Comparative vector competence of Dermacentor variabilis and Ixodes scapularis (Acari: Ixodidae) for the agent of human granulocytic ehrlichiosis

Vector competence of Ixodes scapularis Say and Dermacentor variabilis Say for the agent of human granulocytic ehrlichiosis (HGE) was compared. Five white-footed mice, Peromyscus leucopus Rafinesque, were inoculated intra-peritoneally with blood from a mouse infected with the agent of HGE. Approximately 100 I. scapularis and D. variabilis larvae were placed on each mouse and allowed to feed to repletion. Fed larvae were collected, separated according to species and allowed to molt to nymphs. Twenty-six per cent of I. scapularis (34/131) and 11% of D. variabilis (11/96) tested positive for the agent of HGE by polymerase chain reaction (PCR) after molting to nymphs. Positive I. scapularis nymphs transmitted the agent of HGE to P. leucopus mice in 5 of 5 trials. In contrast, the positive D. variabilis nymphs did not transmit the agent of agent of HGE in any of 6 trials. In a 2nd experiment, 3 P. leucopus mice were infested with I. scapularis nymphs that were infected with the agent of HGE. All 3 mice became infected with the agent of HGE and approximately 300 D. variabilis larvae were placed on each mouse and allowed to feed to repletion. Larvae were collected and allowed to molt to nymphs as before. Approximately 8% (5/60) of the nymphs became positive for the agent of HGE as determined by PCR. Twenty-five of these nymphs were then placed on each of 9 P. leucopus mice and allowed to feed to repletion. Evidence of transmission was not observed in any of 9 mice exposed to D. variabilis nymphs. These results demonstrate that although I. scapularis is a competent vector of the agent of the HGF, D. variabilis is not.     

Natural infection of small mammal species in Minnesota with the agent of human granulocytic ehrlichiosis.

The natural reservoirs for the agent of human granulocytic ehrlichiosis (HGE) are suspected to be the small mammals that host immature stages of Ixodes scapularis ticks. To determine if such small mammals are naturally infected, we collected blood and serum samples from small mammal species in rural and suburban areas of Minneapolis and St. Paul, Minn. Samples were collected from white-footed mice (Peromyscus leucopus), eastern chipmunks (Tamias striatus), southern red-backed voles (Clethrionomys gapperi), and insectivorous shrews (Blarina brevicauda and Sorex cinereus). Blood samples were tested by PCR for active infection with the HGE agent, and sera from P. leucopus mice were tested for serologic evidence of infection by indirect immunofluorescence. PCR analyses revealed the presence of HGE agent DNA in 20 of the 190 samples (10.5%) tested. Of the 119 P. leucopus mouse serum samples that were analyzed, 12 (10.1%) contained Ehrlichia equi antibodies. In 3 of 119 (2.5%) P. leucopus mice from which both blood and serum were collected. HGE agent DNA and antibodies against E. equi were present. Animals with evidence of infection with the HGE agent are widely distributed around the Minneapolis-St. Paul area in regions with known I. scapularis tick activity. Small mammals that are frequent hosts for larval I. scapularis ticks and that are found in areas where HGE occurs are likely to be a major reservoir from which infected ticks that bite humans are derived.     

Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with molecular sizes of 44 to 47 kDa, are immunodominant antigens in human infection. Monoclonal antibodies (MAbs) to the OMPs were made by immunizing BALB/c mice with the purified HGE agent and then by fusing spleen cells with myeloma cells. The immunologic specificities of three MAbs (3E65, 5C11, and 5D13) were examined with five human HGE agent isolates and one tick isolate. By Western blot analysis, all three MAbs recognized the HGE agent but not Ehrlichia chaffeensis, Ehrlichia sennetsu, Ehrlichia canis, or their host cells. MAb 3E65 reacted with a 44-kDa protein in the homologous human isolate but not in the remaining five isolates. The two remaining MAbs recognized proteins with molecular sizes of 44 to 47 kDa in all six isolates. Western blot results with the OMP fraction of the six isolates were consistent with results with the whole HGE agent. Immunofluorescent-antibody staining and immunogold labeling with these MAbs showed that these antigens were primarily present on the membrane of the HGE agent. MAbs 5C11 and 5D13 recognized the recombinant 44-kDa protein by Western immunoblot analysis, but MAb 3E65 did not. Passive immunization with MAb 3E65 was more effective in protecting mice from HGE agent infection than with MAbs 5C11 and 5D13. These MAbs would be useful for analyzing the role of the major OMP antigens in HGE agent infection and for serodiagnosis.     

Infection with Agents of Human Granulocytic Ehrlichiosis, Lyme Disease, and Babesiosis in Wild White-Footed Mice (Peromyscus leucopus) in Connecticut

White-footed mice, Peromyscus leucopus, were captured in southern Connecticut during 1997 and 1998 to determine the prevalence of infections caused by granulocytic Ehrlichia sp., Borrelia burgdorferi, and Babesia microti. Of the 50 mice captured and recaptured, 25 of 47 (53.2%) and 23 of 48 (47.9%) contained antibodies to the BDS or NCH-1 Ehrlichia strains, respectively, as determined by indirect fluorescent antibody (IFA) staining methods. The majority (83.3%) of 48 mice also contained antibodies to B. burgdorferi, as determined by enzyme-linked immunosorbent assay. Moreover, 20 of 26 (76.9%) contained antibodies to B. microti by IFA staining methods. In nested PCR tests using the 16S rRNA gene, the DNA of the human granulocytic ehrlichiosis (HGE) agent was detected in 17 of 47 mice (36.2%), but only 4 (23.5%) of these 17 mice were PCR positive at each capture. Antibody-positive reactions to granulocytic Ehrlichia sp. organisms were detected in 17 of 23 (73. 9%) of the PCR-positive mice. The sequences from PCR products from nine positive blood samples were identical to the HGE agent. Ehrlichia spp. were cultured from three of five mice captured in April 1998, including one that was PCR positive in April 1997. In addition, 2 of 14 larval Ixodes scapularis pools, which were attached to two PCR-positive mice, contained DNA of the HGE agent. A high percentage of white-footed mice are infected or have been infected naturally by the HGE agent with low-level persistent infection or frequent reinfection in some individual mice. However, the changes noted in the presence of DNA and antibodies in repeated blood and serum samples from individual mice over several months of field collection suggests that infection with granulocytic Ehrlichia is transient in most wild P. leucopus.     

Sequence analysis of the ank gene of granulocytic ehrlichiae

The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.     

Acquisition and Transmission of the Agent of Human Granulocytic Ehrlichiosis by Ixodes scapularis Ticks

The purpose of the present study was to investigate the transmission of a human isolate of the agent of human granulocytic ehrlichiosis (HGE agent) from infected mice to larval ticks and to examine the population kinetics of the HGE agent in different stages of the tick life cycle. The HGE agent was quantitated by competitive PCR with blood from infected mice and with Ixodes scapularis ticks. The median infectious dose for C3H mice was 10⁴ to 10⁵ organisms when blood from an infected severe combined immunodeficient mouse was used as an inoculum. Uninfected larval ticks began to acquire infection from infected mice within 24 h of attachment, and the number of HGE agent organisms increased in larval ticks during feeding and after detachment of replete ticks. Molted nymphal ticks, infected as larvae, transmitted infection to mice between 40 and 48 h of attachment. Onset of feeding stimulated replication of the HGE agent within nymphal ticks. These studies suggest that replication of the HGE agent during and after feeding in larvae and during feeding in nymphs is a means by which the HGE agent overcomes inefficiencies in acquisition of infection by ticks and in tick-borne transmission to mammalian hosts.     

Comparison of major antigenic proteins of six strains of the human granulocytic ehrlichiosis agent by Western immunoblot analysis.

The etiologic agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium. In 1996, blood specimens from 53 patients suspected of having HGE were examined by indirect fluorescent antibody (IFA) testing with the HGE agent no. 13 isolate as the antigen, by nested PCR, and by culture. All patients resided in Westchester County, N.Y. Twelve patient specimens were positive for IFA (titer > or = 1:40). Seven of these were also positive by PCR. Of the seven specimens positive by both IFA testing and PCR, the HGE agent was isolated from four (no. 2, 3, 6, and 11) and continuously cultured in HL-60 cells. These were confirmed as the HGE agent by sequencing of 16S rDNA. Both purified whole-cell organisms and the outer membrane fractions of the new isolates were compared with no. 13 isolate and a tick (USG) isolate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis. No. 11 and 13 isolates had identical SDS-PAGE patterns with respect to 49- and 47-kDa proteins. No. 3 and USG isolates lacked the 47-kDa protein, and no. 6 isolate lacked the 49-kDa protein. Both 49- and 47-kDa bands were absent in no. 2 isolate. Western blot results with seven different sera, including five convalescent-phase sera from these patients, one dog anti-USG isolate, and one horse anti-BDS isolate, showed that all major antigens in six isolates were recognized by all sera. However, the molecular sizes and the numbers of major antigens recognized varied among the six isolates. Overall, HGE agent no. 3, 6, 11, and 13, and USG isolates had similar patterns, with 1 or 2 major antigens with molecular masses of around 49 and 47 kDa. No. 2 isolate was quite distinct in having a major antigen of 43 kDa. This indicates that although these antigenic epitopes are all cross-reactive among strains, the HGE agent has a strain pleomorphism in its major antigenic proteins. The major antigen profiles of the outer membrane protein fractions and of whole organisms of six HGE agent isolates were similar, suggesting that 49- and 47-kDa major antigens are the outer membrane proteins of the HGE agent.     

The prevalence of Borrelia burgdorfieri (Spirochaetales: spirochaetaceae) and the agent of human granulocytic ehrlichiosis (Rickettsiaceae: Ehrlichieae) in Ixodes scapularis (Acari:Ixodidae) collected during 1998 and 1999 from Minnesota

We tested 103 adult Ixodes scapularis Say from 12 counties in Minnesota for the presence of Borrelia burgdorferi and the causative agent of human granulocytic ehrlichiosis (HGE), using polymerase chain reaction (PCR). A total of 17 ticks (16.5%) was positive for B. burgdoiferi using nested PCR for the flagellin gene. or both PCR for the ospA gene and nested PCR for the flagellin gene. A total of four ticks (3.8%) was positive for the agent of HGE using nested PCR for 16S rDNA. Counties in Minnesota with established and recently reported populations of I. scapularis both had ticks infected with B. burgdorferi. The agent of HGE was only detected in counties with established I. scapularis populations.     

Characterization of an immunoreactive protein from the agent of human granulocytic ehrlichiosis.

A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.     

Comparison of the nucleotide sequences of 16S rRNA, 444 Ep-ank, and groESL heat shock operon genes in naturally occurring Ehrlichia equi and human granulocytic ehrlichiosis agent isolates from Northern California

We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes. Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444 Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444 Ep-ank gene of the HGE agent and E. equi isolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of the groESL heat shock operon gene fragment is identical among E. equi, the HGE agent, and E. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E. equi isolates from northern California. These results suggest that E. equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.     

Human granulocytic ehrlichiosis in southern Germany: increased seroprevalence in high-risk groups.

To date, human granulocytic ehrlichiosis (HGE), the causative agent of which is likely transmitted by ticks in the Ixodes ricinus-Ixodes persulcatus complex, has not been diagnosed with certainty in patients outside the United States. The presence of a closely related vector tick, I. ricinus, as well as the occurrence of similar Ehrlichia spp. of veterinary importance, suggests that this disease is likely to be present in Europe. The aim of the present study was to compare the prevalence of antibodies against the HGE agent in sera collected from patients in groups at high risk for exposure to I. ricinus with that of a control population. Risk groups consisted of 150 forestry workers and 105 patients with an established diagnosis of Lyme disease. The control group was 103 healthy blood donors without a history of tick bites. We used a patient isolate of the HGE agent from Minnesota (J. L. Goodman, C. Nelson, B. Vitale, J. E. Madigan, J. S. Dumler, T. J. Kurtti, and U. G. Munderloh, N. Engl. J. Med. 334:209-215, 1996) propagated in HL60 cells as the source of antigen for a specific immunofluorescence assay (IFA). Elevated IFA titers (> or = 1:80) were present in 21 of 150 (14%) serum samples from forestry workers and in 12 of 105 (11.4%) serum samples from Lyme disease patients, but in only 2 of 103 (1.9%) serum samples from blood donors (P < or = 0.01 for either of the at-risk groups versus blood donors). The results of this study suggest that the HGE agent or a closely related organism exists in southern Germany and that seroconversion to it is common among groups exposed to Ixodes ticks. Final proof that HGE occurs in Germany will require the isolation of the causative agent from patients. HGE should be considered in the differential diagnosis of febrile illnesses in individuals exposed to Ixodes ticks in Europe as well as in North America.     

New infectious spirochete isolated from short-tailed shrews and white-footed mice.

A spirochete with two periplasmic flagella was isolated from the blood or tissues of spleens and kidneys from short-tailed shrews (Blarina brevicauda) and white-footed mice (Peromyscus leucopus) in Connecticut and Minnesota. After inoculation, the shrew-mouse spirochete infected Swiss mice and Syrian hamsters. This spirochete is morphologically and serologically distinct from the species of Treponema, Borrelia, Leptospira, and Spirochaeta examined.     

Gamma interferon dominates the murine cytokine response to the agent of human granulocytic ehrlichiosis and helps to control the degree of early rickettsemia

The cytokine response to the agent of human granulocytic ehrlichiosis (HGE) was assessed in a murine infection model and the role of gamma interferon (IFN-gamma), a cytokine that is crucial for host defenses against intracellular pathogens, was investigated by using IFN-gamma-deficient mice. The agent of HGE (aoHGE) is an obligate intracellular bacterium that survives within neutrophils: morulae (vacuoles containing HGE organisms) are evident in polymorphonuclear leukocytes of experimentally infected immunocompetent mice for 1 to 2 weeks. We now show that IFN-gamma levels increase during early infection of C3H/HeN or C57BL/6 mice with HGE bacteria. Moreover, in response to aoHGE extracts or concanavalin A, splenocytes from ehrlichia-infected mice produced more IFN-gamma and less interleukin-4 than controls, suggesting that aoHGE partially skewed the immune response towards a Th1 phenotype. Absolute concentration of morulae containing neutrophils in blood was 122 +/- 22 cells/microliter on day 8. The bacterial DNA burden was also highest on day 8 and then declined after IFN-gamma levels peaked. In contrast, IFN-gamma-deficient mice had a markedly elevated HGE bacteria burden with morulae concentration of 282 +/- 48 cells/microliter on day 5 (P = 0.004) and 242 +/- 63 cells/microliter on day 8 (P = 0.005). Rickettsemia resolved in immunocompetent and IFN-gamma deficient mice after 2 weeks, while both the immunocompetent and the IFN-gamma-deficient mice had increased serum antibodies against aoHGE antigens at this time point. These data demonstrate that the HGE agent elicits a prominent IFN-gamma response in mice and that IFN-gamma is important in controlling the degree of rickettsemia during the early phase of infection, while IFN-gamma independent mechanisms play a role at later time points.     

An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.     

ankA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats

Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to Ehrlichia phagocytophila. To identify and characterize E. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equi antibodies. Two clones, one each from HGE agent and E. equi, that were recognized specifically by antibodies to the E. phagocytophila group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6% identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was named ank (for ankyrin). When the encoded protein, named AnkA, was expressed in Escherichia coli, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE, and sera from horses convalescent from HGE and E. equi infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected cytoplasmic antigen in E. phagocytophila group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These results suggest that this Ehrlichia protein may influence host cell gene expression.     

Granulocytic ehrlichiosis in mice deficient in phagocyte oxidase or inducible nitric oxide synthase

Mice deficient in phox (gp91(phox-/-)) or NOS2 (NOS2(-/-)) were infected with the agent of human granulocytic ehrlichiosis (HGE) to evaluate the importance of these pathways in the eradication of HGE bacteria. NOS2(-/-) mice had delayed clearance of the HGE agent in comparison to control or gp91(phox-/-) mice, suggesting that reactive nitrogen intermediates play a role in the early control of HGE.     

Prevalence of Antibodies against the Human Granulocytic Ehrlichiosis Agent in Lyme Borreliosis Patients from Germany

 To contribute to the discussion of whether or not human granulocytic ehrlichiosis (HGE) occurs in midwestern Germany, sera from individuals with different risk categories for tick exposure were retrospectively examined by means of an immunofluorescence assay. The seroreprevalence for the HGE agent accounted for 5.5% of the 270 patients tested. Specific antibodies were detected more often in patients with early Lyme infection than in patients with stage III disease or in asymptomatic individuals seropositive for Lyme disease. Investigation of 50 patients with an active or recent syphilis infection revealed no cross-reactivity between Treponema pallidum antibodies and the HGE agent. The prevalence of HGE antibodies (13.1%) among 76 Lyme borreliosis patients from this urban area was significantly higher (P<0.05) than that in the control groups (2.6%). The findings indicate that concomitant or serial infections with Borrelia burgdorferi and the HGE agent or closely related organisms may be a common occurrence in tick-exposed patients from Germany.     

Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis alters murine immune responses, pathogen burden, and severity of Lyme arthritis

Lyme disease and human granulocytic ehrlichiosis (HGE) are tick-borne illnesses caused by Borrelia burgdorferi and the agent of HGE, respectively. We investigated the influence of dual infection with B. burgdorferi and the HGE agent on the course of murine Lyme arthritis and granulocytic ehrlichiosis. Coinfection resulted in increased levels of both pathogens and more severe Lyme arthritis compared with those in mice infected with B. burgdorferi alone. The increase in bacterial burden during dual infection was associated with enhanced acquisition of both organisms by larval ticks that were allowed to engorge upon infected mice. Coinfection also resulted in diminished interleukin-12 (IL-12), gamma interferon (IFN-gamma), and tumor necrosis factor alpha levels and elevated IL-6 levels in murine sera. During dual infection, IFN-gamma receptor expression on macrophages was also reduced, implying a decrease in phagocyte activation. These results suggest that coinfection of mice with B. burgdorferi and the HGE agent modulates host immune responses, resulting in increased bacterial burden, Lyme arthritis, and pathogen transmission to the vector.