The transmission dynamics of Anaplasma phagocytophilum (Ap) and Borrelia burgdorferi (Bb) among Ixodes scapularis (Is) and mammalian hosts was investigated at Camp Ripley, an area representative of central Minnesota. Prevalence of white-footed mouse infection with Ap and Bb were 20% and 42%, respectively, with a coinfection level of 14%. Peak levels of infection with both agents occurred in May. The average levels of seropositivity to Ap and Bb were 29.3% and 48%, respectively. Of the mice infected with Ap, 47.5% were able to eliminate the pathogen as compared with 19.4% of mice infected with Bb. Ap was detected in 88.4% of 43 eastern chipmunks examined and isolated from 44.7% of the animals. Bb was present in 72.7% of 11 chipmunks examined, and 100% of the animals were also infected with Ap. The seasonality of tick activity differs from that reported for the New York area. Is infestation of mice began in May with peak nymphal infestation also occurring in May (7.4 per infested mouse) and overlapping with peak larval infestation in June (77.1 per infested mouse). Infestation ranged from 100% in May to 34.5% in October. Is comprised 98.4% of the ticks infesting the mice. The temporal pattern of the developmental stages of Is infesting chipmunks was the same as for mice, except that the tick burdens were greater. The nymphal stage peaked in May (81.3 per animal), and the larval stage peaked in June (164.7 per animal). Infestation was 100% in May-August, and >99% of the ticks were Is. Antibodies to Ap were present in >80% of the white-tailed deer examined, but they were infected with the Ap-1 variant rather than the Ap strain infecting mice and humans. Antibodies to Bb were detected in >80% of the deer, but Bb DNA was only detected in 1.5% of blood specimens.

The human granulocytic ehrlichiosis agent (HGEa) survives extreme differences between ticks and humans, possibly by use of differential expression of specific antigens for survival in different hosts. The role of the immunodominant p44 antigens is unknown. In this study, HGEa cultured in human or tick cells was probed with human, mouse, and hamster serum and with monoclonal antibodies (MAbs). p44 antigens were strongly expressed in human HL-60 cells but were strikingly reduced in tick cells. In HGEa alternately grown in HL-60 or tick cells, a p44 epitope recognized by MAb R5E4 was expressed in human but not tick cells. This was not a temperature effect, because incubation of infected tick cells at 37 degreesC did not induce expression of the p44 epitope. The p44 antigen predominates in human but not tick cells and may be involved in regulatory changes that mediate survival of the HGEa by immune modulation after tick transmission.

ABSTRACT The p44 gene of the agent of human granulocytic ehrlichiosis (aoHGE) encodes a 44-kDa major outer surface protein. A technique was developed for the typing of the aoHGE based on the PCR amplification of the p44 gene followed by a multiple restriction digest with Hin dIII, Eco RV, and Asp I to generate restriction fragment length polymorphism patterns. Twenty-four samples of the aoHGE were collected from geographically dispersed sites in the United States and included isolates from humans, equines, canines, small mammals, and ticks. Six granulocytic ehrlichiosis (GE) types were identified. The GE typing method is relatively simple to perform, is reproducible, and is able to differentiate among the various isolates of granulocytic ehrlichiae in the United States. These characteristics suggest that this GE typing method may be an important epizootiological and epidemiological tool.

We examined white-footed mice (Peromyscus leucopus) from Minnesota for infection with the etiologic agent of human granulocytic ehrlichiosis (HGE). From April to September 1997, we collected P. leucopus from Washington County, Minnesota, an area enzootic for HGE. Blood was cultivated in HL60 cells for isolation of the HGE agent. Of 59 mice examined, only a single mouse was culture positive for the HGE agent. The 16S ribosomal DNA sequence of the isolate was determined to be identical to that of the HGE agent. The isolate was reactive with monoclonal antibodies to the 44-kDa antigen of the HGE agent and was infectious for laboratory mice.

Immunodominant proteins in the range of 42-45 kD are important for the serodiagnosis of human granulocytic ehrlichiosis (HGE). Antigens from human isolates of the etiologic agent of HGE cultivated in HL-60 cells were used to immunize BALB/c mice and generate a panel of hybridomas secreting monoclonal antibodies. Using an enzyme immunoassay, an immunofluorescent assay (IFA), and Western blotting, we showed that culture supernatants and ascites of these hybridomas were reactive with human isolates of the etiologic agent of HGE, Ehrlichia equi and E. phagocytophila. Following screening and subcloning, we selected three stable hybridomas, R1B10, R5E4, and R5A9, which were determined to be of the isotypes IgG3, IgG1, and IgG2a, respectively. These results suggest that the epitopes of the 42-45-kD protein recognized by these three monoclonal antibodies are conserved among E. equi, E. phagocytophila, and the etiologic agent of HGE. Western blot analysis showed reactivity with the 44-kD protein of human isolates of the HGE agent. None of the monoclonal antibodies were reactive with HL-60 cells that were not infected with the HGE agent. No cross-reactivity with related intracellular pathogens could be detected when undiluted supernatants from hybridoma cultures were allowed to react by IFA with antigens from E. chaffeensis, E. risticii, E. platys, Rickettsia rickettsii, R. prowazekii, or Coxiella burnetii. The additivity index of two antibodies, R5E4 and R1B10 was near zero, suggesting that these two antibodies may compete for the same epitope of the 44-kD protein, while monoclonal antibody R5A9 appears to interact with a different epitope. The antibodies secreted by these hybridomas may be useful as immunologic agents in serodiagnostic, immunohistochemical, and other studies of the etiologic agent of HGE.

ABSTRACT Human granulocytic ehrlichiosis (HGE) is usually diagnosed by immunofluorescent antibody (IFA) serology with Ehrlichia equi-infected neutrophils or HGE agent-infected cultured HL60 cells. The HGE agent and E. equi are antigenically diverse, and interpretation of serologic results is also often variable. Thus, we investigated the sensitivity and specificity of various HGE agent and E. equi antigens used for IFA diagnosis by three different laboratories. Serum samples from 28 patients with well-characterized HGE and 9 patients with suspected HGE who were investigated by PCR, blood smear examinations, and serology were used, along with 9 serum samples from patients with other rickettsial and ehrlichial infections. Each serum sample was tested with up to 10 different antigen preparations. Overall, qualitative IFA results agreed in 70% of the samples. Titers among antigens were similar (r = 0.89 to 0.96), but titers of individual samples varied by fourfold or more in 5 of 81 (6%) of the serum samples. Sensitivity ranged from 100% to 82%, and specificity varied from 100% to 67%, but these differences were not significant, even among those tested in the same laboratory or between two different laboratories. Antibodies were detected in 14 to 44% of acute-phase sera from confirmed HGE patients. Most false-positive reactions resulted with Ehrlichia chaffeensis; when these sera were excluded, the specificity of most antigens was 91 to 100%. These data indicate that IFA results often agree and that IFA is useful for diagnosis of HGE in convalescence. However, without further standardization, variability among serologic tests using E. equi and HGE agent isolates for diagnosis of HGE will occasionally provide discrepant results and confound diagnosis.

ABSTRACTHuman granulocytic ehrlichiosis (HGE) is an emerging infection caused by anEhrlichiaspecies closely related toEhrlichia equiandEhrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused byEhrlichia chaffeensis,Rickettsia rickettsii, andCoxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples fromR. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests forBorrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n= 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure toEhrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.

PURPOSE: To assess possible triggers and cofactors for chronic fatigue syndrome (CFS) and to compare levels of selected cytokines between cafes and an appropriately matched control group.PATIENTS AND METHODS: We conducted a case-control study of 47 cases of CFS obtained through a regional CFS research program maintained at a tertiary care medical center. One age-, gender-, and neighborhood-matched control was identified for each case through systematic community telephone sampling. Standardized questionnaires were administered to cases and controls. Sera were assayed for transforming growth factor-beta (TGF-beta), interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and antibody to Borrelia burgdorferi and Babesia microti.RESULTS: Cases were more likely to have exercised regularly before illness onset than controls (67% versus 40%; matched odds ratio (MOR) = 3.4; 95% CI = 1.2 to 11.8; P = 0.02). Female cases were more likely to be nulliparous prior to onset of CFS than controls (51% versus 31%; MOR = 8.0; 95% CI = 1.03 to 170; P = 0.05). History of other major factors, including silicone-gel breast implants (one Female case and one female control), pre-morbid history of depression (15% of cases, 11% of controls) and history of allergies (66% of cases, 51% of controls) were similar for cases and controls. However, cases were more likely to have a diagnosis of depression subsequent to their diagnosis of CFS compared to a similar time frame for controls (MOR = undefined; 95% CI lower bound = 2.5; P < 0.001). Positive antibody titers to B burgdorferi (one case and one control) and B microti (zero cases and two controls) were also similar.CONCLUSIONS: Further investigation into the role of prior routine exercise as a cofactor for CFS is warranted. This study supports the concurrence of CFS and depression, although pre-morbid history of depression was similar for both groups.

Objective: To determine whether azithromycin or amoxicillin is more efficacious for the treatment of erythema migrans skin lesions, which are characteristic of Lyme disease.Design: Randomized, double-blind, double-dummy, multicenter study. Acute manifestations and sequelae were assessed using a standardized format. Baseline clinical characteristics and response were correlated with serologic results. Patients were followed for 180 days.Setting: 12 outpatient centers in eight states.Patients: 246 adult patients with erythema migrans lesions at least 5 cm in diameter were enrolled and were stratified by the presence of flu-like symptoms (such as fever, chills, headache, malaise, fatigue, arthralgias, and myalgias) before randomization.Intervention: Oral treatment with either amoxicillin, 500 mg three times daily for 20 days, or azithromycin, 500 mg once daily for 7 days. Patients who received azithromycin also received a dummy placebo so that the dosing schedules were identical.Results: Of 217 evaluable patients, those treated with amoxicillin were significantly more likely than those treated with azithromycin to achieve complete resolution of disease at day 20, the end of therapy (88% compared with 76%; P = 0.024). More azithromycin recipients (16%) I than amoxicillin recipients (4%) had relapse (P = 0.005). A partial response at day 20 was highly predictive of relapse (27% of partial responders had relapse compared with 6% of complete responders; P < 0.001). For patients treated with azithromycin, development of an antibody response increased the possibility of achieving a complete response (81% of seropositive patients achieved a complete response compared with 60% of seronegative patients; P = 0.043). Patients with multiple erythema migrans lesions were more likely than patients with single erythema migrans lesions (P < 0.001) to have a positive antibody titer at baseline (63% compared with 17% for IgM; 39% compared with 16% for IgG). Fifty-seven percent of patients who had relapse were seronegative at the time of relapse.Conclusions: A 20-day course of amoxicillin was found to be an effective therapeutic regimen for erythema migrans. Most patients were seronegative for Borrelia burgdorferi at the time of presentation with erythema migrans (65%) and at the time of relapse (57%).

PURPOSE:The purposes of this study were to determine (1) the optimal techniques for and potential diagnostic usefulness of the polymerase chain reaction (PCR) in early Lyme disease, and (2) the true frequency and clinical correlates of PCR-documented blood-borne infection in the dissemination of Lyme disease. PATIENTS AND METHODS:We performed a prospective, controlled, blinded study of PCR, culture, and serology on fractionated blood samples from 105 patients; 76 with physician-diagnosed erythema migrans and 29 controls. Clinical characteristics of the patients were obtained with a standardized data entry form and correlated with results of the laboratory studies. RESULTS:Only 4 of the 76 (5.3%) patients with erythema migrans were culture positive; however, 14 of 76 (18.4%) had spirochetemia documented by PCR of their plasma. None of 29 controls were PCR or culture positive (P = 0.007, versus patients). PCR-documented spirochetemia correlated with clinical evidence of disseminated disease; 10 of 33 patients (30.3%) with systemic symptom(s) were PCR positive compared to 4 of 43 (9.3%) without such evidence (P = 0.02). PCR positivity was more frequent among patients with each of four specific symptoms: fever, arthralgia, myalgia, and headache (all P < 0.05). A higher total number of symptoms (median 2.5 in PCR-positive patients versus 0 in PCR-negative controls; P < 0.01) and the presence of multiple skin lesions (37.5% of patients with multiple, versus 13.3% of patients with single lesions [P = 0.04] were also correlated with PCR positivity. Patients with both systemic symptoms and multiple skin lesions had a 40% PCR-positivity rate; however, 4 of 42 (9.5%) asympatomatic patients with only single erythema migrans lesions were also PCR positive. In multivariate analysis using logistic regression, the number of systemic symptoms was the strongest independent predictor of PCR positivity (P = 0.004). CONCLUSIONS:PCR detection of Borrelia burgdorferi is at least three times more sensitive than culture for identifying spirochetemia in early Lyme disease and may be useful in rapid diagnosis. PCR positivity significantly correlates with clinical evidence of disease dissemination. Bloodstream invasion is an important and common mechanism for the dissemination of the Lyme disease spirochete.

Serum specimens from persons,vith or without Lyme borreliosis were analyzed by indirect fluorescent antibody staining methods for total immunoglobulins to Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain), There was serologic evidence of human exposure to multiple tick-borne agents in 15 (6.6%) of 227 serum samples obtained in Connecticut and Minnesota. Of these, 10 serum samples were from Connecticut patients who had erythema migrans and antibodies to Borrelia burgdorferi (range, 1:160 to 1:40,960). A maximal antibody titer of 1:640 was noted for a B, microti infection, whereas titration end points of 1:640 and 1:1,280 were recorded for E, chaffeensis and E, equi seropositives, respectively. In specificity tests, there was no cross-reactivity among the antisera and antigens tested for the four tick-borne pathogens, On the basis of serologic testing, a small group of persons who had Lyme borreliosis had been exposed to one or more other tick-borne agents, but there was no clinical diagnosis of babesiosis or ehrlichiosis. Therefore, if the clinical picture is unclear or multiple tick-associated illnesses are suspected, more extensive laboratory testing is suggested.

During a special two-day hunt (11, 12 November 1989) in Saint Croix State Park, Minnesota (USA), one side of the neck for each of 146 white-tailed deer (Odocoileus virginianus) was examined for ticks. Of the 5,442 ticks collected, 90% (4,893) were the winter tick, Dermacentor albipictus, and 10% (549) were the deer tick, Ixodes dammini, the primary vector of the causative agent of Lyme disease, Borrelia burgdorferi. Adult males had the greatest frequency of infestation of either D. albipictus (100%) or I. dammini (88%) and had on average more ticks, compared to other deer. Based on an examination of midgut material from 435 I. dammini by polyclonal antibody analysis, spirochetes were observed in 22% of the ticks. Species-specific monoclonal antibody analysis of the spirochetes confirmed that the bacteria were B. burgdorferi.

Skinbiopsy specimens fromtheperipheral aspect oferythema migrans lesions (site 1)andfromclinically normal perilesional areas(site 2)werecompared assourcesofBorrelia burgdorferi. Thisspirochete wasisolated fromtheskinof18of21(86%)patients withuntreated early Lymedisease atone orbothbiopsy sites. Site 1 specimens weresuperior tosite 2specimens fortheisolation ofB.burgdorfieri. Site 1 specimensfrom18(86%) patients wereculture positive, andsite 2specimens from12(57%)patients wereculture positive. Forpatients whosesite 2specimens were culture positive, site 1specimens were alsofoundtobeculture positive. B. burgdorferi was isolated fromtwopatients withatypical lesions andfromtwopatients witherythema migrans lesions that wereless than 5 cm indiameter. Thisstudy demonstrates that thecultivation ofB.burgdorfieri from skinbiopsy specimens fromcutaneous lesions thought tobeerythema migrans canbean efficacious procedure forconfirming thediagnosis ofLymedisease andthatthespirochete ispresent inclinically normalappearing perilesional skin.

PURPOSE:To compare the safety and efficacy of azithromycin, amoxicillin/probenecid, and doxycycline for the treatment of early Lyme disease, to identify risk factors for treatment failure, and to describe the serologic response in treated patients. PATIENTS AND METHODS:Fifty-five patients with erythema migrans and two patients with flu-like symptoms alone and fourfold changes in antibody titers to Borrelia burgdorferi were randomized to receive (1) oral azithromycin, 500 mg on the first day followed by 250 mg once a day for 4 days; (2) oral amoxicillin 500 mg and probenecid 500 mg, three times a day for each for 10 days; or (3) doxcycline, 100 mg twice a day for 10 days. If symptoms were still present at 10 days, treatment was extended with amoxicillin/probenecid or doxycycline for 10 more days. Evaluations were done at study entry and 10, 30, and 180 days later. RESULTS:Three of the patients who initially had symptoms suggestive of spread of the spirochete to the nervous system, one from each antibiotic treatment group, subsequently developed neurologic abnormalities, but symptoms in the other 54 patients resolved within 3 to 30 days after study entry. Six of the 19 patients (32%) (95% confidence interval, 13% to 57%) given amoxicillin/probenecid developed a drug eruption, whereas none of the patients given azithromycin or doxycycline had this complication. The presence of dysesthesias at study entry was the only risk factor significantly associated with treatment failure (p less than 0.001). By convalescence, 72% of the patients were seropositive, and 56% still had detectable IgM responses to the spirochete 6 months later. CONCLUSIONS:The three antibiotic regimens tested in this study were generally effective for the treatment of early Lyme disease, but the regimens differ in the frequency of side effects and in ease of administration.

Theinvitro andinvivosusceptibilities oftheLymedisease pathogen Borrelia burgdorferi tocefuroxime were compared withthose ofseveral other antibiotics commonly usedtotreat this disease. Cefuroxime demonstrated ahigher MBC invitro (1.0 ,ug/ml) thanceftriaxone (0.08 ,Ig/ml) orerythromycin (0.32 ,ug/ml), buttheMBC wassimilar tothat ofamoxicillin (0.8 ,ug/ml) anddoxycycline (1.6,ig/ml). B.burgdorferi was considerably less susceptible totetracycline (3.2 ,ug/mI) andpenicillin G (6.4,ug/ml). Ofthethree other Borrelia species tested, two(Borrelia turicatae andBorrelia anserina) also demonstrated susceptibility tocefuroxime, while thethird (Borrelia hernsii) was less susceptible. Results obtained withfourantimicrobial agentsintheinvivohamster modelparallel theantibiotic susceptibilities intheinvitro study. Thethree antibiotics withsimilar MBCsin vitro, i.e., cefuroxime, doxycycline, andamoxicillin, demonstrated comparable activities inpreventing borreliosis inB.burgdorferi-chalHenged hamsters (50%curative doses = 28.6,36.5and45.0mg/kg, respectively). Penicillin G,whichdemonstrated thehighest MBC invitro, hadveryweakprotective activity in thehamster modelsystem. Theseresults indicate thattheinvitro andinvivoactivities ofcefuroxime against B.burgdorfefi arecomparable tothose ofseveral oral antibiotics currently being usedinthetreatment ofearly Lymedisease andsuggest thattheoral formofthis cephalosporin may bean effective alternative therapy for this disease.

During a studyofmigrating landbirdsin1987, we examinedover 9,200individual birdsrepresenting 99 species fromtheSaint CroixRiverValley, a Lyme disease-endemic area ofeastcentralMinnesotaand northwestern Wisconsin. We foundthat250deertick(Ixodes dammini) larvae andnymphsinfested 58birds from15migrant species; 56ticks (22.4%) were positive fortheLymedisease spirochete Borrelia burgdorferi. Fiveground-foraging migrant birdspecies favoring mesichabitats, veery (Catharus fuscescens), ovenbird (Seiurus aurocapillus), northern waterthrush (S.novaboracensis), common yellowthroat (Geothlypis trichas), andswamp sparrow (Melospiza georgiana), accounted fornearly three-quarters ofparasitized individuals. Nearly half ofthespirochete-positive ticks were removedfrommigrating birdstakenina riparian floodplain forest. Recaptured migrants withinfected ticks indicate thattheytransmit B.burgdorferi tohexapodlarvae. We suggest thatbirds may bebothan important localreservoir intheupper Mississippi Valleyand long-distance dispersal agentsforB.burgdorferi-infected ticks tootherregions ofthecontinent. Landbirdsthatmigrate forlongdistances may travel seasonally up to7,000 kmfromtheir breeding habitats inthe middle andhighlatitudes ofNorthAmerica totheir winteringareasintheneotropics. Manymigrants travel through a variety ofhabitats andregions during their seasonal passage, stopping atsome torestandreplenish nutrient reserves before continuing (15,19).Whilestopped ata resting site, thebirds may acquire locally abundant cysts,seeds, spores, larvae, or otherpropagules, whichmay besubsequently dispersed daysor weekslater. Forexample, spores of pathogenic fungi common on agricultural crops havebeen isolated from63species oflandbirds migrating fromMexico totheCentral Plains (G.Warner, Ph.D.dissertation, University ofMinnesota, Minneapolis, 1976). Similar studies of bothresident andmigrant birds inhabiting theconifer forests ofcentral NorthAmericademonstrated thatthebirds were importantvectorsindispersing dwarfmistletoe (Arceuithobiuimspp.) seeds(20,21).InNorthAfrica, 40species of autumn-migrating landbirds were foundtotransport ticks, including Ixodesricinius (14), a European vectorforthe Lymedisease pathogen, Borrelia b-rgdorferi (5). Recent studies inNorthAmerica on winterandsummer bird populations showthatbirds serveashosts forI.dammini, one of several knownacaridan vectorsforB.burgdoiferi (2,3,7). Although birds are nottheonlyvertebrate hosts, theyoffer aready means ofwidespread dispersal ofticks infected with Borrelia spp.(3,7,24,25). Thiscosmopolitan prevalence of B.burgdorferi (2,25)provides an opportunity toexplore its webofrelationships withdiverse arthropod vectors, their hosts, andthenatural environments inwhichthesecomplex interactions takeplace. During a studyofhabitat use bymigrating birdsin1986, ectoparasites foundon captured birds were removedand preserved forlater study. Examination oftheixodid ticks removedshowedthat a majority were deerticks(I.damnmini). Ixodid ticks were again collected frommigrating birds during thecontinuing habitat study in1987, andtheseticks were examined forthespirochete B.bmrgdo,iferi.

The antibody and complement requirements for killing of Borrelia burgdorferi 297 by normal human serum (NHS) and NHS plus immunoglobulin G (IgG) were examined. B. burgdorferi activated both the alternative and classical complement pathways in NHS. In NHS chelated with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 4 mM MgCl2 (Mg-EGTA) to block classical pathway activation, consumption (activation) of total hemolytic complement, complement component 3 (C3), and C9 by B. burgdorferi was observed. Furthermore, challenge of unchelated NHS with 297 cells resulted in the consumption of C4, in addition to an increase in C3 and C9 consumption over that observed in chelated serum. In spite of complement activation, B. burgdorferi was resistant to the nonspecific bactericidal activity of NHS. The addition of human anti-B. burgdorferi IgG to NHS, however, resulted in the complete killing of 297 cells. Bactericidal activity of this serum was abrogated if NHS was immunochemically depleted of C1, indicating that killing was mediated by the classical pathway. The manifestation of bactericidal activity was accompanied by a large increase in total complement and C3 consumption over that observed in NHS alone. Under similar conditions, only a minimal increase in C9 consumption was observed. No increase in total complement consumption was observed if NHS plus anti-B. burgdorferi IgG was treated with Mg-EGTA prior to challenge. The results of these experiments demonstrate that B. burgdorferi is resistant to the nonspecific bactericidal activity of NHS, in spite of classical and alternative complement pathway activation. B. burgdorferi is sensitive to serum, however, in the presence of IgG, which mediates bacterial killing through the classical complement pathway.