Tick-borne relapsing fever in British Columbia, Canada: first isolation of Borrelia hermsii.

The spirochete that causes tick-borne relapsing fever, Borrelia hermsii, was isolated in pure culture during 1995 and 1996 from three acutely ill human patients infected in southern British Columbia, Canada. The geographic area of exposure is a known focus of this disease dating back to 1930 when the first case was recognized in a human. Analyses of plasmid DNA, protein profiles, and reactivity with a species-specific monoclonal antibody identified the new isolates of spirochetes as B. hermsii, all of which were most similar to an isolate of this spirochete from northern California described previously. These are the first reported isolates of B. hermsii from Canada.

Tick-borne relapsing fever in the northwestern United States and southwestern Canada.

Records from 182 cases of tick-borne relapsing fever (TBRF) were reviewed. In confirmed cases, there was febrile illness, and spirochetes were identified on peripheral blood preparations. In probable cases, there were clinical features of TBRF and either the same exposure as a confirmed case or serological (indirect fluorescent antibody test and western blotting [WB]) evidence of infection with Borrelia hermisii. Sera also were tested for antibody to Borrelia burgdorferi. We identified 133 confirmed and 49 probable cases of TBRF. A Jarisch-Herxheimer reaction was reported in 33 (54.1%) of 61 cases for which this information was available. Most patients who had antibodies to B. hermsii were serologically positive for B. burgdorferi, and WB demonstrated false positivity of testing for B. burgdorferi. Thirty-five (21%) of 166 cases were unreported to public health authorities. In 52 cases, there were more than two relapses before the diagnosis. This study demonstrates that TBRF is underrecognized and underreported and may be falsely identified as Lyme disease.

Evaluation of three commercial tick removal tools.

OBJECTIVE: To evaluate three commercially available tick removal tools against medium-tipped nontissue tweezers. METHODS: We evaluated three commercially available tick removal tools against medium-tipped tweezers. Three inexperienced users randomly removed attached American dog ticks (Dermacentor variabilis Say) and lone star ticks (Amblyomma americanum L.) from laboratory rabbits in a university animal facility using all tools during one removal session. RESULTS: Tick damage occurring from removal and quantity of attachment cement were compared. No tool removed nymphs without damage and all tools removed adults of both species successfully. American dog ticks proved easier to remove than lone star ticks, whose mouthparts often remained in the skin. CONCLUSIONS: Nymphal ticks were consistently removed more successfully with commercial tools when compared with tweezers but with more difficulty than adults were removed. The commercial tick removal tools tested are functional for removal of nymphs and adults and should be considered as viable alternatives to medium-tipped tweezers.

Rickettsia peacockii sp. nov., a new species infecting wood ticks, Dermacentor andersoni, in western Montana.

Rickettsia peacockii, a new species of spotted fever group rickettsiae, was identified from Rocky Mountain wood ticks (Dermacentor andersoni) collected in the Sapphire Mountain Range on the eastern side of Bitterroot Valley, Montana. DNA from R. peacockii SkalkahoT (T = type strain) in naturally infected tick tissue was amplified by a PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA), rickettsial citrate synthase, and 190-kDa surface antigen (rOmpA) genes. Partial 16S rDNA and rOmpA gene sequences exhibited levels of similarity of 99.7 and 93.2%, respectively, with the sequences of the spotted fever agent Rickettsia rickettsii R. By using Gimenez staining, fluorescent antibody tests, a PCR assay, and a restriction fragment length polymorphism analysis, 76 of 115 female ticks (minimal field infection rate, 66.1%) collected between 1992 and 1995 were found to be infected. The organism is passed transstadially and transovarially (minimal vertical transmission rate, 73.3%), and infections are localized in ovarial tissues. Attempts to cultivate R. peacockii were unsuccessful.

DNA typing of rickettsiae in naturally infected ticks using a polymerase chain reaction/restriction fragment length polymorphism system.

We used the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) rickettsial typing system of Regnery and others to rapidly identify rickettsiae in naturally infected ticks. Unlike previously described methods, our PCR assays type rickettsiae directly from tick tissues without first isolating the organisms. We collected 226 adult Dermacentor andersoni ticks in the Bitterroot Mountains of western Montana and analyzed them for possible rickettsial infection by hemolymph test using the Gimenez stain. Thirteen (5.8%) of these ticks were positive by hemolymph test and selected for further analysis using the above PCR/RFLP typing system. The PCR assays performed using the first primer set (RpCS) resulted in amplification of fragments of the predicted size from nine of the 13 hemolymph test-positive tick samples. Only four of these nine tick samples were also positive in similar PCR assays performed with a second primer set (Rr190) that is presumed to be spotted fever group specific. The RFLP analyses of material amplified from these four ticks indicated they were infected with Rickettsia rickettsii (one sample) and R. rhipicephali (three samples). The PCR/RFLP analyses of the five PCR-positive tick samples that were positive only in assays performed with the RpCS primer set indicated that these ticks were infected with R. bellii. The remaining four of 13 hemolymph test-positive tick samples gave negative PCR results with both the RpCS and Rr190 primer sets. Infected hemocytes from these PCR-negative ticks contained organisms of distinctive bacillary morphology that appeared similar to those described previously as long forms, and it is possible that these organisms belong to a genus other than Rickettsia. We also examined established laboratory isolates of tick-borne rickettsiae from different regions of North America to determine whether this typing system produces consistent results. Multiple isolates of R. montana (nine isolates), R. bellii (five isolates), R. rickettsii (Hlp-like) (four isolates), and R. canada (two isolates) were tested and no significant variations in PCR/RFLP patterns were observed between members of the same serotypes. However, among the five isolates of R. rhipicephali tested, two slightly different RFLP patterns were noted. Our results suggest that this PCR/RFLP typing scheme has wide applicability for identifying rickettsiae directly from D. andersoni or D. variabilis tick tissues.

Experimental infection of Columbian black-tailed deer with the Lyme disease spirochete.

The course of Borrelia burgdorferi-infection in Columbian black-tailed deer. (Odocoileus hemionus columbianus), its effect on the health of these animals, and their reservoir competence for fleas were evaluated experimentally. Four yearling females inoculated intramuscularly with 10(8) organisms of the CA4 strain of B. burgdorferi, and two yearling males unexposed to spirochetes, were monitored daily for 3 mo. Spirochetes were reisolated from the blood of three does at 14 or 70 days postinjection, and from several tissues of the fourth doe at necropsy. Considerable antigenic heterogeneity was observed among the reisolates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only two of the four infected deer developed significant antibodies (> or = 1:128) to B. burgdorferi with titers persisting for < or = 2 mo. Hematological values were highly variable and the degree of variation observed was much greater than that reported previously for Columbian black-tailed deer or other subspecies of mule deer. Infected deer did not manifest signs of Lyme disease. On histologic examination of eight tissues per deer, we observed a minimal hepatic lesion in all animals exposed to B. burgdorferi. No spirochetes were detected in 367 fleas (Pulex irritans) that had naturally infested these deer; thus this flea probably is an inefficient host of B. burgdorferi.

Confirmation that Rickettsia helvetica sp. nov. is a distinct species of the spotted fever group of rickettsiae.

We propose the name Rickettsia helvetica sp. nov. for a rickettsial serotype of unknown pathogenicity isolated in 1979 in Switzerland from Ixodes ricinus ticks and designated the Swiss agent. The growth characteristics and the results of microimmunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting (immunoblotting) with specific mice sera, and a polymerase chain reaction followed by restriction fragment length polymorphism analysis confirmed previously reported preliminary findings which suggested that this rickettsia, to which a name was given provisionally, does represent a new member of the spotted fever group of rickettsiae. The type strain is C3 (Reference Center for Rickettsioses, Marseille, France).

Comparison of latex agglutination, indirect immunofluorescent antibody, and enzyme immunoassay methods for serodiagnosis of Rocky Mountain spotted fever in dogs.

Indirect immunofluorescent antibody (IFA), latex agglutination (LA), and enzyme immunoassay (EIA) methods were compared for evaluation of the serum antibody responses of dogs experimentally and naturally exposed to spotted fever-group rickettsiae. Selected sera (obtained on days 1, 42, 53, 124, 145, 236, 255, 264, and 292) were examined from three 8-month-old female Beagles inoculated with Rickettsia rickettsii on days 34 and 250 of the study. A second group of dogs comprised three 8-month-old female Beagles inoculated with R montana on days 34 and 102. Subsequently, these dogs were inoculated with R rickettsii on day 250. Serum samples were obtained from the second group of dogs on days 1, 96, 103, 132, 180, 215, 292, and 494. A third group consisted of 21 naturally exposed dogs, from which sequentially obtained serum samples were available, and which had clinical signs compatible with Rocky Mountain spotted fever. Clinical signs of disease in dogs of the third group resolved after treatment with tetracycline (22 mg/kg of body weight, PO, q 8 h) was instituted. At least 2 sequentially obtained serum samples from each dog were tested. In general, the first sample was obtained just prior to treatment and the convalescent serum samples were obtained at weekly or greater intervals thereafter. For correlation and reactivity data, an IFA test for IgG/IgM (using heavy and light chains-specific conjugate) was used as the reference standard for comparison of results with those of the other tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Lyme borreliosis: ten years after discovery of the etiologic agent, Borrelia burgdorferi.

Since the recovery of its causative agent, Borrelia burgdorferi, in 1981, Lyme borreliosis has become the most prevalent tick-borne disease in the United States as well as in Europe. Its steadily increasing clinical spectrum now includes erythema migrans, acrodermatitis chronica atrophicans, lymphadenosis beniga cutis, arthritis, myocarditis, progressive meningoencephalitis, myositis, and various ocular and skin disorders. The true incidence of Lyme borreliosis in the world is unknown. In the United States, it has increased from 2,000 cases in 1987, to more than 8,000 in 1989. It occurs now in regions where the tick vectors, Ixodes dammini and Ixodes pacificus, are absent and where other species of ticks may be responsible for maintaining and distributing the spirochete. In Europe, Lyme borreliosis has been reported from 19 countries; its occurrence coincides with the distribution of the vector tick, Ixodes ricinus and possibly Ixodes hexagonus. Specific and dependable serological tests are still not available, but development of probes for specific antigens and the polymerase chain reaction appear promising in detecting ongoing infections and in identifying B. burgdorferi in ticks, animal, and human hosts. Brief reference is made to advances in the preparation of whole cell and genetically engineered vaccines.

Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals.

Borrelia burgdorferi expresses a conserved, species-specific 39-kDa protein (P39) that can stimulate antibodies during human infection. To confirm that anti-P39 antibodies are produced consistently in animals exposed to infectious spirochetes, white-footed mice, Peromyscus leucopus, and laboratory white mice, Mus musculus (strain BALB/c), were experimentally inoculated with either infectious or noninfectious B. burgdorferi and the antibody response to P39 was determined by immunoblot at 21 days postinoculation. All mice inoculated with approximately 10(7) infectious B. burgdorferi produced anti-P39 antibodies and were cultured positive for this spirochete. Mice inoculated with similar numbers of inactivated or viable noninfectious B. burgdorferi still producing P39 did not induce anti-P39 antibodies. By contrast, putative antiflagellin antibodies were detected in less than 18% of the infected animals, which supports the notion that antibody reactive with flagellin may not be reliable as a marker for B. burgdorferi exposure as was originally thought. Mice infected with B. burgdorferi following exposure to ticks (Ixodes dammini) produced anti-P39 antibodies no later than 7 days postinfection, indicating that P39 is an effective immunogen in natural infections. Notably, anti-P39 antibodies were the predominant B. burgdorferi reactive antibodies detected early in the infection. Our results indicate that anti-P39 antibodies are produced in response to an active infection and are therefore reliable markers for infection in experimentally and naturally inoculated animals.

Lyme borreliosis: a relapsing fever-like disease?

To determine by xenodiagnosis length and concentrations of spirochetemias produced by Borrelia burgdorferi in white-footed mice (Peromyscus leucopus), laboratory reared mice were inoculated with either spirochete-containing tick suspensions or BSK II spirochete culture and were exposed for as long as three months to larval Ixodes dammini. Upon development to the nymphal stage, ticks were evaluated for spirochetal infections by direct immunofluorescence. All mice were found to circulate spirochetes for at least three months in concentrations sufficient to infect ticks. The percentage of infected ticks alternated from low to high, suggesting occurrence of episodes of mild and heavy spirochetemias. The results suggest that B. burgdorferi in its animal hosts and possibly also in humans causes prolonged spirochetemias characterized by episodes of alternating high and low concentrations of spirochetes as reflected by similar percentages of infected ticks. The long persistence of spirochetes in the peripheral blood stream and the cyclical form of Lyme borreliosis appear to be related, as in relapsing fevers, to the capacity of B. burgdorferi to undergo antigenic variations.

Relationship of Borrelia burgdorferi to its arthropod vectors.

At the IV International Conference on Lyme Borreliosis, a workshop was held to identify the unique development of the Lyme disease spirochete, Borrelia burgdorferi, in its established and suspected arthropod vectors. The following is a summary of the panel's discussions of research aspects concerning relationship(s) of this borrelia to its vectors, and the mode(s) of its transmission to animal hosts.

Kinetics of IgM and IgG responses to experimental and naturally acquired Rickettsia rickettsii infection in dogs.

The kinetics of specific IgM and IgG antibody response was characterized in four 9-month-old Beagles after inoculation of 2 x 10(2) plaque-forming units (PFU) of Sheila Smith strain of Rickettsia rickettsii. Immunoglobulin M antibodies were first detected by indirect immunofluorescence on postinoculation (PI) day 9, peaked by PI day 20, and were no longer detectable by PI day 80. Immunoglobulin G antibodies became detectable between PI days 22 and 28, peaked by PI day 42, and decreased gradually through PI day 130. Subsequent challenges with R rickettsii on PI days 216 (2 x 10(2) PFU/dog) and 1,029 (5 x 10(4) tissue culture infective dose [TCID50]/dog) resulted in slightly different serologic responses. The initial challenge exposure failed to increase the concentration of IgG antibodies and induced only low concentrations of IgM antibodies. After the second challenge inoculation, IgM antibodies were not detectable and the concentration IgG antibodies increased slightly. Clinical abnormalities and seroconversion were documented in control dogs following each challenge exposure. Examination of acute and convalescent serum samples from 55 dogs in which Rocky Mountain spotted fever was suspected clinically suggested that sole evaluation of IgM antibodies in acute-phase serum would result in inaccurate diagnoses because of false-positive and -negative results. Use of a composite conjugate that detects IgM and IgG antibodies to R rickettsii appears to be satisfactory for diagnostic purposes; however, concurrent quantitation of IgM antibodies may facilitate serodiagnosis in a select group of dogs in which a four-fold increase in convalescent antibody titer is not detected by use of the composite conjugate.(ABSTRACT TRUNCATED AT 250 WORDS)

Hispid cotton rats (Sigmodon hispidus) as a source for infecting immature Dermacentor variabilis (Acari: Ixodidae) with Rickettsia rickettsii.

Hispid cotton rats (Sigmodon hispidus Say and Ord) were susceptible to infection with Rickettsia rickettsii Wolbach under laboratory conditions and were capable of serving as sources for infecting ticks with rickettsiae. Cotton rats developed rickettsemias that could be detected for as long as 6 h following intraperitoneal inoculation of 10(5) plaque-forming units (PFUs) of R. rickettsii (Morgan strain). An estimate of the median infectious dose (ID50) was made by inoculating six groups (eight animals per group) with serial dilutions of 10(1) to 10(6) PFUs. In this experiment, cotton rats became infected after exposure to relatively few organisms (ID50 = 37 PFUs). None of the 48 cotton rats succumbed to infection, and only 6 of the 48 rats failed to seroconvert. Dermacentor variabilis (Say) larvae were fed on rickettsemic cotton rats to determine whether this species could serve as a source for infecting ticks with R. rickettsii. A small percentage (0.9-3.0%) of larval ticks that fed on three of the four cotton rats had R. rickettsii in their tissues when examined as nymphs. A fourth cotton rat died 7 d after inoculation with R. rickettsii and infected a much higher percentage of ticks (64.0%) than the other three animals.

Vector/host relationships of the Lyme disease spirochete, Borrelia burgdorferi.

Lyme borreliosis is now occurring on several continents where its causative agent, Borrelia burgdorferi, is maintained and transmitted by ticks of the "Ixodes ricinus complex" namely I. dammini, I. pacificus, and possibly I. scapularis in North America, I. ricinus In Europe, and I. persulcatus in Asia. Because all developmental stages of these ticks feed on a large variety of hosts including humans, the vector/host relationships of this spirochete is highly complex as indicated by the voluminous literature reviewed in this article. The association of B. burgdorferi with ticks parasitizing exclusively rabbits and birds, suggests that the geographic distribution of this agent may be far greater than assumed and may include areas where the disease in humans is absent. Finally, the persistence of the Lyme disease spirochete in the midgut of its tick vectors and its invasion of other tissues during the ticks' feeding, are unique and differ from the behavior of all other arthropod-borne borreliae.

Pathophysiology of the Lyme disease spirochete, Borrelia burgdorferi, in ixodid ticks.

The pathophysiology of Borrelia burgdorferi, the Lyme disease spirochete, is unique in tick/vector relationships, differing substantially from that of other spirochetes, e.g., Borrelia duttonii, the agent of tick-borne relapsing fever, and Borrelia recurrentis, the agent of louse-borne relapsing fever, in their respective vectors. Following ingestion by a tick, B. burgdorferi lodges in the midgut diverticula, in some instances penetrating the gut wall and invading various tissues. Certain investigators suggest that transmission of the spirochete occurs via infectious saliva, although, in light of the fact that only 5% of adult ticks are systemically infected, this mechanism is open to question. Alternatively, transmission may occur via periodic regurgitation of gut fluids during the feeding process. While ticks of the genus Ixodes were once thought to be the only vectors, it now appears that other genera, and possibly other hematophagous arthropods, may also be involved.

Concurrent neuroborreliosis and Alzheimer's disease: analysis of the evidence.

Several recent reports have claimed a possible association between Borrelia burgdorferi infection and Alzheimer's disease (AD). Herein, we describe our search for additional evidence of neuroborreliosis in AD. Brain tissue from neuropathologically confirmed cases of AD was cultured for B burgdorferi using standard microbiologic methods. Material derived from culture was further examined using electron microscopy, direct immunofluorescence and acridine orange fluorescence. Previous studies have shown high titers of antiborrelia antibodies in CSF in all cases of confirmed neuroborreliosis; therefore, we tested CSF from neuropathologically confirmed cases of AD by indirect immunofluorescence and enzyme-linked immunoassay. In addition, imprint preparations from AD and control brain tissues were studied by direct immunofluorescence using a monoclonal antiborrelia antibody. Finally, a Western blot method was used to analyze protein extracts from cultures and AD brain tissue for the presence of borrelia antigen. Contrary to previous studies, our results do not support an association between infection with B burgdorferi and AD.