Molecular interrogation of Alzheimer Autopsy Brain tissues for
evidence of NonHuman (microbial) DNA of Borrelia burgdorferi
by Alan MacDonald MD (copyright 2005)
Syphilis in its Late Tertiary form causes dementia (General Paresis)
which has its onset 20 to 30 years after Primary Infection with
Treponema pallidum. Spirochetes in General Paresis autopsy brains
were found by Noguchi and Moore.(1913, J Exp Med)
Lyme Disease infection has Primary, Secondary, and Tertiary
manifestations; and an example of Tertiary Lyme Disease is the skin
condition Acrodermatitis Chronica Atrophicans (ACA). Biopsies of skin
from ACA lesions have demonstrated Borrelia  organisms 20 to 30 years
after Primary Infection. In 2003 Fikrig and colleagues
( ) demonstrated in primate
model that multiple transcriptomes of Borrelia could be recovered at
autopsy in animals whose sole exposure to spirochete was a hypodermic
injection (very analogous to arthropod infection of man. The Flagellin b
transcriptome for p41 protein was prominently represented in the infected
Based on the Tertiary Syphilis model, the ACA model,  and the primate
model ,a molecular interrogation of Alzheimer Brain tissue was
undertaken to seek evidence of Borrelia burgdorferi  Flagellin b DNA.
Borrelia burgdorferi have been previously grown in laboratory cultures of
Alzheimer disease autopsy tissue.
Borrelia burgdorferi have been previously seen in microscopic sections of
Alzheimer disease autopsy tissue using specific monoclonal antibodies
and immunoflourescence techniques,
Quality Control  - Tissues utilized
1. Alzheimer's disease tissues: All specimens in this investigation
were obtained from Harvard Medical School, McLean Hospital,
Brain Tissue Resource Center ( The
primary investigator applied for and was accepted as an Affiliated
Investigator at the Harvard Brain Bank. The diagnosis of
Alzheimer,s disease was established by faculty board certified
Neuropathologists using the CERAD criteria. Ten specimens of
fresh frozen autopsy hippocampus were shipped to the primary
investigator and evaluated as designated below.
2. One Autopsy brain specimen from a patient not diagnosed with
Alzheimer's dementia was studied in parallel.
Quality control - PCR Primers

1.  The complete nucleotide sequence for Borrelia burgdorferi
(Chromosome and all plasmids) from the TIGR nucleotide database
was utilized. (
2.  PCR primers were designed using the Primer Premiere software
from Premiere Biosoft Inc( design) to
probe for the nucleotide sequences of Open Reading Frame BBO 147
which codes for the Flagellin B proteins ( corresponding to the 41kd
band on Western Blot preparations of whole cell Borrelia burgdorferi)
3.  Manufacture of the PCR primers was completed at the Yale
University School of Medicine Keck Laboratory
( using strict purification
Quality Control - Polymerase Chain Reaction

Preparation of DNA extractions from Alzheimer brain slices was accomplished
as follows:
Sigma Kit , Extract-N-Amp PCR(tm) for DNA extraction from tissues
( was utilized according to the manufacturer's
All specimens were handled individually using strict aseptic technique in a
Laminar Air Flow hood to eliminate any possibility of cross contamination.
PCR was accomplished using the Perkin Elmer Thermocycler apparatus.
Experiments were completed in duplicate.
Agarose gels ( Metaphor Agarose 2%) ( were utilized.
Lambda DNA ladders ( were run with each gel to identify
the gel position of the expected 500 nucleotide PCR product
Picogreen ( was used to stain the gels according to the
manufacturer's instructions. The stained gels were examined with the Dark
Reader transilluminator (tm) ( according to the
manufacturer's instructions and photographed  (using photographic filters
according to the manufacturer's instructions )for documentation of results.
Upon completion of PCR , each gel band of approximately 500 base pair size
was excised using strict aseptic technique in a Laminar Airflow Hood and
transferred to a sterile plastic vial which was sealed and refrigerated in
preparation for transport.
Quality Control - DNA Sequencing
Sequencing of all 500 bp PCR products was completed at Genaisance in,.Lark
Technologies Houston Texas,using a BigDye(R) terminator v1.1 cycle sequencing
kit (Applied Biosystems). and the reactions were analyzed on ABI PRISM 3730xl
DNA Analyzers using Applied Biosystems analysis Software (
Electropherograms and alignment overviews are available for review
from Lark Genaissance, Houston, Texas
Quality Control - GenBank Deposit
All PCR products obtained from this study were deposited in the National
Center for Biotechnology (NCBI) GenBank
( in 1995. (see home page of this site for
GenBank Accession numbers)

Comparison of the
results of a PCR study
of primate autopsy
brains after
experimental infection
with Borrelia
burgdorferi by
cutaneous inoculation
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