Longitudinal study of infection with Borrelia burgdorferi in a population of Peromyscus leucopus at a Lyme disease-enzootic site in Maryland.

The maintenance of Borrelia burgdorferi in a population of Peromyscus leucopus was investigated from 202 mark and recapture mice and 61 mice that were removed from a site in Baltimore County, Maryland. Borrelia burgdorferi infection was detected by culture and polymerase chain reaction (PCR) of ear tissue, and exposure to the spirochete was quantified by serology. Overall prevalence of B. burgdorferi, as determined by culture and PCR of ear tissue at first capture, was 25% in the longitudinal sample and 42% in the cross-sectional sample. Significantly more juvenile mice were captured in the longitudinal sample (18%) than in the cross-sectional sample (0%). Among 36 captured juvenile mice, only one was infected with B. burgdorferi; this contributed to a significant trend for infection with B. burgdorferi with age. Recovery from infection with B. burgdorferi was not detected among 77 mice followed for an average of 160 days. The incidence rate of infection with B. burgdorferi was 10 times greater in mice captured during two periods of high risk of exposure to nymphal Ixodes scapularis ticks compared with a period of low risk. Maintenance of B. burgdorferi in this population was dependent on indirect transmission of the organism from infected ticks to susceptible mice and development of chronic infection with the spirochete, which had no measurable effect on the survival of infected mice.

Ear biopsy location influences detection of Borrelia burgdorferi by PCR, but not by culture in naturally infected Peromyscus leucopus.

We determined if the ear biopsy location affected detection of Borrelia burgdorferi when either culture or the polymerase chain reaction (PCR) was used among 50 white-footed mice (Peromyscus leucopus), live-captured in a Lyme disease enzootic area in Maryland (USA) between March and October of 1991 and 1992. The infection status of individual mice was determined by organ culture; ear biopsy samples were obtained from the peripheral and central part of the ear for detection of B. burgdorferi by culture and by PCR. Overall, B. burgdorferi was cultured from one or more tissue samples in 33 (66%) of 50 captured mice. Among infected mice, B. burgdorferi was detected by culture in 29 (88%) of 33 peripheral and 28 (85%) of 33 central ear biopsy samples. By PCR it was detected in 24 (73%) of 33 peripheral and all 33 central samples (P = 0.002). Detection of B. burgdorferi by culture was independent of the ear biopsy location; however, the organism was detected by PCR with greater frequency in central ear biopsy samples as compared to peripheral samples. Agreement between culture and PCR was moderate (Kappa = 0.64) on peripheral ear samples and excellent (Kappa = 0.79) on central samples. We propose that when ear biopsy samples are used to detect B. burgdorferi by PCR in wild-caught P. leucopus, removal of biopsy samples from the central part of the ear will achieve maximum sensitivity and will achieve the highest concordance between assays when both culture and PCR of ear biopsy samples are conducted in parallel.

The adrenocortical response of greater sage grouse (Centrocercus urophasianus) to capture, ACTH injection, and confinement, as measured in fecal samples.

Investigators of wildlife populations often utilize demographic indicators to understand the relationship between habitat characteristics and population viability. Assessments of corticosterone may enable earlier detection of populations at risk of decline because physiological adjustments to habitat disturbance occur before reproductive diminutions. Noninvasive methods to accomplish these assessments are important in species of concern, such as the greater sage grouse (GRSG). Therefore, we validated a radioimmunoassay that measures immunoreactive corticosterone metabolites (ICM) in fecal samples and used it to characterize the adrenocortical response of 15 GRSG exposed to capture, intravenous injection of 50 IU/kg adrenocorticotrophic hormone (ACTH) or saline, and 22 h of confinement. Those animals injected with ACTH exhibited a more sustained (P = 0.0139) and less variable (P = 0.0012) response than those injected with saline, indicating different levels of adrenocortical activity. We also found that potential field-collection protocols of fecal samples did not alter ICM concentrations: samples held at 4 degrees C for up to 16 h contained similar levels of ICM as those frozen (-20 degrees C) immediately. This study demonstrates a multiphasic adrenocortical response that varied with the level of stimulation and indicates that the assay used to measure this phenomenon is applicable for studies of wild GRSG.

Analysis of Borrelia burgdorferi sequentially isolated from Peromyscus leucopus captured at a Lyme disease enzootic site.

Thirty isolates of Borrelia burgdorferi sequentially cultured from 15 naturally infected white-footed mice (Peromyscus leucopus) were examined for variability in protein and plasmid profiles. Heterogeneity was detected in OspB and OspC and in proteins between 18.0 and 28.0 kDa by PAGE. Plasmid profiles were heterogeneous in the first isolate from 11 mice (73%) and between the first and last sequential isolate from 13 mice (87%). Comparison of the first and last isolates showed increased expression of OspC in 6 mice (40%) and was associated in each case with a shift in mobility of a 16.0-kb plasmid, suggesting that regulatory elements of ospC may reside on this plasmid. Hybridization studies suggested that individual mice may have been infected by a heterogeneous population of spirochetes and that changes in the protein and plasmid profiles between the first and last sequential isolates from some mice may have been the result of clonal selection.

Comparison of polymerase chain reaction and culture for detection of Borrelia burgdorferi in naturally infected Peromyscus leucopus and experimentally infected C.B-17 scid/scid mice.

Culture and the polymerase chain reaction (PCR) were compared for detection of Borrelia burgdorferi infection in wild-caught Peromyscus leucopus and experimentally inoculated C.B-17 scid/scid (severe combined immunodeficient) mice. PCR targeted highly conserved regions of the ospA gene and could detect one to five cultured organisms and 10 to 50 copies of molecularly cloned ospA DNA. Organs (kidney, spleen, and urinary bladder) and/or ear biopsy samples were obtained from 108 captured P. leucopus mice, and tissues were obtained from 7 experimentally inoculated mice. A simple sample-processing procedure with proteinase K and detergent treatment was used in the PCR analysis. Overall, B. burgdorferi was detected in 29 of 108 (27%) P. leucopus mice by culture and in 31 of 108 (29%) mice by PCR. As assessed by the kappa statistic, agreement between PCR and culture was high for ear and bladder (kappa = 0.80 and 0.65, respectively) and low for kidney and spleen (kappa = 0.37 and 0.03, respectively). While concordant results were obtained from 98 animals, PCR detected B. burgdorferi from 6 additional mice for which cultures were negative and culture detected B. burgdorferi from 4 animals which were PCR negative. Further phenol-chloroform extraction of DNA in a limited number of samples improved the sensitivity of PCR compared with that of culture. These results indicate that PCR may be as sensitive as culture for detecting B. burgdorferi in ear samples and that PCR analysis is suitable for establishing the infection status of animals in mark-release-recapture studies.

Population dynamics of a naturally occurring heterogeneous mixture of Borrelia burgdorferi clones.

Two unique isolates of Borrelia burgdorferi, differing in plasmid content and outer surface protein C expression, were cultured on sequential captures of a single free-living Peromyscus leucopus mouse and were examined for differences in transmissibility. Both isolates were transmissible from inoculated C.B-17 mice to larval Ixodes scapularis ticks and, subsequently, from infected nymphal ticks to C3H/HeJ mice. Plasmid and protein analyses suggested that the original isolates were a mixed population of B. burgdorferi, and cloning by limiting dilution resulted in the identification of two clonal groups. In addition to being heterogeneous in plasmid and genomic macrorestriction analyses, the clones varied with respect to the electrophoretic mobilities and antigenicity of their OspC proteins, as shown by their reactivity to a panel of monoclonal antibodies. Plasmid analysis of sequential isolates from C3H mice experimentally infected with the primary isolate or various mixtures of its subclones showed an apparently random fluctuation in clonal dominance in the majority of mice. Surprisingly, mice infected with each subclone were permissive to superinfection with the heterologous subclone, despite the presence of anti-B. burgdorferi antibodies at the time of the secondary challenge. These results show conclusively that mice captured at Lyme disease enzootic sites may be infected by mixed populations of genetically and antigenically distinct B. burgdorferi clones and that these infections can be acquired by coinfection or by sequential infection. The lack of cross-immunization between clones existing within a naturally occurring population may play a role in the maintenance of the genetic heterogeneity of B. burgdorferi in nature.

Isolation of a new subspecies, Bartonella vinsonii subsp. arupensis, from a cattle rancher: identity with isolates found in conjunction with Borrelia burgdorferi and Babesia microti among naturally infected mice.

Bacteremia with fever due to a novel subspecies of Bartonella vinsonii was found in a cattle rancher. The subspecies shared major characteristics of the genus Bartonella in terms of most biochemical features and cellular fatty acid profile, but it was distinguishable from other subspecies of B. vinsonii by good growth on heart infusion agar supplemented with X factor and by its pattern of enzymatic hydrolysis of peptide substrates. DNA relatedness studies verified that the isolate belonged to the genus Bartonella and that it was genotypically related to B. vinsonii. The highest level of relatedness was observed with recently characterized strains from naturally infected mice that were coinfected with Borrelia burgdorferi and Babesia microti. We propose the name Bartonella vinsonii subsp. arupensis subsp. nov. as the new subspecies to accommodate these human and murine isolates.

Gestational attenuation of Lyme arthritis is mediated by progesterone and IL-4.

Infection of different strains of laboratory mice with the agent of Lyme disease, Borrelia burgdorferi, results in arthritis, the severity of which has been correlated with the dominance of Th1 cytokines. In this study, we demonstrate that changes in B. burgdorferi-specific immunologic responses associated with pregnancy can alter the outcome of Lyme arthritis in mice. Whereas nonpregnant female C3H mice consistently developed severe Lyme arthritis, pregnant mice had a marked reduction in arthritis severity that was associated with a slight reduction in IFN-gamma and markedly increased levels of IL-4 production by B. burgdorferi-specific T cells. Similar reductions in arthritis severity and patterns of cytokine production were observed in nonpregnant, progesterone-implanted mice. Ab neutralization of IL-4 in progesterone-implanted mice resulted in severe arthritis. Our results are consistent with the known shift toward Th2 cytokine expression at the maternal-fetal interface, and are the first to show a pregnancy-related therapeutic effect in an infectious model.

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.

Cosegregation of a novel Bartonella species with Borrelia burgdorferi and Babesia microti in Peromyscus leucopus.

During surveillance for various tickborne pathogens in the upper Midwest during the summer and early fall of 1995, a Bartonella-like agent was detected in the blood of mice that were concurrently infected with Borrelia burgdorferi or Babesia microti (or both). The organism was isolated in pure culture after inoculation of blood from wild-caught mice into C.B-17 scid/scid mice. Phylogenetic analysis of the 16S rRNA and the citrate synthase genes showed that the novel Bartonella species and a Bartonella isolate from a mouse captured on Martha's Vineyard, Massachusetts, were closely related to each other and secondarily related to Bartonella grahamii and Bartonella vinsonii. Further analysis of Peromyscus leucopus blood and tissue samples demonstrated that the novel Bartonella species was exclusively found in conjunction with B. burgdorferi and B. microti. Patent coinfection with these agents may be relatively frequent in naturally infected mice.