Ronald F Schell and Steven M Callister, Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Washington, USA
Although the clinical presentation of borreliosis, especially relapsing fever, has fascinated physicians for centuries, the mainstream of immunologic research bypassed the disease for many years. The advent of penicillin and DDT discouraged researchers anxious to study the immunology of borreliosis. After World War II, except for a few devotees whose laboratories continued to work on some problems related to the microbiology and immunology of relapsing fever, research on Borreliae was ignored. Scientific publications plunged from 50 a year in the 1950s to approximately 10 a year in the 1980s.
The increased recognition of Lyme disease renewed interest in the Borreliae. Lyme disease was first recognized in Europe during the early 1900s. However, this illness was not reported in the USA until 1969, when a grouse hunter from Wisconsin contracted the first reported case of Lyme disease in North America. Subsequently, additional cases of Lyme disease were reported from the upper Atlantic Coastal states and the upper midwest of the USA. Human cases have since become increasingly recognized throughout the world. Lyme disease is the most common tick-associated illness in the USA.
During the 1980s, the spirochetal bacterium Borrelia burgdorferi was implicated as the causative agent of Lyme disease. Since that time, molecular characterization studies have demonstrated at least several Borrelia subsp. capable of causing Lyme disease. The three most common are B. burgdorferi sensu stricto, B. afzelii and B. garinii. To date, only B. burgdorferi sensu stricto spirochetes have been detected in the USA, while these three genospecies are found throughout Europe.
Borreliae are entirely host-associated. Spirochetes have been found only in arthropods or host vertebrates that the arthropods have fed upon. B. burgdorferi sensu lato spirochetes are transmitted to human hosts by ixodid ticks of the Ixodes ricinus complex. Lyme disease is a multisystem disorder which usually begins with localized infection of the skin manifested by an expanding skin lesion, erythema migrans (EM), and constitutional symptoms such as fatigue, headache, mild stiff neck, arthralgia, myalgia and fever. Subsequent dissemination of the spirochete can cause more severe clinical manifestations, including secondary annular skin lesions, meningitis, Bell's palsy, radiculoneuritis and atrioventricular heart blockage. Arthritis or nervous system manifestations are hallmarks of chronic Lyme disease. An additional late manifestation, acrodermatitis chronica atrophicans, is seen primarily in Europe. Differences among the genospecies of B. burgdorferi sensu lato may account for geographic and regional variations in the clinical presentations.
In contrast to Lyme disease, relapsing fever caused by B. hermsii confirms that this illness is primarily a result of infection in the bloodstream. The clinical presentation is characterized by periods of fever lasting several days, separated by week-long intervals of normal temperatures. Diseases caused by Borreliae are curable with antimicrobial agents and are rarely fatal.
Spirochetes are not closely related to either gram-negative or gram-positive bacteria. B. burgdorferi sensu lato spirochetes are 0.2-0.3 by 20-30 pm. These organisms are highly flexible, left-handed (rotate in counterclockwise direction) helical cells, composed of 3-10 loose coils. They are susceptible to drying, but can survive for several months in medium at 4°C. The Lyme spirochete is composed of an outer cell membrane (outer sheath), protoplasmic cylinder and numerous flagella.
Some Borreliae are also surrounded in vivo by an amorphous slime layer. The slime layer, likely composed of host components, is weakly attached to the spirochete and is lost upon washing. The presence of host proteins in the slime layer may explain the inability of humans or animals to eliminate the Lyme disease spirochete despite a vigorous immune response. Thus, the spirochete may evade direct cell killing, antibody-dependent phagocytosis or anti body-mediated lysis with a coating of protective 'self molecules' that prevent immune recognition.
A similar outer envelope exists on Treponema pallidum. Treatment of treponemes with detergents to remove this outer envelope is required to achieve high reactivity in immunologic assays. The polysaccharide 'capsular' material appears to prevent phagocytosis. Similarly, there is evidence that carbohydrates are present in the slime layer of B. burgdorferi sensu lato, which may also influence the destruction of spirochetes by host factors.
The outer cytoplasmic membrane of Lyme disease spirochetes has a trilaminar organization of 45-62% protein, 23-50% lipid and 3-4% carbohydrate. The fluid membrane can be easily separated from the underlying protoplasmic cylinder with dilute solutions of sodium dodecyl sulfate or nonionic detergents. The outer membrane can move to one end of the spirochete by a phenomenon called 'patching' or 'capping'. The outer envelope layers also form blebs when B. burgdorferi sensu lato are incubated with specific antibody and complement. Bleb formation is a prelude to cell death.
The major surface proteins are located in the outer cell membrane of Borreliae. To date, outer surface proteins (Osp), designated A, B, C, D, E and F, have been demonstrated in B. burgdorferi sensu lato organisms; however, their functions remain unknown. These proteins are heterogeneous, especially among European isolates. Initial investigations demonstrated that the Osp A of USA isolates was homogeneous; however, more recent investigations have confirmed significant heterogeneity. The genes for Osp A and Osp B are located on a 40 kb double-stranded linear plasmid. Linear plasmids also encode the variable major proteins (VMP) of B. hermsii, which undergo antigenic variation. These antigenic changes allow survival of spirochetes in the human host for extended periods of time. Lyme disease spirochetes also appear to upregulate or down-regulate individual Osps, especially Osp A and Osp C, during infection of humans.
A variety of other antigens can also be detected in the outer membrane of B. burgdorferi sensu lato. These include proteins with molecular weights of 16, 27, 55, 60, 66 and 83 kDa. Antibodies against these proteins are readily detectable in chronic and complicated cases of Lyme disease. However, their relationship to the chronic nature of the disease and development of autoimmune immunologic reactions has not been elucidated. No single or combination of polypeptides has been directly associated with the different clinical manifestations of Lyme disease. The development of persistent or chronic Lyme arthritis that is not responsive to antimicrobial therapy has been associated with an immune response to Osp A and Osp B and class II major histocompatibility complex molecules HLA-DR4 and HLA-DR2.
The flagella, responsible for motility, are located within the outer membrane and are generally not exposed to the surface. Between 7 and 11 flagella are inserted subterminally and bipolarly to the protoplasmic cylinder of B. burgdorferi sensu lato. Numbers of flagella vary among other Borreliae. The flagella run parallel to the long axis and overlap in the middle of the spirochete. Flagellin, with a molecular weight of 41 kDa, is the predominant flagellar protein. An antibody response to flagellin is a consistent feature in all stages of Lyme disease. Despite the presence of high concentrations of flagellar antibodies, Lyme disease continues to progress, suggesting that flagellin does not induce a protective immune response.
The structure and importance of the protoplasmic cylinder remains undefined. The cell wall contains muramic acid and ornithine as part of the peptido-glycan. Another component may be lipopolysaccha-ride. The Jarisch-Herxheimer reaction (characterized by a transient high fever), which can occur in Lyme disease patients after treatment with antibiotics, may be due to the sudden release of lipopolysaccharide from lysed organisms. Many patients have experienced a Jarisch-Herxheimer reaction following antimicrobial therapy.
Borreliae can be cultivated in artificial medium; however, the bacteria multiply slowly. Generation times range from 8 to 15 h (B. burgdorferi) to 26 h (B. recurrentis). Continuous passage of Borreliae in artificial medium alters the antigenic structure and renders the bacteria noninfectious.
The clinical presentation alone is usually sufficient to diagnose relapsing fever. In contrast, diagnosis of Lyme disease is often difficult because of the variety of symptoms that can develop. Visualization of B. burgdorferi sensu lato in blood has not been successful because Lyme disease spirochetes rapidly disseminate into tissues. Silver staining has been used to demonstrate spirochetal forms in both biopsy and autopsy tissues; however, spirochetes cannot be positively identified.
Lyme disease spirochetes have been successfully cultured in vitro from blood, cerebrospinal fluid and synovial fluid but only a small number of these cultures have yielded organisms. In contrast, recovery of B. burgdorferi sensu lato from EM skin lesions has been more successful. Polymerase chain reaction (PCR) technology has also been used to directly detect Lyme disease spirochetes in patients. To date, PCR testing appears to be useful for recovery of B. burgdorferi sensu lato DNA from atypical EM
lesions, cerebrospinal fluid and synovial fluid, provided the laboratory has sufficient expertise with the technology.
In many cases, serologic evidence of infection with Lyme disease spirochetes is the only option available to clinicians. Unfortunately, gross inaccuracies caused by the lack of specificity of conventional diagnostic assays and their subjectivity have made misdiagnosis and overdiagnosis common. These factors have contributed to confuse the general public and make many clinicians distrust serologic testing. During the past several years, however, diagnostic assays have been greatly improved. Unfortunately, there is currently no single assay that offers sufficient sensitivity and specificity to become the 'gold standard' of Lyme disease testing, although detection of borrel-iacidal antibodies in Lyme disease patients offers promise. A flow cytometric borreliacidal antibody test that measures the killing of live spirochetes after they were inoculated in the patient's serum and complement showed a sensitivity of 72% and more than 98% specificity. Regardless, quality laboratory procedures are available, provided results are obtained from experienced laboratories and interpreted correctly and the clinician understands the advantages and disadvantages of each laboratory procedure.
In early studies of the pathogenesis of Lyme disease, a major obstacle was the lack of suitable animal models. Rabbits and guinea pigs infected with B. burgdorferi develop skin lesions which histologically resemble human EM. These lesions, however, are not consistently induced and are the only clinical manifestations detected. Disseminated infection occurs in mature hamsters, rats and mice but clinical manifestations similar to those seen in human Lyme disease do not occur, despite persistence of spirochetes in the tissues. The hallmark of chronic B. burgdorferi sensu lato infection in humans, Lyme arthritis, appears to be observable in rodent models only when the immune system is immature or compromised. Rhesus monkeys have been shown to develop clinical signs and symptoms similar to localized and disseminated human infection; however, development of clinical arthritis only occurred in a small percentage of infected animals. In addition, the high costs associated with these animals has precluded widespread use of this Lyme disease model.
Despite these imperfections, animals models have been used extensively to elucidate pathogenic mechanisms of B. burgdorferi sensu lato. Lyme disease spirochetes have been shown to stimulate various inflammatory cytokines, including interleukin 1 (IL-1), IL-6 and tumor necrosis factor a (TNFot), and several autoimmune mechanisms which appear to play a role in pathogenesis.
Animal models also continue to play a vital role in efforts to develop an effective Lyme disease vaccine. The role of cell-mediated immunity remains largely unknown; however, an important role for antibody-mediated immunity after vaccination has been established. The induction of antibodies, termed borreliacidal, that can specifically kill B. burgdorferi sensu lato are often responsible for anti-body-mediated protection. Vaccination of animals with several individual Osps, including Osp A, Osp B, Osp C, and a 39 kDa protein have provided protection against Lyme disease spirochetes. Concomitantly, borreliacidal antibodies against Osp A, Osp B and the 39 kDa protein have been detected in vitro.
Osp A has emerged as the leading Lyme disease vaccine candidate and the efficacy of several recombinant Osp A vaccines is being investigated in animals and humans. Clinical trials have demonstrated safety in human volunteers; however, several significant obstacles must be overcome before induction of long-term comprehensive protection can be demonstrated. Most importantly, the success of Osp A vaccination appears to be dependent on the induction and long-term maintenance of high concentrations of borreliacidal antibodies. The present formulations of the Osp A vaccine have not maintained and sustained high concentrations of borreliacidal antibodies.
An anamnestic response will likely occur too slowly to prevent infection. Shortly after infection with B. burgdorferi sensu lato, the spirochetes become refractory to killing by borreliacidal antibodies. High levels of borreliacidal antibodies are detectable in sera from humans with all stages of Lyme disease; however, spirochetes are not eliminated. A recently developed flow cytometric test for detecting these highly specific borreliacidal antibodies is useful for confirming a Lyme disease diagnosis, and widespread availability of this test should greatly improve Lyme disease testing. However, induction of protective levels of anti-Osp A borreliacidal antibodies for extended periods of time has been difficult, even after vaccination of animals with high concentrations of Osp A in combination with adjuvants.
In addition, experimental Osp A vaccines comprise a single protein, despite the fact that Osp A is anti-genically polymorphic. Thus, vaccination provides little or no protection against heterologous B. burgdorferi sensu lato isolates. Significant frequencies of anti-Osp A escape mutants have also been observed in animals after vaccination with a single Osp A protein. More recently, researchers demonstrated down-regulation of Osp A expression and upregulation of other Osps shortly after infection of the vertebrate host. Thus, the incorporation of multiple Osp A proteins and other B. burgdorferi sensu lato Osps, especially Osp C, appears necessary for comprehensive protection against the Lyme disease spirochete.
See also: Antigenic variation; Bacterial cell walls; Endotoxin (lipopolysaccharide (LPS)); Rheumatolog-ical disorders.
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