lyme disease

December 23, 2002

The SNR archives has a paper on Lymes disease, i think it is by Val

Brown when he was doing the " Five Phase Model " .

At 8:58 AM -0500 12/23/02, Van Deusen wrote:

>,

>

>If you do a search on Google for Lyme disease and neurofeedback or

>biofeedback, you should find at least one article which I have seen several

>times in which NF was used successfully. Can't recall if it was a group or

>case study, and I don't have the link.

>

>Pete

> Re: Lyme disease

>

> Has NF ever been used for alleviating symptoms of Lyme disease? My

>brother

> has advanced Lyme and recently had a spect scan, as some areas of his

>brain

> have been affected. I will be seeing him over the holidays and would love

>to

> know if NF would offer relief. Some of his symptoms include: insomnia,

> chronic fatigue, brain fog and mental confusion at times, joint pain,

>muscle

> stiffness.

> Thanks,

> D

>

December 23, 2002

Hi

http://www.mindfitness.com/brown.htm

Here is the Lyme disease article

Re: Lyme disease

>

> Has NF ever been used for alleviating symptoms of Lyme disease? My

> brother

> has advanced Lyme and recently had a spect scan, as some areas of his

> brain

> have been affected. I will be seeing him over the holidays and would

love

> to

> know if NF would offer relief. Some of his symptoms include: insomnia,

> chronic fatigue, brain fog and mental confusion at times, joint pain,

> muscle

> stiffness.

> Thanks,

> D

>

January 21, 2003

In a message dated 1/21/2003 11:54:55 PM Eastern Standard Time, AngelBear1129 writes:

Lyme disease is the most common tick-borne disease in the United States, with approximately 16,000 new cases reported each year. The disorder was first identified in 1975 when a group of children in Lyme, Connecticut, experienced mysterious arthritis-like symptoms. The deer tick carrying the bacterium B. burgdorferi is responsible for the spread of the disease in the United States. Cases have been reported in nearly all states, and the disease is also on the rise in large areas of Asia and Europe.

Signs and Symptoms

Lyme disease is accompanied by the following signs and symptoms:

Red rash that appears within a few weeks of a tick bite, initially as a small red spot at the site of the bite. The spot expands over time, forming a circle or oval and sometimes resembling a bull's eye. The rash can range in size from that of a dime to the entire width of a person's back. As the infection spreads, rashes can appear at different places on the body.

Flu-like symptomsâ€”fever, headache, stiff neck, body aches, and fatigue.

Arthritisâ€”60 percent of people not treated with antibiotics develop recurring attacks of arthritis, most commonly in the knees, that last a few days to a few months. About 10 to 20 percent of untreated individuals will develop ongoing arthritis.

Neurological symptomsâ€”stiff neck and severe headache (may indicate meningitis), temporary paralysis of muscles in the face (Bell's palsy), numbness, pain or weakness in the limbs, or poor motor coordination. Symptoms can develop weeks, months, or even years following an untreated infection, and can last for weeks or months. Symptoms usually resolve completely, but they may recur.

Heart problemsâ€”heart abnormalities such as palpitations, lightheadedness, fainting, chest pain, and shortness of breath are uncommon. Symptoms may appear several weeks after infection and last a few days or weeks.

What Causes It?

Ixodes ticks carrying the bacterium B. burgdorferi bite people. The bacteria enter the skin at the site of the bite, after the infected tick has been in place 36 to 48 hours. Symptoms are primarily due to the body's response to this invasion.

Who's Most At Risk?

The following factors increase the risk for developing Lyme disease.

Environment: exposure to heavily wooded areas

Season: infection is most likely during the summer and fall

Age: most common in children and young adults

Location: 90 percent of cases occur in the coastal northeast, as well as in Wisconsin, Minnesota, California, and Oregon

Lyme disease is the most common tick-borne disease in the United States, with approximately 16,000 new cases reported each year. The disorder was first identified in 1975 when a group of children in Lyme, Connecticut, experienced mysterious arthritis-like symptoms. The deer tick carrying the bacterium B. burgdorferi is responsible for the spread of the disease in the United States. Cases have been reported in nearly all states, and the disease is also on the rise in large areas of Asia and Europe.

Signs and Symptoms

Lyme disease is accompanied by the following signs and symptoms:

Red rash that appears within a few weeks of a tick bite, initially as a small red spot at the site of the bite. The spot expands over time, forming a circle or oval and sometimes resembling a bull's eye. The rash can range in size from that of a dime to the entire width of a person's back. As the infection spreads, rashes can appear at different places on the body.

Flu-like symptomsâ€”fever, headache, stiff neck, body aches, and fatigue.

Arthritisâ€”60 percent of people not treated with antibiotics develop recurring attacks of arthritis, most commonly in the knees, that last a few days to a few months. About 10 to 20 percent of untreated individuals will develop ongoing arthritis.

Neurological symptomsâ€”stiff neck and severe headache (may indicate meningitis), temporary paralysis of muscles in the face (Bell's palsy), numbness, pain or weakness in the limbs, or poor motor coordination. Symptoms can develop weeks, months, or even years following an untreated infection, and can last for weeks or months. Symptoms usually resolve completely, but they may recur.

Heart problemsâ€”heart abnormalities such as palpitations, lightheadedness, fainting, chest pain, and shortness of breath are uncommon. Symptoms may appear several weeks after infection and last a few days or weeks.Â

What Causes It?

Ixodes ticks carrying the bacterium B. burgdorferi bite people. The bacteria enter the skin at the site of the bite, after the infected tick has been in place 36 to 48 hours. Symptoms are primarily due to the body's response to this invasion.

Who's Most At Risk?

The following factors increase the risk for developing Lyme disease.

Environment: exposure to heavily wooded areas

Season: infection is most likely during the summer and fall

Age: most common in children and young adults

Location: 90 percent of cases occur in the coastal northeast, as well as in Wisconsin, Minnesota, California, and Oregon

October 13, 2003

>

> After 20 years of " CFS " , last Dec. it was discovered by DNA that I

have

> Lyme. >

> Beck Spelce

Hi Beck,

What lab tested you for Lyme DNA? Also, What have you heard about

Sameneto , an herb that fights Lyme ?

Al

January 6, 2004

JAson wrote:

I did their 3 day

LDA/PCR with two abx to provoke dead spirochetes to end up in bladder to be

collected in the urine samples and yield a pos. I still got negative. I only

regret having used a rife machine a few weeks before I did that test though,

as

who knows if I may have killed off spiros so that none were around to give a

pos

on the test.

Adrienne:

I don't know if you have Lyme or not. I know you got benefit from Rife. But

not enough so that you were so well you needed no further testing.

So, if you were in fact still sick, you could not (?) imagine that you had

killed all the spiros. I mean, if you had Lyme and then killed it, you would be

all well? Or do you think it is possible to just kill off enough to escape pcr

detection?

Were you being rifed specifically for Lyme? I don't recall.

Adrienne

March 14, 2005

Dear Ken,

Yes, ozone will take care of it.

We have a South African distributor.

I will back channel his info to you.

Best of Health!

Dr. Saul Pressman

HBOT vs Ozone

>

> Saul,

> I must admit my opinion of your scientific knowledge of fact just

> dropped a few points. It is obvious your knowledge of the subject of

> hbot is deeply lacking or else your desire to sell ozone equipment is

> overiding common sense and good judgement. These statements do not even

> warrant a good debate.

> Hartsoe

>

> Saul Pressman wrote:

>

> >Dear Jill,

> >

> >In each of these conditions ozone therapy would be much superior, with

> >fewer problems of application, and far lower cost.

> >

> >For stroke or heart attack, it could be applied in the ambulance,

> >avoiding any loss of time in transport to the hospital chamber and then

>

> >a wait for the red tape to be cut.

> >

> >For viral and bacterial infections, ozone therapy is also far superior

> >to hyperbaric oxygen.

> >

> >Best of Health!

> >Dr. Saul Pressman

> >

> >----- Original Message -----

> >From: " jill1313 " <jill1313@...>

> ><oxyplus >

> >Sent: Sunday, March 06, 2005 7:43 AM

> >

> >... I'd like stroke victims when rushed to the hospital to be put in

> >the chamber in the hospital (many have them), but it's not done. I

> >believe a few sessions of hbot after a stroke could minimize damage

> >because you would prevent spread of injury into the penumbra

> >surrounding the damaged tissue. I'd like to see it for heart attacks

> >too. I'd like to see it in hospital acquired bacterial infections where

>

> >people are put on lots of IV abx -- maybe adding in hyperbaric could

> >lessen the amount of abx required.

>

> OxyPLUS is an unmoderated e-ring dealing with oxidative therapies, and

other

> alternative self-help subjects.

>

> THERE IS NO MEDICAL ADVICE HERE!

>

> This list is the 1st Amendment in action. The things you will find here

are

> for information and research purposes only. We are people sharing

> information we believe in. If you act on ideas found here, you do so at

your

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ability to

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>

March 14, 2005

I think there are various borrelia species (like lyme) in ticks

around the world, and relapsing fevers tend to be spirochetes and

thus similar. But I think there are also viral tick fevers as well.

And here in U.S. ticks carry babesia (malarial type parasite) and

erlichia and others stuff. So the pathogen is more important than the

vector (ie tick)

>

> > I just spoke to a friend in South Africa and her brother has been

very

> > ill with suspected " Tick bite fever " . How does this differ from

Lyme

> > disease (If it does). I realize that there are a large variety

of

> > ticks in the world but are all the diseases they cause similar to

lyme?

> > Any suggestions for him to start doing would be appreciated?

> > I am suggesting ozone therapy to him but have no contacts in the

field

> > in South Africa. Any contacts also appreciated!

> > Best wishes and much love,

> >

> > <http://www.nod32.com>

>

March 14, 2005

Hi Ken. Check out http://www.lymephotos.com It shares a simple salt

and vit c solution. Could be tried without much expense.

Blessings

Donna

http://www.excellentthings.com

iri2 wrote:

> I just spoke to a friend in South Africa and her brother has been very

> ill with suspected " Tick bite fever " . How does this differ from Lyme

> disease (If it does). I realize that there are a large variety of

> ticks in the world but are all the diseases they cause similar to lyme?

> Any suggestions for him to start doing would be appreciated?

> I am suggesting ozone therapy to him but have no contacts in the field

> in South Africa. Any contacts also appreciated!

> Best wishes and much love,

>

> <http://www.nod32.com>

March 14, 2005

Thank you Jill.

What or other resources would you recommend for my friend to read

up about it and help him make decisions. Many thanks.

Best wishes and much love, Ken

Ken Gullan

Institute for Research Integration (IRI), San Diego, CA 92106-2424

IRI is a 501C(3) non-profit corporation established to help children with

developmental difficulties.

To contact me off-list use kengullan@... or call 619-222-1104

Re: Lyme disease

I think there are various borrelia species (like lyme) in ticks

around the world, and relapsing fevers tend to be spirochetes and

thus similar. But I think there are also viral tick fevers as well.

And here in U.S. ticks carry babesia (malarial type parasite) and

erlichia and others stuff. So the pathogen is more important than the

vector (ie tick)

>

> > I just spoke to a friend in South Africa and her brother has been

very

> > ill with suspected " Tick bite fever " . How does this differ from

Lyme

> > disease (If it does). I realize that there are a large variety

of

> > ticks in the world but are all the diseases they cause similar to

lyme?

> > Any suggestions for him to start doing would be appreciated?

> > I am suggesting ozone therapy to him but have no contacts in the

field

> > in South Africa. Any contacts also appreciated!

> > Best wishes and much love,

> >

> > <http://www.nod32.com>

>

March 15, 2005

What or other resources would you recommend for my friend to

read up about it and help him make decisions. Many thanks.

Best wishes and much love, Ken

Hi Ken:

I am not Jill but know of 2 groups that deal with Lyme.

Lyme-and-rife/

Lyme-and-rife

Don't know if it will help you but you can try and see.

Love and Light

Christel

March 15, 2005

I'm not sure they know much about it here but I believe there is a

European lyme disease group and you could post there. If you

put " lyme " into the search, you should come up with it. I'd

think the European group might know something.

> >

> > > I just spoke to a friend in South Africa and her brother has

been

> very

> > > ill with suspected " Tick bite fever " . How does this differ

from

> Lyme

> > > disease (If it does). I realize that there are a large

variety

> of

> > > ticks in the world but are all the diseases they cause

similar to

> lyme?

> > > Any suggestions for him to start doing would be appreciated?

> > > I am suggesting ozone therapy to him but have no contacts in

the

> field

> > > in South Africa. Any contacts also appreciated!

> > > Best wishes and much love,

> > >

> > > <http://www.nod32.com>

> >

September 10, 2005

, have you seen this research (below) on genetic exchange and

plasmid transfers in B. burdorferi?

The Lyme spirochete evolves " variations " within the infected person, so

that the person could have one " strain " in the joints, and a slightly

different strain in the brain, for example. Plus, B. burgdorferi is a

vector illness, a sexually transmittable illness, and can be transmitted

from human to human in other ways. See also the article by Harvey and

Salvato:

http://ilads.org/harvey.html published in Medical Hypotheses in Medical

Hypotheses, 2003, Vol 60: p 742–759

Also B. burgdorferi has 11 plasmids (used to exchange genetic information

from other microbes and produce toxins), more than any other bacteria.

Vickie

Proceedings of the National Academy of Science (PNAS), September 28, 2004

Genetic exchange and plasmid transfers in Borrelia burgdorferi sensu

stricto revealed by three-way genome comparisons and multilocus sequence

typing

http://www.pnas.org/cgi/content/abstract/0402745101v1

Abstract

The three-way genome comparison allowed distinction between polymorphisms

introduced by mutations and those introduced by recombination using the

method of phylogenetic partitioning. Tests for recombination suggested

that patches of high-density nucleotide polymorphisms on the chromosome

and plasmids arise by DNA exchange. The role of recombination as the main

mechanism driving B. burgdorferi diversification was confirmed by

multilocus sequence typing of 18 clinical isolates at 18 polymorphic

loci. A strong linkage between the multilocus sequence genotypes and the

major alleles of outer-surface protein C (ospC) suggested that balancing

selection at ospC is a dominant force maintaining B. burgdorferi

diversity in local populations.

We conclude that B. burgdorferi undergoes genome-wide genetic exchange,

including plasmid transfers, and previous reports of its clonality are

artifacts from the use of geographically and ecological isolated samples.

Frequent recombination implies a potential for rapid adaptive evolution

and a possible polygenic basis of B. burgdorferi pathogenicity.

Full article:

http://www.lymediseaseassociation.org/Achievements.html

Introduction

Comparative genomics of closely related species is a powerful method for

tracking microbial epidemics (1, 2), uncovering microbial virulence

factors (3–5), and annotating genes and other conserved elements in

genomes (6, 7). A powerful method of bacterial genotyping is multilocus

sequence typing (MLST), which is the comparative sequencing of selected

genes (5, 8–10). (Note that in this article by MLST we refer to the

comparative sequencing of multiple loci at large, not necessarily

housekeeping genes.) We have taken a comparative genomics approach to

identify polymorphic ORFs in the genomes of Borrelia burgdorferi sensu

stricto, which is the predominant bacterial species causing Lyme disease

in North America (11). Measuring rates of sequence evolution and

selective constraints (12, 13) is a means of uncovering virulence factors

and candidates for vaccine, diagnostics, and therapeutics.

Early population studies of B. burgdorferi using multilocus enzyme

electrophoresis (MLEE) (14) and DNA sequences (15) found little evidence

for genetic exchange among different isolates. In fact, one report (16)

concluded that B. burgdorferi was among the most clonal of bacterial

species. However, these studies were based on archival strains isolated

from several worldwide locations, and the conclusions on clonality

described above may have reflected a high degree of geographic

structuring of this obligate parasitic species (11, 17) rather than a

lack of genetic exchange within local populations (18). Indeed, analysis

of outer-surface protein C (ospC) genes from local B. burgdorferi

isolates in North America (19) and Europe (20) suggested extensive

recombination at this locus. Crossspecies gene transfer has been

suggested at other loci as well (21), and son and (22)

concluded that intracellular transfers of cp32 plasmids have occurred.

Key questions remain in regard to the rate, extent, and mechanism of

genetic exchanges among B. burgdorferi clones. At issue are questions of

whether there are loci other than ospC that show selectively maintained

and recombination-mediated variation and whether large (>1-kb) pieces of

DNA or whole plasmids can be transferred among clones (20). Such

questions may best be answered by surveying closely related B.

burgdorferi isolates on a genome-wide scale.

Here, we report the identification of highly polymorphic loci identified

by means of a comparison of three closely related genomes and their use

in the study of population structure of B. burgdorferi. We found that

incorporation of divergent alleles through homologous recombination is

the most likely cause of clusters of nucleotide polymorphisms in the B.

burgdorferi genome. This article also provides evidence that genomic

diversity in natural populations of B. burgdorferi is maintained by

balancing selection at ospC predominantly and, to a lesser extent, at a

limited number of other plasmid-borne loci. The discovery of genome-wide

genetic exchange among local B. burgdorferi clones suggests that new

adaptive features, such as its human virulence, could emerge quickly.

Materials and Methods

DNA Methods. We determined 8-fold coverage draft sequences of B.

burgdorferi JD1 and N40 genomes as described (23, 24). Genome assembly of

JD1 and N40 (in particular, the assembly of the highly paralogous cp32

plasmids) is not yet complete. Sequencing of selected genes (both

strands) from additional clinical isolates was performed by the Stony

Brook University Core DNA Sequencing Facility (see Supporting Materials

and Methods, which is published as supporting information on the PNAS web

site).

Identification of Orthologous ORF Pairs. We used NUCMER of the MUMMER

software package (25) to align the JD1 and N40 assemblies with the

finished B31 genome. JD1/N40 scaffolds that uniquely (1:1) match the B31

genome were identified as orthologous genome segments. JD1/N40 plasmids

were named by using the B31 nomenclature when plasmid orthology is

established. JD1/N40 scaffolds matching multiple B31 plasmids (such as

scaffolds homologous to the cp32 plasmids of B31), and scaffolds with no

matches were excluded in this study. For each pair of orthologous genome

segments, we identified nonoverlapping 250-bp ORFs on both genomes by

using GLIMMER (26). Syntenic ORFs in two orthologous genome segments are

considered " orthologous ORFs " (e.g., see Fig. 4, which is published as

supporting information on the PNAS web site).

MLST. We performed multilocus genomic typing (see Supporting Materials

and Methods for procedures) on an additional 18 North American B.

burgdorferi sensu stricto clinical isolates (Table 3, which is published

as supporting information on the PNAS web site) by using a selected

library of 18 polymorphic loci (loci and PCR primers are given in Table

4, which is published as supporting information on the PNAS web site)

identified by means of genome comparisons.

Tests of Recombination. We applied a modification (30) of s' test

(31) and Sawyer's test (32) to distinguish polymorphisms introduced by

recombination and those introduced by mutational substitutions. These

tests identify recombination based on significant clustering of

nucleotide substitutions in each of the three phylogenetic partitions

(see Results). For MLST analysis, multiple alignments of nucleotide

sequences were obtained for each locus according to the CLUSTALW (27)

alignments of translated amino acid sequences. Gene phylogeny at each

locus was estimated by using MRBAYES 2.1 (33), based on a nucleotide

evolution model with site-specific rate of evolution at each codon

position. A majority-rule consensus tree was obtained from the converged

chains. Posterior probabilities were obtained as measures of branch

support. We defined major-group alleles as major lineages on the gene

tree. Recombination events were identified by using the following

criteria. Recombination causes incongruent gene trees among loci (34).

Multiple nucleotide substitutions at one locus between two strains that

are identical in other loci are most likely results of lateral gene

transfer (35). For single-nucleotide differences, alleles unique among

strains are mutational substitutions, whereas alleles shared among

divergent clonal groups are introduced by recombination (35).

Data Availability. The B31 genome has been deposited in the GenBank

database [National Center for Biotechnology Information (NCBI) taxonomy

ID 224326]. Nucleotide sequences of MLST loci have been deposited in

GenBank under the accession nos. AY696304–AY696571. Alignments and

Bayesian trees of MLST sequences are available from the authors.

Results

Strain Choice and Genome Coverage. B. burgdorferi N40 is a tick isolate

obtained from Westchester County, NY (36), and JD1 is a nymphal tick

isolate obtained from Ipswich, MA (37). Together with the completed

genome of strain B31 (23, 24) [a tick isolate obtained from Shelter

Island, NY (38)], these three strains cover a wide range of

population-level genetic diversity. This diversity is based on ospC

typing [b31, JD1, and N40 belong to ospC major groups A, D, and E,

respectively (19)], on rDNA spacer typing [restriction fragment length

polymorphism types I, II, and III (39)], and on chromosomal pulsed-field

gel electrophoresis typing [MluI pulsed-field gel electrophoresis types

B, C, and E (40, 41)].

The draft genome sequences for N40 and JD1 are incomplete, so the

comparisons in this study cover 80% of chromosomal ORFs and approximately

one half of the plasmids in B31 (Tables 1 and 2). Plasmid profiles differ

among the genomes, and the assembly of JD1 and N40 plasmids (cp32s in

particular) is not yet complete. ORFs that are not in our comparison are

those that are missing from one of the sequences (because of either

genetic differences among the three strains or incompleteness of the

draft sequences) or have multiple NUCMER hits (e.g., multiple JD1/N40

matches to cp32 in B31). Nonetheless, the three-way comparison of

plasmidborne ORFs covered a high proportion of the two universally

present plasmids, lp54 (46 of 57 ORFs of 250 bp) and cp26 (21 of 26 long

ORFs).

Genome Divergence. Genomes of B31, N40, and JD1 are closely related

(Tables 1 and 2), and nearly all differences are single-nucleotide

polymorphisms (SNPs) with only two nucleotide states [there are a few

three-state polymorphisms and insertion/deletions (indels)]. The three

genomes are approximately equally distant from each other, with N40

slightly more diverged from B31 (0.77% overall nucleotide difference) and

JD1 (0.80%) than B31 and JD1 are diverged from each other (0.51%). We

categorized each SNP into one of the following three possible

phylogenetic partitions (31). Partition 1 SNPs are those with one state

shared by B31 and JD1, with N40 showing another state, designated as

[(B31, JD1), N40]; partition 2 SNPs are designated as [(B31, N40), JD1];

and partition 3 SNPs are designated as [(JD1, N40), B31]. As a result of

the low ( 0.5%) sequence divergence and the approximately equal distances

among the three genomes (essentially a " star phylogeny " ), partition 1–3

substitutions can be regarded conveniently as lineage-specific

substitutions in N40, JD1, and B31, respectively. The implicit assumption

is that at each SNP, the most common nucleotide state among the three

genomes is the ancestral one.

Plasmid ORFs appear to evolve, on average, 2- to 4-fold faster than the

average chromosomal ORFs, and the apparent accelerated evolution of the

N40 lineage is mainly due to substitutions on the plasmids (Tables 1 and

2). Selective constraint for amino acid replacement, as measured by the

Ka/Ks ratio, for the plasmid ORFs is 2-fold weaker than that for the

chromosomal ORFs (Tables 1 and 2). However, plasmid sequences are not

uniformly more variable or unconstrained than chromosomal ORFs. For

instance, most ORFs on the two universally present plasmids, lp54 and

cp26, are as conserved as chromosomal ORFs, except for BBA24 [encoding

decorin-binding protein (42)], BBA68 [encoding BbCRASP-1, (43)], BBA69,

BBA70, and BBB19 (ospC) (Tables 1 and 2).

Frequency distributions of sequence differences closely follow the

Poisson expectations (Fig. 5, which is published as supporting

information on the PNAS web site), suggesting that most nucleotide

substitutions are selectively neutral. ORFs containing HDNPs were

identified in a small proportion (n = 49, 6.9%) of the chromosomal ORFs

and a much larger proportion (n = 42, 28%) of the plasmid ORFs (Table 5,

which is published as supporting information on the PNAS web site). From

this HDNP list, we chose 11 of the most polymorphic ORFs and six

polymorphic plasmid ORFs found in an earlier study (W.-G.Q., J.F.B., and

B.J.L., unpublished data) as markers for genomic typing of clinical

isolates (described below). These most variable ORFs encompass one B31

chromosomal locus (BB0082) and 17 loci on nine linear and circular

plasmids.

Evidence for Recombination from Three-Way Genome Comparison. Nucleotide

differences of HDNPs among three pairs of genome comparisons are shown in

Fig. 1. The overall pattern is that most HDNPs show high variability in

only two of the three pairwise comparisons. This pattern indicates that

most nucleotide substitutions occurred during the evolution of only one

of the three possible lineages. For example, the most variable loci on

the main chromosome (BB0082, BB0218, BB0348, BB0550, and BB0684) varied

little between B31 and JD1. Likewise, BB0276 and BB0492 are unchanged

between B31 and N40. The high number of clustered nucleotide

substitutions at these HDNP loci, in conjunction with the mostly

single-lineage origin of these polymorphisms, strongly suggests that

most, if not all, of these HDNPs were introduced en masse by genetic

exchange rather than by multiple point mutations. (As examples, the

significant clustering of single-partition polymorphisms at BB0032, the

BB0082–BB0084 region, and BB0833 are shown in Fig. 6, which is published

as supporting information on the PNAS web site.) Indeed, tests of

recombination (31, 32) using three-way alignments of the HDNP loci show

significantly nonrandom runs of single-partition substitutions on 11

chromosomal and 5 plasmid ORFs, supporting the origin of these HDNPs by

recombination. However, such tests of recombination are known to be

conservative and to vastly underestimate the true magnitude of

recombination (44). A few ORFs showing hypervariability in all three

pairwise comparisons [prominent examples are BB0144, coding for a

putative glycine/betaine/L-proline ATP-binding cassette transporter, and

BBB19 (ospC)] are likely to be results of multiple recombination events

at the same loci.

Fig. 1 shows that the largest fraction of the HDNPs was introduced during

the evolution of the N40 lineage (high red and blue bars with a low green

bar). For instance, the HDNPs at the right end of lp54, encompassing

BBA68, BBA69, and BBA70, were apparently introduced by a single event of

recombination into N40. Likewise, part or all of cp26 was introduced by

one event into JD1, and another event converted the lp25 in JD1. The

consecutive runs of HDNPs sharing a single phylogenetic partition on

these plasmids (Fig. 1) suggest that whole-plasmid transfers between B.

burgdorferi strains may well be responsible for these

information-transfer events. Plasmids that are not in our study are

largely those that are homologous to the cp32 plasmids in B31. Had these

plasmids been included, our conclusion of large-scale genetic exchange

mediated by plasmid transfer would likely have been strengthened, given

the recent results of son and (22).

MLST Analysis of Genomes and Clinical Isolates. The central role of

recombination in driving B. burgdorferi adaptive diversification was

confirmed by genomic typing at one chromosomal and seventeen

plasmid-borne polymorphic loci for 18 additional B. burgdorferi sensu

stricto isolates from human skin and blood (Table 3). These isolates were

chosen to include multiple representatives of 11 genetically diverse ospC

types [major groups A–U of ospC (19)]. Many ospC types were represented

twice or more to maximize the chance of detecting recent mutation and

recombination. The MLST system (8, 9) designed for the genomic typing of

pathogenic bacteria typically uses selected housekeeping genes. However,

this strategy is not suitable for genomic typing of B. burgdorferi

because its genome contains a large number of plasmids that carry only

very few housekeeping genes (23, 24, 45). Therefore, identification of

events such as genetic exchanges by way of plasmid transfer requires the

inclusion of nonhousekeeping markers to cover the wide range of

replicons.

Comparison of these 18 genes across 21 strains led us to several

insights. First, none of the 17 non-ospC ORFs that were analyzed shows

hypervariability like ospC. Only a small number (two to five, mostly two)

of alleles are present at each HDNP loci except ospC, which has 11

" major-group " alleles (Fig. 2). Even based on incomplete genome

comparisons and a limited number of isolates, the absence of ospC-like

hypervariability among these most variable markers is significant and

suggests that ospC is under the strongest form of balancing selection

across the genome. [Recombination at vlsE generates transient diversity

during infection (46), unlike the evolutionarily stable balanced

polymorphisms at ospC.] Second, gene trees (shown in Fig. 7, which is

published as supporting information on the PNAS web site) among MLST

loci, especially those on different plasmids, are often incongruent,

strongly indicating gene exchange (34). Other evidence for recombination

includes the presence of all four combinations of alleles (most frequent

alleles, brown and green in Fig. 2) at pairs of loci, such as

BBC02/BBD14, BBD14/BBG32, and BBE17/BBG32. Incongruent gene genealogies

and allele reassortment often occur among loci located on different

plasmids, further supporting the hypothesis that plasmid transfer is the

main mechanism of recombination. Third, strains of the same ospC types

generally share the same MLST genotype (Fig. 2). The fact that genetic

homogeneity among the MLST genotypes sharing the same ospC is disrupted

only rarely by gene exchange at non-ospC markers suggests that B.

burgdorferi population structure is dominated by balanced polymorphisms

at ospC. Therefore, the ospC groups could be viewed as evolutionarily

stable " clonal complexes " (9) within B. burgdorferi populations. Last, we

observed breakdown of the homogeneity of ospC clonal complexes by recent

events of recombination (boxed region in Fig. 2). One such event occurred

at BBQ49 among 132a, 132b, and B31 within the ospC group A complex. Other

events transferred fragments spanning lp54 BBA68, BBA69, and BBA70; a

divergent lp28–4 BBI38 allele into the N40 genome; and an lp36 BBJ19

allele into the 136b genome. The putative direction of these lateral gene

transfers was determined by assuming that the most common allelic type

among the B31, JD1, and N40 genomes is the ancestral type.

Relative Rates of Recombination and Mutation. Rates of recombination

relative to mutation were estimated by counting the number of

recombination and mutation events among strains belonging to the same

ospC clonal complexes (35, 47). All SNPs at BB0082 are shown in Fig. 8,

which is published as supporting information on the PNAS web site. Six

ospC clonal complexes (A, B, E, G, I, and K) are represented with two or

more isolates. Isolates within the A, E, and K complexes show no

polymorphism. Isolates within the complexes B, G, and I are segregated

with one identical SNP at site 17, which is likely introduced by

recombination. Only one mutational event was found within the G complex

(at site 823) because this SNP is not found in any other clonal

complexes. Therefore, there were three events of recombination and one

event of mutation during the recent divergence of isolates within the B,

G, and I clonal complexes at this chromosomal locus, resulting in a

recombination to mutation (r/m) ratio of 3:1. By using SNPs from one

chromosomal locus and 16 plasmid loci (excluding ospC), we found a total

of six mutations and 16 recombination events (r/m, 2.7:1). These values

are smaller than the ratio found in more freely recombining bacterial

species, such as Neisseria meningitidis and Streptococcus pneumoniae

(r/m, 5:1 to 10:1; ref. 48), and larger than the ratio in more clonal

species, such as Staphylococcus aureus (r/m, 1:15; ref. 49).

Discussion

Scale-Dependent Population Structure of B. burgdorferi. Bacterial species

show different degrees of clonality (17, 50). Many bacterial species show

a freely recombining population structure, in which genetic exchange

among bacterial cells is frequent enough that there are no stable clones.

Examples are N. meningitidis, S. pneumoniae, Staphylococcus pyogenes (9),

Helicobacter pylori (51, 52), and the Bacillus cereus species group (53).

Other species show a more clonal population structure, such as

Haemophilus influenzae, Escherichia coli (9), Salmonella enterica (54),

and Sta. aureus (49). An often neglected aspect in the discussion of

bacterial clonality is the choice of isolates used in such an analysis.

(18) recognized the effect of the geographic and ecological

sampling scale in characterizing the population structure of microbial

species. For instance, a critical factor in understanding the role of

recombination in the clonal divergence in E. coli was the sampling of

isolates within the same subgroup (47). MacLeod et al. (55) showed that

proper characterization of the genetic structure of Trypanosoma brucei

depends critically on recognizing population subdivisions due to

geographical and ecological isolation.

Cross-population sampling is likely the main reason most previous studies

of B. burgdorferi population structure (e.g., ref. 14) failed to show

recombination beyond a few selected loci. These studies used laboratory

archival strains that were obtained from diverse locations on two

continents and belonged to different species in the B. burgdorferi

species complex. We were able to observe past genetic exchange in B.

burgdorferi sensu stricto by comparing isolates from endemic regions in

the northeastern United States. B. burgdorferi from these regions forms a

geographically and ecologically uniform metapopulation, which is founded

on a single rather recent ancestral population (56) and maintained in a

similar enzootic transmission cycle with the white-footed mouse

(Peromyscus leucopus) as the main reservoir and the northern lineage of

black-legged ticks (Ixodes scapularis) as the main vector (57).

The scale-dependence of bacterial population structures is expected to be

a more prominent characteristic in obligate parasitic and geographically

structured species (such as B. burgdorferi) than in free-living and more

cosmopolitan species (such as E. coli). B. burgdorferi is a vector-borne

pathogen, further restricting the chance of direct encounter and gene

transfers between local clones. Even among B. burgdorferi clones from the

same geographic region, there could be niche differentiation and

ecological isolation, resulting in a lack of gene flow between sympatric

clones or species. Our study shows that whole-genome sequencing of a

small number of local clones (preferably with different ospC types) will

be an effective means for fine-scale characterization of local B.

burgdorferi population structure in other endemic regions of Lyme

disease. We conclude from this study and previous studies that, although

B. burgdorferi shows a high degree of clonality between geographically or

ecologically isolated populations (15, 20), it is quite capable of

genome-wide genetic exchange within the same ecological populations.

Selective Maintenance of ospC Clonal Complexes. Recombination is a

powerful facilitator of species adaptation. Rare beneficial alleles are

more likely to be fixed in a population by avoiding " Muller's Ratchet "

(58), the mutational meltdown and rapid fitness loss of an asexual

species. More important, beneficial combinations of alleles among loci

can arise quickly through random genome assortment. The most popular MLST

system of detecting recombination in a bacterial species uses neutral

variations of housekeeping genes (8, 59). Use of neutral markers allows

estimation of the rate and fragment size of lateral gene transfers. Feil

et al. (35) defined a bacterial clonal complex as a group of isolates

identical in their MLST genotypes and the immediate descendant variants

of these isolates. The adaptive relevance of clonal complexes defined by

shared neutral variations is generally not known, and the target of

selection, if present, could not be identified. In contrast, we used a

set of mostly nonhousekeeping genes in consideration of the highly

segmented nature of B. burgdorferi genomes and the potential clinical

significance of nonneutral variations. Our study shows that ospC is

likely to be a dominant locus in the selective maintenance of B.

burgdorferi diversity in nature. Previous evidence for the

extraordinarily strong balancing selection at ospC includes the

within-population diversity and geographic uniformity of ospC allele

frequencies (19, 56). Hypervariable sites of ospC have been mapped to its

surface (60). The age of balanced polymorphisms may predate divergence

among B. burgdorferi species (20). Comparative genomics shows that the

balancing selection present at ospC may be the strongest across the

genome. This conclusion is mainly based on the observations that (i) we

failed to find ospC-like hypervariability at this genome-wide, albeit

incomplete, survey of ORF variability, and (ii) MLST genotypes are

largely definable by their ospC types.

We also conclude that, although the strongest form of balancing selection

in B. burgdorferi may be operating at ospC, highly divergent HDNPs at

other plasmid loci are likely maintained by balancing selection as well.

Our reasoning is as follows. On the main chromosome, the HDNPs are

predominantly synonymous polymorphisms, whereas on the plasmids the level

of nonsynonymous substitutions is almost as high as that of synonymous

substitutions (Fig. 3). The high Ka/Ks ratio of plasmid HDNPs may be due

either to positive selection or relaxed selective constraint. However,

one might expect to observe a high number of sequence variants at these

loci if these sequences were to diverge through relaxation of selection.

Instead, we observed only two to three major-group alleles of ORFs in

multiple isolates of these HDNP markers. Sequences of plasmid non-ospC

HDNPs are highly divergent, suggesting that these are ancient alleles

that have been maintained in population for a long time. Maintenance of

divergent alleles in high frequencies is a hallmark of balanced

polymorphisms (e.g., ospC, or Adh in Drosophila melanogaster; ref. 61).

However, balancing selection at these non-ospC plasmid HDNPs is not as

dominant as selection at ospC in influencing the B. burgdorferi

population structure.

Plasmid HDNPs as Virulence and Phylogenetic Markers. Based on the

putative selective prominence of ospC and other plasmid HDNPs, we suggest

that these plasmid ORFs, mostly with unknown functions, may play a role

in interaction with the host and, therefore, may be potential virulence

factors. The HDNPs that we identified include (besides ospC) genes such

as dbpA (BBA24), whose product is involved in host-cell binding (42), and

BBA68, which encodes the complement resistance protein BbCRASP-1 (43),

but not ospA or ospB (BBA15/16) outer-surface protein genes. Associative

studies of the alleles of plasmid HDNPs with Lyme disease symptomology

may clarify the role of potential virulence factors. The present study

provides a library of potential markers for the study of B. burgdorferi

virulence in humans (39, 62).

However, the HDNPs revealed from genome comparisons should not be used to

infer the overall phylogenetic relationships among B. burgdorferi

isolates. Frequent recombination precludes the reconstruction of

intraspecific phylogenies for B. burgdorferi isolates based on such

polymorphisms. Nonetheless, plasmid HNDPs may prove to be valuable

markers for classifying B. burgdorferi isolates immunologically (18) and

clinically.

In summary, comparative genomics and MLST analysis of closely related

isolates revealed genome-wide genetic exchange and plasmid transfers in

B. burgdorferi. The mechanisms performing such transfers remain unproven.

However, we note that B. burgdorferi carries the requisite

homologous-recombination machinery to incorporate genetic material that

it might take up (63, 64), bacteriophage-mediated transduction has been

demonstrated in the laboratory (65), and both ticks and reservoir hosts

are capable of being simultaneously infected by multiple ospC clones (19,

56) (D. Brisson and D. E. Dykhuizen, personal communication). Balanced

polymorphisms at ospC and other plasmid HDNP loci, which appear to be

cornerstones of B. burgdorferi adaptation, would not be maintained

independently if B. burgdorferi were a predominantly clonal species, in

which genetic variability is easily lost by selective sweeps and genetic

drift (66–68).

September 21, 2005

Yes. There is a school of thought that arthritis is caused by bacterial or

other infections; I too have noted an improvement in arthritic conditions

with KT and other anti-infectives such as Vitamin C and Cayenne.

In the case of Lyme Disease, Molecular Biologist Garth Nicholson, PhD has

written over 500 papers

and is of the opinion that Lyme Disease, Gulf War Illness, and CFS are the

same infection, a mycoplasma created by the Dept. of Defense. Our tax

dollars at work.

Mycoplasma infections are cured by the long term usage of antibiotics.

Unfortunately, most doctors will not prescribe them, and antibiotics create

their own problems. Since Russian and German medical research has

demonstrated that KT is a broad spectrum antiviral, antifungal, and

antibacterial agent. The downside of KT or any other treatment is that

eventually the body will build a tolerance from it, which means it will stop

working until you take a rest from it for a couple weeks or so. You will

have to use another anti-infective agent at that time because the mycoplasma

may come back.

Other anti-infectives would include echinacea, large doses of Vitamin C,

Goldenseal, Grapefruit Extract, bentonite clay packs, internal usage of

bentonite if no obstructions are present, and colloidal silver.

Hope that helps,

Lyme Disease

> Hi All,

> I brought my mechanic a gallon of KT the other day and he said that he

> thinks his wife has lyme disease and asked me if the tea would help

> it. I couldn't give him an answer but told him that I would bring it

> up on the group and find out if anyone has had lyme disease. They both

> thought it was arthritis because she is having to walk with a cane and

> all her joints hurt, but she is only 32 years old.

>

> Can anyone elaborate on this?

>

> Thanks,

> Judy

>

September 21, 2005