pooled flea samples from fox dens. Bartonella vinsonii berkhoffii was identified in 30% of pooled flea samples collected from 20 geese. Both B. vinsonii berkhoffii (n = 2) and B. rochalimae (n = 1) were identified in the blood of foxes. CONCLUSIONS: We confirm that B. henselae, B. vinsonii berkhoffii and B. rochalimae circulate in the Karrak Lake ecosystem and that nest fleas contain B. vinsonii and B. henselae DNA, suggesting that this flea may serve as a potential vector for transmission among Arctic wildlife.

Development and validation of a droplet digital PCR assay for the detection and quantification of Bartonella species within human clinical samples.

This report describes the development, optimization, and validation of a ddPCR assay for the detection of Bartonella spp. DNA within several sample matrices, including clinical blood samples from patients with or without documented Bartonella spp. bacteremia. The Bartonella spp. ddPCR assay was developed based upon previously published TaqMan-based qPCR assays that can amplify DNA of over 25 Bartonella spp. Host DNA (housekeeping gene) amplification serves as a reference target to facilitate quantification. The efficiency, sensitivity, and specificity of the Bartonella spp. ddPCR assay was assessed by direct comparison with the current qPCR methods used by the Intracellular Pathogens Research Laboratory (North Carolina State University, North Carolina, USA), and Galaxy Diagnostics (Research Triangle Park, North Carolina, USA). Bartonella spp. ddPCR assay parameters were successfully optimized to detect Bartonella concentrations equivalent to 0.5 bacterial genome copies per microliter of blood (0.001 pg/ul of bacterial DNA). The number of droplets detected (resolution) for each concentration was consistent across each of four assessed time points. The Bartonella spp. ddPCR assay detected 16 species/strains including B. henselae; B. quintana; B. vinsonii subsp. berkhoffii (genotypes I, II, III and IV); B. vinsonii subsp. vinsonii; B. melophagi; B. volans; B. monaki; B. alsatica; B. bovis; B. elizabethae; B. clarridgeiae; and B. koehlerae. Bartonella DNA was detected in only one previously negative patient sample (119/120 negative; 99% specificity). The ddPCR sensitivity (53/112) was significantly better than qPCR (6/112) when testing patient blood and enrichment blood culture samples. The development of commercial ddPCR systems with integrated technologies has significantly streamlined the DNA detection process, making it more efficient and standardized for clinical diagnostic testing. The assay described in this work is the first step toward the development of a multiplex ddPCR assay (i.e., using the QX One from Bio-Rad) for the simultaneous detection and absolute quantification of multiple vector-borne pathogens (such as Babesia, Bartonella and Borrelia) within clinical samples.