SummaryBorrelia burgdorferi , the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surfaceexposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi . Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.

SummaryBorrelia burgdorferi, the causative agent of Lyme disease, has a limited set of genes to combat oxidative/nitrosative stress encountered in its tick vector or mammalian hosts. We inactivated the gene encoding for superoxide dismutase A (sodA, bb0153), an enzyme mediating the dismutation of superoxide anions and examined the in vitro and in vivo phenotype of the mutant. There were no significant differences in the in vitro growth characteristics of the sodA mutant compared with the control strains. Microscopic analysis of viability of spirochaetes revealed greater percentage of cell death upon treatment of sodA mutant with superoxide generators compared with its controls. Infectivity analysis in C3H/HeN mice following intradermal needle inoculation of 10 3 or 10 5 spirochaetes per mouse revealed complete attenuation of infectivity for the sodA mutant compared with control strains at 21 days post infection. The sodA mutant was more susceptible to the effects of activated macrophages and neutrophils, suggesting that its in vivo phenotype is partly due to the killing effects of activated immune cells. These studies indicate that SodA plays an important role in combating oxidative stress and is essential for the colonization and dissemination of B. burgdorferi in the murine model of Lyme disease.

BackgroundDisease risk maps are important tools that help ascertain the likelihood of exposure to specific infectious agents. Understanding how climate change may affect the suitability of habitats for ticks will improve the accuracy of risk maps of tick-borne pathogen transmission in humans and domestic animal populations. Lyme disease (LD) is the most prevalent arthropod borne disease in the US and Europe. The bacterium Borrelia burgdorferi causes LD and it is transmitted to humans and other mammalian hosts through the bite of infected Ixodes ticks. LD risk maps in the transboundary region between the U.S. and Mexico are lacking. Moreover, none of the published studies that evaluated the effect of climate change in the spatial and temporal distribution of I. scapularis have focused on this region.MethodsThe area of study included Texas and a portion of northeast Mexico. This area is referred herein as the Texas-Mexico transboundary region. Tick samples were obtained from various vertebrate hosts in the region under study. Ticks identified as I. scapularis were processed to obtain DNA and to determine if they were infected with B. burgdorferi using PCR. A maximum entropy approach (MAXENT) was used to forecast the present and future (2050) distribution of B. burgdorferi-infected I. scapularis in the Texas-Mexico transboundary region by correlating geographic data with climatic variables.ResultsOf the 1235 tick samples collected, 109 were identified as I. scapularis. Infection with B. burgdorferi was detected in 45% of the I. scapularis ticks collected. The model presented here indicates a wide distribution for I. scapularis, with higher probability of occurrence along the Gulf of Mexico coast. Results of the modeling approach applied predict that habitat suitable for the distribution of I. scapularis in the Texas-Mexico transboundary region will remain relatively stable until 2050.ConclusionsThe Texas-Mexico transboundary region appears to be part of a continuum in the pathogenic landscape of LD. Forecasting based on climate trends provides a tool to adapt strategies in the near future to mitigate the impact of LD related to its distribution and risk for transmission to human populations in the Mexico-US transboundary region.

Carbon storage regulator A (CsrA) is an RNA binding protein that has been characterized in many bacterial species to play a central regulatory role by modulating several metabolic processes. We recently showed that a homolog of CsrA in Borrelia burgdorferi (CsrA Bb , BB0184) was upregulated in response to propagation of B. burgdorferi under mammalian host-specific conditions. In order to further delineate the role of CsrA Bb , we generated a deletion mutant designated ES10 in a linear plasmid 25-negative isolate of B. burgdorferi strain B31 (ML23). The deletion mutant was screened by PCR and Southern blot hybridization, and a lack of synthesis of CsrA Bb in ES10 was confirmed by immunoblot analysis. Analysis of ES10 propagated at pH 6.8/37°C revealed a significant reduction in the levels of OspC, DbpA, BBK32, and BBA64 compared to those for the parental wild-type strain propagated under these conditions, while there were no significant changes in the levels of either OspA or P66. Moreover, the levels of two regulatory proteins, RpoS and BosR, were also found to be lower in ES10 than in the control strain. Quantitative real-time reverse transcription-PCR analysis of total RNA extracted from the parental strain and csrA Bb mutant revealed significant differences in gene expression consistent with the changes at the protein level. Neither the csrA Bb mutant nor the trans-complemented strain was capable of infection following intradermal needle inoculation in C3H/HeN mice at either 10 3 or 10 5 spirochetes per mouse. The further characterization of molecular basis of regulation mediated by CsrA Bb will provide significant insights into the pathophysiology of B. burgdorferi.

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Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.Lyme disease is the most prevalent arthropod-borne infection in the United States and remains a significant public health issue in certain geographic loci (3,46). It is a multiphasic disorder with clinical symptoms involving the cutaneous, musculoskeletal, cardiovascular, and nervous systems (62). Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to several vertebrate hosts, including humans, by the bite of infected Ixodes ticks. When ticks consume a blood meal from mammalian hosts, there is a rapid alteration of gene expression in B. burgdorferi, facilitating adaptation of the spirochetes to the highly disparate environmental conditions that exist between the tick vector and the vertebrate host (4, 9, 10, 15, 29, 30-32, 44, 53, 57-59, 63, 67). This adaptive gene expression may aid in the efficient trafficking of the spirochetes from the tick vector to the mammalian host and subsequently facilitate dissemination and colonization of various host tissues (16,22).Whole-genome transcriptional analyses using B. burgdorferi, propagated under in vitro growth conditions that mimic either the tick vector or mammalian host environment, have revealed preferential expression of plasmid-encoded genes (4,44,53,66). Among the several linear and circular plasmids present in B. burgdorferi, linear plasmid 54 (lp54) encodes the largest number of open reading frames (ORFs) that exhibit differential gene expression in respon...

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BackgroundIn the United States, Tick‐Borne Relapsing Fever (TBRF) in dogs is caused by the spirochete bacteria Borrelia turicatae and Borrelia hermsii, transmitted by Ornithodoros spp. ticks. The hallmark diagnostic feature of this infection is the visualization of numerous spirochetes during standard blood smear examination. Although the course of spirochetemia has not been fully characterized in dogs, in humans infected with TBRF the episodes of spirochetemia and fever are intermittent.ObjectivesTo describe TBRF in dogs by providing additional case reports and reviewing the disease in veterinary and human medicine.AnimalsFive cases of privately‐owned dogs naturally infected with TBRF in Texas are reviewed.MethodsCase series and literature review.ResultsAll dogs were examined because of lethargy, inappetence, and pyrexia. Two dogs also had signs of neurologic disease. All dogs had thrombocytopenia and spirochetemia. All cases were administered tetracyclines orally. Platelet numbers improved and spirochetemia and pyrexia resolved in 4 out of 5 dogs, where follow‐up information was available.Conclusion and Clinical ImportanceTBRF is likely underdiagnosed in veterinary medicine. In areas endemic to Ornithodoros spp. ticks, TBRF should be considered in dogs with thrombocytopenia. Examination of standard blood smears can provide a rapid and specific diagnosis of TBRF when spirochetes are observed.