Quantitative PCR Analysis of the Bartonella henselae carD Gene during Bacterial Stress

<p>The Bartonella genus is comprised of arthropod-borne, intracellular bacterial pathogens that colonise the mammalian bloodstream. A large number of mammalian species are hosts for one or more Bartonella species, as either reservoir or incidental hosts. Bartonella species are only able to invade and replicate in host red blood cells in the reservoir host, and to be taken up by an associated haematophagous arthropod vector to complete transmission and the bacterial life cycle. Humans are the reservoir hosts for B. quintana and B. bacilliformis, and are incidental hosts for more than 16 additional zoonotic Bartonella species, including B. henselae, which is normally carried by cats. B. henselae infection, usually acquired through cat scratches or bites, can result in several clinical manifestations, with varying degrees of severity; the most common of these is cat scratch disease, where symptoms commonly range from enlarged lymph nodes to severe fever. Although usually a mild illness, B. henselae infection can occasionally lead to severe symptoms, affecting neurological and other major organ systems. During their life cycle the Bartonellae must adapt to various toxic host environments; such adaptation is mediated by several bacterial stress pathways, which modify bacterial transcription. However, many gaps remain in the understanding of B. henselae stress response pathways. The object of this study, the carD gene, was identified as a possible component of the Bartonella stress response. The carD gene has been shown to be critical for stress defence in other bacterial species, including Mycobacterium tuberculosis, Thermus thermophilus, and Myxococcus xanthus. Our study aimed to investigate whether carD played as significant a role during the B. henselae response to stresses as it does in other bacterial genera. We first attempted to perform growth comparisons between a B. henselae carD mutant strain and a wild type strain during exposure to stress conditions; however, our mutagenic carD plasmid interfered with bacterial growth of Escherichia coli cultures, which hindered transformation and generation of a B. henselae carD mutant. As an alternative, we investigated the expression of B. henselae carD under stress conditions, comparing carD expression during stress against a non-stressed B. henselae control, using quantitative PCR. We found no significant difference of expression of the carD gene between the control and any of our conditions, although a trend of increased carD expression was found in several stress conditions. We believe that these findings merit further study into the role of carD in the B. henselae stress response.</p>

Quantitative PCR Analysis of the Bartonella henselae carD Gene during Bacterial Stress

<p>The Bartonella genus is comprised of arthropod-borne, intracellular bacterial pathogens that colonise the mammalian bloodstream. A large number of mammalian species are hosts for one or more Bartonella species, as either reservoir or incidental hosts. Bartonella species are only able to invade and replicate in host red blood cells in the reservoir host, and to be taken up by an associated haematophagous arthropod vector to complete transmission and the bacterial life cycle. Humans are the reservoir hosts for B. quintana and B. bacilliformis, and are incidental hosts for more than 16 additional zoonotic Bartonella species, including B. henselae, which is normally carried by cats. B. henselae infection, usually acquired through cat scratches or bites, can result in several clinical manifestations, with varying degrees of severity; the most common of these is cat scratch disease, where symptoms commonly range from enlarged lymph nodes to severe fever. Although usually a mild illness, B. henselae infection can occasionally lead to severe symptoms, affecting neurological and other major organ systems. During their life cycle the Bartonellae must adapt to various toxic host environments; such adaptation is mediated by several bacterial stress pathways, which modify bacterial transcription. However, many gaps remain in the understanding of B. henselae stress response pathways. The object of this study, the carD gene, was identified as a possible component of the Bartonella stress response. The carD gene has been shown to be critical for stress defence in other bacterial species, including Mycobacterium tuberculosis, Thermus thermophilus, and Myxococcus xanthus. Our study aimed to investigate whether carD played as significant a role during the B. henselae response to stresses as it does in other bacterial genera. We first attempted to perform growth comparisons between a B. henselae carD mutant strain and a wild type strain during exposure to stress conditions; however, our mutagenic carD plasmid interfered with bacterial growth of Escherichia coli cultures, which hindered transformation and generation of a B. henselae carD mutant. As an alternative, we investigated the expression of B. henselae carD under stress conditions, comparing carD expression during stress against a non-stressed B. henselae control, using quantitative PCR. We found no significant difference of expression of the carD gene between the control and any of our conditions, although a trend of increased carD expression was found in several stress conditions. We believe that these findings merit further study into the role of carD in the B. henselae stress response.</p>

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

Title Prevalence of Bartonella Species in Shelter Cats and their Ectoparasites in Southeastern Brazil

Background: Feline Bartonella can be transmitted to humans through cat scratches or bites, and between cats by Ctenocephalides felis flea. Methods: The occurrence of Bartonella DNA was assessed in ectoparasites and their cat hosts living in shelters based on the ITS region and gltA gene. Results: Bartonella DNA was detected in 47.8% of cat blood samples, in 18.3% of C. felis fleas,13.3% of flea eggs pools and 12.5% of lice pools. B.henselae and B. clarridgeiae DNA were detected in cat fleas, while B. henselae, B. clarridgeiae, and B. koehlerae in blood samples from bacteremic cats. Cats infested by positive ectoparasites showed approximately twice the chance of being infected. Conclusions: Our results indicate shelter cats have a high prevalence of Bartonella species known to be human pathogens and highlight the importance of controlling their infestation by ectoparasites to avoid cat and human infection.

Hedgehogs and Squirrels as Hosts of Zoonotic Bartonella Species

Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by ‘Candidatus B. rudakovii’ and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.

Evaluating Transmission Paths for Three Different Bartonella spp. in Ixodes ricinus Ticks Using Artificial Feeding

Bartonellae are facultative intracellular alpha-proteobacteria often transmitted by arthropods. Ixodes ricinus is the most important vector for arthropod-borne pathogens in Europe. However, its vector competence for Bartonella spp. is still unclear. This study aimed to experimentally compare its vector competence for three Bartonella species: B. henselae, B. grahamii, and B. schoenbuchensis. A total of 1333 ticks (1021 nymphs and 312 adults) were separated into four groups, one for each pathogen and a negative control group. Ticks were fed artificially with bovine blood spiked with the respective Bartonella species. DNA was extracted from selected ticks to verify Bartonella-infection by PCR. DNA of Bartonella spp. was detected in 34% of nymphs and females after feeding. The best engorgement results were obtained by ticks fed with B. henselae-spiked blood (65.3%) and B. schoenbuchensis (61.6%). Significantly more nymphs fed on infected blood (37.3%) molted into adults compared to the control group (11.4%). Bartonella DNA was found in 22% of eggs laid by previously infected females and in 8.6% of adults molted from infected nymphs. The transovarial and transstadial transmission of bartonellae suggest that I. ricinus could be a potential vector for three bacteria.

Exposure of Domestic Cats to Three Zoonotic Bartonella Species in the United States

Cat-associated Bartonella species, which include B. henselae, B. koehlerae, and B. clarridgeiae, can cause mild to severe illness in humans. In the present study, we evaluated 1362 serum samples obtained from domestic cats across the U.S. for seroreactivity against three species and two strain types of Bartonella associated with cats (B. henselae type 1, B. henselae type 2, B. koehlerae, and B. clarridgeiae) using an indirect immunofluorescent assay (IFA). Overall, the seroprevalence at the cutoff titer level of ≥1:64 was 23.1%. Seroreactivity was 11.1% and 3.7% at the titer level cutoff of ≥1:128 and at the cutoff of ≥1:256, respectively. The highest observation of seroreactivity occurred in the East South-Central, South Atlantic, West North-Central, and West South-Central regions. The lowest seroreactivity was detected in the East North-Central, Middle Atlantic, Mountain, New England, and Pacific regions. We observed reactivity against all four Bartonella spp. antigens in samples from eight out of the nine U.S. geographic regions.