6.13. CHARACTERIZATION OD SPIROCHETES CULTIVATED FROM THE BRAIN OF ALZHEIMER'S PATIENTS IN BSK MEDIUM

As serological and diagnostic tests are available for Borrelia burgdorferi, a multifaceted analysis was done in order top answer the question whether Borrelia burgdorferi may be involved in the AD cases where spirochetes were cultivated in BSKII medium. Positive identification of the agent as Borrelia burgdorferi sensu stricto (s. s.) was based on genetic and molecular analyses. We have analyzed the sequence of the 16S rRNA gene of spirochetes cultivated from the brain of three of the 14 AD cases, grown in medium BSKII, selective for Borrelia burgdorferi (Miklossy, 1993) and carried out morphological characterization by transmission electron microscopy. We correlated this with post mortem serological analysis of blood and cerebrospinal fluid (CSF). The detection of Borrelia burgdorferi specific antigens and genes in the brains of these patients provided further evidence that they suffered from chronic Lyme neuroborreliosis. Borrelia antigens and genes were co-localized with beta-amyloid deposits in these three AD cases. 
         
The pathological findings observed in the 3 AD cases were similar to those described in dementia paralytica caused by Treponema pallidum (Jahnel, 1916, 1917, 1920, Pacheco e Silva, 1926, 1927). They are consistent with primary parenchymatous involvement of tertiary Lyme neuroborreliosis. Similar to the observations of Noguchi and Moore (1913) with respect to Treponema pallidum, our results show that Borrelia burgdorferi may also persist in the brain in chronic Lyme neuroborreliosis and cause dementia, cortical atrophy and amyloid deposition. The present findings reinforce the similarity between the clinical and pathological manifestations of syphilis and Lyme disease and suggest that Borrelia burgdorferi may also be involved in the pathogenesis of several chronic neuro-psychiatric disorders.  


Consistent with the present findings, the genospecies Borrelia garinii and Borrelia burgdorferi s. s. have been reported to be predominantly involved in neuroborreliosis (Wilske et al., 1994).
In endemic areas of Lyme disease, the wide distribution of other spirochetes (e.g. oral spirochetes), which were found to be associated with AD, may mask a clustering of an association of Borrelia burgdorferi with AD. Careful epidemiological studies will be necessary to analyze this point.                                              


The molecular analysis of spirochetes cultivated from the blood of a clinically asymptomatic forester who showed positive serology for Lyme disease was also performed. The case of this healthy forester, where the 16S rRNA analysis also defined the spirochetes cultivated from the blood as Borrelia burgdorferi s. s., indicates that it could represent a latent stage of the disease. A clinical follow-up and repeated serological tests and cultures would be necessary to answer this question.

In agreement with our previous findings and anticipation that several types of spirochetes are involved in Alzheimer's disease, including oral spiroxhertes, Riviere et al., (2002) using species-specific PCR and monoclonal antibodies, detected oral Treponema in 14/16 AD cases and 4/18 non-AD controls, indicating that in a high percentage of AD cases oral spirochetes, namely Treponema pectinovorum and Treponema socranskii which are known periodontal pathogens may be involved in AD. This is in harmony with the fact that oral Treponema spirochetes are widely present in the population at large, particularly in the older population. Intestinal spirochetes were also cultivated from human subjects.  Positive serology against these, "commensal spirochetes" is known to occur, indicating that these spirochetes were in contact with the host immune system.  


Reports of associations between infection and AD are not confined to spirochetes. The presence of Herpes virus type 1 (HSV-1) in the AD brain has been reported (Jamieson, 1991; Itzhaki, 1997, 2004).  Chlamydia pneumoniae was also found to be associated with AD (Balin, 1998) and mice exposed to Chlamydia developed AD-like amyloid plaques (Little et al. 2004). The clinical and pathological hallmarks of Alzheimer’s disease (AD) are present in the atrophic form of general paresis caused by spirochetes (Jahnel, 1916, 1917, 1920; Pacheco e Silva, 1926, 1927, Lubarsh et al., 1958). Co-infection of spirochetes with other bacteria, including Chlamydia and also with Herpes viruses and cytomegalovirus (CMV), or even fungi (Candida albicans) is well known. Propionibacterium acnes in the cortex of patients with Alzheimer's disease was also reported by Kornhuber (1996). Actinomyces, has been reported to be present in the brain of patients with an incidence that was four times higher than in other pathological or normal conditions (Howard and Pilkington, 1993) .  Recently Helicobacter pylori was also reported to be involved in AD.              


An infectious etiology of Alzheimer’s disease would not be in controversy with the genetic defects shown to be present in AD. In chronic bacterial infections or in experimental amyloidosis the bacterial infection or bacterial exposure, always precede amyloid deposition. In patients with a genetic defect that facilitates infection, the genetic problem would be the primary step in the cascade of events, followed by infection, then by amyloid deposition.