Using clotted, pelleted blood samples for direct molecular detection of Bartonella spp. in small mammal wildlife surveillance studies.

OBJECTIVE: Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals. RESULTS: DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.

Epidemiological survey and genetic diversity of Bartonella in fleas collected from rodents in Fujian Province, Southeast China.

BACKGROUND: Fleas, considered to be the main transmission vectors of Bartonella, are highly prevalent and show great diversity. To date, no investigations have focused on Bartonella vectors in Southeast China. The aim of this study was to investigate the epidemiological and molecular characteristics of Bartonella in fleas in Southeast China. METHODS: From 2016 to 2022, flea samples (n = 1119) were collected from 863 rodent individuals in seven inland and coastal cities in Southeast China. Flea species, region, gender, host species and habitat were recorded. The DNA samples from each individual flea were screened by real-time PCR for the Bartonella ssrA gene. All positive samples were confirmed by PCR based on the presence of the gltA gene and sequenced. The factors associated with Bartonella infection were analyzed by the Chi-square test and Fisher's exact test. ANOVA and the t-test were used to compare Bartonella DNA load. RESULTS: Bartonella DNA was detected in 26.2% (293/1119) of the flea samples, including in 27.1% (284/1047) of Xenopsylla cheopis samples, 13.2% (5/38) of Monopsyllus anisus samples, 8.3% (2/24) of Leptopsylla segnis samples and 20.0% (2/10) of other fleas (Nosopsyllus nicanus, Ctenocephalides felis, Stivalius klossi bispiniformis and Neopsylla dispar fukienensis). There was a significant difference in the prevalence of Bartonella among flea species, sex, hosts, regions and habitats. Five species of Bartonella fleas were identified based on sequencing and phylogenetic analyses targeting the gltA gene: B. tribocorum, B. queenslandensis, B. elizabethae, B. rochalimae and B. coopersplainsensis. CONCLUSIONS: There is a high prevalence and diversity of Bartonella infection in the seven species of fleas collected in Southeast China. The detection of zoonotic Bartonella species in this study, including B. tribocorum, B. elizabethae and B. rochalimae, raises public health concerns.

Bartonella species in dromedaries and ruminants from Lower Shabelle and Benadir regions, Somalia.

BACKGROUND: Bartonellosis, caused by bacteria of the genus Bartonella, is a zoonotic disease with several mammalian reservoir hosts. In Somalia, a country heavily reliant on livestock, zoonotic diseases pose significant public health and economic challenges. To the best of our knowledge, no study has been performed aiming to verify the occurrence of Bartonella spp. in Somalia. This study investigated the occurrence and molecular characterization of Bartonella in dromedary (Camelus dromedarius, Linnaeus, 1758), cattle, sheep, and goats from Somalia. MATERIALS AND METHODS: 530 blood samples were collected from various animals (155 dromedary, 199 goat, 131 cattle, and 45 sheep) in Benadir and Lower Shabelle regions. DNA was extracted for molecular analysis, and a qPCR assay targeting the NADH dehydrogenase gamma subunit (nuoG) gene was used for Bartonella screening. Positive samples were also subjected to PCR assays targeting seven molecular markers including: nuoG, citrate synthase gene (gltA), RNA polymerase beta-subunit gene (rpoB), riboflavin synthase gene (ribC), 60 kDa heat-shock protein gene (groEL), cell division protein gene (ftsZ), and pap31 and qPCR targeting the 16-23S rRNA internal transcribed spacer (ITS) followed by Sanger sequencing, BLASTn and phylogenetic analysis. RESULTS: Out of 530 tested animals, 5.1% were positive for Bartonella spp. by the nuoG qPCR assay. Goats showed the highest Bartonella occurrence (17/199, 8.5%), followed by sheep (6/44, 6.8%), cattle (4/131, 3.1%), and dromedary (1/155, 1.9%). Goats, sheep, and cattle had higher odds of infection compared to dromedary. Among nuoG qPCR-positive samples, 11.1%, 14.8%, 11.1%, and 25.9% were positive in PCR assays based on nuoG, gltA, and pap31 genes, and in the qPCR based on the ITS region, respectively. On the other hand, nuoG qPCR-positive samples were negative in the PCR assays targeting the ribC, rpoB, ftsZ, and groEL genes. While Bartonella bovis sequences were detected in cattle (nuoG and ITS) and goats (gltA), Bartonella henselae ITS sequences were detected in dromedary, goat, and sheep. Phylogenetic analysis placed gltA Bartonella sequence from a goat in the same clade of B. bovis. CONCLUSION: The present study showed, for the first time, molecular evidence of Bartonella spp. in dromedary and ruminants from Somalia and B. henselae in sheep and goats globally. These findings contribute valuable insights into Bartonella spp. occurrence in Somali livestock, highlighting the need for comprehensive surveillance and control measures under the One Health approach.

Detection of Bartonella spp. in farmed deer (Artiodactyla: Cervidae) using multiplex assays in the Qinghai-Tibet Plateau, China.

In this study, we investigated the prevalence of Bartonella in deer from Qilian County, Qinghai Province, China. Blood samples were collected from 69 red deer, 40 white-lipped deer, and 27 sika deer. The detection of Bartonella spp. has been conducted. The overall prevalence of Bartonella was 33.6% (46/135). Species-specific prevalence was 50.72% in red deer (35/69), 20.00% in white-lipped deer (8/40), and 11.11% in sika deer (3/27). There were significant differences in the prevalence rates among the different species of deer. The amplicon sequence comparison revealed a high homology of the ruminant-associated Bartonella spp. Nanopore sequencing further confirmed the results. Bartonella reads were presented in each of the qPCR-positive samples. Phylogenetic analysis indicated that the Bartonella sequences detected in deer blood were closely related to ruminant-borne Bartonella spp. In summary, we reported the Bartonella prevalence of different deer species in Qinghai, and there were at least one species of ruminant-associated Bartonella, B. schoenbuchensis. IMPORTANCE: This is the first report about Bartonella infections in the deer population from China. We found that there were two species of Bartonella and an unidentified species of Bartonella among the unculturing strains carried by these deer populations. We first used Nanopore sequencing to detect Bartonella from deer blood samples and indicated that Nanopore sequencing is beneficial to detect pathogens due to its advantage of real-time and high sensitivity.

Anaplasma bovis and Bartonella spp. in Libyan Jirds (Meriones libycus) from China.

Anaplasma bovis (1), Bartonella krasnovii (3), and Bartonella sp. (17) were detected in 80 Libyan jirds (Meriones libycus) from China. These findings extend the known host and geographic ranges of these pathogens, with neither A. bovis nor B. krasnovii previously confirmed in Libyan jirds.

Sneaky tactics: Ingenious immune evasion mechanisms of Bartonella.

Gram-negative Bartonella species are facultative intracellular bacteria that can survive in the harsh intracellular milieu of host cells. They have evolved strategies to evade detection and degradation by the host immune system, which ensures their proliferation in the host. Following infection, Bartonella alters the initial immunogenic surface-exposed proteins to evade immune recognition via antigen or phase variation. The diverse lipopolysaccharide structures of certain Bartonella species allow them to escape recognition by the host pattern recognition receptors. Additionally, the survival of mature erythrocytes and their resistance to lysosomal fusion further complicate the immune clearance of this species. Certain Bartonella species also evade immune attacks by producing biofilms and anti-inflammatory cytokines and decreasing endothelial cell apoptosis. Overall, these factors create a challenging landscape for the host immune system to rapidly and effectively eradicate the Bartonella species, thereby facilitating the persistence of Bartonella infections and creating a substantial obstacle for therapeutic interventions. This review focuses on the effects of three human-specific Bartonella species, particularly their mechanisms of host invasion and immune escape, to gain new perspectives in the development of effective diagnostic tools, prophylactic measures, and treatment options for Bartonella infections.

Molecular evidence of Bartonella spp. in tropical wild birds from the Brazilian Pantanal, the largest wetland in South America.

Despite the worldwide occurrence of bartonellae in a broad range of mammal species, in which they usually cause a long-lasting erythrocytic bacteremia, few studies reported Bartonella spp. in avian hosts. The present work aimed to investigate the occurrence and molecular identity of Bartonella spp. infecting birds in the Pantanal wetland, central-western Brazil using a multigene approach. For this purpose, blood samples were collected from 517 individuals from 13 avian orders in the states of Mato Grosso and Mato Groso do Sul. DNA was extracted from avian blood and 500/517 (96.7%) samples were positive in a conventional PCR targeting the avian ß-actin gene. Nineteen (3.8%) out of 500 avian blood samples were positive in a qPCR assay for Bartonella spp. based on the nuoG gene. Among 19 avian blood DNA samples positive in the qPCR for Bartonella spp., 12 were also positive in the qPCR for Bartonella based on the 16S-23S RNA Intergenic region (ITS). In the PCR assays performed for molecular characterization, one 16S rRNA, three ribC, and one nuoG sequences were obtained. Based on BLASTn results, while 1 nuoG, 2 ribC, and 2 ITS sequences showed high identity to Bartonella henselae, one 16S rRNA and 2 ITS showed high similarity to Bartonella machadoae in the sampled birds. Bartonella spp. related to B. henselae and B. machadoae were detected, for the first time, in wild birds from the Brazilian Pantanal.

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.

Epidemiology of Bartonella henselae infection in pet and stray cats in Croatia with risk factors analysis.

BACKGROUND: Cats are the primary reservoirs of the bacterium Bartonella henselae, the main cause of cat-scratch disease in humans. The main vector of the bacterium is the cat flea, Ctenocephalides felis. In southeastern Europe, data are lacking on the prevalence of B. henselae infection in cats, the strains of B. henselae involved and the risk factors associated with the infection. METHODS: Blood samples collected in ethylenediaminetetraacetic acid-containing tubes from 189 domestic cats (156 pet cats and 33 stray cats) from Zagreb, the capital city of Croatia, and 10 counties throughout Croatia were cultured for Bartonella spp. Following culture, bacterial isolates were genotyped at eight loci after using PCR to amplify 16S ribosomal RNA (rRNA) and the internal transcribed spacer region between the 16S and 23S rRNA sequences. Univariate and multivariate logistic regression were used to identify risk factors for B. henselae infection in cats. RESULTS: Bartonella spp. was detected in 31 cats (16.4%), and subsequent genotyping at the eight loci revealed B. henselae in all cases. Thirty complete multilocus sequence typing profiles were obtained, and the strains were identified as four sequence types that had been previously reported, namely ST5 (56.7%), ST6 (23.3%), ST1 (13.3%) and ST24 (3.3%), as well as a novel sequence type, ST33 (3.3%). The univariate analysis revealed a significantly higher risk of B. henselae infection in cats residing in coastal areas of Croatia (odds ratio [OR] 2.592, 95% confidence interval [CI] 1.150-5.838; P = 0.0191) and in cats with intestinal parasites (OR 3.207, 95% CI 1.088-9.457; P = 0.0279); a significantly lower risk was identified in cats aged > 1 year (OR 0.356, 95% CI 0.161-0.787; P = 0.0247) and in cats sampled between April and September (OR 0.325, 95% CI 0.147-0.715; P = 0.005). The multivariate analysis that controlled for age showed a positive association with the presence of intestinal parasites (OR 4.241, 95% CI 1.243-14.470; P = 0.0119) and coastal residence (OR 2.567, 95% CI 1.114-5.915; P = 0.0216) implying increased risk of infection, and a negative association with sampling between April and September (OR 0.379, 95% CI 0.169-0.848; P = 0.018) implying a decreased risk of infection. After controlling for the season, an increased risk of infection remained for the coastal region (OR 2.725, 95% CI 1.200-6.186; P = 0.012). CONCLUSIONS: Bartonella henselae is prevalent throughout Croatia and is a public health threat. Environmental and host factors can significantly affect the risk of infection, and these should be explored in more detail. The presence of intestinal parasites highlights the need to eliminate the flea vector, Ctenocephalides felis, as the most effective approach to control infections in cats and humans.

Genetic diversity of Bartonella rpoB haplotypes in domestic cats from Chile.

The study aimed to determine the inter and intra-host Bartonella spp. genetic diversity in cats from Chile. 'Seventy-nine cats' blood DNA samples qPCR Bartonella spp. positive were subjected to T-A cloning of Bartonella spp. rpoB partial gene (825 bp), and sequencing by Sanger method. The sequences were submitted to phylogenetic and polymorphism analysis. Thirty-six (45.6%) samples were successfully cloned, generating 118 clones of which 109 showed 99.6%-100% identity with Bartonella henselae whereas 9 showed 99.8-100% identity with Bartonella koehlerae. Haplotype analysis yielded 29 different rpoB-B. henselae haplotypes, one (hap#2) overrepresented in 31 out of 33 cats, and 4 rpoB-B. koehlerae haplotypes, with hap#2 represented in all 3 B. koehlerae infected cats. More than one rpoB -B. henselae and B. koehlerae haplotypes were identified in individual cats, reporting by first time coinfection by different B. henselae/B. koehlerae rpoB variants in cats from Chile.

Environmental and sociodemographic factors associated with zoonotic pathogen occurrence in Norway rats (Rattus norvegicus) from Windsor, Ontario.

AIMS: Rat-associated zoonotic pathogen transmission at the human-wildlife interface is a public health concern in urban environments where Norway rats (Rattus norvegicus) thrive on abundant anthropogenic resources and live in close contact with humans and other animal species. To identify potential factors influencing zoonotic pathogen occurrence in rats, we investigated associations between environmental and sociodemographic factors and Leptospira interrogans and Bartonella spp. infections in rats from Windsor, Ontario, Canada, while controlling for the potential confounding effects of animal characteristics (i.e., sexual maturity and body condition). METHODS AND RESULTS: Between November 2018 and June 2021, 252 rats were submitted by collaborating pest control professionals. Kidney and spleen samples were collected for L. interrogans and Bartonella spp. PCR and sequencing, respectively. Of the rats tested by PCR, 12.7% (32/252) were positive for L. interrogans and 16.3% (37/227) were positive for Bartonella species. Associations between infection status and environmental and sociodemographic variables of interest were assessed via mixed multivariable logistic regression models with a random intercept for social group and fixed effects to control for sexual maturity and body condition in each model. The odds of L. interrogans infection were significantly higher in rats from areas with high building density (odds ratio [OR]: 3.76; 95% CI: 1.31-10.79; p = 0.014), high human population density (OR: 3.31; 95% CI: 1.20-9.11; p = 0.021), high proportion of buildings built in 1960 or before (OR: 11.21; 95% CI: 2.06-60.89; p = 0.005), and a moderate number of reports of uncollected garbage compared to a low number of reports (OR: 4.88; 95% CI: 1.01-23.63; p = 0.049). A negative association was observed between median household income and Bartonella spp. infection in rats (OR: 0.26; 95% CI: 0.08-0.89; p = 0.031). CONCLUSIONS: Due to the complexity of the ecology of rat-associated zoonoses, consideration of environmental and sociodemographic factors is of critical importance to better understand the nuances of host-pathogen systems and inform how urban rat surveillance and intervention efforts should be distributed within cities.

Zoonotic bacterial pathogens in bats samples around the world: a scoping review.

The aim of this scoping review was to describe the zoonotic bacterial pathogens already reported and their frequency in different bat species. Six databases were searched, without restriction on the year or location where the studies were carried out. Based on the inclusion and exclusion criteria, 146 studies that were published between 1964 and 2020 (most after 2005) were selected. In these studies, 102 zoonotic bacterial genera were described in different samples of fourteen bat families in 55 countries, suggesting the possible role of bats as hosts for these pathogens. The pathogens mainly identified in bats were Bartonella spp., Leptospira spp. and Staphylococcus spp. In conclusion, the information provided by this scoping review expands the knowledge about zoonotic bacterial pathogens already identified in bats, which can guide epidemiological surveillance policies for these pathogens in different countries.

Molecular detection and characterization of Bartonella spp. in small mammals in the Amazonia and Cerrado biomes, midwestern Brazil.

Although Bartonella spp. have been worldwide described in rodents and bats, few studies have reported these agents in marsupials. The present work aimed to investigate the occurrence and genetic diversity of Bartonella in small mammals (rodents, marsupials, and bats) and associated ectoparasites in two ecoregions (Amazonia and Cerrado biomes) in midwestern Brazil. For this purpose, DNA samples from 378 specimens of small mammals (128 rodents, 111 marsupials, and 139 bats) and 41 fleas (Siphonaptera) were screened for the Bartonella genus employing a quantitative real-time PCR assay (qPCR) based on the nuoG (nicotinamide adenine dinucleotide dehydrogenase gamma subunit) gene. Then, positive samples in qPCR were submitted to conventional PCR (cPCR) assays targeting the gltA, ftsZ, and rpoB genes. One (0.78 %) rodent, 23 (16.54 %) bats, and 3 (7.31 %) fleas showed positive results in the qPCR for Bartonella sp. After cPCR amplification and sequencing, 13 partial Bartonella DNA sequences of the following genes were obtained only from bats´ blood samples: 9 gltA (citrate synthase), 3 ftsZ (cell division protein), and 1 rpoB (RNA polymerase beta subunit). The maximum likelihood inference based on the gltA gene positioned the obtained sequences in three different clades, closely related to Bartonella genotypes previously detected in other bat species and bat flies sampled in Brazil and other countries from Latin America. Similarly, the ftsZ sequences clustered in two different clades with sequences described in bats from Brazil, other countries from Latin America, and Georgia (eastern Europe). Finally, the Bartonella rpoB from a specimen of Lophostoma silvicolum clustered with a Bartonella sp. sequence obtained from a Noctilio albiventris (KP715475) from French Guiana. The present study provided valuable insights into the diversity of Bartonella genotypes infecting bats from two ecoregions (Amazonia and Cerrado) in midwestern Brazil and emphasized that further studies should be conducted regarding the description and evaluation of different lineages of Bartonella in wild small mammals and their ectoparasites in different Brazilian biomes.

Pleural and pericardial effusions with fever and altered mental status: an atypical presentation of bartonellosis.

A male in his 60s presented to the emergency department (ED) with a 3-week history of fever and progressive confusion. Initial laboratory and radiographic workup was largely unremarkable except for moderate bilateral pleural effusions. The patient was admitted on broad-spectrum antibiotics and further workup for fever of unknown aetiology. The differential diagnosis was broadened to different zoonotic infections, and subsequent laboratory testing showed a markedly elevated Bartonella henselae IgG and Bartonella quintana IgG (1:4096 and 1:512, respectively) in addition to positive B. henselae IgM titre (>1:20). During hospitalisation, the patient became more hypoxic and was found to have enlarging pleural effusions as well as a new pericardial effusion. The patient was treated with intravenous then oral doxycycline 100 mg two times per day and oral rifampin 300 mg two times per day for 4 weeks with subsequent improvement in clinical status as well as both effusions. This case highlights a unique presentation of Bartonella and its rare manifestation of pleural and pericardial effusions.

Detection of Bartonella bovis DNA in blood samples from a veterinarian in Mexico.

The genus Bartonella encompasses 38 validated species of Gram-negative, facultative intracellular bacteria that colonize the endothelial cells and erythrocytes of a wide spectrum of mammals. To date, 12 Bartonella species have been recorded infecting humans, causing diseases of long historical characterization, such as cat scratch fever and trench fever, and emerging bartonellosis that mainly affect animal health professionals. For this reason, this study aimed to report a documented case of Bartonella bovis infecting a veterinarian from Mexico by the amplification, sequencing and phylogenetic reconstruction of the citrate synthase (gltA) and the RNA polymerase beta-subunit (rpoB) genes, and to report the natural course of this infection. To our knowledge, this work is the first to report the transmission of B. bovis via needlestick transmission to animal health workers in Latin America.

Bartonella henselae as a putative trigger for chronic type 2 leprosy reactions.

Leprosy reactions are an acute inflammatory phenomenon that can arise before diagnosis, during treatment, or after cure of leprosy. These reactions are considered one of the main diseases that cause physical disabilities. Immunosuppressive treatment for these immune responses makes these patients susceptible to coinfections, which can trigger new leprosy reactions. The main objective of this study was to evaluate the occurrence of infection by Bartonella sp. in blood samples from 47 patients who had untreatable episodes of type 2 leprosy reactions for more than six months, comparing them with a control group. Cultures and molecular methods (PCR) were used. Amplicons from species-specific reactions and sequencing showed a higher prevalence of Bartonella henselae infection in patients, 19/47 (40.4 %), compared to control, 9/50 (18.0 %), p = 0.0149. Five patients accepted treatment for coinfection, and all showed improvement in leprosy reactions with treatment for B. henselae infection. We conclude that these bacteria can trigger chronic reactions of type 2 leprosy and should be investigated in these patients. SUMMARY LINE: Patients who have chronic type 2 leprosy reactions are more susceptible to Bartonella henselae infection than controls: 19/47 (40.4 %) compared 9/50 (18.0 %), p = 0.0149.

Acute perimyocarditis associated with Bartonella henselae infection.

Perimyocarditis involves inflammation of the heart muscle and surrounding tissue, causing reduced left ventricular ejection fraction. Typically viral, but occasionally bacterial, this condition can arise from Bartonella henselae, a rare yet potentially serious pathogen that can lead to cardiac inflammation and subsequent heart failure. Since this bacterium is mainly associated with cat scratch disease-which is self-limiting and has a mild disease course-B. henselae's potential role in cardiac disease is underestimated. We present a mid-30s man, immunocompetent, who presented to the emergency department with acute heart failure due to B. henselae-associated perimyocarditis. Despite not recalling any scratches or bites from cats, the patient had been living with cats, which likely exposed him. This case highlights the varied clinical presentations of B. henselae-associated heart disease and underscores the importance of considering this pathogen as a potential cause of perimyocarditis, particularly in individuals with exposure to cats.