A Division III Undergraduate Thesis Literature Review

1.
LYME DISEASE:
The Mystery, Science, Controversy, and Evidence
A Division III Undergraduate Thesis Literature Review
Justice Erikson
Submitted for the completion of the degree of
Bachelor of Arts
Hampshire College
Amherst, MA
May 2​nd​
2017
Faculty Committee:
Charles Ross (Chair)
John Castorino
Christopher Jarvis
Lynn Miller, Professor Emeritus

2. Acknowledgements
I would like to thank my fiancée Beatrice Evelyn for encouraging me to strive for greatness,
listening to me read sections that I wasn’t sure about, putting up with me covering the entire
living room in sticky notes to organize these pages, and reminding me to take care of myself
when needed.
I would like to thank my mother Cara LeBlanc for helping me when I got stuck, being my
cheerleader, and teaching me that I can do anything I set my mind to.
I would like to thank my father Josiah Erikson for his quiet confidence in my abilities and for
making sure that my material needs were met during not just this year but indeed most of my
life thus far.
I would like to thank Kat Mclellan for encouraging words and a sympathetic ear.
I would like to thank Naya Gabriel for her unwavering support and friendship, and for helping
me with all kinds of laboratory dilemmas.
I would like to thank Sarah Steely for listening to my struggles and laughing with me.
I would like to thank Iris Everill for reminding me that there is a world outside of academia.
I would like to thank Sam Jackson for his enthusiastic help in the lab and interest in my work,
which inspired me to keep going on hard days.
I would like to thank Thomas Varley for helpful comments in the early stages.
I would also like to thank Meghan McGarry, Josia Gertz DeChiara, Yvonne Thomas, Griffin
Harmon, Autumn Phaneuf, Bram Baxter, Flavia Nwankwo, Sarah Hunter, Emma Opitz, Julia
Rauch, Alex de Strulle, and many others for their moral support.
Finally, I would like to thank all of the myriad faculty members that I have worked with over the
years, whether in an advising relationship or just in classes. Your support and encouragement
has been invaluable.
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3. Abstract​: This thesis is a literature review spanning many topics related to Lyme disease. It
seeks to answer questions such as: how does Lyme disease cause illness? What medical
treatments are available, and what alternatives are there when those treatments fail? Is Lyme
disease a chronic autoimmune disorder? Analysis spans socio-political spheres, epidemiology,
physiology, and molecular biology.
Table of Contents
Introductions
Introduction for the Non-Scientist
Personal Introduction
1: What is Lyme Disease?
1.1 Zoonosis and Ecology: Ticks and Deer
1.2 ​History of Introduction to New England
2: How Does Lyme Disease Make You Sick?
2.1 Phases of Infection: Two Models
2.2 Inflammation and Autoimmunity
2.3 Detailed Pathogenesis and Immune Evasion Techniques
2.4 Neurological effects of Lyme borreliosis
2.5 Biofilms: A Method of Persistent Infection
3: What Medical Treatments Are There For Lyme Disease?
3.1 Diagnostic tests of borreliosis
3.2 Treatment of the CDC Model
3.3 Treatment of Chronic Lyme
4: What Should Be Done About Lyme Disease in The Future?
5.1 Unanswered Questions for Future Research
5.2 My protocol
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4. Introduction for the Non-Scientist
Lyme Disease (or Lyme borreliosis, the distinction between which we will discuss
in the medical chapter) is a ​zoonotic arthropod-borne infectious disease​: ticks carry the
disease-causing microbes and transmit them to the mammals that contract the disease such as
humans, dogs, and horses. There are a number of regional ticks that may transmit the disease,
depending on the area. These microbes responsible are called ​Borrelia sp., ​small spirochetal
(corkscrew-shaped) bacteria, with a wide variety of elusive host defense mechanisms that make
it uniquely suited for what it does. The species ​Borrelia burgdorferi​ is most commonly
attributed with causing Lyme disease, but we now know that there are several other species of
Borrelia​ that also cause slight variations of Lyme disease.
(Continued)
Personal Introduction
Since I began my research on Lyme Disease last year, I have encountered dozens of
people whose lives have been affected by it. They often stop me mid-sentence, cutting me off
with a sincere sense of urgency, to tell me about the time that they, their mother, their best
friend, or their neighbor had Lyme Disease. I work as a college admissions tour guide, and I talk
with families from all around the US every day. One girl, during a walk around my garden plot
that is quickly being taken over by Japanese Knotweed, tells me that she has had chronic Lyme
for years, and had to give up many of her favorite activities including hiking and sports, because
she was just too exhausted to do anything more than the bare minimum of physical activity. She
also expressed to me great concern that her grades had dropped because of her reduced energy
to engage with her schoolwork due to the fatigue that comes with Lyme; as well as a feeling of
invisibility and hopelessness.
Friends and acquaintances have come to me in a panic after receiving a Lyme disease
diagnosis or finding a tick bite. They tell me that they know that something about Lyme disease
treatment is inadequate. They worry that if the antibiotic doesn’t clear the infection it could get
much worse. They want to know what really helps and what they can do. They are scared. Others
still tell me about their doctor in rural Connecticut, Massachusetts, or New York: How this
doctor really knows how to treat Lyme and was able to “cure” them or their loved ones using
ozone therapy, high-dose intravenous vitamin C, herbal therapies, or other methods. They ask if
I know about Plum Island, if I have read ​Lab 257​ or ​Healing Lyme​. Older people tell me calmly
that they had Lyme once, but they don’t really believe that it ever went away. Sometimes their
knees still hurt a little more than they should, or they get very very tired for a few months. They
tell me that what I’m trying to do is very important, and that they are glad someone is brave
enough to do it. Nobody has told me that they think that Lyme isn’t serious or that our
treatments are enough.
And so I nod my head, I tell them that they are probably right, that something is wrong. I
will read the books, if I haven’t already. I’ll try to work out my piece of this puzzle, because we
need to. Too many people are suffering, and too many people aren’t listening.
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5. This is a young person’s epidemic as much as an older person’s epidemic; the Centers for
Disease Control and Prevention (CDC) (2015a) reports that in the last decade, the two groups
with the highest prevalence of Lyme Disease diagnoses were five year olds and fifty year olds.
Researchers with the CDC estimate that around 329,000 cases of Lyme Disease occur in the
United States every year (Nelson et al. 2015), and while these are primarily in the Northeast part
of the country, almost every state in the union reports some diagnoses every year. It has become
abundantly clear to me that more research and understanding of this disease is a critical public
health issue.
And so in this thesis, I will aim to discuss the truth about Lyme disease. There are many
sides to the story and not many of them agree on very much of what Lyme disease is, how it
functions, or how to treat it. I will attempt to discuss all sides of the story, but let me introduce
the major players: there is the Center for Disease Control (CDC) and the Infectious Diseases
Society of America (IDSA), whose statements on Lyme disease tend to agree with and, in fact be
written by, the “founding fathers” of Lyme disease - Steere, Malawista, Barbour, Burgdorfer, and
others (I will typically just refer to them as the CDC). There are a variety of people that disagree
with this group. There are chronic Lyme patients, their doctors, and their advocates who are sick
and tired of being told that their lived experiences are not real. Some of them call for long-term
antibiotic treatment. Some of them call for alternative therapies like ozone therapy or high-dose
intravenous vitamin C. And there are the rebellious doctors and researchers who are doing
research to show that Lyme disease may not work in the way the CDC says it does, writing
academic papers to propose new models, and risking their medical licenses to treat their
patients with whatever modality they have seen work. Everyone wants to eradicate Lyme
disease, but everyone has a different idea of what needs to be done.
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6. Chapter 1: What is Lyme Disease?
It seems to me that when most people think of Lyme disease, they think of deer ticks and
New England forests. Next they’ll think of a bullseye rash and getting a quick round of
antibiotics from their doctor. To many, that’s it. But there are also many for whom this world is a
lot bigger. Perhaps they know someone, or many people, who say they have chronic Lyme
disease. Maybe they’ve done some research and know how debilitating and scary chronic Lyme
can be. And maybe they also know that ​chronic Lyme disease​ technically doesn’t exist
according to the Center for Disease Control (“Post-Treatment Lyme Disease Syndrome”, 2016).
Before you read further, think about what you know about Lyme disease. Where did that
information come from? What are your unanswered questions?​ In this chapter I will begin to
explore what exactly Lyme disease is, what it isn’t, what it might be, and what some people want
you to believe that it isn’t.
~
The history of Lyme disease is one of the most interesting stories of an infectious disease,
particularly in the United States. There is much controversy and debate over exactly how and
when it got here, but most can agree on why it stayed and took hold of the New England region,
and spread from there. In order to to understand this process, one has to consider the full
ecology of the region, and the life cycle of ​Borrelia​ within its various hosts - ticks, mice, deer,
humans, and more. As you’ll see, the middle of the 20th century was a perfect storm of
ecological factors in the Connecticut/Massachusetts bay region present to facilitate a boom of
the Lyme borreliosis life cycle. Thanks to the alertness of people living in the area, and the way
they recruited local health departments and academics, the disease was able to be identified and
named, and researchers began the ongoing work of figuring out how this potentially devastating
disease manages to evade treatment so effectively.
The Discovery of Lyme Arthritis and the Contributions of Dr. Allen Steere
In the late 1960s, it became clear to Polly Murray and Judith Mensch that something was
very wrong in their small town of Lyme, Connecticut. Their children and neighbors were
developing a disease that they were told was juvenile rheumatoid arthritis at an alarming rate.
Judith Mensch reported that 12 children, 4 of whom lived on the same road (out of a community
of 5000), had all been diagnosed with Juvenile Rheumatoid Arthritis (Steere et al., 1977b). Polly
Murray, as well as her husband and children, had developed the disease and were suffering
greatly as she collected extensive notes and stories of the spread of this mysterious disease in
Lyme. She was possibly the first patient to get the diagnosis that has been all-too-common
among lyme patients: that it was all in her head (Grann, 2001). But she persevered and
remained involved in the discovery of Lyme and she eventually published a book detailing her
experiences in 1996. She was no stranger to the medical literature and spent long hours is the
library repeating the question “What is wrong with me?” (Murray, 1996). Surely, she thought, it
was statistically impossible that their neighborhood would have an incidence rate of “juvenile”
rheumatoid arthritis 100 times higher than what is expected for the disease. And right she was.
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7. Polly Murray and Judith Mensch finally reported their well-documented observations in
October of 1975 to the Connecticut State Health department, who in turn recruited the Yale
University School of Medicine to assist in solving this mystery that was much larger than any of
them had bargained for.
Thus begins the journey of Dr. Allen Caruthers Steere. He was a young fellow with Yale
University and had just joined the Division of Rheumatology, which was headed by Dr. Stephen
Malawista. Steere had a keen interest in the pathology of arthritis and joined the team sent to
investigate this strange incidence in Lyme. Along with David Snydman of the health department
and others, the team began to monitor Lyme, CT and two surrounding towns with a total
population of twelve thousand. They published their first findings in 1977, and went on to
publish many papers together. Many of the team members are still regarded as the foremost
experts on Lyme disease today. However, many people have critiqued Dr. Allen Steere and his
colleagues in the discovery of Lyme disease for their model of pathogenicity, restrictive
diagnostic criteria, and their continued refusal to acknowledge what is now known as chronic
Lyme disease (persisting after antibiotic treatment).
The Steere group’s first paper described the first look into the immunology of erythema
migrans - the “bullseye” skin lesion (Figure 1) often characteristic of early Lyme disease
infection; and suggested that the disease must have a common infectious agent (Steere et al.,
1977a). (Please note that Fig. 1 shows an “ideal” example of erythema migrans, it exhibits
differently in every patient and doesn’t always look like a bullseye at all.) This wasn’t the very
first time that erythema migrans had been described. However, in 1909, Arvid Afzelius had
described the same rash in Sweden, and associated it with the bite of ​Ixodes ricinus​ ticks, which
are now known to be capable of transmitting Lyme disease in Europe. It was later called
erythema chronicum migrans in Europe and associated with neurologic symptoms termed
meningopolyneuritis (inflammation of the brain and pathology of the peripheral nerves) (Reik et
al., 1979). These and other nervous system presentations would later become the scariest and
most controversial symptoms of late-stage or chronic Lyme disease.
Figure 1: Erythema migrans. CDC 2016
The second paper from the Steere group studied 39 children and 12 adults in Lyme, CT
with recurring bouts of arthritic symptoms in the joints, particularly the knees (Steere et al.,
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8. 1977b). It is notable that there was an extremely high incidence in a grouping of four adjacent
streets, and many families had more than one member affected. The disease was clearly highly
localized, although no particular common exposure such as a vaccine or common swimming
spot could be identified. The authors reported that one quarter of patients reported having an
erythema migrans rash before developing symptoms, and they knew of only two people out of
159 surveyed who developed the rash but not arthritis. One patient associated the rash with a
recent tick bite at the same location. This paper first identified the basic symptoms of fever,
fatigue and malaise, and myalgias; all now considered typical of Lyme disease (Table 1). The
paper concludes by naming this new disease Lyme arthritis, so that it may be distinguished from
the juvenile rheumatoid arthritis diagnosis that the children studied had been receiving.
The research group continued to follow patients affected in the Lyme area and expanded
the studied communities to 12 contiguous communities for their third paper (Steere et al. 1978).
In this expanded study, 21% of the now 43 patients associated the onset of their disease with a
tick bite, and one was able to bring the tick in to be identified as​ Ixodes scapularis​. They also
observed that incidence on the east side of the Connecticut river was 30 times higher than on the
West side of the river. They reported that it is difficult for ticks and other animals in the life cycle
of lyme disease to cross bodies of water (which may not be entirely true, deer can swim well),
supporting their conclusion that the new Lyme disease is in fact a tick-borne zoonotic disease.
In 1979, Reik, Steere, and others published a paper describing some of the neurological
effects of Lyme disease including lymphocytic meningitis, cranial nerve palsies, and sensory
radiculopathy (Reik et al., 1979). In 1980, Steere’s group first described “lyme carditis” and
noted that complete heart block is more common in Lyme disease-involved carditis than other
forms (Steere et al. 1980a). With these two discoveries, the general symptoms of Lyme disease
had been pinned down and the disease described and established in the medical world on a basic
level. The next steps were to discover the causative agent and establish treatment protocols.
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9. Table 1: Lyme Disease Symptoms
Symptom Source(s)
Erythema Migrans
(Bullseye Rash)
Estimated prevalence: 25%(1), 70-80% (2)
>50% (7)
1:Steere et al, 1977b
2:​https://www.cdc.gov/lyme/signs_symptoms/
3: Reik et al, 1979
4: Steere et al. 1980a
5: Wormser et al. 2006
6: ​Lab 257
7: ​Singh and Girschick, 2004
Fever/Flu-like symptoms 1, 7
Fatigue / Malaise 1, 6, 7
Myalgias (general) 1, 2,
Arthritis / Joint Aches, Stiffness, and
Swelling
1, 2, 5, 6, 7
Nerve Palsies 2, 3, 5, 6, 7
Headaches 2, 6, 7
Stiffness 2,
Dizziness/Shortness of breath 2,
Nerve pain, numbness, tingling,
radiculopathies
2, 3, 7
Meningitis/Encephalitis 2, 3, 6, 7
Lyme Carditis: Heart Palpitations or Irregular
Heartbeat
2, 4, 6, 7
Short-term memory problems 2,
Borrelial lymphocytoma Goc et al. 2016, 7
Depression 6
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10. 1.1 Zoonosis and Ecology: Ticks and Deer
As we have outlined already, Lyme borreliosis infections in mammals come from bites
from infected ticks. But where did the tick get it? Figure 2 shows a diagram created by the CDC,
illustrating the life cycle of ​Borrelia​ infection. The tick has a two-year life span over which there
are opportunities for the tick to both become infected and infect mammals. Tick eggs are laid in
spring, and when they hatch in summer they find mice and birds to feed on, at which point they
may get infected by those small animals (who were previously infected by other ticks), and carry
that infection on to larger mammals when they feed again as nymphs and adults the following
year. Humans are particularly likely to be bitten by nymphs in spring and summer. Deer are the
more common targets of adults in the fall. The females will lay eggs in spring and continue this
life cycle. You’ll notice that the ticks feed a total of three times, and if they get infected the first
time, they have the opportunity to spread it to two other animals, thus exponentially spreading
the disease.
Figure 2: Life Cycle of Lyme Borreliosis (CDC 2016)
1.2 History of Introduction to New England
The history of how Lyme disease came to New England is often debated and largely
obfuscated by the shadows of time. The CDC and affiliated researchers have evidence that they
believe shows that ​Borrelia​ have existed in North America for millions of years. This comes from
the recent microscopic analysis of a 15-20 million year old ​Amblyomma​ tick larva preserved in
Dominican amber. Let’s take a moment to investigate that. This paper states that the
tick-containing amber was collected from a region of the Dominican Republic, which is over 900
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11. miles from the continental United States. So even if the tick did contain ​Borrelia​, it is by no
means proof that it has been in the United States, let alone New England, for hundreds of years.
Figure 3:​ ​Brachispira pilosicoli ​vs ​Borrelia burgdorferi ​vs Amber spirochetes (bottom tw0)
​Image 1: ​Brachyspira pilosicoli​ spirochetes: ​https://en.wikivet.net/Brachyspira_pilosicoli
Image 2: ​Borrelia mayonii​ spirochetes: http://phil.cdc.gov/phil/details_linked.asp?pid=20517
Images 3-4: Poinar, 2015
The images of the supposed​ Borrelia​ are low-resolution and generally unclear because of
everything else in frame (Figure 3, Image 3). The images of the spirochetes that have been taken
out of the original image and laid on a white background (Figure 3, Image 4) certainly resemble
Borrelia ​(Figure 3, Image 2). But they also certainly resemble ​Brachispira pilosicoli​, spirochetal
bacteria that colonize pig guts (Figure 3, Image 1) (Poinar, 2015). It seems to me like very
limited evidence to base such an impactful conclusion on.
On the other hand, Alan Barbour (2015 p 1), one of the researchers who worked on the
identification of Lyme’s causative agent; introduced the history of Lyme with a case of an old
woman with Lyme-like symptoms that were supposed to be due to the bite of a European sheep
tick (​Ixodes ricinus​) from Germany sometime before 1909. This story is frequently cited, and it
appears to be likely that borreliosis existed as a minor disease in Germany not supported by the
ecology of the area. The book “Lab 257” by Michael Carroll (2004, p1-38) has popularized a
story that may account for ​Borrelia’​s sudden appearance in the Northeast United States. It
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12. discusses the recruitment of scientists who had been working on biowarfare for Nazi Germany
under project ​PAPERCLIP​, the founding of a secret Germ Laboratory called Plum Island, and the
huge breaches in laboratory safety that have been documented and covered up since its founding
in 1956.
According to Michael Carroll, Dr. Erich Traub, a Nazi virologist of much renown, was
recruited swiftly under project ​PAPERCLIP​ after WWII​ ​to work with the USDA and the newly
founded Plum Island laboratories. Under the pretense of protecting the US from potential
biowarfare, the lab experimented with numerous infectious animal diseases of interest to the US
(and Germany) for biowarfare purposes. Although all definitive documentation on tick research
and Traub has been destroyed, numerous people involved remember those times and have
reported that outdoor tick trials more than likely occurred on Plum Island, and that their
laboratory safety was laughable at best, with one even reporting open holes in the roofs of lab
spaces.
Plum Island itself is just two miles by ferry from Long Island, and about twelve miles as
the crow flies from Old Lyme, CT. It is populated by all sorts of wildlife, including known Lyme
vectors such as birds, deer, and small rodents. White-tailed deer are great swimmers and could
easily (and have been seen doing so) traverse the distance between plum island and long island,
if not directly to Old Lyme. Birds in the area migrate right through Plum Island, often stopping
along their way. All it would take is one infected tick let loose, and it could be the start of the
pandemic we know today.
Desowitz (1981 p146-158), in his description of the zoonosis of babesiosis (a common
Lyme coinfection, and an obnoxious tick-borne infectious disease in its own right) describes how
in the nineteenth century, there were dramatic ecological changes to the Nantucket/Martha’s
Vineyard/Long Island/Shelter Island/Plum Island etc. area due to human settlement - ones that
not-so-serendipitously created and ideal environment for ticks - and with an early nineteenth
century deer repopulation effort, it became a whole lot easier for tick-borne bacteria to make it
from mice, to deer, to grass, and to the clothes of humans. In addition to sharing a common
vector, the presumed zoonosis and identification of babesiosis and Lyme disease both occurred
around the seventies and eighties in the same areas; one can presume that the same or similar
ecological factors impacted both. Because Lyme disease is caused by ​B. burgdorferi​ , ​B.
burgdorferi​ follows ​I. scapularis​ deer ticks, deer ticks follow white tailed deer (Bosler et al.
1984; Duffy et al. 1994), and white tailed deer exist across the United States (Leopold, Sowls &
Spencer 1947), it wasn’t long before Lyme Disease became a national problem.
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13. Chapter 2: How Does Lyme Disease Make You Sick?
Phases of Infection: Two Models
It has become increasingly evident as the conversation around Lyme disease develops
that the CDC’s model for how Lyme disease presents symptomatically and acts within the body
is simply not good enough for us to be able to either do effective and useful research on the
subject, or diagnose and treat patients effectively. However, in order to challenge the status quo,
we must first discuss the status quo.
According to the CDC, Lyme disease transmission effectively works as follows: Assuming
you are in the northeastern, mid-atlantic, or north-central United States, you get bitten by a
blacklegged/deer tick (​Ixodes scapularis​). If it stays attached for longer than 36-48 hours you
may become infected with ​B.burgdorferi ​(cdc.gov/lyme, 3/17). Then we enter the phases of
infection defined by the CDC and the Infectious Diseases Society of America (IDSA) (Wormser
et al., 2006). While the CDC gives signs and symptoms on its website for the use of concerned
citizens, the IDSA has published a more extensive and effectually definitive document that
details the definition of Lyme disease, the approved model of infection and pathogenesis, and
approved treatment protocols.
So according to them, lyme disease infection works as follows: The clinical description of
Lyme disease is generally broken up into two stages. Early infection occurs in the first month
after a tick bite and is characterized by the erythema migrans rash. Approximately three to thirty
days after ​B. burgdorferi ​has successfully moved into the skin tissues, development of erythema
migrans (EM) may occur. The CDC website currently reports that this occurs in 70-80% of all
infected persons. The definitions between the stages are mostly defined by their symptoms, and
the symptoms for early Lyme disease are generally flu-like symptoms such as fever, chills, aches,
swollen lymph nodes, fatigue, etc. as well as erythema migrans. At this point the infection is
assumed to exist only in the bite-surrounding tissues and to have not yet disseminated into the
body at large.
During this stage, the body is responding to the invasion of ​Borrelia​ bacteria with its
frontline response - the innate immune system. The innate immune system includes
macrophages (“big eaters”), white blood cells that are able to engulf and kill ​Borrelia​ spirochetes
(Montgomery & Malawista 1996). This crucial mechanism, as well as many other immune
responses to ​Borrelia​, have been the focus of much study, and may lend later insights into the
validity of various treatment methods. We will discuss them in more detail shortly.
Early infection can in most cases be cured with a short round of antibiotics, and in fact
the Infectious Diseases Society of America research group, Wormser et al. (2006) only
recommends various oral or intravenous short-term rounds of antibiotics for any stage of Lyme
Disease or its most common co-infections (Table 2). At the first clinical observation of EM, the
physician is advised by the CDC to prescribe two weeks of basic antibiotics such as doxycycline.
In the CDC’s diagnosis guidelines, neurological symptoms such as meningitis and cerebral nerve
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14. palsy still fall under “early Lyme disease”; and the standard treatment for these does not extend
beyond nearly the same two-week course of antibiotics. For carditis and lymphocytoma, the
recommendation is again two weeks of antibiotics. If the infection goes untreated or is
unsuccessfully treated early on, late stage Lyme disease develops.
Table 2: Official Lyme Disease Treatment Protocols​. From Wormser et al., 2006.
Showing that a short 14-21 day oral regimen of doxycyline is almost always the only approved
treatment for Lyme disease. Superscript letters correspond to elaboration of treatment protocols
in the text.
Late infection is when the more serious symptoms of Lyme develop: the broad
neurological degradation, arthritis, heart irregularities, facial palsy, and severe fatigue. Here we
see a minor step up in the CDC treatment protocols. For Lyme arthritis, a four-week course of
antibiotics is recommended. And for patients with significant and debilitating neurological
symptoms, there is the option of 2-4 weeks of intravenous antibiotics, but if symptoms do not
subside, re-treatment is not recommended. Limited symptomatic treatment beyond this is
suggested, especially anti inflammatory medications for arthritic symptoms, but no further
treatment unless there is “objective” evidence of reinfection (another tick bite). The CDC and
National Institutes of Health call any lingering symptoms beyond their limited treatment
protocols “Post-Treatment Lyme Disease Syndrome” (Abbreviated PTLDS) (CDC 2015c) and
deny the existence of persistent infection or true Chronic Lyme Disease. In fact, they specifically
go out of their way to list treatments for PTLDS that are not recommended, citing a “lack of
biologic possibility, lack of efficacy, absence of supporting data, or the potential to harm the
patient” including long-term antibiotic therapy, and vitamins and nutritional managements. The
same guidelines go on to thoroughly discuss their evidence for why most PTLDS/chronic Lyme
does not exist, dismissing it as depression, chronic fatigue (which they also dismiss), etc. What I
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15. find most distressing about this is that the authors primarily cite their own research groups as
positive evidence, and everyone else as bad evidence. Conflicts of interest are everywhere in this
field.
These CDC classifications are highly restrictive and do not account for many ways in
which Lyme disease could exist. The CDC model often mostly and sometimes entirely excludes:
Patients who don’t exhibit a classical EM rash living in a non-endemic area, patients with
subclinical symptoms; patients who don’t test positive on the current gold-standard IgG/IgM
western blot tests or present false negatives in serological testing, and patients who may have
been infected by an expanding list of Bb sensu lato agents. In almost all of these cases, examples
of horizontal or non-zoonotic infection such as sexual or gestational transfer, complex immune
evasion problems, and other possible non-arthropod vectors are not accounted for by the model
or given any mention or consideration in CDC-approved publications. Harvey and Salato’s paper
on Borreliosis Pandemic (Harvey and Salato, 2003) is an excellent and extensive review of why
and how this model of Lyme Disease needs to be expanded to include patients with illnesses
caused by ​Borrelia​ infection that do not meet current criterion for Lyme disease as defined by
the CDC. In addition to resulting in a lack of care for these patients, this paradigm also excludes
these atypical patients from almost all borreliosis research. The CDC’s model has become a
self-fulfilling paradigm, and wrongfully shuts out a huge portion of the borreliosis-affected
population from their right to know what is happening to their bodies, and to receive proper
medical treatment for it. This is beyond negligent. It is immoral.
Harvey and Salato also detail extensive reasoning for their distrust of both western blot
and serological testing for the diagnosis of Lyme disease, outlining situations in which the tests
produce false negatives. What is probably the most unsettling place where the CDC and the
actual body of literature don’t match up, is the notion that Lyme borreliosis is not a persistent
infection, and therefore any post-treatment symptoms must be due to reinfection, past damage,
or autoimmune responses. Harvey and Salato find that this is entirely premise-less or based on
indirect examples from unrelated diseases. However, there is an extensive and growing body of
evidence that very strongly supports the formation of cystic and biofilm forms of Bb that can
remain dormant for months to years (Sapi et al. 2012. Goc et al., 2015). Based on this and
studies of other spirochetal pathogens, we know that a dormant cystic infection can persist
indefinitely and continue to produce symptoms (or present as asymptomatic infection), transmit
infection, and still not invoke enough of an active immune response to test positive on
immunological assays. Additionally, ​Borrelia burgdorferi​ is known to possess numerous other
mechanisms of evading and possibly suppressing the immune response (Berndtson, 2013.
Bhattacharjee et al., 2013). In light of these and similar findings, Harvey and Salato propose a
new model of borreliosis that places “Lyme Disease” as a small part of a much larger global
borreliosis pandemic (Table 3). This extensively challenges the current medical model of
borreliosis, and rightfully so. If we are to find a way to cure this awful pandemic, we must first
recognize it for what it is.
Table 3: Harvey and Salato (2003) proposed model of “Epidemic Borreliosis”
14

16. Some of the most paradigm-shifting differences between the CDC model and the Harvey
and Salato Epidemic Borreliosis model are the epidemiological factors, placing the disease in a
much larger worldwide context. The idea that the primary vector is humans and that this is an
ancient and worldwide epidemic, rather than a new and localized zoonotic disease, in itself
warrants a much larger and more serious investigation on the part of medical science. Other
major differences include the previously discussed inclusion of a broader consideration of
Borrelia​ agents and their vectors, characterization of persistent infection, more holistic
diagnosis criteria, a revised clinical timeline, and recognition of symptoms to fit this expansive
consideration of the disease. This model is a great step towards a better understanding of
borreliosis, and it and similar work will hopefully open the doors for patients to find recognition
and understanding of their symptoms, as well as allow researchers and practitioners to be
supporting in developing effective and sustainable treatment protocols for all forms of
borreliosis.
Inflammation and Autoimmunity
Lyme disease is incredibly complex in its interactions with the immune system, and
while I will be explaining most mechanisms as they come up, it’s a good idea to have a basic
familiarity with the immune system before reading the remainder of Chapter 2. Table 4 serves as
15

17. an introduction to some of the basic immunology vocabulary that will help you to understand
the discussion of how exactly Lyme disease causes illness.
Table 4: Immune System Basics
Macrophages​ are a part of the innate
immune response. They are frequently
the first responders to infection and are
able to consume (​phagocytose​) and kill cells
that they recognize as non-self. Afterwards,
they present pieces (​antigens​) of the invaders
they have encountered to specialized immune
cells to notify them of the threat. They have
many receptors on their surface to recognize
microbial threats, including toll-like receptors
(TLRs).
MHC​ (major histocompatibility
complex) molecules and the peptides
they display are how the immune
systems recognize what proteins are
normal or “self” and which belong to
invaders. MHC class I molecules are on
almost every cell in the body, whereas MHC
class II molecules are primarily used by
antigen-presenting cells to display peptides
from the potential invaders they have
encountered.
Dendritic Cells ​are another major
antigen-presenting cell of the innate
immune system. They take up antigens
in peripheral sites and bring them to
development areas of the adaptive immune
system to train specialized cells to attack that
antigen.
Antibodies ​come in many classes that
handle different kinds of immunity. Their
role is to bind to pathogens to prevent them
from harming other cells (​neutralization​),
coat pathogens in preparation for
phagocytosis (​opsonization​), and activate
the complement system.
B-Cells ​specialized cells of the
adaptive immune system and are
the primary route through which
vaccines work. Their receptors are randomly
generated forms of ​antibodies​ that
correspond to possible non-self peptides.
When they are presented with antigens that
match their receptors they can mature into
plasma cells and proliferate antibodies.
Complement ​is an innate
chemical cascade that assists
with many functions of the
immune system. Complement
can inherently recognize
common features of microbes and bind to the
surfaces of microbial invaders, kill them
directly, opsonize pathogens, and signal other
cells of the immune system to attack.
T-Cells ​are specialized cells of the
adaptive immune system that serve a
variety of activated functions based on
their class. CD4 “helper” T cells bind to MHC
class II and assist with the activation of
infected macrophages, enhancing innate
immune functions, and helping B cells. CD8
“killer” T cells bind to MHC class I and are
responsible for killing infected cells. There are
also T “memory” cells and T “regulatory”
cells.
Monocytes ​are precursors to macrophages,
mast cells, and dendrites. Too many in the
tissues can indicate chronic disease or stress.
Chemokines/Cytokines ​are chemical
messengers between immune cells that can
help activate cell functions.
Images and information from Janeway’s Immunobiology, 8th ed. (Murphy and Weaver, 2012)
16

18. Most of the symptoms of Lyme borreliosis are caused by various manifestations of
inflammation (Table 1). Inflammation is the immune system’s response to anything it identifies
as foreign and/or a threat to the body, from tissue damage to microbial infection. After the
threat has been tagged, white blood cells flood the area to assist with killing pathogens and
repairing tissue. This is useful and necessary in many cases, but inflammation itself can be very
harmful to the body if infection is prolonged or if inflammation goes unregulated, such as in
autoimmune disease.
There have been studies of the immune mechanisms involved with borreliosis in mice
that can give us some insight into how human immune systems respond to borreliosis. One
study looked at Interleukin-10 (IL-10), an inflammatory regulator. IL-10 is there to say “enough
is enough” and reduce inflammation after an infection has been cleared under normal
circumstances. IL-10 utilizes the STAT3 pathway to downregulate inflammatory cytokines, MHC
class II costimulatory molecules, etc. in macrophages as well as CD4 T cells, dendritic cells, and
polymorphonuclear neutrophils. It also induces anti-inflammatory effects such as the anergy
(inactivation after antigen encounter) of T cells, CCR5 expression, and the induction of T
regulatory cells, all acting to shut down the immune response (Mege et al., 2006). The study of
IL-10 in murine borreliosis (Brown et al., 1999) showed that lack of IL-10 and the subsequent
uncontrolled inflammation actually correlated with lower numbers of infecting spirochetes in
tissues. This indicated that while the inflammation was undoubtedly painful and damaged
tissues, it was effective at clearing the infection. However, this study also used a mouse strain
that was known to have a naturally overactive immune response to borreliosis, and these mice
suffered the worst inflammation without the same clearing of spirochetes seen in the otherwise
normal IL-10 knockout species. This suggests that inflammatory response to borreliosis and
immune efficacy may have a critical genetic component regardless of other factors.
Such an effect has also been proposed in humans: a genetic predisposition where a
person’s Major Histocompatibility Complex II allele (HLA-DRB1*0401) causes the immune
system to aggressively attack OspA (Outer Surface Protein A) (Bergstrom et al., 2002. Drouin et
al. 2008). This gene has also been linked to genetic predisposition to arthritis in general
(MacGregor et al., 1995). Even Steere has posited that there could be a genetic predisposition to
autoimmune responses to Lyme disease (Steere, 2009).
The immune system (T-cells specifically) is able to identify infection by ​Borrelia​ and
other pathogens largely based on small pieces of their surface (termed antigens, to which
antibodies bind) that are recognized as not being a part of the host’s body. Therefore, it is
important to be familiar with these proteins specifically. Much research on the molecular
biology of Borrelia has been done since its discovery, and the proteins/lipoproteins that have
been identified as important to the immune response have been named as Outer Surface
Proteins (Osps) A-F (Bergstrom et al., 2002).
However, microbes are sometimes able to change which proteins they express in
different situations by rearranging or altering expression of their own DNA, and this can make
identification by the immune system and scientists more difficult. ​Borrelia​ move from ticks
which do not have an antibody-based immune system into mammals, which do. This dramatic
shift of environment necessitates flexibility of the organism. ​Borrelia​ has an unusually low
17

19. density outer surface of lipoproteins, which may contribute to the fact that it is able to readily
change its array of expressed outer surface proteins (Bergstrom et al., 2002).
Each of these Osps have their own functions in transmission and pathogenicity, many of
which have been described in the literature (Bhattacharjee et al., 2013; Brooks et al. 2006; Suk
et al. 1995; Fingerle et al. 1995). Osp A is primarily expressed when the organism is in the unfed
tick’s midgut and helps it bind to the epithelium of the gut. Both OspA and OspB have also been
shown to be important for binding to and penetrating cells in the host, allowing them to
establish infection. OspB is especially important in infectivity, possibly because it is responsible
for penetrating cells (Bergstom et al., 2002).
Detailed Pathogenesis and Immune Evasion Techniques
Borrelia​ is well known as being incredibly good at evading the immune system and
producing an infection that is difficult to treat once established. This is the reason why a round
of antibiotics is recommended after any tick bite that may have transmitted Lyme disease. The
Borrelia​ genome coupled with assistance from tick saliva prove formidable opponents against
the immune system.
As discussed in the previous section, ​Borrelia​ express OspA while in the tick gut. During
tick feeding, the Borrelia begin to multiply and face their first challenges from the host immune
system. The blood that the tick ingests could contain OspA antibodies (such as if the host has
received an OspA vaccine) and prevent infection. The blood meal also introduces complement -
the immune system’s helper chemicals which can kill bacteria directly in some cases, or coat and
deactivate them in preparation for killing by immune cells (leukocytes), and recruit leukocytes.
In addition to the Osps unique to ​Borrelia​, there are other more generic surface proteins that
may be targeted by the host immune system, including CD14 and TLR-2. Recognition of these
proteins as being non-self by the host immune system results in rapid killing and engulfing of
the bacteria (Rupprecht et al., 2008; Singh and Girschick, 2004).
18

20. Figure 4: ​How ​Borrelia​ evades the immune system. “​Borrelia are recognized
by immune cells through TLR2 and CD14 and attacked by complement and antibodies.
Therefore, the borrelia downregulate their surface proteins, hide in the extracellular
matrix, and use complement-neutralizing proteins like Salp, CRASPs, or ISAC/IRAC or
induce the formation of immune complexes by secreting soluble antigens to be protected
from recognition and subsequent killing.​” Rupprecht et al. 2008.
Despite all of these threats, Borrelia are still remarkably good at evading the host
immune system (Figure 4). They can downregulate the outer surface proteins that the host
immune system responds to, interfere with immune cells ability to attack them through
downregulation of their effector mechanisms, or even physically hide in places like the
extracellular matrix (Rupprecht et al., 2008).
Many of the mechanisms through which the host immune system is able to recognize
Borrelia​ and mount a defense is through the recognition of OspA. To avoid this, OspA
expression is rapidly downregulated once the tick’s blood meal begins so that ​Borrelia​ can make
it into the host’s body and avoid being wiped out before it has the opportunity to establish
infection. Taking the place of OspA is OspC, which is upregulated during the blood meal in
preparation for entrance into the host bloodstream. Interestingly, OspC can actually bind a
protein in tick saliva, Salp15, which inhibits complement binding to the bacteria (sort of acting
like chemical armor). This makes OspC essential for ​Borrelia​’s survival during early infection.
However, the host immune system will eventually build antibodies and specialized cells to attack
OspC, so it too is downregulated after the first few weeks of infection (Rupprecht et al., 2008;
Singh and Girschick, 2004).
Borrelia​ doesn’t just use Salp15 from tick saliva to defend against the complement
system. It also uses Salp20, ISAC, and IRAC. In addition to these defensive proteins from tick
saliva, ​Borrelia​ also has some of its own proteins that bind complement called
19

21. complement-regulator-acquiring surface proteins (CRASPs). As mentioned earlier, ​Borrelia​ is
able to upregulate IL-10 production in mononuclear leukocytes, resulting in decreased
inflammation/immune response in the host. Additionally, ​Borrelia​ is able to give off soluble
antigens for which ​Borrelia​-specific antibodies to bind, apparently diverting them away from
the organism itself. And finally, ​Borrelia​ also has mechanisms of hiding in the extracellular
matrix away from the bloodstream and circulating lymphocytes. It is able to break down
elements of the extracellular matrix and attaches to components of connective tissue, such as
decorin (an element of connective tissue that binds to collagen) (Rupprecht et al., 2008). This is
probably a major reason why Lyme disease sometimes presents like many other connective
tissue disorders and especially affects the joints, heart, and skin.
Neurological Effects of Borreliosis
One of the most devastating manifestations of late-stage or chronic Lyme disease is
Lyme neuroborreliosis: when ​Borrelia ​cross the blood-brain barrier and begin to cause
degenerative effects on the nervous system. Symptoms can include meningoradiculitis
(inflammation of the meninges and nerve roots) and lancinating (piercing or stabbing
sensations), radicular pain (pain radiating into lower extremities along nerve roots),
lymphocytic meningitis, and forms of cranial and peripheral neuritis (inflammation of nerves
causing pain and loss of function) (Rupprecht et al., 2008).
We are just beginning to understand the pathogenesis of this particularly terrifying form
of borreliosis. There are several theories as to how ​Borrelia​ is able to cross the blood-brain
barrier, considering its abilities to both survive the bloodstream and invade the extracellular
matrix and connective tissues. It may travel directly along nerves via connective tissues, or it
may travel through the bloodstream and latch onto the endothelial layer of the cerebral or spinal
vessels. It seems unlikely that it would be able to travel along nerves effectively, however that
could explain some of the symptoms of Lyme neuroborreliosis such as meningoradiculitis. It is
still debated whether Borrelia passes between or through endothelial cells, but what is certain is
that it is able to cross the blood-brain barrier and can be found in the cerebrospinal fluid of
patients with disseminated Lyme disease (Rupprecht et al., 2008).
Interestingly, the differences seen in the symptoms of Lyme disease between the US and
Europe could be due to the differences between ​B.burgdorferi​ and ​B.garinii ​in dissemination
technique. In the US, symptoms such as dispersed erythema migrans and meningitis tend to
suggest dissemination via blood vessels (hematogenous dissemination). Conversely, symptoms
seen more frequently in Europe such as meningoradiculitis where symptoms originate near the
site of the tick bite and spread from there suggest microbial migration along peripheral nerves to
the nerve roots or through lymphatic vessels. Therefore the differences seen in symptoms and
proposed mechanisms of pathogenesis between the US and Europe could simply be due to the
individual adaptations of local ​Borrelia​ species (Rupprecht et al., 2008).
20

22. Figure 5:​ “​The inflammatory B-cell response in the CSF in response to the CNS infection.
Borrelia are recognized by monocytic cells (1), which produce the B-cell–attracting chemokine CXCL13
(2). B cells immigrate into the CSF (3) and mature to plasma cells (4). These plasma cells can produce
B.b.-specific antibodies (5) that can eventually destroy the invaded spirochetes (6).” Rupprecht et al.,
2008.
Once the ​Borrelia​ enters the central nervous system, it is faced with local nonspecific
immune cells such as monocytes, macrophages, and dendrites. These cells are are likely to
produce high amounts of inflammatory mediators such as IL-6, IL-8, IL-12, IL-18, and IFN-𝛾 as
well as produce chemokines to recruit the specific immune response. While there are over 50
different possible types of chemokines, Lyme neuroborreliosis appears to recruit B-cells more
than any other infection of the central nervous system. This indicates that the chemokines
involved are probably the few specific chemokines capable of recruiting B-cells in large
quantities: CCL19, CCL21, CXCL12, and CXCL13. CXCL13 have been found in high
concentrations in the cerebrospinal fluid of patients with Lyme neuroborreliosis, suggesting
active B-cell recruitment to the cerebrospinal fluid. B-cells are the primary producers of
antibodies in the body, and once they have learned an antigen (like a borrelial surface protein)
and matured into plasma cells they can produce mass amounts of antibody specific to that
antigen (Rupprecht et al., 2008).
Despite being downregulated before the spirochetes even enter the body, it appears that
OspA is the major antigen dealt with in the cerebrospinal fluid and is expressed there but not in
blood serum. OspA is very useful to the cell for adhesion, and in this case adhesion to neurons
and endothelium. Additionally, it appears that CD8​+​
T cells are also recruited via other
chemokines such as CCL4, CCL5, CXCL10, and CXCL11. CD8​+​
“killer”​​
T cells are other highly
specialized immune cells whose primary role is to kill cells that have become infected or
cancerous (Rupprecht et al., 2008).
21

23. Figure 6:​ ​“The neural dysfunction in neuroborreliosis. Three principal mechanisms that lead to
the injury of neuronal cells: (1) the secretion of cytotoxic substances by leucocytes and glial cells, (2) direct
cytotoxicity, and (3) autoimmune-triggered processes through molecular mimicry.” Rupprecht et al.,
2008.
Knowing all of this, how does borreliosis actually harm the nervous system to produce
symptoms? There are three main methods: indirect cytotoxicity, direct cytotoxicity, and
molecular mimicry (Figure 6). Through research in mouse models, it has been shown that
Borrelia can attach directly to neurons and glial cells, probably through OspA. OspA has been
shown to cause apoptosis and astrogliosis (increase in astrocytes resulting from the destruction
of neurons). These are considered direct cytotoxic effects. ​Borrelia​ could also damage neurons
indirectly through inducing cells in the nervous system to produce damaging chemicals. In
rhesus monkeys, Schwann cells have been observed producing high levels of Nitrous Oxide (NO)
in response to contact with ​Borrelia​. Studies of rat brain cells cultured alongside ​B. burgdorferi
have also shown high levels of NO. Macrophages have been shown to produce quinolonic acid
(which can be neurotoxic) in response to ​Borrelia​. Lastly, ​Borrelia​ can induce the production of
inflammatory mediators such as IL-6 or TNF-α in glial cells, and this induction of inflammation
could lead to autoimmunity. It is also possible that the immune response (particularly the
overpopulation of B-cells) to ​Borrelia​ could produce antibodies that cross-react to self antigens,
also producing an autoimmune response. This same mechanism could also account for why the
Osp-A vaccine was purported to produce an autoimmune response: OspA antibodies may also
recognize some self antigens in the host, causing immune cells to attack a patient’s own tissues
(Rupprecht et al., 2008).
Ultimately, there are not many certainties about how Lyme disease produces such
devastating effects on the nervous system when it manages to cross the blood-brain barrier, but
there are a lot of potential mechanisms. It appears to generally wreak havoc on the immune
system by spreading aggressively, damaging cells, over-recruiting immune cells, and co-opting
immune functions to further damage the host. These mechanisms could account for the varied
22

24. neurodegenerative, inflammatory, and autoimmune symptoms seen in patients with late-stage
or chronic Lyme disease.
Biofilms: A Method of Persistent Infection
One of the major current theories for how ​Borrelia​ is able to evade the immune system is
through biofilms. Biofilms are complex aggregates of bacteria that have a number of
mechanisms and structures to help large numbers of bacteria live in an efficient, compact, and
nearly undetectable manner (Sort of like a secret refuge city). Biofilms are involved in almost all
infections in humans, especially infections acquired from surgery, thereby making those
infections difficult to diagnose and treat (Wu et al. 2015). Biofilm formation occurs in several
stages, with just a small cluster of cells sticking together and attaching to a surface at first,
followed by an increasingly complex arrangement of cells held together by the self-produced
extracellular polymeric matrix. The bacterial colony uses quorum-sensing to synchronize genetic
expression in cells, making their organized structure possible. Eventually the biofilm can
disperse and produce planktonic (in their free-moving single forms) cells. Biofilms are
significantly more antibiotic-and-immune resistant than planktonic cells. This has been
determined through a variety of techniques which have not been well-characterized in Borrelia,
but have been characterized in microbes such as ​Pseudomonas aeruginosa​. Through research
on these biofilms, it has been proposed that their resistance comes from: the architecture of the
biofilm itself and the metabolic conditions it creates (ex. low oxygen), the ability to mutate more
rapidly within the biofilm, quorum-sensing techniques, protection by the extracellular polymeric
matrix itself, the ability to produce antibiotic-disabling enzymes ​en masse​, and the simple fact
that such a high density of cells would require antibiotic concentrations that would cause major
toxicity in a patient (Hoiby et al. 2010).
Most of the research on borreliosis has been under the assumption that the bacteria
remain planktonic while causing infection in mammals. Researchers realized early on that
Borrelia had multiple morphological forms, primarily planktonic, cystic, and aggregate, but the
link between Borrelial biofilms and chronic lyme disease appears to be more recent (Kurtti et al.
1987; Brorson & Brorson, 1998). However, new research has shown that ​Borrelia​ does readily
form biofilms. Sapi et al. (2012) designed a trial that involved plating ​B. burgdorferi​ on a wide
variety of substrates and letting it incubate stationarily to develop biofilms. They noticed that ​B.
burgdorferi​ were able to form biofilms rapidly (Figure 5) on every surface they presented as well
as form floating biofilms. A stress trigger prompted bacteria to form a biofilm, such as
temperature change or a chemical threat. They found that Borrelial biofilms had the same kind
of polymeric extracellular matrix with eDNA that can be expected from other biofilms.
23

25. Figure 7: ​B.burgdorferi ​biofilm formation observed using dark field microscopy
from Sapi et al. 2012.
Sapi et al. (2016) made huge strides again when they showed the presence of ​Borrelia
biofilms in human tissues, using methods similar to those in their earlier paper, including
immunohistochemical staining, fluorescent​ in situ​ hybridization, and PCR analysis. They used
archived samples from ​Borrelia​ lymphocytomas, one of the rarer but debilitating symptoms of
Lyme disease where lymph nodes swell and become painful due to the infection. This is the first
evidence of ​Borrelia burgdorferi​ biofilms ​in vivo​, and is therefore a key link to the discovery of
the full workings of chronic Lyme disease.
This brings a new dimension to our consideration of ​Borrelia​’s immune evasion
techniques. If ​Borrelia​ forms biofilms within the human body, then that could explain why
many patients report their illnesses reoccuring some time after antibiotic treatment. The
antibiotics could stress the bacteria such that they are stimulated to form biofilms, which can
then “hide out” in body tissues unscathed by the immune system or further antibiotics. The
biofilm can then mature and begin proliferating planktonic bacteria when conditions become
favorable. This would produce what would appear to be a new infection, with symptoms
resuming. This may be part of why many patients who consider themselves to have chronic
Lyme will be diagnosed with a new infection of Lyme disease, even though they do not
remember a recent tick bite. I believe this lack of consideration of the biofilm is much of why
chronic borreliosis has been dismissed for so long. Because we know that doxycycline is typically
effective against planktonic ​Borrelia​, but until recently it had not been tested against ​Borrelia
biofilms.
Goc et al. (2015) has been one of the first (if not the first) papers to really look at
Borrelia​’s resistance to a variety of antimicrobials in its biofilm form. They tested a wide variety
of natural substances that have been suggested for the treatment of Lyme disease, and most of
them showed no significant effect. They did have moderate success with baicalein, monolaurin,
luteolin, cis-2-decenoic acid, and kelp compared to doxycycline (Figure 8). While these
compounds are not the focus of my research, this paper laid the groundwork for the
investigation of alternative treatments of Lyme disease using the biofilm model.
24

26. Figure 8: ​ Anti-biofilm actions of baicalein, monolaurin, luteolin, cis-2-decenoic acid, and kelp
against doxycycline. From Goc et al. 2015.
25

27. Chapter 3: What Medical Treatments Are There For Lyme
Disease?
Testing and Diagnosis
While erythema migrans and/or a remembered tick bite in an endemic area remain the
most commonly relied upon diagnostic criteria for early Lyme disease, there are laboratory tests
that can be used to confirm an infection once an immune response has been established. The
most commonly used diagnostic test for Lyme disease is with an enzyme-linked immunosorbent
assay (ELISA). The ELISA tests for antibodies to ​B. burgdorferi​ in either blood serum or the
cerebrospinal fluid (Mayo Clinic Staff, 2016). Blood serum is by far the most common, but
cerebrospinal fluid may be tested in more serious disseminated cases or outside of New
England, due to the actions of ​Borrelia​ in the CSF discussed in Chapter 2 (Schwartz et al. 1989).
However, ELISA tests can give false-positives, due to a previous infection that built up an
antibody response, or cross-reactive proteins. The test could also pick up on antibodies to other
related spirochetes like syphilis or some oral bacteria (Barbour, 2015).
Western blot tests may also be used to confirm a diagnosis of Lyme disease. Western
blots can test for antibodies to several ​B.burgdorferi​ outer surface protein antibodies (Mayo
Clinic Staff, 2016). It is a much more specific test, as it can identify which parts of B.burgdorferi
a patient may have developed antibodies for. However, its specificity means that it is more
time-consuming and therefore expensive to perform. For that reason it is generally only used
when ELISA testing is inconclusive or other clinical factors don’t necessarily indicate a Lyme
disease infection.
There are also more direct ways of testing for the organism itself, including culturing,
microscopy, and PCR techniques. These can be useful when trying to determine whether a
treatment has been effective, since spirochetes may no longer be present but antibodies will
remain after infection. Direct culturing involves growing the organism in the laboratory from a
sample from the patient - either a skin biopsy from the erythema migrans rash or whole blood in
a disseminated case. Polymerase Chain Reaction (PCR) can be used to test for pieces of the
microbes in the skin or blood, by amplifying (creating many copies of) any ​Borrelia​-specific
genes found in the sample. Microscopy can also be used to visualize spirochetes in a sample
using stains that can color the microbes. These are all highly specific but again, not often used
(and indeed not approved in a general Lyme protocol) due to time and money.
Of course, these thests are only specific for B. burgdorferi, not any of the other strains of
Lyme disease-causing ​Borrelia​ such as ​B. garinii ​or ​B. afzelli​. This is fine for most cases
originating within the US, but what if a patient was bitten by a tick years ago while traveling in
Europe, but is just now seeking treatment for symptoms home in the US? Even if their doctor
suspected Lyme disease, they might not think to ask about travel from years ago or test for other
26

28. strains of ​Borrelia​. The above tests are not particularly good for what the are meant to do,
either. The tests available to clinicians lack specificity and reliability. Many doctors and
researchers have devoted their careers to “Lyme awareness” and are calling for the CDC and
IDSA to more thoroughly consider the problem of Lyme disease testing and open the possibility
of developing new tests rather than denying all claims made by the “Lyme awareness” camp
(Phillips et al. 2006, Woodcock 2006).
Even if there are better tests developed, would it be possible to test for biofilms on a
system-wide level when it isn’t known where biofilms might be hiding out in tissues? Could
affected tissues be biopsied and stained for biofilm markers? How could they be distinguished
from other forms of biofilms? And even then, how to treat a chronic Lyme disease infection due
to biofilms?
Treatments
Although the mainstream medical community has not yet accepted a model of borreliosis
that accounts for persistent infection and a broader range of possible routes of infection and
pathologies, alternative healthcare practitioners and disillusioned doctors have been working to
develop treatment protocols for patients ineffectively treated by the medical industry’s current
Lyme Disease model. These practitioners are required to look beyond antibiotics and consider
the nuances of the realities of borreliosis - taking into consideration its immune evasion tactics
including biofilm forms, as well as holistically treating the wide range of complex symptoms that
come along with epidemic borreliosis infections that fall outside of the CDC model. In this
section I will talk about how a couple of practitioners have approached this in their practices.
Antioxidants and Thomas Levy
Thomas Levy is well-known for his promotion of high-dose vitamin C for almost
everything - or at least, all diseases that are caused by oxidative stress, which he reports
accounts for much of the diseases that we deal with today. These include: cancer,
atherosclerosis, autoimmune and infectious diseases, etc. Although he does not discuss Lyme
Disease specifically in his book, Levy cohesively describes the history and use of vitamin C to
prevent and treat disease in Primal Panacea (2011). He makes extensive and fantastic claims
about the extent to which this treatment can be useful, but seems to back it up with
well-rationalized and broad scientific base. In fact, he cites over 1,250 sources throughout the
book, most of them peer-reviewed articles.
Figure 9: Ascorbic acid molecular structure and free radical reaction.
27

29. Levy’s claims may be dramatic, but the molecular and clinical biology supports his
theory. Vitamin C, also known as ascorbic acid, is uniquely good at “scavenging” free radicals.
Denisov and Afanas' ev (2005) describe that as an acid, ascorbic acid readily loses protons and is
able to stabilize the resulting negative charge across its ketone and alcohol groups (Figure 9),
making the reaction favorable. Because of this it is readily able to donate protons to “free
radicals” - compounds with an unpaired electron in their outer shell - and balance that radical
electron like it does negative charge. Scandalios (2007) reports that ascorbic acid is also able to
regenerate itself via other biochemical processes within the cell so that it can go on to scavenge
more free radicals. These properties make ascorbic acid a very efficient and useful antioxidant.
Valko et al. (2006) describes the role that antioxidants and free radicals play in normal human
health and disease states. They state that free radicals are produced by a variety of normal
cellular processes, and they play vital roles in some parts of signaling and even immune
responses. However, too many of them in the wrong places can cause serious damage. Pratt and
Cornely (2013) report the biochemical basis for the fact that free radicals cause chain reactions
that can lead to DNA and other cellular damage. This is known to cause a huge range of health
problems including cancer, cardiovascular disease, hypertension, neurodegenerative diseases,
rheumatoid arthritis, and even aging.
The destructive mechanisms of borreliosis are not well-known, but Pancewicz et al.
(2001) and others state that free radicals and antioxidants definitely play a crucial role in how
the bacteria cause disease. Primarily, ​Borrelia​ seems to be activated by free radicals and
produces free radicals. Garcia-Monco and Benach (1997) find that this causes a massive immune
response, which creates tissue-damaging inflammation, and accounts for many borreliosis
symptoms such as erythema migrans and arthritis. However, the immune response’s primary
way of attempting to kill the bacteria is through the use of localized free radicals, and therefore
small doses of vitamin C have been both shown to suppress this immune response on a
molecular level, as seen by Goldschmidt (1991) and stimulate it on a cellular level, as seen by Li
and Lovell (1985), and Leibovitz and Siegel (1977). Researchers such as Miller (1969) have also
investigated potential direct bactericidal mechanisms of ascorbic acid. So the idea is that if we
can utterly overwhelm the system with ascorbic acid, it is able to quench these free radical chain
reactions from the ​Borrelia​, but it won’t be counter productive by intercepting the useful
bactericidal immune response, because ascorbic acid is also potentially capable of killing the
Borrelia​ on its own. A brand new study from Goc et al. (2015) finds that ascorbic acid is an
effective bactericidal agent against ​B.burgdorferi​ and ​B.garinii​ spirochetes as compared to
doxycycline and other plant-derived compounds, but found no susceptibility against latent
forms of ​Borrelia​. My research is in the same vein: testing the bactericidal effects of ascorbic
acid on ​Borrelia burgdorferi​ spirochetes and biofilms. There is still a lot to be researched on
these mechanisms, but at the end of it all we might finally find a treatment that works for all
forms of borreliosis.
Herbalism and Stephen Harrod Buhner
Herbalism as it exists in the United States today primarily helps those who the
biomedical institutions and industry have failed. It picks up slack where mainstream medicine is
28

30. unable to fulfill a need in people’s medical experiences, and therefore often needs to develop its
own protocols without the assistance of much medical science. Because usually the science, for
one reason or another, just isn’t there.
Stephen Harrod Buhner is a prominent author in Natural Medicine, having many
popular titles under his belt such as ​The Secret Language of Plants​, which many herbalists will
refer to regularly for an understanding of plant medicine that most of the scientific community
just doesn’t address. Plants are more than just their “primary” or “active” chemical constituents.
They are living systems, every bit as varied and mysterious as any other organism. So why does
the biomedical community act as if it can “prove” or “disprove” a plant medicine by extracting its
active constituent and performing double-blind randomized placebo-controlled clinical trials?
They aren’t disproving herbal medicine if they aren’t actually using it. That being said,
laboratory research can give us crucial information in trying to understand ​why​ these medicines
might be working.
In ​Healing Lyme​, Buhner (2005) discusses his experiences with and perspectives on
what he calls “The Lyme Wars” - many of the controversies I have discussed, and more. He then
details his protocol for treating patients that have not received adequate care from mainstream
physicians. His core protocol contains: ​Andrographis paniculata​, Japanese Knotweed
(resveratrol, ​Polygonum cuspidatum​), Cat’s Claw (​Uncaria tomentosa​), and the optional
Astragalus and Smilax (sarsaparilla). These herbs are discussed individually in detail for their
uses, history, role in Lyme disease, chemistry, etc. They are, supposedly, ​both antispirochetal
and bolstering to the immune system​. I believe that this particular concert of effects is what may
make them a better treatment option than antibiotics in many cases. Despite being a much less
studied topic than vitamin C therapies, ​Healing Lyme​ still contains many references to primary
literature. Buhner has published a book titled ​Herbal Antibiotics​ (2012), which covers some of
the herbs used in his protocols in greater depth. I will evaluate some of this research as a
representative example.
Example: Andrographis
The first and core herb in the protocol is andrographis, ​Andrographis paniculatum​.
Buhner (2005) describes the primary mechanisms of andrographis as immunostimulant,
antibacterial, antiinflammatory, and analgesic (pain-relieving), among other things. He justifies
its use in the protocol with descriptions of its antispirochetal, nervous calming and protecting,
and liver protecting and enhancing. He emphasizes its ability to readily cross the blood-brain
barrier. It is also notable that it is supposedly clinically effective for a variety of neglected
tropical diseases. A cursory survey of the bibliography indicates that there may be good evidence
for the actions of andrographis and its “active” compound andrographolide against the neglected
tropical diseases as found by Dutta and Sakul (1985), some bacteria, as an antiinflammatory
agent as seen by Balu and Alagesaboopathi (1993), as an active agent against free radical damage
in the liver as found by Koul and Kapil (1994), and as an immunostimulant as shown by Puri et
al. (1993). It seems that most of the logic of its use in treating borreliosis must be inferred from
these actions. Which is not unreasonable, as ​Borrelia​ does behave much like a parasite in many
ways, such as its immune evasion techniques. And of course, immune stimulation against other
bacteria, parasites, and viruses can somewhat reasonably be assumed to be helpful against
29

31. Borrelia​ as well. Overall, this research and logic holds up reasonably well, but there is still a sore
lack of dedicated research on alternative and especially herbal treatments for borreliosis.
30

32. Chapter 4:
What Should Be Done About Lyme Disease in The Future?
There is still a long way to go until the Lyme disease epidemic can be eradicated. There
needs to be better institutional and governmental support for and openness towards other
models of approaching borreliosis. When the CDC and IDSA can be open to new ideas regarding
pathogenesis and treatment, it will open up a world of possibilities for research collaboration
between the researchers that have been working on Lyme disease since the very beginning, and
researchers that are bringing new ideas to the field.
There are some major questions that this research would need to tackle:
1. Is borreliosis solely a tick-borne zoonotic disease, or can it be transferred in other ways,
such as congenitally and sexually?
2. Why did the Lyme vaccine cause arthritic symptoms?
3. How can we both expand and narrow down our Lyme disease models to fit both endemic
and non-endemic regions? How might diagnostic and treatment protocols vary between
these areas?
4. What diagnostic tools can be used to accurately and quickly diagnose an early stage
borreliosis infection?
5. What diagnostic tools can be used to accurately diagnose an active late-stage borreliosis
infection that does not rely on antibody tests, which cannot distinguish between a
previously cleared infection and an active one?
6. What are all of the possible effects and symptoms of borreliosis? How many organ
systems does it effect and in what way?
7. To what extent is persistent borreliosis infection due to biofilm formation, or other forms
of immune evasion?
8. Does borreliosis cause autoimmune “echoes” after infection that may persist in causing
symptoms?
9. What are the properties of ​Borrelia​ biofilms and how might they be targeted effectively?
10. What have practitioners experienced in the treatment of borreliosis? What treatment
have they found effective? Are they effective in a laboratory setting?
11. Given this information, can an effective and flexible treatment protocol be developed?
12. Is an effective and safe borreliosis vaccine possible?
My Research Protocol
Based on my initial research of the literature in Fall of 2015, which has been expanded
upon in this thesis, I developed a research plan to address a piece of what I believe needs to be
done to find an effective treatment for chronic Lyme disease. Inspired by the claims of Stephen
Buhner and Thomas Levy that ascorbic acid and resveratrol may be effective treatments for
31

33. chronic Lyme, I sought to investigate whether they might have a direct effect on ​B. burgdorferi
biofilms. It is also possible that these chemicals may work through a more complex mechanism
involving the immune system.
My research seeks to provide a link between ​B. burgdorferi​ biofilm research and
potential alternative treatments for chronic Lyme disease. Based primarily on research done by
Sapi et al. (2012, 2015) on ​B. burgdorferi​ biofilms and resistance to antibiotics and Goc et al.
(2015) on alternative phytochemical treatments for Lyme, I developed a protocol to test ​B.
burgdorferi​ biofilm resistance to antioxidants such as ascorbic acid and resveratrol using
doxycycline as a control.
My hypothesis was that antioxidants in high concentration might have the ability to
break down the unique protective layer of biofilms and open the biofilm up to attack by both the
immune system and antimicrobial agents including the antioxidants themselves. Since
antioxidants are generally safe for humans at high concentrations and may have a beneficial
effect of the immune system, they would be a better choice than drugs that may have negative
effects in high doses and have limited effectiveness against biofilms, such as doxycycline.
This protocol was based on the Minimum Biofilm Eradication Concentration assay
(MBEC). This assay first forms biofilms on pegs with coating specifically designed for optimum
biofilm growth, and then includes a number of protocols for quantifying various challenge
(antimicrobial) concentrations to biofilm growth. The MBEC assay is designed to quantitatively
determine at what concentrations the challenges inhibit growth, kill cells, and eradicate
biofilms. My protocol uses ascorbic acid, resveratrol, doxycycline, and pH-matched media as
challenges. It also utilized the LIVE/DEAD assay to test for biofilm death in response to these
challenges.
The second phase of the protocol was to evaluate microscopically the healthy and
challenged biofilms in large format, staining for biofilm characteristics such as calcium
complexes (using Alizarin Red) and extracellular DNA (using the red fluorophore DDAO). I was
going to use immunofluorescence to confirm the identity of the constituent microbes as ​B.
burgdorferi​. Finally, I was going to use confocal microscopy to better document the effects of
the antioxidants on biofilm structure.
32

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