Lyme Disease

 

By Laura Torrentera

 

Borreliosis and Neuroborreliosis (Lyme disease)

 

 Ethiological agent Borrelia burgdorferi

 

Transmission hosts and vector:

The Lyme Borreliosis is transmitted to humans by the bite of a tick. The vector life cycle include small and large mammals as reservoirs like the white-footed mouse, squirrels, raccoons, the white tail deer, horses, dogs and other domestic animals. All these hosts carrying ticks infected with B. burgdorferi can transmit the disease to humans. In the U. S. the most common vector of the disease in the northeastern and north-central United States, is the blacklegged tick (the deer tick) Ixodes scapularis. In the Pacific coastal United States, the disease is spread by the western blacklegged tick Ixodes pacificus. Although other species are also known to spread the disease, including; the lone star tick, fleas, the biting flies (e.g. green-headed fly) (1, 2, 3).

 

Borrelia burgdorferi Characteristics:

The family Spirochaetales is a distinguished group of bacteria that have helical or coiled shape. The spirochetes coiled morphology posses two or more axial filaments (endoflagella) enclosed in the space between an outer sheath and the body of the cell. There are three genera: Treponema, Leptospira and Borrelia.  Because of the characteristics of their cell spirochetes are too thin to be visualized under light microscopy. They can be observed using dark-field microscopy or by staining the samples with a fluorescent dye observed by a fluorescent microscope (3). Spirochetes, including Borrelia burgdorferi possess an outer membrane protein (OMP). It has been determined for several spirochetes that OMPs function as adhesins, porins or to have roles in complement resistance (6). Several studies of the membrane properties concluded that Borrelia burgdorferi changes its membrane surface antigenic expression in response to host immune responses (7). The axial filaments (endoflagella) of B. burdorgferi produce the Flagellin. This protein has been shown to bind the host neural tissue (8). Several studies found that B. burgdorferi has the ability to make “cysts(spheroplast L-form). Researchers and infectologists are founded that the dormant cysts are loaded when the patient receive treatment (adverse environmental conditions for the bacteria) and probably are the cause of resistance to some antibiotics (due to the low metabolic activity of the speroblast L-form) and therefore this dormant stage is responsible for the frequent failures of antibiotic treatments and the commonly relapses of Lyme disease (9). Borrelia spirochetes have a very complex life cycle in the human host which includes intracellular and extracellular pathogenesis: corkscrew shaped forms, uncoiled filamentous forms, L-forms lacking a cell wall, cystic forms, and granular forms (9).

It is necessary to increase the research of all the pathogenesis forms of Borrelia such as the OMPs, flagellin, membrane surface antigenic expression, changes and mechanisms of cyst formation and resistance, etc. in the design of new vaccines, drug treatments and a better understanding of Lyme borreliosis life cycle.

 

Testing for Lyme disease identification

The most recommended tests to detect Lyme disease are blood samples to measure antibodies made in response to the infection. The CDC recommends a two-step process when testing blood for evidence of Lyme disease. Both steps can be done using the same blood sample. The firs step tests are: ELISA and IFA. These test are “very sensitive” that means that everyone with Lyme, and also people that does not have the disease will test positive. For these disadvantages the CDC in case of ELISA or IFA positives recommends a second step by using Western Blot, this test is designed to be “specific” that means that usually the test is positive only if the person is infected. If Western Blot is negative that means that ELISA or IFA were “false positive” or the patient where tested to early before antibodies are generated. When Western Blot is performed IgM and IgG antibodies are measured. IgM positive antibodies indicate a new infection, while IgG positive antibodies indicate chronic disease or previous infection. CDC guide lines do not recommend testing directly by Western Blot without testing first by ELISA or IFA. These measurements are dangerous do to the false positive or false negative results. Such results increase the possibility of develop chronic disease if the patient is not treated promptly or in cases of false positives results may lead to people being treated for Lyme disease when they don't have it. Some laboratories offer Lyme testings that are not officially established. For example urine antigen test, immonofluorescent staining for cell-wall-deficient forms of B. burgdorferi and the lymphocyte transformation tests (4). The most accurate test but lest commonly used because is expensive and it is not jet recommended for the CDC is the PCR test. This test detects the genetic material of Borrelia burgdorferi. PCR can identify a current (active) infection (5)

 

Historical Information

The first cases of Lyme disease are thought to be described as early as the late 1700’s in Europe. During the 1900’s the Europeans characterized the disease as a systemic illness associated with a rash, named erythema migrans and they considered that probably was associated with ticks bites. In the U. S. the first officially recognized cases of Lyme disease occurred in the towns of  the Old Lyme in Southeastern Connecticut in 1979. Physicians characterized a group of children who show an unusual rash and associated arthritis. The large number of cases and geographic proximities of the outbreak, indicate an epidemic. Soon after the disease causal organism and the vector of transmission were identified at the University of New York at Stony brook in 1982 by the PhD and MD Willy Burgdorfer. Dr.  Burgdorfer isolated the spirochete causative of Lyme disease from the gut of the deer tick Ixodes scapularis. The Lyme Disease Foundation nominates Dr. Burgdorfer as an honor member of its Board and named the spirochete genus Borrelia and burgdorferi as a species name. The CDC began surveillance for Lyme disease since 1982, the cases increases dramatically and in 1991 Lyme disease was classify as an emerged disease reportable nationally (1).

 

Signs and Symtoms

Lyme disease is a systemic infection that can affect almost any part of the body. The disease signs and symptoms vary in relation of the stage of the disease (from acute,  chronic and Lyme-neuroborreliosis). According to the International Lyme and Associated Diseases Society (ILADS), "Lyme disease is the latest great imitator and should be considered in the differential diagnosis of MS, ALS, seizure and other neurologic conditions, as well as arthritis, CFS, Gulf war syndrome, ADHD, hypochondriasis, fibromyalgia, somatization disorder and patients with various difficult-to-diagnose multi-system syndromes." (11).

This is the list of signs and symptoms that a patient with Lyme can develop:

  1. Fewer than 50% of patients with Lyme disease recall a tick bite.
  2. Fewer than 50% of patients with Lyme disease recall any rash. Although the erythema migrans (EM) or “bull’s-eye” rash is considered classic, it is not the most common dermatologic manifestation of early-localized Lyme infection.
  3. If the patient does not show these two “classic symptoms” the recognition of the disease is very difficult and the patient could get chronic with multiple sing and symptoms and multisystem affectation.
  4. These could be: Fever, sweat, chills, flushing, stiffness (joints, neck, back), unexplained weight change (weight loss, weight gain), twitching (face, other muscles), fatigue, headache, hair loss, swollen glands, sore throat, tingling, numbness, burning feeling, testicular or abdominal pain, ear buzzing, ringing, sexual dysfunction, irritable bladder, menstrual irregularities, endometriosis, breast pain, nausea, vertigo, motion sickness, join pain and swelling, poor valance, facial paralysis, confusion, double vision, blurry vision, constipation, diarrhea, chest pain, ribs soreness,  shortness of breath, heart palpitations, pulse skips, heart block, heart murmur or valve prolapsed, difficulty with concentration, difficulty reading, forgetfulness, poor short term memory, disorientation, getting lost.
  5. Neuroboreliosis:

The Lyme Neuroborreliosis (LNB) clinical presentation according with neurologist and psychiatrist allows to the inclusion of diagnosis of facial neuritis, multiple enhancing cranial nerves, symmetric orbital myositis with cranial neuritis, noncompressive radiculitis, pediatric leptomeningitis. The inflammatory reaction of neuroborreliosis is postulated by some Lyme specialist to be one of the etiologies for a very broad spectrum of neurologic disorders. These include the amyloidal deposition of Alzheimer disease, Multiple Schlerosis, Autism, and neuropsychiatric disorders (anxiety, depression, bipolar disorder, and schizophrenia).  The brain white matter hyperintensities (brain lesions) observed on MR imaging in patients with LNB and multiple sclerosis display sufficient similarity to suggest a common autoimmune pathogenesis for both (10).

 

Microbial Virulence Mechanisms contributing to the disease process

It is a particular characteristic observed in spirochetes outer membrane proteins, OMPs atypical signal peptide cleavage sites. It has been isolated four membrane surface lipoproteins: protein A (DbpA), protein C (OspC), protein BBF01, and VlsE. This membrane proteins were analyzed by quantitative reverse transcription-PCR under various immune conditions demonstrated that B. burgdorferi changes its membrane lipoprotein surface antigenic expression in response to immune attack. The protein C (ospC) expression changed by 446 times and increased BBF01 and vlsE expression up to 20 and 32 times, respectively, under the influence of immune pressure generated in immuno-competent B-cells during infection. Borrelia burgdorferi changes its surface Antigenic expression in response to host immune responses. (7). It was found that Antibodies in sera of 7 patients with neurologic manifestations of Lyme borreliosis a particular monoclonal antibody (mAb H9724) making complex to the flagellin of B. burgdorferi that bind neural tissue. To identify the antibody binding site common to B. burgdorferi flagellin and the neural tissue, the researchers made recombinant fusion proteins expressing epitopes of flagellin. They found that antibodies in patients' sera and mAbH9724 bound within 18-amino acids in the central region of flagellin. They found also antibodies in patient’s sera and mAb H9724 bounding to a human neuroblastoma cell line. Patients sera absorption with a A peptide (EGVQQEGAQQPA), corresponding to amino acids numbered as: 213-224 of flagellin, inhibited binding to the neuroblastoma cell line. These results suggested that the immune response to a specific B-cell epitope within flagellin, shared by a human neuroblastoma cell line, may be involved in the pathogenesis of neuroborreliosis (8). Lyme neuroborreliosis (LNB) is the result of secondary to systemic infection by B. burgdorferi in the United States and by B. garinii and B.afzelii species in Europe. The mechanism of neurologic injury probably includes vasculitis, cytotoxicity, neurotoxic mediators, or autoimmune reaction via molecular mimicry (9, 10, 11).

 

 

Control and treatments 

The infectious Disease Society of America (IDSA), published a guideline for Lyme disease treatment. A recent review paper updated the information about antibiotics treatments. Based on IDSA treatment recommended for children and pregnant women is doxycycline (100 mg, twice daily), amoxicillin for children under 9 years old (50 mg, per day orally). For adults amoxicillin (500 mg orally, three times a day), Cefuroxine-axetil (500 mg orally, twice daily; or 30 mg/kg per day divided, twice daily), erytrhomicin (250 mg orally, 4 times daily; or 30 mg/kg per day divided in 3 to 4 doses). Those allergic to tetracycline and penicillin should be avoiding them. The CDC recommends that antibiotic treatment should be maintained only for 14 to 21 days. In Europe people with acrodermatitis are treated for 30 days. In U. S. patient that develop arthritis should be treated for 30 to 60 days. In cases of severe or late stage of the disease; neuroborreliosis or cardiovascular damage required intravenous antibiotics (eftriaxone 2g/day), or cefotaxime (2 g every 8 hours) or penicillin G (5 millions Units, every 6 hours). The IV treatments should be used for 4-6 weeks. In more than 15% of patients during antibiotic treatment develop (aproximatelly after 24 hours of initiated treatment) patient develop an apparent worsening of symtoms, including fever, sweding, headache, general malaise, etc). These reactions related with the release of cytokines and receive the name of

“ Jarisch-Herxheimer-type reaction” (1, 15). There are a lot of controversies and issues of the type of drugs, duration of treatment and the CDC does not agrees with the group of physicians that apply long term treatments. In some states physicians that threat patients during long time are prosecuted. There are stages in wish Lyme disease organizations are fighting to obtain permissions for long term treatments (for example, in Maryland, Connecticut and New York physicians are able to treat patients for long term) (12).  IDSA has convened a review panel to examine whether the Society’s Lyme disease guidelines, published in 2000 should be revised or updated based on a rigorous review of the medical and scientific evidence on the diagnosis and treatment of Lyme disease. The Review Panel will hold an open public hearing July 30, 2009 in Washington, DC. 

 

 

Current Cases of Outbreaks in U. S.

According with the CDC MMWR the Northeastern and north-central United States, and the Pacific coastal regions are considered as Lyme disease endemic regions. Since Lyme disease became nationally reported as an emerged disease in 1991, the annual number of reported cases has more than doubled. The CDC is considering only the laboratory-positive reports specially on local and state health departments in areas where Lyme disease is endemic. Because several factor mentioned before Lyme disease surveillance is complicated by both underreporting and over diagnosis of cases. There are other logistic complications among states regarding differences in patient demographics data formats to report for example; age and sex, check list of symptoms, type of treatment, response, chronic stage, disagreements of doses of antibiotic to use and length of treatments among states. Including above-average and below-average incidence suggest variation in diagnostic and reporting practices among states. Often clinical information on symptoms is not verified independently and often is incomplete (16). Fig. 1A shows the cases reported in 2005 notice that the states more affected for the disease are the states considered Lyme endemic (Connecticut, Delaware, Maryland, Massachusetts, Minnesota, New Jersey, New York, Pennsylvania, Rhode Island, and Wisconsin) but there is a trend of spreading out the infection to the all country. Fig. 1B shows the worldwide outbreak (I explained in the next section). Figure 2 shows the reported cases of Lyme by year, from 2003 to 2005 by sex and age group. This information was published by CDC, MMWR during 2005, Notice that the two groups of age more affected are children from 5 to 14 years old (in this group, males are more affected than females). The second group is in adults from 40 to 59 years old. More than 200,000 cases of Lyme disease in the United States have been reported to the CDC, resulting in a national incidence of 9.7 cases per 100 000 individual (10, 16).

 

 

Figure 1 A. Lyme disease reported cases in the United States in 2005. Notice that the two regions more affected are these considered as Lyme disease endemic regions (Connecticut, Delaware, Maryland, Massachusetts, Minnesota, New Jersey, New York, Pennsylvania, Rhode Island, and Wisconsin).

 

Figure 1 B. Reported Outbreaks of Lyme disease worldwide. The Yellow-shaded areas indicate the geographic distribution of recorded clinical cases. The colored ellipses indicate the distribution of the various Borrelia subspecies.

 

 

Figure 2

 

Figure 2.  Cases of Lyme disease reported by sex and age. Notice that the two groups of age more affected are childrens from 5 to 14 years old (in this group, males are more affected than females). The second group is in adults from 40 to 59 years old.

 

Current Cases Worldwide

In Canada, the Center for Disease Control reported that the black-legged tick, Ixodes scapularis, is wide distributed in Ontario, with a range of extension at least as far north of five regions of Ontario. The Canadian CDC reported the Lyme spirochete in the gut of 12.9% of I. scapularis adult ticks. The Canadian authorities are connecting the presence of B. burgdorferi in Canada as acommon strains of Lyme Disease found in the U. S.

The Canadian Vector-Borne Disease Laboratory in British Columbia, declared British Colombia as an endemic region for Lyme Borreliosis. Lyme disease has been found in rabbit ticks in the state of Alberta. In Montreal, The McGrill University Department of Medicine in a recent report said that Borrelia burgdorferi is spreading out widely in Canada, predominantly found in parts of British Columbia, southern and eastern Ontario, southeastern Manitoba, and parts of Nova Scotia. In the United Kingdom, the Health Protection Agency (HPA) reported an increase of Lyme Borreliosis cases by 90% since 2006 across the UK, considering the endemic areas in UK: Hampshire, Dorset, and Berkshire. The British attribute the phenomena of Lyme Borreliosis due to the characteristic humid and mild summer in this country. This climatic conditions increase the tick population. The tick hot-spot in England are the UK, and New Forest, South Downs, Dorset, and Berkshire (18). In Sweden, The Department of Molecular Biology at Umeå University, studies reported in 2007 that Lyme disease geographical distribution shows a constant increase in Sweden. This Swedish study report that birds migration spread out ticks due to their long distance dispersal and their role as reservoir hosts for Borrelia. The Swedish researchers also found that the Arctic region sea birds of Norway carry Ixodes uria ticks infected with Borrelia garinii, this bacteria is the strain that cause Lyme disease in Europe. Russian scientists in collaboration with the U. S. CDC researchers were working in the identification of Russian Borrelia strains. They found that 27.7% of ticks, Ixodes persulcatus were infected with at least one microorganism, in ticks from Arkhangel'sk province, Novgorod province and St. Petersburg, the prevalence of Borrelia. burgdorferi DNA was 16.6%, 5.8%, and 24.5% respectively (18). German researches reported that children from forested regions of Germany are in highest risk of ticks bites and Lyme disease than children from urban areas. In Croatia, were reported 3,317 cases of Lyme disease from 1987 to 2003. The more affected area was the Northwestern. The Department of Public Health reported that in Croatia Lyme Borreliosis is dominated by erythema migrans, with a high incidence of neurological manifestations. In Switzerland, since 2004 the cases of tick-borne encephalitis are increasing caused mostly by Lyme disease. In Italy, were documented 24 cases of Lyme over the last year (18). Considering the lack of information of Lyme disease from the developing countries such as Latin America, Asia, Africa, India, China, etc. The actual number of cases worldwide is probably much, much higher that we can expect. Because of unreported cases, lack of research and inadequate testing and diagnosis we really do not known what is the exact situation of Lyme disease worldwide .

 

Prevention

The most obvious method of prevention is avoid ticks bites! If people are exposed to endemic areas of Lyme disease, they most wear protective clotting and boots, Use ticks repellent containing (N,N di-ethyl-m-toluamide), DEET. Practice frequent skin inspections. If a tick is found in the skin a prompt removal of ticks an immediate medical supervision is recommended. Other environmental recommendation in tick areas is a frequent cleaning and clearing of woods, jars, gardens, burning of removed vegetation, spread out of acaricides, control, checks and cleaning from ticks of the deer population, mice, raccoons, rabbits, and several other mammals that carry ticks. Some studies showed that the control of growing of the deer population in endemic areas of ticks resulted in a reduction of the 94% of the Ixodes scapularis population. During 2002 Smithkline Beecham, Biological, Laboratories, Philadelphia, PA, released to the market a vaccine, LYMErix, that is a recombinant vaccine directed toward a B. burgdorferi  lipoproteinouter-surface protein A. It was used in a selected group of people at risk of Lyme disease. The vaccine displayed a limited efficacy, high price and possible link with autoimmune induced arthritis, or the development of Lyme disease itself. So, LYMErix is no longer available in the market. At the present people are eager to know when an effective vaccine is forthcoming (1).

 

Current Research

The Institute of Allergy and Infectious Diseases (NIH) in collaboration with several research institutions and Universities have several projects in development regarding the several aspects of Lyme disease. The main goals are to find better and accurate diagnosing, effective treatments, and methods of prevention. NIAID Lyme disease research programs include a broad range of activities of systematic studies on: Animal models to study the disease, Borrelia strains physiology, Mechanisms of pathogenesis (Molecular, Genetic, and Cellular).  Immunity mechanisms, identification of vectors, vector competency, and their relationships on the transmission of Lyme, antibiotic therapy efficacy and modes of action, development of accurate and reliable diagnostic tests for acute and chronic Lyme disease. Regarding antibiotics some studies found that persisting or chronic Lyme could be alleviated by the anti-inflammatory properties of doxycycline and tetracyclines, these as well as several beta-lactam and cephalosporin antibiotics, including ceftriaxone that now are commonly used to treat chronic Lyme disease. These antibiotics have neuroprotective properties that can positively influence various neurological disorders, this group of antibiotics have beneficial effects that can alleviate some of the musculoskeletal and neurological symptoms generally associated with chronic Lyme disease (17). A non-NIH funded pilot study was conducted to test the benefits of Gabapentin for the symptomatic treatment of chronic neuropathic pain in patients with late-stage Lyme borreliosis. Gabapentin was administered orally for 1-2 years, improvement was noted in both pain quality and pain quantity (9 of 10 patients), as well as a positive effect on mood, general feeling of health, and quality of sleep (5 of 10 patients). These promising results suggested that Gabapentin could be used to treat patients with neuroborreliosis (17). A very interesting research (very important for Texas!) in NIAID laboratories in collaboration with CDC is related with the causes of Southern Tick-Associated Rash Illness (STARI). STARI is manifested as a rash and similar symptoms to that of Lyme disease and occurs in the southeastern and south-central United States. It is associated with the bite of the lone star tick and the bacteria strain received the name of Borrelia lonestari (17). Another interesting project is related to Borrelia gene regulatory expression focusing on three important regulatory proteins: BosR, a Zn-dependent transcriptional activator that regulates key antioxidant enzymes; Ø 54, an alternate sigma factor that also regulates certain parts of the oxidative stress response and regulates the osmotic stress response; and Ø S which controls the stationary phase of growth and the expression of genes that are critical to the pathogenesis of Lyme disease (19). NIAID also is funding preclinical studies on developing and testing other candidate vaccines, for example, decorin-binding protein A or DbpA) that is in research from MedImmune, Inc. and Sanofi-Aventis Pharmaceuticals, this group is working in a combined vaccine composed of the DbpA and OspA of B. burgdorferi, in experimental animals they found that is more effective than either given alone in preventing the development of Borreliosis. On the basis of these findings, both companies have an agreement to develop a new, more effective second-generation vaccine to prevent Lyme disease in humans (20). Some research suggest the possibility that B. burgdorferi may play a role in the etiology of Alzheimer’s disease; NIAID intramural scientists are examined this issue in greater detail (20). In two publications of 2007, the researcher  using a very sensitive PCR assay capable of amplifying a Borrelia-specific DNA target sequence from all strains of B. burgdorferi sensu lato species that are known to cause disease in humans. Dr. Allan MacDonald a Neuro-pathologist working with brain necropsies of patients who died from Alzheimer’s disease donated from the McLean Hospital Brain Bank found in several brain necropsies positive signals for infectious Borrelia DNA in a pilot study. He hypothesized that “These Alzheimer's diseased neurons analyzed with DNA probes, produced little dots of positive staining.  That “dots” are Granulovacuolar bodies in Alzheimer's diseased neurons (little dots in a bubble), are one of the expected microscopic profiles of Alzheimer's disease. Grabulovacuolar bodies inside nerve cells”(14). In a set of experiments Dr. MacDonald describe that Borrelia display different shapes: corkscrew shaped forms, uncoiled filamentous forms, L-forms lacking a cell wall, cystic and granular forms (9). In a recent paper Dr. Mac Donald describe that “Alzheimer's neuroborreliosis is the result of latent Borrelia infections ascending neural circuits through the hippocampus to the higher brain centers, creating a trail of neurofibrillary tangle injured neurons in neural circuits of cholinergic neurons by trans-synaptic transmission of infection from nerve to nerve”(13).

(See pictures bellow taking from Dr. MacDonald, with his permission)

  

 

Figure 1. Borrelia spirochetes stained with Barbour

monoclonal H9742 – Borrelia Flagellin specific -Fetal death in

utero.

 

Figure 2. Cystic form of Borrelia

burgdorferi grown in culture from

spinal fluid after 16 months of

incubation from patient with positive

Western Blots in Spinal Fluid and

Serum 

Figure 3. Borrelia burgdorferi strain B31

in culture - variable corkscrew,

Straighteneds, and cystic forms

 

 

 

Figure 4. Cystic and spirochetal

emerging from B31 strain of Borrelia

burgdorferi in culture.

 

 References:

 

1.      Robert L. Bratton, MD; John W. Whiteside, MD; Michael J. Hovan, MD. Richard L. Engle, MD; and Frederick D. Edwards, MD. 2008. Review:  Diagnosis and Treatment of Lyme disease. Mayo Clin Proc: 83(5); 566-571.

http://www.mayoclinicproceedings.com/content/83/5/566.full.pdf+html?sid=174f8b3e-105d-4cc5-ba13-1dba421e43b0

 

2.      LYME DISEASE OVERVIEW. 2002-2005 LymeInfo.Net Last Updated: Feb 2005.  http://www.lymeinfo.net/lyme.html

 

3.      The Merck Manual Online Medical Library. http://www.merck.com/mmpe/sec14/ch174/ch174a.html

 

4.      Testing for Lyme disease identification CDC. Human diseases diagnosis. http://www.cdc.gov/ncidod/dvbid/lyme/ld_humandisease_diagnosis.htm

 

5.      Detection of Borrelia burgdorferi by PCR-Overview. IGeneX, Inc.
795 San Antonio Rd., Palo Alto, CA 94303 http://www.igenex.com/lymeset5.htm

 

6.      Paul A. Cullena, David A. Haakec, and Ben Adlera. 2004. Outer membrane proteins of pathogenic spirochetes. FEMS Microbiol Rev. 28(3): 291–318. http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2666356&blobtype=pdf

7.      Fang Ting Liang, Jun M. Lamine,  Mbow, Steven L. Sviat, Robert D. and Gilmore, Mark Mamula, and Erol Fikrig. 2004. Borrelia burgdorferi Changes Its Surface Antigenic Expression in Response to Host Immune Responses. Infection and Immunity Vol. 72 (10): 5759–5767 . http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=517580&blobtype=pdf

8.      E. Fikring, R. Berland, M. Chen, S.Williams, L. H. Sigalt, and R. A. Flavell. 1993. Serologic response to the Borrelia burgdorferi flagellin demonstrates an epitope common to a neuroblastoma cell line. Proc. Nati. Acad. Sci. USA.Vol. 90: 183-187.  http://www.pnas.org/content/90/1/183.full.pdf?ck=nck

9.      V. Preac Mursic et al. 1996. Lyme Disease Studies on the Cystic Form of Borrelia burgdorferi Mechanisms of Persistence. Infection Vol. 24 No. 3 (chapters 1-12). http://www.samento.com.ec/sciencelib/4lyme/studiesoncyst.pdf

10.  P. Hilildenbrand, D.E. Craven, R. Jones and P. Nemeskal. 2009. Lyme Neuroborreliosis: Manifestations of a Rapidly Emerging Zoonosis. Neuroradiol. (1-9) http://www.ajnr.org/cgi/reprint/ajnr.A1579v1?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=Lyme+Neuroborreliosis&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&resourcetype=HWCIT

11.  Lyme Info Net. 2002-2005. Last Updated:Feb.2005 http://www.lymeinfo.net/lyme.html

12.  International Lyme and Associated Diseases (ILADS) http://www.ilads.org/

13.  MacDonald A. B. 2007. Alzheimer's disease Break Stage progressions: reexamined and redefined as Borrelia infection transmission through neural circuits. Med Hypotheses 68(5):1059-1064.

http://www.ncbi.nlm.nih.gov/pubmed/17113237?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

 

14. MacDonald A. B. 2007. Alzheimer's neuroborreliosis with trans-synaptic spread of infection and neurofibrillary tangles derived from intraneuronal spirochetes. Med Hypotheses 68(4):822-825.

http://www.ncbi.nlm.nih.gov/pubmed/17055667?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

 

15. Gary P. Wormser, Robert B. Nadelman, Raymond J. Dattwyler, David T. Dennis,Eugene D. Shapiro, Allen C. Steere, Thomas J. Rush, Daniel W. Rahn,

Patricia K. Coyle,  David H. Persing, Durland Fish,8 and Benjamin J. Luft. 2000. Practice Guidelines for the Treatment of Lyme Disease. Clinical Infectious Diseases. 31(Suppl 1):S1–14 by the Infectious Diseases Society of America. http://www.journals.uchicago.edu/doi/pdf/10.1086/314053?cookieSet=1

16. CDC, MMWR. Lyme Disease; United States, 2003-2005. http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5623a1.htm

17. National Institute of Allergy and Infectious Diseases. NIH. http://www3.niaid.nih.gov/topics/lymeDisease/research/antibiotic.htm

 

18. Brian Rosner, et al. 2008. Lyme Disease Manual Report. Chap 2. Is Lyme Disease Worldwide? http://www.lymebook.com/africa-europe-canada-sweden-england-uk-united-kingdom

 

19. National Institute of Allergy and Infectious Diseases. NIH. http://www3.niaid.nih.gov/topics/lymeDisease/research/research.htm

 

20. National Institute of Allergy and Infectious Diseases. NIH. http://www3.niaid.nih.gov/topics/lymeDisease/research/vaccine.htm