MEDLINE Abstracts: Lyme Disease and Other Tick-Borne Diseases

May 01, 2002

MEDLINE Abstracts: Lyme Disease and Other Tick-Borne Diseases

John F. Anderson, PhD, Connecticut Agricultural Experiment Station

West Nile Virus (WNV) occurs naturally in birds and is transmitted by mosquitoes. It is primarily tropical/subtropical and is widely distributed through Africa, parts of western Asia, and has been recovered from several European countries. It was first isolated from a febrile woman living in the West Nile District, Northern Province of Uganda in 1937. While this virus has been isolated from a sick rock pigeon and a dead goose in the Old World, major bird mortality has not been documented. Outbreaks of human disease caused by WNV have been reported in Europe, but this virus seemingly did not persist.
An outbreak of arboviral encephalitis in humans was recognized to be occurring in late August 1999 in New York City and was initially reported on 4 September. We began trapping mosquitoes for the testing of viruses the following day on 5 September in Greenwich, the Connecticut town closest to New York City. Traps were subsequently placed in 13 other towns in southwestern, CT. American crows were reported dying in southwestern Connecticut in the second week of September.
Virus isolations were attempted from mosquitoes and from brain tissues from dead birds. All isolation attempts were made in Vero cells grown in 5% CO2 at 37°C. Isolates were initially screened serologically against known North American viruses associated with mosquitoes, and subsequently their genomes were sequenced and analyzed genetically by comparative phylogenetic analysis.
Virus was isolated from two species of mosquitoes, Culex pipiens and Aedes vexans, captured on the Greenwich/Stamford town line on 14 September. Virus was also isolated from brain tissues of 28 of 31 crows that died from 13 September through 12 October and from brain tissues of a Cooper's hawk. CDC isolated virus from the brain of a sandhill crane that died at the Beardsley Zoo in Bridgeport, CT. The birds all died along a 62-mile corridor from Greenwich on the New York border eastward to Madison, CT, in towns bordering directly on Long Island Sound or island about 15 miles.
All virus isolations were similar to one another and were closely related to a WNV isolate from mosquitoes captured in Romania in 1996. CDC demonstrated their isolates from birds and mosquitoes also to be related to the Romanian isolate but to be almost identical to a then unreported WMV isolate from a goose that died in Israel in 1998.
No human or horse cases of West Nile Virus Fever were recorded in Connecticut.
Our isolates and those by CDC are the first WNV isolates to be recorded in North America. It is unknown how WNV was introduced into the New World, but, if established in North American this virus will likely have severe effects on the health of humans, horses, and birds.

Christopher Paddock, MD, Centers for Disease Control and Prevention

In contrast to Lyme disease, rickettsial and ehrlichial infections assume relative obscurity among infectious diseases recognized by most physicians. Although existing state and national surveillance systems for tickborne illnesses report substantially fewer cases of Rocky Mountain spotted fever (RMSF) and ehrlichiosis than of Lyme disease, these diseases are important with respect to their broad geographic distributions throughout the US and their potential for severe and fatal illnesses in otherwise healthy persons. The differential diagnoses for these diseases are extensive, and the early clinical signs and symptoms resemble various other infectious and noninfectious etiologies. These infections respond quickly to appropriate treatment. Most antimicrobials used as empirical therapies for presumed bacterial infections in febrile patients are characteristically ineffective in treating rickettsial and ehrlichial diseases; appropriate treatment is seldom provided unless the diagnosis is suspected. Physician awareness of these diseases and early recognition of their salient epidemiologic and clinical features form the foundation of successful patient outcomes.
RMSF has been recognized as a clinical entity for over a century, and cases have originated from almost every state in the continental US. During the last decade, approximately 400 to 800 cases of RMSF were reported each year; >90% of these cases occurred during April through September. Age-specific incidence is highest in children < 10 years, although case-fatality ratios are highest in persons >60 years. Hospitalization occurs in 50%-70% of patients. During 1981-1996, the mean annual case-fatality ratio for RMSF was 3.4%. The ehrlichioses comprise at least 3 clinically similiar but epidemiologically distinct diseases in the US, caused by Ehrlichia chaffeensis, E. phagocytophila, and E. ewingii. These diseases have only been recognized in the last 14 years and fall under the rubric of emerging infections. Survellance data for the ehrlichioses are sparse.
The current best estimates report approximately 1,200 US cases during 1986 through 1997. Cases have been reported from almost every state, although these diseases are most frequently identified in the southeastern, northeastern and upper midwestern regions of the US. Different tick vectors transmit different species of ehrlichia, reflected by the generally distinct geographic distribution of each type of ehrlichiosis. Age-specific incidences for the ehrlichioses are highest in persons >40 years of age, although severe and occasionally fatal cases have occurred in young adults and children. Laboratory confirmation of RMSF and the ehrlichioses requires serologic, molecular, or culture-based methods. Indirect immunofluorescence assays (IFA) remain the most available and best recognized method of confirming these diseases; however, diagnostic antibody levels typically do not appear until 7-14 days after the onset of illness. In this context, treatment decisions should be based on a presumptive diagnosis developed from epidemiologic and clinical clues and should never be delayed while waiting for laboratory confirmation. Tetracyclines (especially doxycycline) are the antibiotics of choice in treating most adult and pediatric patients with these infections.

Peter Krause, MD, University of Connecticut School of Medicine

Babesiosis is an emerging malaria-like disease caused by an intraerythrocytic protozoan that is transmitted by the deer tick, the same tick that transmits Lyme disease. The causitive protozoan was first described in cattle in 1891 by the Hungarian microbiologist Babes. At least three Babesia species have been found to cause disease in humans, B. microti (a rhodent species), B. gibsoni (WA-1, a dog species),and B. divergens (B. bovis, a cattle species). Since the first documented case of human babesial infection was reported in 1957, infection by B. divergens has been demonstrated in Europe and infection by B. microti has been documented increasingly in the northeastern and upper midwestern United States. A parasite very closely related to B. gibsoni (WA-1), appears to infect humans along the Pacific coast. The clinical manifestations of babesiosis range from subclinical illness to fulminant disease resulting in death. The case fatality rate has been estimated at 5%. Although infection is common in endemic areas, most is mild or asymptomatic. In clinically apparent cases, symptoms of babesiosis begin after an incubation period of one to nine weeks from the beginning of tick feeding. Typical symptoms include intermittent temperature to as high as 104°F and one or more of the following: chills, sweats, myalgia, arthralgia, nausea, and vomiting. The findings on physical examination generally are minimal, often consisting only of fever. Mild splenomegaly, hepatomegaly, or both are noted occasionally but rash seldom is reported. Abnormal laboratory findings include moderately severe hemolytic anemia, an elevated reticulocyte count, thrombocytopenia, proteinuria, and elevated blood urea nitrogen and creatinine. The symptoms of babesiosis usually last for a few weeks to several months. Patients at risk for severe disease include those who lack a spleen, are infected with B. divergens, are over the age of 50 or are coinfected with the agents of HIV or Lyme disease. Moderate to severe babesiosis may occur in children but infection generally is less severe than in adults. Diagnosis of B. microti infection is made by microscopic demonstration of the organism using Giemsa-stained thin blood films. In experienced laboratories, the polymerase chain reaction (PCR) can be a sensitive and specific test for detection of Babesia DNA but great care must be taken to prevent false positive test results. Serological testing is useful, particularly in diagnosing B. microti infection. The indirect immunofluorescence serologic assay (for both IgG and IgM antibody) is sensitive and specific when performed in qualified laboratories. The current therapy of choice for babesiosis consists of the combination of clindamycin and quinine for 7 to 10 days. Adverse reactions are common, however, especially transient hearing impairment and abdomenal distress. Exchange blood transfusion should be reserved for patients with severe infections including those with high parasitemia (over 5%) and coma, hypotension, congestive heart failure, pulmonary edema or renal failure. In combination with clindamycin and quinine, it is the treatment of choice for all cases of B. divergens babesiosis.

Ellen Stromdahl, MS, United States Army Center For Health and Preventative Medicine

Ticks removed from humans are sent by Department of Defense medical personnel/clinics to the Tick-Borne Disease Laboratory of the U.S. Army Center for Health Promotion and Preventive Medicine (USACHPPM) for identification and testing for pathogens by polymerase chain reaction (PCR). Results of tick identification are reported by telephone within 1 day to the tickbite patient's healthcare provider and results of analysis are subsequently reported by telephone within 3-8 working days. Amblyomma americanum are tested for Ehrlichia chaffeensis and Borrelia burgdorferi, Ixodes scapularis are tested for Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis (HGE), and Dermacentor variabilis are tested for Rickettsia rickettsii. Since different species of ticks transmit different diseases, and since most tick-borne diseases have very similar early symptoms, knowing the species and infection status of the tick enhances the physician's ability to accurately diagnose and treat the patient. In 1999, analysis by PCR found 1/444 (0.2%) A. americanum positive for B. burgdorferi and 7/444 (2%) positive for E. chaffeensis. One hundred twelve of 390 (29%) I. scapularis were positive for B. burgdorferi and 26 (7%) were positive for the agent of HGE. Ten (3%) I. scapularis were coinfected with both organisms. Fourteen of 304 (5%) D. variabilis were positive for spotted fever group (SFG) rickettsiae. Restriction fragment length polymorphism (RFLP) analysis identified all as Rickettsia montana, a non-pathogenic SFG rickettsia.

Angela James, PhD, Centers for Disease Control and Prevention

Lyme disease is the most common tick-borne disease reported in the United States. The majority of the cases occur in northeastern and mid-Atlantic regions. Borrelia burgdorferi sensu lato, the etiological agent, is transmitted by Ixodes spp. ticks. Borrelia burgdorferi has been isolated from several locales throughout the southern United States and these isolates appear to be genetically more diverse than isolates from the northeastern regions. Borrelia sp. isolates have been made from mammals, birds, and ticks from Georgia, Florida, North Carolina, South Carolina, Virginia, Missouri, Oklahoma, and Texas. Borrelia sp. isolates have been cultured in BSKII from Ixodes scapularis, I. affinis, I. dentatus, and I. minor. It appears that I. affinis, I. minor, and I. dentatus maintain enzootic cycles and that I. scapularis acts as a bridge vector to humans. A variety of vertebrate species serve as reservoirs of Borrelia sp., including the cotton mouse, cotton rat, eastern woodrat, and cottontail rabbit. However, few human cases have been reported from this region. This low number may be a result of ecological factors such as the variety and number of hosts, precipitation, and the one or two year life cycle of the black-legged tick (I. scapularis) in this region. Moreover, accurate diagnosis of Lyme disease and tick-associated erythemas in general may be complicated by the possibility of multiple ixodid tick/Borrelia sp. transmission cycles in the southern US. For example, B. lonestari was recently described; it was detected via PCR in the metastriate tick, Amblyomma americanum. The lone star tick (A. americanum) is the most prevalent tick species found in the southeastern United States and it frequently bites humans. The prevalence and association of Borrelia burgdorferi, B. lonestari, and other, possibly new Borrelia genospecies to human illness remains unclear in the southern United States. Thus, a reevaluation of Lyme disease in the south is warranted.

Louis A. Magnarelli, PhD, Connecticut Agricultural Experiment Station

Humans, domesticated animals, and wildlife, such as white-tailed deer and white-footed mice, may be exposed to one or more infectious agents transmitted by Ixodes scapularis ticks. Although Lyme borreliosis is more prevalent in Connecticut and in other northeastern and upper midwestern states, monocytic and granulocytic ehrlichiosis, and babesiosis also can occur there.
Serologic analyses were conducted to determine prevalence of infections. Results revealed the presence of antibodies to different pathogens in humans, horses, dogs, white-tailed deer, and white-footed mice in Connecticut.
Coexistence of antibodies to Borrelia burgdorferi and the human granulocytic ehrlichiosis agent was most prevalent. In many instances, however, it was unclear if there were simultaneous infections by multiple agents or if hosts were exposed to different disease organisms in separate incidents that occurred over several weeks or months.
If one of these diseases is clinically suspected or diagnosed, laboratory testing should be extended to determine if there are other tick-borne infections.

in vitro
and in vivo
findings of hyperbaric oxygen treatment in experimental Bb
infection

Charles Pavia, PhD, NY Medical College School of Medicine; NYCOM Microbiology and Immunodiagnostic Laboratory of NYIT

In these studies, we evaluated repeated HBOT for its ability to kill Bb in vitro, and in vivo, in a murine model of Lyme disease. Several North American tick-derived and recently obtained patient isolates were studied separately in our assay systems. To test for in vitro susceptibility, one-half to one million Bb were cultured in a small volume (0.1 - 0.2 ml) of BSK media using small snap-cap test tubes. With the caps removed, these cultures were then exposed, for one hour (twice daily for 2 consecutive days), to pure, filtered oxygen pressurized to 2-3 times normal atmospheric conditions. This was achieved using a specially constructed, miniaturized cylindrical chamber (length = 12 inches; diameter = 8 inches), equipped to accept any pressurized gas mixture through its portal opening. After the final HBOT, all cultures received an additional 0.5 ml of BSK media (making the final volume now 0.6 - 0.7 ml), and their caps were snapped shut. Matching control cultures received no HBOT. All cultures were incubated at 33oC for 2-3 days and were examined microscopically for live Bb.
Our results showed that 14 of 17 strains of Bb had their growth inhibited by 33-94%, while there was little or no inhibition of 3 Bb strains. For the in vivo studies, separate groups of C3H or CD1 mice were infected intradermally with 100,000 Bb. Two to 4 weeks later, one group of infected mice received two, 1.0-1.5 hour HBO exposures, for two consecutive or alternating days. The treated mice were sacrificed one day after the last treatment, and extract cutlures of their urinary bladders were prepared in BSK media. It was found that no Bb grew out of 80% of these extract cultures, whereas live Bb organisms were recoverable from 90% of extract cultures prepared from matched, infected control mice not treated with HBO. These data suggest that HBOT may be considered as a clinically useful form of adjunct therapy in the treatment of Lyme disease.

B. burgdorferi
in the Rabbit

Presenter: James N. Miller, PhD, Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA

Major Contributors: Ellen S. Shang, PhD; David R. Blanco, PhD, Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA; Michael A. Lovett, MD, PhD, Division of Infectious Diseases, Department of Medicine, UCLA School of Medicine, LA, CA

Studies by Barthold and his colleagues have underscored the significance of host-adapted B. burgdorferi in the attempt to understand the immunology and pathogenesis of Lyme disease. Utilizing the rabbit model and the B. burgdorferi B31 strain, we have demonstrated by skin implantation challenge, a complete infection-derived immunity to large numbers of host-adapted homologous spirochetes (1.38 x 108 ). Further, we have shown that heterologous challenge with host-adapted organisms from strain 297 results in rapid clearance of skin and disseminated infection. Immunization of rabbits with outer membrane vesicles (OMV) isolated from the B31 strain and its avirulent derivative lacking OspA and DbpA (B313) conferred highly significant protection against challenge with 6 x 104 cultivated B31 spirochetes but not against challenge using host-adapted organisms. Analysis of the antibody responses to OM proteins among infected rabbits immune to skin implant challenge suggests that the remarkable protection induced by infection is due to antibodies directed against antigens unique to or markedly up-regulated in host-adapted B. burgdorferi. The rabbit model thus provides the opportunity to identify B. burgdorferi surface molecules up-regulated during rabbit infection and ultimately to determine their relationship to the basis of infection-derived immunity.

B. burgdorferi
Antigenic Variation Protein

Steve J. Norris, PhD, University of Texas -- Houston Medical School

Lyme disease borrelia can persistently infect Lyme disease patients and infected animals for months to years despite a vigorous host immune response. Our laboratory recently discovered the vls (VMP-like sequence) antigenic variation system in B. burgdorferi B31 that may in part explain the ability of this organism to evade an active immune response. The vls locus consists of the expression site for a surface lipoprotein, VlsE, and 15 'silent' cassettes that represent variations of the central cassette region of the vlsE gene. Segments of the silent cassettes begin to recombine into the vlsE gene within 4 days following infection, and this ongoing process could produce as many as 1030 different sequence variations. Lyme disease patients consistently produce a strong antibody response against VlsE, which may actually be useful in the diagnosis of Lyme disease. Although immunization with a single form of VlsE fully protects mice against infection with B. burgdorferi expressing the identical protein, it is only partially protective against variants expressing slightly different versions of VlsE; this finding demonstrates the role of VlsE sequence variation in immune evasion. DNA sequences homologous to vlsE have been identified in every infectious Lyme disease isolate examined to date, and may represent a common immune evasion mechanism in Lyme disease borrelia. Further characterization of the vls system may lead to improved immunodiagnosis and additional immunoprotective vaccines against Lyme disease.

Borrelia burgdorferi
: Their Application in Diagnosis and Immunoprophylaxis of Lyme Disease

Mario Philipp, PhD, Tulane University Medical Center; Tulane Regional Primate Research Center, Department of Parasitology

Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable antigens such as the variable major protein (Vmp) of Borrelia hermsii and the variant surface glycoprotein (VSG) of African trypanosomes include an immunodominant variable domain and one or more invariable domains which are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain.
VlsE (variable major protein-like sequence), the variable surface antigen of Borrelia burgdorferi, the Lyme disease spirochete, also contains both variable and invariable domains. In addition, interspersed within the VlsE variable domain there are six invariable regions (IR11-6) which together amount to one half of this portion's primary structure. We show here that these IRs are conserved among strains and genospecies of the B. burgdorferi sensu lato complex. Unlike the invariable regions of Vmp and VSG, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B. burgdorferi. The utilization of IR6 as a universal diagnostic reagent for Lyme disease and the possible application of other IRs as vaccines will be discussed.

R. K. Straubinger, A. F. Straubinger, B. A. Summers, R. H. Jacobson, James A. Baker Institute for Animal Health; Department of Pathology; and Department of Population Medicine and Diagnostic Science, NYS College of Veterinary Medicine Cornell University, Ithaca, New York 14853

Objective: Determine whether therapy with the antibiotics amoxicillin, ceftriaxone, doxycycline and azithromycin, commonly used in Lyme disease patients alleviate clinical signs of canine Lyme borreliosis, and whether the infectious agent, Borrelia burgdorferi, can be eliminated from the host.
Design: In three separate experiments over a four-year period, 24 beagle dogs infected with B.burgdorferi by tick exposure were treated with antibiotics for 30 consecutive days. Ten infected dogs were not treated and served as positive controls. After treatment, dogs were housed in P-2 units for an additional 70 to 350 days. Serum samples were collected in 2-week, and skin biopsy samples in monthly intervals. Detection of B.burgdorferi was attempted by culture and PCR in tissue samples.
Results: After tick exposure, 33 of 34 dogs became infected as shown by serology, culture, and PCR. Clinical signs of Lyme borreliosis included elevated temperature for one to two days and lameness, caused by acute mono- or oligoarthritis of large joints such as the shoulder, elbow, carpal, and knee joints. After antibiotic therapy, dogs improved clinically, and only once was post-treatment lameness observed in a dog. At the end of the experiments, 25 tissue samples of each dog were cultured. Live spirochetes were recovered from three single tissues samples from three treated dogs, while multiple tissues samples were positive in all non-treated dogs. By PCR 20/24 treated and 10/10 non-treated dogs were positive for B.burgdorferi DNA. Serologically, the level of peak antibody titers was dependent on the time when therapy was initiated. Short time intervals between infection and treatment (50 days) resulted in moderate peak antibody titers, and during the post-therapeutic observation period antibodies against B.burgdorferi disappeared almost completely. However, treatment initiated 120 days post infection allowed antibody levels to develop to maximum titers and beyond 300 days following therapy dogs still had moderate antibody titers. Histologically, 3/24 treated dogs showed mild monarthritis or meningitis in just single tissue samples, while 8/10 non-treated dogs had mild non-suppurative polyarthritis, sometimes combined with periarteritis.
Conclusions: Treatment with high doses with commonly used antibiotics for a 30 day period resulted in clinical improvement and arthritis was prevented or cured. However treatment was not sufficient to eliminate the persistent infection in dogs and impeded the development of maximum antibody titers.

Borrellia burgdorferi
Repetitive Antigen that Confers Protection against Experimental Lyme Disease

Jon T. Skare, PhD, Texas A & M University System Health Science Center

We have previously described the expression cloning of nine Borrelia burgdorferi antigens, using rabbit serum enriched for antibodies specific for infection-associated antigens, and determined that seven of these antigens were associated with infectious B. burgdorferi strain B31. One of these infection-associated antigens encoded a 451 amino acid lipoprotein containing 21 consecutive and invariant 9 amino acid repeat sequences near the amino terminus that we have designated VraA for virulent strain associated repetitive antigen A. The vraA locus (designated BBI16 by The Institute for Genomic Research [TIGR]) maps to one of the 28 kilobase linear plasmids (designated lp28-4) that is not present in noninfectious isolates. The absence of lp28-4 alone from B. burgdorferi strain B31 results in a 15-fold decrease in the infectious dose (ID50) relative to wild-type B. burgdorferi, suggesting that genetic loci found on lp28-4 are involved in the pathogenesis of B. burgdorferi infection. We have also determined that VraA is a surface exposed lipoprotein based on protease accessibility assays of intact whole cells. Furthermore, VraA is synthesized greater when cells are grown at 37° C relative to cells grown at either 32°C or 23°C, suggesting that VraA is a host inducible antigen. Homologues cross-reactive to B. burgdorferi strain B31 VraA with distinct molecular masses were identified in several B. burgdorferi sensu lato isolates as well as other Borrelia spp. including B. andersonii, suggesting that the immunogenic epitope(s) present in VraA are conserved between Borrelia spp. In protection studies, five of six mice immunized with recombinant glutathione-S-transferase (GST)-VraA were protected from infectious challenge with 102 infectious B. burgdorferi strain B31 whereas naïve mice or mice immunized with GST alone were completely susceptible. Furthermore, the protection elicited by VraA immunization provides an additional testable vaccine candidate to protect against Lyme disease and perhaps other Borrelia spp related diseases.

Jarmo Oksi, MD, PhD, Turku University Central Hospital, Department of Medicine, Finland

The pathogenetic mechanisms of Lyme neuroborreliosis (LNB) are poorly understood. They may be caused either by direct action of Borrelia burgdorferi (B.b.) or by indirect immunologic reactions of the host. The diagnosis of LNB has usually been based on nonspecific findings, serologic testing and other indirect methods. However, evidence for the presence of B.b. can also be obtained by culture or polymerase chain reaction (PCR) of cerebrospinal fluid, plasma, or brain tissue specimens. Furthermore, the direct demonstration of B.b. in the brain lesions indicates that direct invasion of the spirochetes has obviously been the pathogenetic mechanism in these cases. The low number of spirochetes in tissue samples and body fluids is one of the reasons for the difficulties to demonstrate B.b. by culture or PCR. Therefore, a negative result obtained by these methods can never exclude LNB. However, a positive result can confirm the diagnosis or treatment failure independently of results of antibody tests. PCR offers a practical means to differentiate patients with "Post-Lyme syndrome" or with "serological scars" from those who definitely need retreatment.
We report on 13 patients with clinical relapse of disseminated LB after antibiotic treatment and culture or PCR positivity. These patients were a subgroup of a total of 165 patients with disseminated infection with B.b., which were followed after antibiotic treatment. Before the initial antibiotic treatment 11 of the 13 patients had neurologic symptoms or findings including meningitis in 3, encephalitis in 3, radiculitis or neuritis in 2, and other neurologic symptoms in 5 of the patients. Brain MRI was abnormal in 5 of 8 studied. 8 of the patients had culture or PCR-proven initial diagnosis; and the diagnosis of the remaining 5 patients was based on positive serology only. The microbiologic confirmation of relapse was based on blood culture in 3 patients, on brain biopsy PCR in 1 patient, and on plasma PCR in 9 patients. All 13 patients were initially treated for more than 3 months with iv and/or oral antibiotics. Antibody levels decreased or changed to seronegative in 6 of the 13 patients after the first treatment. However, this did not guarantee a succesful eradication of the spirochete as shown by PCR and culture. None of the patients was PCR or culture positive after the retreatment.

Willy Burgdorfer, PhD, MD, National Institutes of Health

Of the approximately 850 ticks identified so far, about 100 have been shown to maintain and transmit pathogens of animal and human diseases.
Brief historical accounts are presented of the first recognized involvement of ticks in transmitting pathogens. They are credited to T.S. Smith and F.L. Kilborne for demonstrating that the larvae of Boophilus annulatus are the vectors of Babesia bigemina, the causative agent of Texas Fever, and to H.T. Ricketts for identifying the wood tick, Dermacentor andersoni as the vector of Rocky Mountain spotted fever. Ricketts also was the first to show that the causative agent - unknown to him - was passed via eggs to the progeny of infected female ticks. Subsequent quantitative studies including those by your speaker revealed up to 100% of infected filial ticks. However, maintenance of rickettsial strains by transovarial passage for as many as 12 tick generations was found to interfere with the ticks' biological processes.
Transstadial and transovarial transmission occurs in most ticks infected with spotted fever group rickettsiae. For the recently described Rickettsia peacockii, transovarial transmission is the only means for survival; it fails to infect salivary glands and is nonpathogenic for its hosts.
The recognition of the East African argasid tick, Ornithodoros moubata as the vector of the relapsing fever spirochete Borrelia duttonii, led to the detection of additional argasid ticks as vectors of spirochetes on several continents. Most of them are efficient in transmitting their spirochetes transstadially as well as transovarially. A detailed account on the development of Borrellia duttonii in Ornithodoros moubata is presented and compared with the development of the Lyme disease spirochete, Borrellia burgdorferi in its tick vector, Ixodes dammini (=I. Scapularis). In this tick, ingested spirochetes remain in the midgut until a blood meal initiates their passage via gut epithelium into other tissues including salivary glands. Massive invasion into developing oocytes prevents normal development of infected eggs.
Continuous transovarial transmission of spirochetes without occassional passage though a susceptible host leads to a degradation of the spirochete's pathogenicity - a phenomenon said to be responsible for the gradual disappearance of tick-borne relapsing fever from previously endemic areas in East Africa.
Finally, reference is made to the suspected tick vectors of the recently described agents of ehrlichiosis. Observations simialr to those for the development of Ehrlichia canis in its tick vector, Rhipicephalus sanguineus are not as yet available.

Richard Marconi, PhD, Medical College of Virginia

The ospE gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The ospE genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box or UHB element. Earlier analyses in our lab demonstrated that ospE related genes are characterized by defined hyper-variable domains (domains 1 and 2) that are predicted to be hydrophillic, surface exposed and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to ospE diversity and thus ultimately to antigenic variation. Using an isogeneic clone of B. burgdorferi B31G (designated B31Gc1) we demonstrate that the ospE related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE related proteins in immune evasion and suggest that multiple plasmid carried gene families may be involved in the maintenance of chronic infection.

George Perides, PhD, Beth Israel Deaconess Medical Center (Harvard)

The major clinical features of Lyme borreliosis involve the nervous system and the joints. Very little is known about the proteolytic enzymes involved in the pathogenesis of Lyme disease. We are investigating the ability of Borrelia burgdorferi to induce the expression of host proteolytic enzymes and their role in the pathogenesis of Lyme disease.
Regarding the nervous system, we have reported that a matrix metalloproteinase (MMP) with electrophoretic mobility corresponding to 130 kDa, (an MMP-9 complex), is found in the cerebrospinal fluid (CSF) of 78% patients with Lyme disease. Similarly, this MMP was found in the CSF of 62% of patients with post-treatment chronic Lyme disease. This MMP is seen in only 6% of CSF samples from healthy individuals or patients with other neurologic diseases. To determine whether B. burgdorferi can induce MMP expression by cells of the nervous system we used primary human and rat neural cultures. Borrelia burgdorferi induces the expression of MMP-9 in a dose and time dependent manner. Primary astrocytes express the 130 kDa metalloproteinase when cultured in the presence of the spirochete. The MMPs may play a role in a) the break-down of the blood brain barrier, b) the degradation of the brain extracellular matrix and, c) the digestion of myelin basic protein. The may thus, mimic the presence of MMPs seen in other chronic neuro-inflammatory and neurodegenerative diseases.
Regarding Lyme arthritis, we investigated whether MMPs are present in the synovial fluid from patients with joint involvement due to Lyme disease. The synovial fluid of patients with Lyme arthritis contained MMP-3, MMP-9, and "aggrecanase" (a metalloproteinase that digests the main cartilage proteoglycan, aggrecan). We used a cartilage explant culture system, to determine whether B. burgdorferi induces the expression of these enzymes. When bovine or monkey cartilage explants were infected with B. burgdorferi they expressed MMP-3, MMP-9 and "aggrecanase". This induction was associated with a dramatic increase in proteoglycan and collagen degradation. This cartilage digestion was inhibited by plasmin- and MMP-inhibitors. We hypothesize that the presence of B. burgdorferi in the joint results in the induction and activation of metallopeptidases and fibrinolytic enzymes, that lead to joint destruction and clinical arthritis.
We suggest that B. burgdorferi induces the expression of host proteolytic enzymes and these enzymes play a key role in the development of the clinical symptoms associated with Lyme disease.

R. K. Straubinger, P. Florian, I. Braumiller, James A. Baker

Institute for Animal Health, NYS College of Veterinary, Medicine, Cornell University, Ithaca, New York 14853; College of Veterinary Medicine, University of Leipzig, Germany; College of Veterinary Medicine, Ludwig-Maximilians-University Munich, Germany

Objective: In previous work we had shown that the chemokine Interleukin (IL)-8 is up-regulated in the synovia of dogs with acute Lyme arthritis. IL-8 is a proinflammatory chemokine, which probably is responsible for the accumulation of polymorphonuclear neutrophils in affected joints. The detrimental effect of proinflammatory cytokines and chemokines and therefore the extent of the inflammation are controlled by the host with inhibitory factors such as IL-10. Since Lyme arthritis in dogs is episodic and self-limiting, we investigated whether there is an up-regulation of IL-10 mRNA in synovial membranes of dogs showing clinical signs of acute Lyme arthritis.
Design: Tissues had been collected previously from Borrelia burgdorferi-infected dogs at the time when the first clinical signs of arthritis were apparent or during the following three days. Messenger RNA was recovered from frozen tissues and the amount of IL-10-specific mRNA was measured with a real-time PCR technique using the ABI7700 Sequence Detection System. Quantities of IL-10 mRNA in various tissues were compared to those of tissues with no signs of inflammation.
Results: Tissues evaluated for IL-10 mRNA content consisted of the skin from both sides of the chest; six lymph nodes (axillary, superficial cervical, and popliteal lymph nodes); six joints (shoulder, elbow, and knee joints); four muscle and four fascia tissue samples representing each limb; and samples from the peritoneum, pericardium and meninges. Our data show that IL-10 was up-regulated very early during joint inflammation (first day of lameness), predominately in synovia and lymph nodes of infected dogs. Over the following days, IL-10 seemed to be down-regulated and was only detectable in small quantities in all tissues.
Conclusions: The cytokine IL-10 seems to be involved in the pathogenesis of acute Lyme arthritis. Its early up-regulation during arthritis probably limits the detrimental effects of inflammatory factors such as IL-8, IL-1a, IL-1b, and TNF-a. Further studies should help to define the role of IL-10 and possibly other inhibitory factors in Lyme borreliosis.

Borrelia burgdorferi

Epitopes and Molecular Mimics in Lyme Disease

Adriana Marques, MD, National Institutes of Health

The study of the immune response to infectious agents is a prerequisite for understanding disease pathogenesis. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. The use of a novel, unbiased methodology to effectively identify both microbial epitopes and candidate autoantigens will be described. This approach combines analysis of T cell clone response to positional scanning peptide combinatorial libraries and the analysis of the results by generation of scoring matrices followed by large-scale database searches. Using this new method, the peptide specificities of one T cell clone derived from the CSF was determined. These studies documented for the first time that this novel strategy can be applied to identify target epitopes for clones that have been expanded from an organ compartment in an infectious disease.
Furthermore, it was shown that the T cell clone can recognize with high affinity multiple epitopes from one infectious organism as well as multiple other peptides derived from viral proteins and autoantigens that are potentially relevant for the disease process. Despite this degenerate T cell recognition, the T cell clone recognized B. burgdorferi epitopes with highest affinity, and bioinformatic data analysis revealed that the clone discriminates between B. burgdorferi and a closely related spirochete, Treponema pallidum. These observations have profound implications for our understanding of how T cell specificity is encoded and will be important for future studies.

Alan B. Frey, T. Dharma Rao, Eugene Davidson, and Reinhard Straubinger, Department of Cell Biology New York University School of Medicine, Department of Biochemistry Georgetown University School of Medicine, and James A. Baker Institute of Veternary Medicine Cornell University

In an effort to develop a safe and effective vaccine for prevention of Lyme disease, dogs were vaccinated with a multiantigenic preparation of sonicated Borrelia burgdorferi, strain N-40. 20 days later animals were boosted, rested for 50 or 198 days, and then challenged by feeding of field-isolated ticks. Control nonvaccinated dogs, dogs vaccinated with sonicate, or dogs vaccinated with a commercial rOspA formulation were monitored for development of infection by: histopathologic assessment of tissues at necropsy; measurement of serum anti-B. burgdorferi antibodies using ELISA, immunoblot analysis, and spirochete growth inhibition in vitro; growth of spirochete in vitro from skin punch biopsy; and PCR analysis of tissues. Sonicate-vaccinated dogs were completely protected from infection by all criteria utilized, developed high-titer anti-spirochete serum antibodies and growth inhibitory activity which persisted for over 200 days, and did not demonstrate any untoward consequence of vaccination. These data demonstrate that a multiantigenic vaccine is effective in preventing Lyme disease via the natural vector.

Edwin Masters, MD, Regional Primary Care Physicians

Typical and atypical erythema migrans lesions and their possible significance are presented and discussed, with particular emphasis on erythema migrans in the southern United States. Explanations and possible etiologies are presented, along with comparisons to culture proven erythema migrans lesions.
Possible explanations for Borrelia associated erythema migrans lesions in the Southern United States include:

  1. Tick to tick transmission of co-feeding ticks on an infected or uninfected animal (if one of the ticks is already infected). (Mather, et al. 1996 International Lyme Conference, San Francisco; Ogden, et al. 12/97 J Parasit; and Partican, et al. 11/97 Am J Trop Med Hyg).

  2. Possible 'bridge vectors' analogous to California epidemiology, involving ticks feeding on an infected animal reservoir host (Oliver, et al. J Clin Microbiol, Jan 1998).

  3. Demonstrated, albeit limited, transtaadial AA transmission of B.b. (Ryder, et al. J Med Entomol 1992;29:525-530).

  4. Adaptation of related, but possibly atypical B.b. variants to different ticks, (e.g. Amblyomma amoricanum or lone star ticks), not unlike HGE Ixodes vectored vs. HME Lone Star tick vectored Ehrlichiosis due to E. chaffeensis (Masters, Arch Intern Med, 1998; 158:2162-2164). Already documented is marked southern B.b. sensu stricto and even one AA Missouri B.b. isolate.

  5. Human infection with pathogenic B.b. sensu lato.

Dorothy M. Pietrucha, MD, Jersey Shore Medical Center, Medidan Health System

The central and peripheral nervous systems may both be involved in Lyme. Patients may present with meningitis, encephalitis, encephalomyelitis, radiculitis, cranial neuritis, and neuropathy.
Cases have been reported with pseudo-tumor cerebri. Rarely, there have been reports of stroke. Patients have had seizures.
Frequently, there may be an encephalopathy with memory deficits, short attention span, difficulty learning new material. This causes significant difficulties in school.
The skeletal muscles may be involved with elevated muscle enzymes and muscle pain and weakness.
In addition to treating the underlying Lyme Disease with therapy medications may have to be used to control the symptoms of these neurologic manifestations such as diuretics for the increased intracranial pressure, anticonvulsant medications for seizures and analgesics for the pain, physical therapy for the weakness and appropriate educational intervention and support for those who are having academic difficulties.

Marian Rissenberg, PhD, and Dana Leonardi, MA, Columbia Presbyterian College of Physicians & Surgeons

Patients with Lyme disease (LD) experience varying degrees of cognitive dysfunction, from mild concentration difficulties to more severe impairment that interferes with daily functioning and job performance. Children with LD also experience cognitive difficulties, though few studies have addressed the issue. These may be expressed as a decline in school performance or a slowing of academic progress. Frequent absences, fatigue, and discomfort make it difficult to keep up with instruction and assignments, while specific processing deficits may interfere directly with learning. Thus, the appropriate and effective education of children with LD, especially when the illness is chronic, presents a distinct challenge to school districts, which have been uninformed and ill-equipped to deal with the physical, emotional and cognitive impact of LD on their students.
Neuropsychological evaluation can identify cognitive deficits and learning difficulties in children with LD, and provide information essential to the development of appropriate educational programs and services. The current study demonstrates that children with Lyme disease may have significant cognitive deficits that interfere with their academic progress, and further, that aggressive antibiotic treatment can ameliorate at least some of these deficits.
Nine children with LD, referred by their treating physicians, underwent repeated neuropsychological evaluation, once soon after their initial diagnosis and again between twelve and thirty-four months later, having been treated with antibiotics continuously during the interim. At the time of the initial evaluation, all children demonstrated significant variability in their cognitive profile, reflecting cognitive dysfunction, and as a group, a significant discrepancy between Verbal and Performance IQ, the latter being lower. On repeat testing, tests sensitive to speed of mental and motor processing showed the greatest increases. Mental tracking and reasoning also improved following treatment. On academic tests, reading comprehension improved significantly on two separate measures. On one, scores increased significantly, relative to each child's grade expectation, in eight out of nine children.
Neuropsychological evaluation can reveal cognitive deficits in children with LD that have direct and important implications for education. Extended antibiotic treatment can result in significant gains in both cognitive and academic performance. More studies are needed of the impact of LD on learning, its long-term effects on overall development and the effectiveness of various types of treatment. Particularly in areas where the incidence of Lyme disease is high, school districts will need to establish programs for the identification and remediation of Lyme-related academic problems.

Brian A. Fallon, MD, Columbia University College of Physicians & Surgeons

Structural Brain Imaging
MRI scans among patients with neurologic Lyme disease may demonstrate punctate white matter lesions on T2 weighted images, similar to those seen in demyelinating or inflammatory disorders, such as multiple sclerosis, systemic lupus erythematosus or cerebrovascular disease. In early neurologic Lyme disease, hyperintensities may be seen in as many as 50% of patients with evidence of meningitis or encephalitis. Comparable to meningo and cerebrovascular syphilis, European authors suggest that CNS micro- and macrovasculitis may cause both clinical symptoms and MRI changes in patients with CNS borreliosis.
The usefulness of MRI scans in American chronic Lyme encephalopathy is less clear, with abnormalities seen 15% to 41% of the time. After treatment, roughly half of the patients may show resolution of the signal hyperintensity. In late stage encephalomyelitis, MRI scanning often demonstrates focal areas of inflammation, most commonly in the white matter and occasionally in the cortical and subcortical gray matter of the brain.
Combined MRI and PET studies can help to examine the pathophysiology of these hyperintense areas (perfusion, reactivity to hypercapnia, metabolism) and whether they have prognostic significance. Do these hyperintensities represent demyelination or perivascular inflammation? Is the disease process underlying the hyperintensities primarily neuronal metabolic or vascular? FLAIR sequence and magnestization transfer techniques can be used to maximize the yield on identifying white matter hyperintensities.
Functional Brain Imaging
Single photon emission computerized tomography (SPECT) and positron emission tomography (PET), provide a dynamic picture of the brain's functioning: metabolism, blood flow, and chemistry. In comparison to SPECT scans, PET scanning is able to provide better spatial resolution images (4-6 mm vs 6-9 mm) and can be used to provide an absolute quantitative assessment of regional perfusion or metabolic abnormalities. SPECT studies of patients with Lyme disease reveal multifocal areas of decreased perfusion in both the cortex and the subcortical white matter. Logigian reported that patients with definite Lyme encephalopathy had significantly more perfusion deficits that patients with possible LE who in turn had significantly more deficits than normal controls. After treatment with one month of IV ceftriaxone, a partial reversal in brain perfusion deficits was observed, raising the question of whether longer antibiotic therapy may have resulted in even fewer perfusion deficits.
Hypoperfusion defects visualized on SPECT scans may result from any process that alters the radiotracer distribution, including vascular delivery to neurons, transport of the tracer into the cells, and retention of the radioactive tracer in the cells. Problems may arise secondary to direct infection of neurons, from cellular dysfunction due to the indirect effects of neurotoxic immunomodulators such as cytokines, or from decreased perfusion through arterioles secondary to vasculitis. In other words, areas of hypoperfusion may result from a cellular-metabolic and/or a vascular problem.
How may clinical SPECT scans be useful? First, a scan with diffuse abnormalities may confirm that an objective abnormality is present in a patient considered to have a factitious disorder. Second, a normal scan in a patient with prominent neuropsychiatric symptoms may suggest that a psychiatric disorder is the primary cause of a patient's distress and not Lyme disease. Third, improvement after treatment provides objective evidence of physiologic change.
How may clinical SPECTs be abused? One cannot conclude from a SPECT scan that a patient has Lyme disease, as similar patterns of abnormality may be seen with other diseases as well. Other disease processes that demonstrate a heterogeneous tracer uptake include vascular dementia, chronic fatigue syndrome, CNS Lupus, HIV encephalopathy and chronic or acute stimulant abuse.
Future Studies
Studies combining MRI and PET technology, MR Spectroscopy and functional MRI will each contribute significantly to our understanding of the patholophysiology of chronic neurologic Lyme disease.

Sam T. Donta, MD, Boston University Medical Center

This presentation will review the pharmacologic properties of several different types of antibiotics that have potential utility in the treatment of Lyme disease, and try to address the pros and cons of the various antibiotics in treating Lyme disease from its onset to its chronic phase. Available clinical information will also be reviewed to help guide selection of appropriate antibiotics.
Antibiotic Classes and Mechanisms of Action

  1. Pharmacokinetic Principles: Absorption, Protein Binding, Distribution, Elimination, Toxicity.

  2. The Penicillins, Cephalosporins, and other beta-lactam antibiotics

  3. The Tetracyclines

  4. The Macrolides

  5. The Sulfonamides

  6. Vancomycin

  7. The Aminoglycosides

  8. The Quinolones

  9. Clindamycin

  10. Metronidazole

In vitro,

B. burgdorferi

is sensitive to various antibiotics, including the penicillins, tetracyclines, and macrolides. The most active antibiotics are ceftriaxone, cefotaxime, azithromycin, clarithromycin, erythromycin, and amoxicillin. The significance and in vivo relevance of these in vitro results are uncertain because of several mitigating circumstances. First, the standards for in vitro antibiotic sensitivities, MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration), are for organisms grown over a 24-48 hour period of time. For organisms requiring longer growth times, the results of time/kill studies (i.e., the change in numbers of organisms over sequential periods of time) may be more relevant. Nonetheless, it should be expected that antibiotics that are active in vitro should be active in vivo against these organisms. If, however, the spirochetes are in tissues or cells inaccessible to the antibiotic, or they are not metabolically active at the time the antibiotic is present, the organisms will not be killed or damaged by the antibiotic. In this regard, the studies of Klempner et al. that demonstrate that ceftriaxone has little or no activity against

B. burgdorferi

replicating or residing in fibroblasts in tissue culture may be particularly relevant; ceftriaxone and other beta-lactams are highly active in vivo against extracellular organisms, but are not very active against intracellular organisms. If the Lyme spirochetes take up intracellular residence, then beta-lactam antibiotics may not be very effective, and antibiotics with good intracellular penetration may be the most effective agents.


The results of studies related to the treatment of the earliest phase of Lyme disease, i.e. erythema migrans, demonstrate that most antibiotics are effective in relieving patients of symptoms and preventing later complications. Treatment with tetracyclines or beta-lactams for 10-20 days has been shown to be associated with a high degree of success. In contrast, treatment with erythromycin or azithromycin has been less successful. Despite the reported high rates of successful treatment of early Lyme disease, there are many patients whose symptoms were incompletely relieved or whose symptoms developed in the subsequent few months following a 10-20 day treatment course. These typically non-specific symptoms include arthralgias, fatigue, and paresthesias, among a number of other complaints. Such patients have been given diagnoses of fibromyalgia, chronic fatigue syndrome, or post-Lyme syndrome. Similar diagnoses are often also given to patients who are treated for later symptoms and signs of Lyme disease with a 2-4 week course of antibiotics who subsequently have relapsing or persistent symptoms. In such patients, no controlled studies have been conducted to evaluate whether a longer course of treatment would result in a better outcome, or whether one class of antibiotic is any better than any other. Anecdotally, many patients and physicians report improvement when antibiotics are continued for several months.


Treatment Issues


  1. Is the organism extracellular or intracellular?

  2. What is the role of the blood-brain barrier?

  3. What is the proper duration of therapy?

  4. Does antibiotic resistance develop with time and therapy?

  5. Would a combination of antibiotics be more effective?

Conclusions


There are many patients with late symptoms of Lyme Disease who appear to benefit by antibiotic therapy. Controlled clinical trials will be needed to adequately address questions such as duration of therapy, efficacy of one class of antibiotics vs another (eg, tetracyclines vs macrolides vs beta-lactams), efficacy of a combination of antibiotics, and use of adjunctive agents. Such trials will likely also be helpful in gaining a better understanding of the pathophysiology of Lyme disease.

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