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Abstract Background Rhipicephalus sanguineus is a ubiquitous tick responsible for transmitting Ehrlichia canis and most likely Anaplasma platys to dogs, as either single or co-infections. The objective of this study was to assess the effects of either simultaneous or sequential experimental infections with E. canis and A. platys on hematological and serological parameters, duration of infection, and efficacy of doxycycline therapy in dogs infected with one or both organisms. Six dogs per group were either uninfected, A. platys infected, E. canis infected, A. platys and E. canis co-infected, A. platys infected and E. canis challenged or E. canis infected and A. platys challenged at day 112 post-infection (PI). Doxycycline treatment was initiated at 211 days PI, followed by dexamethasone immunosuppression beginning 410 days PI. Results Initially, transient decreases in hematocrit occurred in all groups infected with E. canis, but the mean hematocrit was significantly lower in the A. platys and E. canis co-infected group. All dogs except the controls developed marked thrombocytopenia after initial infection followed by gradually increased platelet counts by 112 days PI in groups with the single infections, while platelet counts remained significantly lower in the A. platys and E. canis co-infected group. Both sequential and simultaneous infections of A. platys and E. canis produced an enhanced humoral immune response to A. platys when compared to infection with A. platys alone. Likewise, co-infection with E. canis and A. platys resulted in a more persistent A. platys infection compared to dogs infected with A. platys only, but nearly all A. platys infected dogs became A. platys PCR negative prior to doxycycline treatment. E. canis infected dogs, whether single or co-infected, remained thrombocytopenic and E. canis PCR positive in blood for 420 days. When treated with doxycycline, all E. canis infected dogs became E. canis PCR negative and the thrombocytopenia resolved. Despite immunosuppression, neither A. platys nor E. canis DNA was PCR amplified from doxycycline-treated dogs. Conclusions The results of this study demonstrate that simultaneous or sequential infection with A. platys and E. canis can alter various pathophysiological parameters in experimentally infected dogs, and because natural exposure to multiple tick-borne pathogens occurs frequently in dogs, awareness of co-infection is important in clinical practice.

Respiratory tract disease can be associated with primary or secondary bacterial infections in dogs and cats and is a common reason for use and potential misuse, improper use, and overuse of antimicrobials. There is a lack of comprehensive treatment guidelines such as those that are available for human medicine. Accordingly, the International Society for Companion Animal Infectious Diseases convened a Working Group of clinical microbiologists, pharmacologists, and internists to share experiences, examine scientific data, review clinical trials, and develop these guidelines to assist veterinarians in making antimicrobial treatment choices for use in the management of bacterial respiratory diseases in dogs and cats.

As worldwide vectors of human infectious diseases, ticks are considered to be second only to mosquitoes. Each tick species has preferred environmental conditions and biotopes that determine its geographic distribution, the pathogens it vectors, and the areas that pose risk for tick-borne diseases. Researchers have identified an increasing number of bacterial pathogens that are transmitted by ticks, including Anaplasma, Borrelia, Ehrlichia, and Rickettsia spp. Recent reports involving humans and canines suggest that ticks should be considered as potential vectors of Bartonella spp. To strengthen this suggestion, numerous molecular surveys to detect Bartonella DNA in ticks have been conducted. However, there is little evidence that Bartonella spp. can replicate within ticks and no definitive evidence of transmission by a tick to a vertebrate host. [ABSTRACT FROM AUTHOR]

In conjunction with efforts to assess pathogen exposure in feral pigs from the southeastern United States, we amplified Bartonella henselae, B. koehlerae, and B. vinsonii subsp. berkhoffii from blood samples. Feral pigs may represent a zoonotic risk for hunters or butchers and pose a potential threat to domesticated livestock. [ABSTRACT FROM AUTHOR]

Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: In conjunction with Dr. S. Sontakke and North Carolina State University, E. B. Breitschwerdt holds US Patent No. 7,115,385 Media and Methods for Cultivation of Microorganisms, which was issued on October 3, 2006. He is a co-founder, shareholder, and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. The remaining authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Mild Cognitive Impairment (MCI) is a neurological disorder at the transition between normal cognitive decline and dementia. Despite the potential role of neuroinflammation in the pathogenesis of MCI, infectious triggers remain mostly unknown. Infection with Bartonella spp., a zoonotic bacterium, has recently been associated with diffuse neurological and psychiatric symptoms. Given the preferential endothelial localization of Bartonella spp. and the role of vascular changes in neurocognitive decline, we hypothesized that there is an association between Bartonella spp. infection and pathologically accelerated decline in cognitive function in aging. To test this hypothesis, we collected serological and molecular markers of past and present Bartonella spp. infection in a sample of older people with and without MCI. Samples were processed in a blinded way to exclude laboratory biases. Contrary to our hypothesis, people with MCI were not more likely than people without MCI to have an active Bartonella spp. infection as measured by droplet digital PCR (p = 0.735) and quantitative PCR (p = 1). In addition, there was no significant difference in positive serological results between cases and controls (p = 0.461). Overall, higher-than-expected active Bartonella spp. infection (37% by ddPCR) and seroreactivity (71% by indirect fluorescent antibody assay) were found in people without MCI. Conclusions require caution, as our study was limited by the small number of cases with MCI. Overall, our results identified a higher than previously recognized rate of exposure and infection with Bartonella spp. in this older study population but does not support a specific role for such infection in MCI., Competing Interests: In conjunction with Dr. S. Sontakke and North Carolina State University, E. B. Breitschwerdt holds US Patent No. 7,115,385 Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006. He is a co-founder, shareholder and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. Dr. Ricardo Maggi is a co-founder and the Chief Technical Officer for Galaxy Diagnostics Inc. Drs Breitschwerdt and Maggi are full-time employees of North Carolina State University. Dr. Frohlich is a full-time employee of the University of North Carolina – Chapel Hill. Dr. Frohlich is the lead inventor of IP issued to UNC and licensed to: Electromedical Products International (EPI). In the last twelve months, Dr. Frohlich has received consulting honoraria from the following entities: EPI, Insel Spital, and the University of Michigan. Dr. Frohlich is a shareholder of EPI. In the last twelve months, Dr. Frohlich has received royalties from Academic Press and the University of North Carolina at Chapel Hill. Ms. Guirguis, Pupillo, and Rodrigues report no financial relationships with commercial interests. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Guirguis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: In recent years, Babesia and Bartonella species co-infections in patients with chronic, nonspecific illnesses have continued to challenge and change the collective medical understanding of "individual pathogen" vector-borne infectious disease dynamics, pathogenesis and epidemiology. The objective of this case series is to provide additional molecular documentation of Babesia odocoilei infection in humans in the Americas and to emphasize the potential for co-infection with a Bartonella species., Methods: The development of improved and more sensitive molecular diagnostic techniques, as confirmatory methods to assess active infection, has provided increasing clarity to the healthcare community., Results: Using a combination of different molecular diagnostic approaches, infection with Babesia odocoilei was confirmed in seven people suffering chronic non-specific symptoms, of whom six were co-infected with one or more Bartonella species., Conclusions: We conclude that infection with Babesia odocoilei is more frequent than previously documented and can occur in association with co-infection with Bartonella spp., (© 2024. The Author(s).)

Background: Lyme disease, caused by infection with Borrelia burgdorferi, is the most common vector-borne disease in the United States. The standard two-tier testing (STTT) algorithm suffers from low sensitivity, misinterpretation, and long turnaround time, preventing timely detection and treatment. To address these challenges, we hypothesized that the canine point-of-care (PoC) SNAP 4Dx Plus test used to detect Borrelia burgdorferi antibodies could be employed for human diagnosis. Materials and Methods: The SNAP 4Dx Plus testing was conducted in accordance with the manufacturer's instructions, with results read by manual inspection. All analyses were conducted using R version 4.3.1, and agreement between the PoC assay and the STTT was assessed using kappa statistics with GraphPad software. Results: We included 102 previously-tested human serum samples, of which 19 samples (18.6%) were STTT positive. Compared to the STTT, the SNAP 4Dx Plus test demonstrated a low sensitivity of 0.16 (95% CI 0.03 to 0.40). Conclusion: Overall, our results do not support the use of the SNAP 4Dx Plus LD assay for the diagnosis of human Lyme disease. Differences in antibody concentrations between human and canine samples may partly explain our findings.

Introduction: The potential role of pathogens, particularly vector-transmitted infectious agents, as a cause of psychosis has not been intensively investigated. We have reported a potential link between Bartonella spp. bacteremia and neuropsychiatric symptoms, including pediatric acute onset neuropsychiatric syndrome and schizophrenia. The purpose of this study was to further assess whether Bartonella spp. exposure or infection are associated with psychosis., Methods: In a blinded manner, we assessed the presence of anti- Bartonella antibodies by indirect immunofluorescence assays (IFA), and infection by amplification of bacterial DNA from blood by quantitative polymerase chain reaction (qPCR), digital PCR (dPCR), and droplet digital PCR (ddPCR) in 116 participants. Participants were categorized into one of five groups: 1) controls unaffected by psychosis ( n = 29); 2) prodromal participants ( n = 16); 3) children or adolescents with psychosis ( n = 7); 4) adults with psychosis ( n = 44); and 5) relatives of a participant with psychosis ( n = 20)., Results: There was no significant difference in Bartonella spp. IFA seroreactivity between adults with psychosis and adult controls unaffected by psychosis. There was a higher proportion of adults with psychosis who had Bartonella spp. DNA in the bloodstream (43.2%) compared to adult controls unaffected by psychosis (14.3%, p = 0.021). The Bartonella species was determined for 18 of the 31 bacteremic participants, including infection or co-infection with Bartonella henselae (11/18), Bartonella vinsonii subsp. b erkhoffii (6/18), Bartonella quintana (2/18), Bartonella alsatica (1/18), and Bartonella rochalimae (1/18)., Discussion: In conjunction with other recent research, the results of this study provide justification for a large national or international multi-center study to determine if Bartonella spp. bacteremia is more prevalent in adults with psychosis compared to adults unaffected by psychosis. Expanding the investigation to include a range of vector-borne and other microbial infections with potential CNS effects would enhance knowledge on the relationship between psychosis and infection., Competing Interests: In conjunction with Dr. S. Sontakke and North Carolina State University, EB holds US Patent No. 7,115,385 Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006. He is a co-founder, shareholder and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. RM is a co-founder and the Chief Technical Officer for Galaxy Diagnostics Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Delaney, Robveille, Maggi, Lashnits, Kingston, Liedig, Murray, Fallon and Breitschwerdt.)

Background: Feline-associated hemotropic Mycoplasma (hemoplasmas) are believed to be transmitted by two primary mechanisms: (1) direct transmission via fighting and (2) vector-borne transmission by the cat flea (Ctenocephalides felis). While the efficiency of transmission by C. felis appears low, most manuscripts focus on the prevalence of hemoplasmas in wild-caught fleas and report either a very low (< 3%) or a high (> 26%) prevalence. Therefore, we aimed to assess the influence of sample processing and PCR methods on C. felis hemoplasma infection prevalence., Methods: A systemic review of PubMed articles identified 13 manuscripts (1,531 fleas/flea pools) that met the inclusion criteria (performed PCR for >1 hemoplasma on C. felis collected from cats). Risk of bias was assessed utilizing the ROBINS-E tool. Meta-analysis performed in R of these manuscripts found that not washing samples and a common set of 16S rRNA primers first published in Jensen et al. 2001 were associated with increased hemoplasma prevalence. To evaluate the influence of washing on newly collected fleas, we assessed the hemoplasma status of 20 pools of 5 C. felis each, half of which were washed and half not washed., Results: Flea washing did not influence the detection of hemoplasma but instead amplified Spiroplasma. To assess non-specific amplification with the Jensen et al. 2001 primers, 67 C. felis samples (34% previously reported hemoplasma infected) were subject to PCR and sequencing. By this method, hemoplasma was detected in only 3% of samples. In the remaining "hemoplasma infected" fleas, PCR amplified Spiroplasma or other bacteria., Conclusions: Therefore, we concluded that hemoplasma infection in C. felis is rare, and future flea prevalence studies should sequence all positive amplicons to validate PCR specificity. Further investigation of alternative methods of feline-associated hemoplasma transmission and the ability of C. felis to maintain hemoplasma infection is necessary., (© 2024. The Author(s).)

There is currently sparse information on the possible effect of long-term storage of serum specimens for the retrospective serodiagnosis of canine monocytic ehrlichiosis (CME). The aim of this study was to assess the agreement between the original serologic outcome and the results of a repeat indirect fluorescent antibody (IFA) assay for the detection of IgG antibodies against E. canis. A secondary aim was to compare the diagnostic performance of two commercially available point-of-care (POC) immunochromatographic (IC) assays. Archived serum samples originally tested as positive (n=66) or negative (n=19) for E. canis IgG antibodies and kept frozen at -20°C for a median of 22 years, were retrospectively examined by IFA and by two POC IC assays. Cohen's Kappa coefficient (0.748, p < 0.0001), indicated a substantial agreement between the original and repeat serologic testing results. An almost identical high sensitivity and moderate specificity were established for the two POC IC assays. Canine serum specimens on long-term storage may still be of value for seroepidemiologic surveys investigating the exposure to E. canis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Vector-borne infections may underlie some rheumatic diseases, particularly in people with joint effusions. This study aimed to compare serum and synovial fluid antibodies to B. burgdorferi and Bartonella spp. in patients with rheumatic diseases. This observational, cross-sectional study examined paired synovial fluid and serum specimens collected from 110 patients with joint effusion between October 2017 and January 2022. Testing for antibodies to B. burgdorferi (using CDC criteria) and Bartonella spp. via two indirect fluorescent antibody (IFA) assays was performed as part of routine patient care at the Institute for Specialized Medicine (San Diego, CA, USA). There were 30 participants (27%) with positive two-tier B. burgdorferi serology and 26 participants (24%) with IFA seroreactivity (≥1:256) to B. henselae and/or B. quintana . Both B. burgdorferi IgM and IgG were detected more frequently in synovial fluid than serum: 27% of patients were either IgM or IgG positive in synovial fluid, compared to 15.5% in serum ( P = 0.048). Conversely, B. henselae and B. quintana antibodies were detected more frequently in serum than synovial fluid; overall only 2% of patients had positive IFA titers in synovial fluid, compared to 24% who had positive IFA titers in serum ( P < 0.001). There were no significant associations between B. burgdorferi or Bartonella spp. seroreactivity with any of the clinical rheumatological diagnoses. This study provides preliminary support for the importance of synovial fluid antibody testing for documenting exposure to B. burgdorferi but not for documenting exposure to Bartonella spp., Importance: This study focuses on diagnostic testing for two common vector-borne diseases in an affected patient population. In it, we provide data showing that antibodies to B. burgdorferi , but not Bartonella spp., are more commonly found in synovial fluid than serum of patients with joint effusion. Since Lyme arthritis is a common-and sometimes difficult to diagnose-rheumatic disease, improving diagnostic capabilities is of utmost importance. While our findings are certainly not definitive for changes to practice, they do suggest that synovial fluid could be a useful sample for the clinical diagnosis of Lyme disease, and future prospective studies evaluating this claim are warranted., Competing Interests: During the course of these studies, Dr. Jennifer Miller was the Director of Research, Development and Laboratory Operations for Galaxy Diagnostics Inc. She is currently employed as a Principal Investigator/Manager I at BioAgilytix Labs. In conjunction with Dr. S. Sontakke and North Carolina State University, E.B. Breitschwerdt holds US Patent No. 7,115,385 Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006. He is a co-founder, shareholder, and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections.

Ctenocephalides felis, the cat flea, is among the most prevalent and widely dispersed vectors worldwide. Unfortunately, research on C. felis and associated pathogens (Bartonella and Rickettsia spp.) lags behind that of other vectors and vector-borne pathogens. Therefore, we aimed to review fundamental aspects of C. felis as a vector (behavior, epidemiology, phylogenetics, immunology, and microbiome composition) with an emphasis on key techniques and research avenues employed in other vector species. Future laboratory C. felis experimental infections with Bartonella, Rickettsia, and Wolbachia species/strains should examine the vector-pathogen interface utilizing contemporary visualization, transcriptomic, and gene-editing techniques. Further environmental sampling will inform the range and prevalence of C. felis and associated pathogens, improving the accuracy of vector and pathogen modeling to improve infection/infestation risk assessment and diagnostic recommendations., Competing Interests: Declaration of interests In conjunction with Sushama Sontakke and North Carolina State University, E.B.B. DVM holds US Patent No. 7,115,385: ‘Media and Methods for Cultivation of Microorganisms’, issued 3 October 2006. He is a co-founder, shareholder, and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella species infections as well as a scientific consultant to IDEXX Laboratories (Westbrook, ME, USA). All other authors declare no potential conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Edward B. Breitschwerdt, DVM holds U.S. Patent No. 7115385; Media and Methods for cultivation of microorganisms, which was issued October 3, 2006. He is a co‐founder, shareholder and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella species infections. All other authors declare no potential conflicts of interest., (Copyright: © 2024 Bullard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microbial infections of the brain can lead to dementia, and for many decades microbial infections have been implicated in Alzheimer's disease (AD) pathology. However, a causal role for infection in AD remains contentious, and the lack of standardized detection methodologies has led to inconsistent detection/identification of microbes in AD brains. There is a need for a consensus methodology; the Alzheimer's Pathobiome Initiative aims to perform comparative molecular analyses of microbes in post mortem brains versus cerebrospinal fluid, blood, olfactory neuroepithelium, oral/nasopharyngeal tissue, bronchoalveolar, urinary, and gut/stool samples. Diverse extraction methodologies, polymerase chain reaction and sequencing techniques, and bioinformatic tools will be evaluated, in addition to direct microbial culture and metabolomic techniques. The goal is to provide a roadmap for detecting infectious agents in patients with mild cognitive impairment or AD. Positive findings would then prompt tailoring of antimicrobial treatments that might attenuate or remit mounting clinical deficits in a subset of patients., (© 2023 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

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.

A validated second-generation SNAP 4Dx Plus (Idexx) incorporates new peptides for improved detection of antibodies against Anaplasma and Ehrlichia tick-borne pathogens in dogs. We compared the first- and second-generation SNAP 4Dx Plus using dogs naturally infected with Anaplasma or Ehrlichia species, or dogs seroreactive by an E. canis indirect fluorescent antibody test (IFAT). The second-generation immunoassay was more sensitive than the first-generation for dogs infected with A. phagocytophilum (51.1% and 29.2%, respectively), A. platys (63.6% and 35.3%, respectively), E. canis (96.2% and 88.3%, respectively), or E. ewingii (73.7% and 70.8%, respectively), and for dogs seroreactive by E. canis IFAT (87.3% and 83.9%, respectively). The second-generation immunoassay detected significantly more Anaplasma - or Ehrlichia -infected dogs that were Anaplasma ( p  < 0.001) or Ehrlichia ( p  = 0.031) seroreactive, respectively, than did the first-generation test. When Ehrlichia seroreactivity by E. canis IFAT and both immunoassays was compared, significantly more E. canis -infected dogs were seroreactive by E. canis IFAT than the first-generation ( p  = 0.006) but not the second-generation ( p  = 0.125) immunoassay. Significantly more E. ewingii -infected dogs were seroreactive by the first- ( p  = 0.011) and second-generation ( p  = 0.049) immunoassays than the E. canis IFAT. Medical records available for 7 dogs that were Anaplasma seroreactive by the second-generation but not the first-generation immunoassay revealed case management decisions that might have been different with an immediate anaplasmosis diagnosis, including earlier doxycycline therapy and less hospitalization. The second-generation SNAP 4Dx Plus test offered improved serologic detection of Anaplasma and Ehrlichia in naturally infected dogs.

Background: Exposure to zoonotic diseases is a significant occupational risk in veterinary medicine. In this study, we characterized personal protective equipment use, injury frequency, and Bartonella seroreactivity in Washington State veterinary workers. Methods: Using a risk matrix developed to reflect occupational risk factors for exposure to Bartonella and multiple logistic regression, we explored determinants of risk for Bartonella seroreactivity. Results: Depending on the titer cutoff used, Bartonella seroreactivity was between 24.0% and 55.2%. No significant predictors of seroreactivity were found, although the relationship between high-risk status and increased seroreactivity for some Bartonella species approached significance. Serology for other zoonotic and vector borne pathogens did not identify consistent cross reactivity with Bartonella antibodies. Conclusion: The predictive power of the model was likely limited by the small sample size and high level of exposure to risk factors for most participants. Given the high proportion of veterinarians seroreactive to one or more of the three Bartonella spp. known to infect dogs and cats in the United States, as well as seroreactivity to other zoonoses, and the unclear relationship between occupational risk factors, seroreactivity, and disease expression, more research is needed in this area.

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., Competing Interests: The authors declare a conflict of interest. Ricardo G. Maggi is a co-founder and the Chief Technical Officer for Galaxy Diagnostics Inc. In conjunction with S. Sontakke and North Carolina State University, E. B. Breitschwerdt holds US Patent No. 7,115,385 Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006. He is a co-founder, shareholder and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. All other authors claim no competing interests.

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., Competing Interests: In conjunction with S. Sontakke and North Carolina State University, EB holds US Patent No. 7,115,384 Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006, and also co-founder, shareholder, and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. The handling editor MA declared a past co-authorship with the EB and RM. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Moore, Breitschwerdt, Kim, Li, Ferris, Maggi and Lashnits.)

The effect of Bartonella henselae on the microbiome of its vector, Ctenocephalides felis (the cat flea) is largely unknown, as the majority of C. felis microbiome studies have utilized wild-caught pooled fleas. We surveyed the microbiome of laboratory-origin C. felis fed on B. henselae -infected cats for 24 h or 9 days to identify changes to microbiome diversity and microbe prevalence compared to unfed fleas, and fleas fed on uninfected cats. Utilizing Next Generation Sequencing (NGS) on the Illumina platform, we documented an increase in microbial diversity in C. felis fed on Bartonella -infected cats for 24 h. These changes returned to baseline (unfed fleas or fleas fed on uninfected cats) after 9 days on the host. Increased diversity in the C. felis microbiome when fed on B. henselae -infected cats may be related to the mammalian, flea, or endosymbiont response. Poor B. henselae acquisition was documented with only one of four infected flea pools having B. henselae detected by NGS. We hypothesize this is due to the use of adult fleas, flea genetic variation, or lack of co-feeding with B. henselae -infected fleas. Future studies are necessary to fully characterize the effect of endosymbionts and C. felis diversity on B. henselae acquisition.

Rationale: Discovery proteomics has been popularized to be essential in the investigator's biological toolbox. Many biological problems involve the interplay of multiple organisms. Herein, a bottom-up proteomics workflow was developed to study a system containing multiple organisms to promote a thorough understanding of how each interacts with the others., Methods: A label-free quantification proteomics workflow was developed with nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). This protocol describes a bottom-up proteomics workflow used to study differential protein expression in the context of fleas (Ctenocephalides felis felis) experimentally infected by the bacterium Bartonella henselae, the etiological agent of Cat Scratch Disease (CSD)., Results: Step-by-step instructions are provided for protein extraction, protein cleanup, total protein measurement, nanoLC-MS/MS data acquisition, and data analysis using Proteome Discoverer software. Comprehensive and exhaustive details are included to promote the adoption of this proteomics workflow in other laboratories., Conclusion: A proteomics protocol is detailed for a system containing multiple proteomes from different taxonomic lineages using CSD (cats bitten by fleas infected with Bartonella henselae) as a model. The operating protocol can be readily applied to other label-free proteomics work involving multiple proteomes from taxonomically distinct organisms., (© 2022 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

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., (© 2022. The Author(s).)

Veterinarians often test for serologic evidence of vector-borne infections in sick dogs presenting with clinical signs or to screen for subclinical chronic infections. Additional peptide targets for the detection of antibodies to Anaplasma phagocytophilum, Anaplasma platys, and Ehrlichia canis were added to an existing point-of-care (POC) ELISA test (SNAP 4Dx Plus Test, IDEXX Laboratories, Westbrook, ME). This second-generation, multi-analyte test detects Dirofilaria immitis antigen and antibodies to Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp. The second-generation test is expected to better meet the needs of practicing veterinarians and their patients. To assess this expectation, the second-generation POC test was evaluated with serum samples from experimentally infected dogs and a broader field population of dogs. Compared to the first-generation test, most dogs experimentally infected with A phagocytophilum (n = 7/8), A platys (n = 4/6), or E canis (n = 4/6) had detectable antibody responses 3-22 days earlier post-infection; these results demonstrated better alignment with polymerase chain reaction (PCR) amplification results and the onset of clinical signs. Using a convenience sample set of 510 sera from both academic and commercial veterinary diagnostic laboratories, the second-generation test had sensitivities greater than 90% for Anaplasma spp. (94.1%), B burgdorferi (95.5%), Ehrlichia spp. (93.4%) and D immitis (98.0%). Specificity ranged from 96.8% - 100% across the four assays. Results from this study demonstrate that the second-generation POC ELISA had an improved ability to detect serologic responses during the acute phase of A phagocytophilum, A platys, and E canis experimental infections. The results from the broader field samples support overall high sensitivity and specificity, consistent with the historical performance of the first-generation POC ELISA test., (Copyright © 2022 IDEXX Laboratories, Inc. Published by Elsevier Inc. All rights reserved.)

Lyme disease (LD) is a tick-transmitted bacterial infection caused by Borreliella burgdorferi and other closely related species collectively referred to as the LD spirochetes. The LD spirochetes encode an uncharacterized family of proteins originally designated p rotein f amily t welve (PF12). In B. burgdorferi strain B31, PF12 consists of four plasmid-carried genes, encoding BBK01, BBG01, BBH37, and BBJ08. Henceforth, we designate the PF12 proteins f amily t welve l ipoprotein (Ftl) A (FtlA) (BBK01), FtlB (BBG01), FtlC (BBH37), and FtlD (BBJ08). The goal of this study was to assess the potential utility of the Ftl proteins in subunit vaccine development. Immunoblot analyses of LD spirochete cell lysates demonstrated that one or more of the Ftl proteins are produced by most LD isolates during cultivation. The Ftl proteins were verified to be membrane associated, and nondenaturing PAGE revealed that FtlA, FtlB, and FtlD formed dimers, while FtlC formed hexamers. Analysis of serum samples from B. burgdorferi antibody (Ab)-positive client-owned dogs ( n  = 50) and horses ( n  = 90) revealed that a majority were anti-Ftl Ab positive. Abs to the Ftl proteins were detected in serum samples from laboratory-infected dogs out to 497 days postinfection. Anti-FtlA and FtlB antisera displayed potent complement-dependent Ab-mediated killing activity, and epitope localization revealed that the bactericidal epitopes reside within the N-terminal domain of the Ftl proteins. This study suggests that FtlA and FtlB are potential candidates for inclusion in a multivalent vaccine for LD.

Background: Reference intervals for platelets and white blood cell (WBCs) counts are lower in greyhounds than other breeds. Proteinuria is common. Vector-borne diseases (VBD) cause thrombocytopenia, leukopenia, and proteinuria. Racing greyhounds are commonly exposed to vectors that carry multiple organisms capable of chronically infecting clinically healthy dogs., Hypothesis/objectives: Vector-borne disease prevalence is higher in retired racing greyhounds than in show-bred greyhounds. Occult infection contributes to breed-related laboratory abnormalities., Animals: Thirty National Greyhound Association (NGA) retired racing and 28 American Kennel Club (AKC) show-bred greyhounds., Methods: Peripheral blood was tested for Anaplasma, Babesia, Bartonella, Ehrlichia, hemotropic Mycoplasma, and Rickettsia species using PCR. Antibodies to Anaplasma, Babesia, Bartonella, Ehrlichia, and Rickettsia species and Borrelia burgdorferi were detected using immunofluorescence and ELISA assays. Complete blood counts, semiquantitative platelet estimates, and microalbuminuria concentration were determined., Results: Seven of 30 NGA and 1/28 AKC greyhounds tested positive for ≥1 VBD (P = .05). More positive tests were documented in NGA (10/630) than in AKC dogs (1/588; P = .02). Exposure to Bartonella species (3/30), Babesia vogeli (2/30), Ehrlichia canis (1/30), and infection with Mycoplasma hemocanis (3/30) occurred in NGA dogs. Platelet counts or estimates were >170 000/μL. White blood cell counts <4000/μL (4/28 AKC; 5/30 NGA, P > .99; 1/8 VBD positive; 8/51 VBD negative, P = .99) and microalbuminuria (10/21 AKC; 5/26 NGA, P = .06; 1/8 VBD positive; 14/25 VBD negative, P = .41) were not associated with VBD., Conclusions and Clinical Importance: The prevalence of thrombocytopenia and B. vogeli exposure was lower than previously documented. Larger studies investigating the health impact of multiple VBD organisms are warranted., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

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., Competing Interests: RGM is a co-founder and the Chief Technical Officer for Galaxy Diagnostics Inc. In conjunction with Dr. S. Sontakke and North Carolina State University, EBB holds US Patent No. 7,115,385 Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006. He is a co-founder, shareholder and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 André, Neupane, Lappin, Herrin, Smith, Williams, Collins, Bai, Jorge, Balbuena, Bradley, Maggi and Breitschwerdt.)

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.

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., (© 2021. The Author(s).)

Background: Improved understanding of Bartonella spp. serology in dogs may aid clinical decision making., Objective: Describe demographic and geographic patterns of Bartonella spp. seroreactivity in dogs, and describe hematologic and serum biochemical abnormalities in Bartonella spp. seroreactive and nonseroreactive dogs., Animals: Serum samples from 5957 dogs in the United States, previously submitted to IDEXX Reference Laboratories., Methods: Serum was tested using 3 indirect ELISAs for B. henselae, B. vinsonii subsp. berkhoffii, and B. koehlerae. Complete blood count and serum biochemistry panel results were reviewed retrospectively., Results: Overall, 6.1% of dogs were Bartonella spp. seroreactive. Toy breeds were less likely to be seroreactive (3.9%) than mixed breeds (7.5%; adjusted odds ratio [aOR], 0.48; 95% confidence interval [CI], 0.32-0.72), and dogs <1 year old were less likely to be seroreactive (3.4%) than dogs 1 to 5.5 years of age (7.3%; aOR, 0.42; 95% CI, 0.23-0.72). Dogs in the West South Central (9.8%) and South Atlantic (8.8%) regions were more likely than dogs elsewhere in the United States to be seroreactive (aOR, 2.22; 95% CI, 1.31-3.87; aOR, 2.44; 95% CI, 1.38-4.36)., Conclusions and Clinical Importance: Demographic and geographic findings for Bartonella spp. exposure were broadly comparable to previously reported patterns., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

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.

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.

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., Competing Interests: AGR is a scientific advisor to NeonMind Biosciences and ETHA Natural Botanicals. In conjunction with Dr. S. Sontakke and North Carolina State University, EBB holds US Patent No. 7,115,385; Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006. He is a co-founder, shareholder and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. and Borrelia infections. RM is a co-founder, shareholder and the Chief Technical Officer for Galaxy Diagnostics. RTM is a paid consultant/speaker for Zoetis and receives license related income from Zoetis. MJS serves on the Scientific Advisory Board for the Global Lyme Alliance and has been issued a patent [US Patent No. 10 481 165] for “Elevated CCL19 after completion of therapy for acute Lyme disease identifies patients at risk for development of post-treatment Lyme disease syndrome who will benefit from further antibiotic therapy”. AO has pending patent applications on the development of Lyme disease diagnostic tests. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bobe, Jutras, Horn, Embers, Bailey, Moritz, Zhang, Soloski, Ostfeld, Marconi, Aucott, Ma'ayan, Keesing, Lewis, Ben Mamoun, Rebman, McClune, Breitschwerdt, Reddy, Maggi, Yang, Nemser, Ozcan, Garner, Di Carlo, Ballard, Joung, Garcia-Romeu, Griffiths, Baumgarth and Fallon.)

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.