Excerpt:

Can Resveratrol, the so-called miracle molecule found in red wine, peanuts and an obnoxious plant called Chinese Giant Knotweed, actually improve your brain function?

Scientists at Northumbria University in the UK have just published a double-blind, peer-reviewed study in the journal, American Journal of Clinical Nutrition, which concludes that a relatively modest dose of this compound in the form of one or two capsules may just do that. In their study, 24 subjects were given one capsule of a supplement called Biotivia Bioforte Resveratrol containing 250 mg of the trans-Resveratrol isomer in each dose.

In previous studies of Resveratrol the compound has been shown to have potent anti-aging properties in animals, as well as the ability to protect cells against a remarkable range of diseases. Resveratrol seems to perform much the same function in animals the same way it does in plants; that is, act as the organism’s immune and defense system. The supplement’s ability to activate certain genes called sirtuins was discovered by Dr. David Sinclair at Harvard about three years ago and since that time has been the subject of hundreds of studies and trials. Albert Einstein Medical School, for example, has applied to present the results of its trial on diabetes patients to the American Diabetes Association Science Meeting in June.

In the Northumbria study, Bioforte resveratrol increased the brain blood flow in the human subjects while they were performing mental tasks by a substantial amount. The effect seemed to be turned on by the increased demand caused by the cognitive tasks being performed by the student subjects. This implies that the effect of Resveratrol on cerebral blood flow is activated on demand when the brain is called upon to perform a challenging mental task. Could this mean that the compound can be taken in the morning before taking an exam, for example, and spring into action when needed to boost the brain’s ability to cope with the exam? Possibly; however, much more study is needed to determine the proper dosage and timing for this application.

Lyme disease is the most prevalent tick-borne infection in North America and Eurasia. It is caused by the bacterium Borrelia burgdorferi and is transmitted to humans via the bite of infected ticks. These spirochetes can cause both acute and chronic infection and inflammation of the skin, joints, heart, and central nervous system. The persistence of infection despite the presence of an active immune response is dependent upon antigenic variation of VlsE, a 35 kDa surface-exposed lipoprotein. A large number of different VlsE variants are present in the host simultaneously and are generated by recombination of the vlsE gene with adjacent vls silent cassettes. To try to identify factors important in vlsE recombination and immune evasion, we selected mutants in genes involved in DNA recombination and repair and screened them for infectivity and vlsE recombination. Mutants in genes encoding RuvA and RuvB (which act together to promote the exchange of strands between two different DNA molecules) had reduced infectivity and greatly diminished vlsE recombination. In immunodeficient mice, ruvA mutants retained full infectivity, and no vlsE recombination was detected. Our findings reinforce the importance of vlsE variation in immune evasion and persistent infection.

Tao Lin^1#, Lihui Gao^1#, Diane G. Edmondson¹, Mary B. Jacobs², Mario T. Philipp², Steven J. Norris^1,3*

1 Department of Pathology and Laboratory Medicine, Medical School, University of Texas Health Science Center at Houston, Houston, Texas, United States of America, 2 Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of America, 3 Department of Microbiology and Molecular Genetics, Medical School, University of Texas Health Science Center at Houston, Houston, Texas, United States of America

Abstract 

Antigenic variation plays a vital role in the pathogenesis of many infectious bacteria and protozoa including Borrelia burgdorferi, the causative agent of Lyme disease. VlsE, a 35 kDa surface-exposed lipoprotein, undergoes antigenic variation during B. burgdorferi infection of mammalian hosts, and is believed to be a critical mechanism by which the spirochetes evade immune clearance. Random, segmental recombination between the expressed vlsE gene and adjacent vls silent cassettes generates a large number of different VlsE variants within the infected host. Although the occurrence and importance of vlsE sequence variation is well established, little is known about the biological mechanism of vlsE recombination. To identify factors important in antigenic variation and vlsE recombination, we screened transposon mutants of genes known to be involved in DNA recombination and repair for their effects on infectivity and vlsE recombination. Several mutants, including those in BB0023 (ruvA), BB0022 (ruvB), BB0797 (mutS), and BB0098 (mutS-II), showed reduced infectivity in immunocompetent C3H/HeN mice. Mutants in ruvA and ruvB exhibited greatly reduced rates of vlsE recombination in C3H/HeN mice, as determined by restriction fragment polymorphism (RFLP) screening and DNA sequence analysis. In severe combined immunodeficiency (C3H/scid) mice, the ruvA mutant retained full infectivity; however, all recovered clones retained the ‘parental’ vlsE sequence, consistent with low rates of vlsE recombination. These results suggest that the reduced infectivity of ruvA and ruvB mutants is the result of ineffective vlsE recombination and underscores the important role that vlsE recombination plays in immune evasion. Based on functional studies in other organisms, the RuvAB complex of B. burgdorferi may promote branch migration of Holliday junctions during vlsE recombination. Our findings are consistent with those in the accompanying article by Dresser et al., and together these studies provide the first examples of trans-acting factors involved in vlsE recombination.

Author Summary 

Lyme disease is the most prevalent tick-borne infection in North America and Eurasia. It is caused by the bacterium Borrelia burgdorferi and is transmitted to humans via the bite of infected ticks. These spirochetes can cause both acute and chronic infection and inflammation of the skin, joints, heart, and central nervous system. The persistence of infection despite the presence of an active immune response is dependent upon antigenic variation of VlsE, a 35 kDa surface-exposed lipoprotein. A large number of different VlsE variants are present in the host simultaneously and are generated by recombination of the vlsE gene with adjacent vls silent cassettes. To try to identify factors important in vlsE recombination and immune evasion, we selected mutants in genes involved in DNA recombination and repair and screened them for infectivity and vlsE recombination. Mutants in genes encoding RuvA and RuvB (which act together to promote the exchange of strands between two different DNA molecules) had reduced infectivity and greatly diminished vlsE recombination. In immunodeficient mice, ruvA mutants retained full infectivity, and no vlsE recombination was detected. Our findings reinforce the importance of vlsE variation in immune evasion and persistent infection.

Citation: Lin T, Gao L, Edmondson DG, Jacobs MB, Philipp MT, et al. (2009) Central Role of the Holliday Junction Helicase RuvAB in vlsE Recombination and Infectivity of Borrelia burgdorferi. PLoS Pathog 5(12): e1000679. doi:10.1371/journal.ppat.1000679

Editor: Jenifer Coburn, Medical College of Wisconsin, United States of America

Received: June 3, 2009; Accepted: November 4, 2009; Published: December 4, 2009

Copyright: © 2009 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The project described was supported by Award Numbers R01AI37277 and R01AI059048 (to S.J.N.) from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy And Infectious Diseases or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: Steven.J.Norris@uth.tmc.edu

# These authors contributed equally to this work.

Introduction 

Lyme borreliosis is a multi-stage, systemic disease caused by members of the spirochete genus Borrelia, including Borrelia burgdorferi in North America and Borrelia afzelii, Borrelia garinii, and B. burgdorferi in Eurasia [1]. Spirochetes are transmitted to mammalian and avian hosts via the bite of hard-bodied ticks of Ixodes genus [2] and disseminate widely throughout the body in the first weeks of infection. If untreated in early stages, chronic and debilitating disease can develop in the skin, joints, heart, and central nervous system [1]. Infected individuals develop an active immune response to the pathogen yet are unable to clear the infection.

A common mechanism of immune evasion is antigenic variation, a process by which pathogens alter surface exposed antigenic proteins [3],[4]. The resulting variant organisms are immunologically distinct from parental strains and thereby gain a selective advantage over individuals that retain parental antigenic determinants. Bacteria that undergo antigenic variation often cause long term or repeated infections. Examples of such bacteria include Neisseria gonorrhoeae, Neisseria meningitidis, Borrelia hermsii, Treponema pallidum, Campylobacter jejuni, Mycoplasma synoviae, Mycoplasma pulmonis, and Anaplasma marginale.

Lyme disease Borrelia possess the vls (Variable Major Protein (VMP)-like sequence) system, a robust antigenic variation mechanism involving DNA recombination at the locus that expresses the surface exposed lipoprotein VlsE [5],[6],[7],[8],[9]. The vls locus consists of the vlsE expression site and a contiguous array of 15 vls silent cassettes, which have homology to the central region of the expression site. Gene conversion events involving replacement of regions of the vlsE expression site cassette with segments of the silent cassettes occur continuously during mouse infection, resulting in a myriad of vlsE sequence variants in each infected animal [10],[11]. Within each cassette, there are six variable regions (VRs) that display considerable sequence diversity, interspersed with six relatively invariant regions (IRs). The structure of the VlsE polypeptide is predominated by alpha helices that are believed to be important in maintaining protein structure [12]. The variable regions form random coils on the membrane distal surface of the protein, the region most likely to be exposed to the host immune system. VlsE variants have different epitopes when compared with parental VlsE1 polypeptide [10],[13], indicating that sequence changes in vlsE result in true antigenic variation.

In the B. burgdorferi strain B31, the vls locus is located near the telomere of the linear plasmid lp28-1 [10]. Loss of lp28-1 in B. burgdorferi B31 is associated with an intermediate infectivity phenotype in immunocompetent mice, in which infection lasts for less than 3 weeks and is largely restricted to joint tissue [14],[15],[16],[17]. Spirochetes lacking lp28-1 are able to survive and cause disease at all tissue sites in severe combined immunodeficiency (SCID) mice [17],[18], indicating that one or more gene products encoded on lp28-1 play an important protective role against adaptive immunity. Bankhead and Chaconas [19] recently demonstrated that the vls locus is the important mediator of infectivity in lp28-1. Deletion of the locus by telomere-mediated truncation resulted in an intermediate infectivity phenotype similar to that displayed by B. burgdorferi lacking lp28-1, while deletion of the other end of the plasmid had no detectable effect on infectivity.

The mechanisms that regulate vlsE recombination and expression are not well understood. vlsE recombination events have not been detected in ticks, during in vitro culture, or in dialysis membrane chambers embedded in rats, suggesting that non-dialyzable host components play a role in initiation of recombination [20],[21],[22],[23]. In contrast, vlsE recombination is detectable within 4 days of mouse inoculation, and appears to occur throughout the course of mammalian infection [22],[24]. Approximately 50% of recovered spirochetes have recombined vlsE sequences during the first 7 days of mouse infection; by four weeks, no organisms with parental vlsE sequence can be recovered [11],[22]. In immunodeficient SCID mice, spirochetes with the parental vlsE disappear more gradually, indicating that the adaptive immune response is required for the rapid elimination of organisms expressing the parental VlsE [11]. Although analyses of vlsE transcription levels during mammalian infection have yielded variable results, immunoblot analysis indicates that VlsE protein expression is dramatically upregulated during mouse or rabbit infection [25],[26].

Genes involved in DNA recombination and repair have been found to play a role in antigenic variation in other bacteria. For example, in Neisseria gonorrhoeae, pilin antigenic variation requires several trans-acting factors in the RecF-like recombination pathway, including RecA, RecX, RecJ, RecO, RecQ, and RecR [27],[28],[29] as well as RecG and a functional RuvABC complex [30]. However, in B. burgdorferi no genes have been yet identified that play a role in recombination within vlsE, although recA has been shown to be dispensable [31].

To identify proteins that play a role in vlsE recombination, we screened transposon mutants of DNA recombination and repair genes for infectivity and vlsE recombination. We found that mutants in the genes encoding the Holliday junction helicase polypeptides RuvA and RuvB were compromised in their ability to undergo vlsE recombination and displayed a reduced infectivity phenotype. In SCID mice, ruvA mutants retained full infectivity. vlsE sequences recovered from SCID mice were identical to parental vlsE sequence, suggesting that the failure to undergo vlsE recombination was a critical factor in the reduced infectivity phenotype. Furthermore, ruvA mutants had no increase in sensitivity to DNA damage. Nearly identical results were obtained in the accompanying article by Dresser et al. [32].

Results 

Screening of putative DNA recombination and repair mutants for infectivity and VlsE variation

For this study, 9 transposon insertion mutants representing 7 different genes whose homologues are known to be involved in DNA recombination and repair in other bacteria were selected from a large Signature Tagged Mutagenesis (STM) panel to screen for their effect on infectivity and vlsE recombination (Table 1). Independent mutants from two of the genes were included to test the possible role of different insertion sites on infectivity and vlsE recombination. T05P01C02 and T10P01D06 are independent mutants of BB0797 with insertion ratios of 0.73 and 0.97 respectively. (The insertion ratio represents the relative position of the transposon insertion in a coding sequence, with the entire coding sequence having a value of 1.0). Likewise, T08P01E02 and T11P01F09 are independent mutants from gene BBE29 with insertion ratios of 0.33 and 0.30. Each mutant was injected in 5 mice and skin biopsies were taken at days 7 and 14 after inoculation and cultured for B. burgdorferi. Mice were sacrificed on day 28 of infection, and samples taken from skin, tibiotarsal joint, heart, and bladder were cultured to recover infectious organisms. B. burgdorferi maintained in in vitro culture sometimes lose plasmids that are not required for in vitro growth but may be required for infectivity. Therefore, the plasmid content of each mutant was determined and compared with that of the parental clone 5A18NP1. Circular plasmid cp9 was found to be missing in two mutants (T11P01A01 and T11P01F09) and lp21 is lacking in T10P01D09. However, previous studies have shown that neither cp9 nor lp21 are essential for infection of mice [14],[15].

Table 1. Transposon mutants utilized in this study.

doi:10.1371/journal.ppat.1000679.t001

As in previous studies, the parental strain 5A18NP1 was recovered from 29 of 30 tissue samples on days 7, 14, and 28 post inoculation [33]. Three of the nine DNA repair mutants tested (T11P01A01, T03TC051, and T05P01C02) exhibited reduced infectivity and one (MG065) lost infectivity in immunocompetent mice (Table 2). One of the mutS mutants (T05P01C02) displayed a reduced infectivity phenotype, whereas the other mutant (T10P01D06) had normal infectivity. The insertion site for the highly infectious mutant was very close to the end of the coding sequence (0.97), so this mutant may express a functional product. ruvA (T11P01A01) and ruvB (T03TC051) mutants both exhibited intermediate infectivity patterns. While 5/5 skin cultures were positive for both mutants at day 7 post inoculation, by 2 weeks no skin cultures were spirochete positive, and at 4 weeks only 6 of 20 and 10 of 16 tissue sites were culture positive for the ruvA and ruvB mutants, respectively. We also examined the growth rates of the ruvA and ruvB mutants in vitro. Both mutants had growth kinetics very similar to that of the parental strain 5A18NP1 (data not shown), indicating that the reduced infectivity phenotype observed for the mutants was not due to a growth defect.

Table 2. Infectivity of B. burgdorferi DNA repair and recombination mutants in CH3/HeN mice at days 7, 14 and 28 post inoculation.

doi:10.1371/journal.ppat.1000679.t002

Restriction fragment length polymorphism (RFLP) was used to estimate the extent of vlsE variation in recovered organisms. Amplicons corresponding to the vlsE cassette region were generated by PCR from uncloned cultures and digested with HphI. This restriction enzyme recognizes a 4-bp sequence that occurs with varying frequency as a result of vlsE recombination. The vlsE cassette region of 5A18NP1, the parental strain used in this study, has a single HphI site near one end (Figure 1A). As expected, cultures from mice inoculated with 5A18NP1 displayed significant RFLP variability that increased over the course of infection, as indicated by multiple bands or the presence of a smear in the HphI-digested samples (Figure 1A). In contrast, the ruvA and ruvB mutant cultures (Figure 1B and 1C) had vlsE RFLP patterns that were either unchanged or had relatively few bands without smearing, indicating that few vlsE variants had been generated during the course of infection. The other putative DNA recombination and repair mutants had complex vlsE RFLP patterns, indicating that a variety of vlsE variants had been generated during infection and that vlsE recombination likely had occurred normally (data not shown).

Figure 1. RFLP analysis of vlsE cassette region PCR products indicates reduced vlsE variation in ruvA and ruvB mutants of B. burgdorferi.

Representative results obtained following inoculation of C3H/HeN mice with (A) the parental clone 5A18NP1, (B) the transposon mutant T11P01A01 (ruvA::himar1) and (C) T03TC051 (ruvB::himar1) are shown. Cultures from the indicated time points and tissues were used as the source of template DNA for amplification of the vlsE cassette region. The resulting PCR products were either treated (+) or not treated (-) with the restriction enzyme HphI. Presence of multiple bands or a smear in the HphI-treated sample is indicative of the presence of a high number of vlsE variants. The day post inoculation is indicated by D4 through D28. Tissues examined include skin (S), bladder (B), heart (H) and tibiotarsal joint (J).

doi:10.1371/journal.ppat.1000679.g001

Characterization of the ruvA mutant

ruvA and ruvB have been reported to be part of a larger operon containing two additional genes (Figure S1A), queA (BB0021, S-adenosylmethionine:tRNA ribosyltransferase-isomerase) and pfpB (BB0020, diphosphate-fructose-6-phosphate 1-phosphotransferase) [34]. To determine whether expression of other genes in the operon might be disturbed by the transposon insertion, we performed RT-PCR on RNA samples from the parental strain 5A18NP1 and the ruvA mutant T11P01A01. Primer pair 1 generates a PCR fragment from within the ruvA coding region that crosses the transposon insertion site. As expected, this fragment can be generated using RNA from the parental strain, but not using RNA from the ruvA mutant, or RNA untreated with reverse transcriptase (Figure S1B). However, RT-PCR products spanning regions of both ruvB and queA were generated from both strains using primer pair 3 and 4 (Figure S1B). In the ruvA mutant T11P01A01, the transposon is inserted such that a ‘read-through’ transcript could possibly be generated from the flgB_P::aacC1 cassette within the transposable element. To test that possibility we used primer pair 2, which generates a PCR fragment corresponding to the ruvA coding sequence downstream of the transposon insertion site. A product could be generated from both strains (Figure S1B), suggesting that ‘read-through’ may be occurring from either the transposon or the native promoter. Thus, we conclude that T11P01A01 is defective in expression of ruvA, but that transcription of ruvB, queA and pfpB is occurring. However, we cannot rule out the possibility of polar effects resulting from changes in the levels of expression of ruvB, queA and pfpB.

Inactivation of ruvA results in reduced infectivity in immunocompetent (C3H/HeN) mice but full infectivity in immunodeficient (SCID) mice

To further examine the effects of ruvA disruption on infectivity and vlsE recombination, a more detailed analysis of infectivity was performed (Table 3). Groups of ten immunocompetent C3H/HeN mice and ten C3H/scid mice were each inoculated with 10⁵ organisms of either the T11P01A01 (the ruvA mutant), or the parental clone 5A18NP1. Mice were also inoculated with the previously characterized lp28-1^– clone 5A8 [15] as a negative control. Groups of 5 mice were sacrificed at days 14 and 28 post inoculation. Samples of ear, tibiotarsal joint, heart, and bladder were collected for recovery of spirochetes. As expected, the positive control clone 5A18NP1 was fully infectious in both mice strains; spirochetes were isolated from all sites at day 14 and day 28 post inoculation (Table 3). The lp28-1^– clone 5A8 exhibited the expected intermediate infectivity phenotype. In normal mice inoculated with 5A8, spirochetes were recovered from only three joint cultures at day 14, and no organisms could be recovered by day 28 post inoculation. However, all tissue sites were infected in C3H/scid mice inoculated with 5A8. The ruvA mutant exhibited an infectivity phenotype similar to that of lp28-1^– B. burgdorferi at day 14, but resulted in some positive cultures at day 28 post inoculation (Table 3). In C3H/HeN mice infected with the ruvA mutant, only 3 joint cultures of 20 possible cultures were positive for spirochetes at day 14 post inoculation, whereas 20 of 20 cultures were positive in the C3H/scid mouse group. At day 28, the ruvA mutant was recovered from 2/5 ear, 2/5 tibiotarsal joint, 2/5 heart, and 1/5 bladder cultures in immunocompetent mice, while again all cultures were positive in the C3H/scid mouse group. These results indicate that the ruvA mutant is significantly impaired in infectivity compared to the parental strain; it is not, however, as severely compromised as the strain lacking lp28-1.

Table 3. Infectivity phenotype of the ruvA mutant T11P01A01 in immunocompetent C3H/HeN and immunodeficient C3H/scid mice is similar to that of the lp28-1^– clone 5A8.

doi:10.1371/journal.ppat.1000679.t003

RuvAB is not required for survival in ticks

We also tested the ability of B. burgdorferi carrying the ruvA or ruvB mutation to survive in ticks. I. scapularis nymphs were inoculated with the ruvA mutant T11P01A01, the ruvB mutant T03TC051, or the parental strain 5A18NP1 by capillary feeding. Ticks were held for 21-25 days and cultured for B. burgdorferi either before or immediately after feeding on 3 C3H/HeN mice. B. burgdorferi were readily detected in both fed and unfed ticks, as determined by direct fluorescent antibody (DFA) staining and culture (Table 4). This result indicates that the ruvA and ruvB mutations do not abrogate the ability of B. burgdorferi to survive in ticks. In addition, the mutant spirochetes were able to multiply upon tick feeding (Table 4). Fewer spirochetes were recovered from fed ticks carrying the ruvB mutant as compared to the ruvA mutant or the parental strain, but enough spirochetes were present in fed ticks to expect a mouse infection to result. However, tissues collected 4 weeks post tick inoculation with the ruvA or ruvB mutants were consistently culture negative (Table 4). The failure to recover organisms from any cultures derived from tick-inoculated mice (as compared to the sporadically positive cultures from needle-inoculated animals; Table 2) may be due to the lower number of organisms delivered during tick inoculation as compared to needle inoculation, coupled with the decreased survival of ruvA or ruvB mutants in immunocompetent mice at 28 days post inoculation (Table 2).

Table 4. B. burgdorferi ruvA and ruvB mutants survive well in ticks, but do not establish long-term infection in mice following tick-mediated inoculation.

doi:10.1371/journal.ppat.1000679.t004

Inactivation of ruvA or ruvB results in reduced vlsE recombination

Spirochetes recovered from infected mice were isolated by colony formation, and the vlsE cassette region sequences of the resulting clones were determined by PCR amplification and sequencing to examine the effect of ruvA or ruvB mutation on vlsE recombination. For the ruvA mutant T11P01A01, a total of 382 vlsE sequences were analyzed, comprising sequences from both immunocompetent and SCID mice, at three time points and from four tissues. For the ruvB mutant T03TC051, a total of 62 clones isolated from C3H/HeN mice were sequenced, including 22 obtained from skin cultures 7 days post inoculation, and 40 clones from joint, heart, bladder and skin cultures 28 days after inoculation.

The overall pattern of vlsE recombination observed is depicted in Figure 2. For each mouse group and time point, it was determined whether the recovered clones retained the parental vlsE sequence, had a unique vlsE sequence, or shared the same vlsE variant sequence when compared to other clones from the same animal (‘vlsE sequences with siblings’). It has been determined previously that vlsE variation is not detectable during in vitro culture [22]; therefore all inoculated organisms initially contained the parental vlsE sequence.

Figure 2. vlsE sequence variation observed with the parental strain 5A18NP1 and the ruvA mutant T11P01A01 and the ruvB mutant T03TC051 following inoculation of immunocompetent C3H/HeN mice and immunodeficient C3H/scid mice.

The combined results from all tissues examined for each time point are shown. The vlsE cassette regions of individual B. burgdorferi clones were amplified by PCR, sequenced, and characterized as either identical to the parental vlsE sequence, a unique vlsE sequence for that mouse, or two or more clones with an identical vlsE variant sequence from a mouse (vlsE sequences with siblings).

doi:10.1371/journal.ppat.1000679.g002

As observed in prior studies with B. burgdorferi B31 clones, the parental strain 5A18NP1 underwent rapid vlsE recombination in both C3H/HeN and C3H/scid mouse hosts. By 14 days post inoculation, 0/59 clones from C3H/HeN mice had retained the parental sequence. Clones with unique vlsE cassette region sequences predominated after 7 days, although a surprisingly high proportion of sibling variants was observed in C3H/HeN mice inoculated with 5A18NP1 (Figure 2, Table S1). At 14 and 28 days post inoculation, no clones with the parental vlsE sequence were detected. A similar pattern was observed during infection of C3H/scid mice, although the dilution of parental sequence clones occurred at a slower rate; 19/76 clones (25%) still had the parental sequence on day 14. However, even in C3H/scid mice no clones with the parental vlsE sequence were recovered at day 28 post inoculation.

In contrast, sequence analysis demonstrated that very little vlsE sequence variation occurred during infection of mice with the ruvA mutant T11P01A11 (Figure 2, Table S1). In immunocompetent mice, only clones with the parental sequence were recovered up to 14 days post inoculation. By day 28 very few (3 of 114, 3%) of the clones retained the parental sequence; however, the remaining clones were quite restricted in terms of sequence variation, with each animal possessing only 1 or 2 variant types. Surprisingly, only parental sequence clones were recovered from C3H/scid mice on days 14 and 28 post inoculation, indicating that clones that had not undergone vlsE variation predominated in the absence of immune selection. The ruvB mutant T03TC051 also exhibited reduced vlsE sequence variation during infection of C3H/HeN mice (Figure 2, Table S1).

The differences between sequence variation in 5A18NP1 and T11P01A01 are further illustrated in Figure 3. Sequence diversity among the clones isolated at day 28 post inoculation from C3H/HeN mice was analyzed using phylogenetic tree software. The 63 clones from infection with 5A18NP1 were from joint, heart, bladder, and ear tissues of a single mouse, whereas the 114 clones for T11P01A01 were from the 12 culture-positive samples from 6 different mice. With clone 5A18NP1, 31/63 (49%) of the clones characterized possessed unique vlsE sequences; the remaining clones were variant siblings that were isolated from the same mouse tissue. Moreover, closely related sequences were often isolated from the same tissue (Figure 3A, brackets), indicating that vlsE sequences in sibling clones were diverging within those tissues. For the ruvA mutant T11P01A11, only eight different vlsE sequences could be detected among the 114 clones examined in the 6 mice, and no more than two different sequences were found in any one animal (Figure 3B). Moreover, clones with the same sequence were often found in more than one tissue site (Figure 3B, brackets). Three clones that retained the parental vlsE sequence were also isolated. These results provide further evidence that the ruvA mutant is severely compromised in its ability to undergo vlsE recombination.

Figure 3. Reduced vlsE sequence diversity generated following inoculation of C3H/HeN mice with the ruvA mutant T11P01A01.

Clones were isolated from C3H/HeN mice 28 days post inoculation with (A) the parental strain 5A18NP1 or (B) T11P01A01. The vlsE cassette region sequences of each clone were optimally aligned and then analyzed for sequence diversity using a phylogenetic tree program. The 5A18NP1 clones were isolated from a single mouse, whereas the T11P01A01 clones were obtained from all positive cultures of 6 inoculated mice. The groups of clones isolated from each mouse and their tissue source are indicated. In (A), brackets demarcate clusters of related (but often nonidentical) clones that occurred in the mouse infected with 5A18NP1. In the 5A18NP1 group, there were 31 unique vlsE sequences, 10 sequences with siblings, and 0 parental sequences. In (B), brackets indicate clones with identical vlsE sequences that were isolated from the 6 mice, frequently from multiple tissues. In this group of clones from mice inoculated with the ruvA mutant, there were 0 unique sequences, 8 sequences with many siblings, and 3 parental sequences. Trees are rooted with the 5A18NP1 parental vlsE sequence. A larger version of this figure is available at the website http://www.uth.tmc.edu/pathology/borreli​a/ .

doi:10.1371/journal.ppat.1000679.g003

The sequences of 17 bp direct repeats at the 5′ and 3′ ends of vlsE central cassette were examined in all of the vlsE sequences recovered from ruvA mutants. No changes were identified indicating that vlsE recombination was not perturbed in the ruvA mutants due to instability of the direct repeats during mouse infection and/or vlsE recombination.

In E. coli and other organisms, ruvA and ruvB act in concert to promote DNA branch migration. Therefore, we also examined the effects of ruvB mutation on vlsE sequence variation in C3H/HeN mice. As shown in Figures 2 and S3 and in Table S1, the ruvB mutant T03TC051 exhibited reduced vlsE recombination rates similar to those observed for the ruvA mutant. No vlsE variants were observed at 7 days post inoculation. On day 28, the vlsE cassette region sequences of 12 to 14 clones from each of three mice were determined. Each animal was found to have only 1 to 3 vlsE variant sequences, and 5 clones with the parental vlsE sequence were isolated from Animal 3 (Figure S3). Only one clone with a unique vlsE variant sequence was identified (1/40, 2.5%). There were subsets of clones with the same vlsE variant sequence, consistent with the outgrowth of siblings of rare vlsE variants (Figures 2 and S3). Thus the ruvB mutant T03TC051 had an infectivity and vlsE variation phenotype that was quite similar to that observed for the ruvA mutant T11P01A01.

Analysis of vlsE recombination events in the ruvA and ruvB mutants

The non-parental vlsE variants recovered 28 days post infection with the ruvA and ruvB mutants were analyzed to determine the length, location and silent cassette source of the recombination events (Figure 4, Figure S2). Each sequence was aligned with parental vlsE sequence, codon formatted and analyzed using a semi-automated analysis program [11]. The program compares each codon to parental and silent vls sequences and depicts the result (identity or no identity) as colored bars with each silent cassette assigned a unique color. Dark regions represent the sequence changes that may have resulted by recombination with that silent cassette. The lighter colored regions are contiguous codons that match both parental and silent cassette sequences and thus represent the possible boundaries of a recombination event.

Figure 4. Low complexity of vlsE recombination events in ruvA and ruvB mutants.

The positions of the six variable regions (VR1-VR6) and six relatively invariant regions (IR1-IR6) within the vlsE central cassette region are provided at the top of the figure. The locations and silent cassette sources of the most likely recombination events for each clone depicted are indicated. A representative clone from a 28 day infection with the parental strain 5A18NP1 is shown at the top. The remainder of the figure shows the vlsE variant clonotypes isolated from C3H/HeN mice 28 days post inoculation with the ruvA mutant T11P01A01 or the ruvB mutant T03TC051. The possible involvement of each of the silent cassettes in sequence variation was analyzed using an Excel®/Visual Basic program, as described previously [11] and depicted in Fig. S2. The horizontal colored bars represent regions of each silent cassette (vls2 to vls16, top to bottom) that match the sequence changes found in the variant clone. Dark regions in each bar correspond to the actual sequence changes, whereas the lighter portion of the bar represents the maximum possible region of that silent cassette exchanged into vlsE to produce the observed sequence change. In isolates from Animal 2 inoculated with the ruvA mutant, a 9 bp untemplated change (black hatched box) that did not match the vls silent cassettes or any other genomic sequence was present in all clones isolated.

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The most likely recombination events are presented in Figure 4; a more detailed presentation showing all silent cassettes potentially involved in the recombination events is shown in Figure S2. Variants recovered from infections with 5A18NP1 at day 28 of infection are usually difficult to assign to a single silent cassette with confidence and appear to have undergone multiple recombination events ([4] and data not shown). For example, the simplest explanation for the variant recovered from a mouse infected with 5A18NP1 (Figure 4, first panel) appears to involve multiple recombinations of varied lengths with 6 different silent cassettes. In contrast, the results obtained with the ruvA and ruvB mutants were consistent with the occurrence of relatively rare and elongated vlsE recombination events (Figure 4). This pattern is exemplified by the vlsE sequence recovered from animal 1, which likely represents a single recombination event with vls5 spanning almost the entire cassette region. Although many silent cassettes have sequence identity to the variant residues, vls5 represents the only continuous match over the entire region (Figure S2). One variant (animal 2) is predicted to have undergone untemplated changes (not matching any vlsE silent cassette) in VR-I followed by an intermittent recombination event with vls2. Several of the recombination events found in the ruvA mutant appear to be intermittent, i.e. the recombination events were discontinuous over the length of the cassette, alternating between the template cassette and vlsE1. This intermittent recombination has been previously observed in vlsE recombination [11]. In addition, we noted that all recombination events from ruvA mutants included variable regions IV through VI. This bias is not observed with the parental strain [4]. Equally striking is length of the recombination events observed in the ruvA mutant. Coutte et al. [11] analyzed 126 clones that had undergone a single recombination event and identified the minimum and maximum predicted lengths of recombination. The majority of these clones (55%) had minimum recombination lengths of 1-5 nucleotides and only 28% had minimum recombination lengths of >15 nucleotides. The variants obtained from ruvA mutant infections, however, had a median minimum recombination length of 168 bp (range 91 to 479 bp), and a median maximum recombination length of 269 bp (range 111 to 546 bp).

Another unusual feature of the recombination events seen in the ruvA mutant is the distribution of silent cassette usage. Only 3 silent cassettes, vls2, vls4, and vls5 were used in the seven templated variants. vls4 and vls5 were each used twice. vls2, the silent gene most proximal to the expression cassette has been observed to be used very rarely in infections with wild type B. burgdorferi, yet we observed it in three of seven templated ruvA variants. These results suggest that the normal mechanism of silent template selection may be perturbed in ruvA mutants.

The vlsE sequences derived from infection with the ruvB mutant displayed similar features to those from the ruvA mutant (Figures 4 and S2). Most sequences were the result of single recombination events and used few vls silent cassette templates. The recombinations observed were unusually long, with a median minimum recombination length of 151 bp (range 119 to 389 bp) and an average maximum length of 179 bp (range 151 to 561 bp). Recombination events always included most of the 3′ end of the recombination cassette; however, these recombinations did not always include VR6, as observed for the recombination events derived from ruvA mutants.

Antibody responses to vlsE in ruvA and ruvB mutants

To determine whether mice infected with the ruvA and ruvB mutants developed a robust antibody response to VlsE, we performed ELISAs on serum from infected animals. We found that serum from animals infected with the ruvA or ruvB mutants displayed more variable antibody responses to VlsE than animals infected with the parental B. burgdorferi strain (data not shown). Some animals displayed similar reactivity to sera from animals infected with parental strain while others displayed lower anti-VlsE activity. However, low anti-VlsE titers did not necessarily correlate with positive B. burgdorferi cultures from harvested organs. It is speculated that low quantities of anti-VlsE antibodies in some mice may correspond to early clearance of B. burgdorferi, thus resulting in a limited stimulation of the immune response against Borrelia antigens, including VlsE.

Attempted complementation of the ruvA mutant

In an attempt to fully demonstrate the role of ruvA in vlsE sequence variation and infectivity, we complemented the ruvA mutant T11P01A01 with the shuttle vector pKFSS1 [35] containing either ruvA alone, ruvAruvB, or ruvAruvBqueApfpB (the four genes in the predicted operon; Figure S1). A 264-bp region upstream of ruvA predicted to include the transcriptional promoter was included in all three constructs. The integrity of the resulting plasmids was confirmed by sequencing the inserts, and transformation of T11P01A01 was demonstrated by streptomycin selection followed by PCR using one primer specific for the pKFSS1 vector sequence and one primer corresponding to the B. burgdorferi DNA insert. Groups of three mice were each inoculated intradermally (10⁵ organisms/mouse) with one of two clones containing each complementation construct, yielding 3×6 = 18 mice inoculated with complemented lones. The two control groups (3 mice each) were inoculated with positive control 5A18NP1 and the non-complemented ruvA mutant T11P01A01. All strains yielded positive cultures from each of 3 skin sites taken distant from the inoculation site on day 7, indicating that the infection was successful in each case. Six representative day 7 cultures were tested for the presence of the complementing plasmids by PCR, and yielded the expected amplicons. All cultures except those from 5A18NP1-inoculated mice were negative on day 14 post inoculation, and on day 28 the proportions of positive clones for the complemented clones were similar to that obtained for the ruvA mutant T11P01A01 (data not shown). In addition, 17 complemented clones isolated on day 7 post inoculation were examined for vlsE recombination, and none exhibited vlsE sequence changes; this result was similar to what had been observed previously with uncomplemented ruvA mutant clones at this time point (Figure 2, Table S1). Therefore attempts to restore infectivity and vlsE sequence variation to wild type levels by complementation have thus far been unsuccessful.

Inactivation of ruvA does not result in increased sensitivity to DNA damage

In many microorganisms, ruvA is important in the repair of DNA damage resulting from UV irradiation or chemical mutagens. To assess the role of B. burgdorferi ruvA in DNA repair we monitored the survival of the ruvA mutant T11P01A01 subjected to DNA damaging conditions. Log phase cultures were irradiated with increasing doses of UV (254nm) light, and cultured to determine spirochete survival by colony counting. Figure 5A shows the results of a representative experiment. The parental strain 5A18NP1, and ruvA mutants had a similar number of colonies surviving at all UV doses, indicating that ruvA mutants do not have increased sensitivity to UV irradiation.

Figure 5. The ruvA mutant T11P01A01 does not exhibit enhanced sensitivity to UV light or mitomycin C.

(A) Survival of ruvA mutant and parental strain 5A18NP1 after exposure of UV light. The dosages of UV irradiation (254 nm) applied to the bacterial suspensions are indicated. (B) Survival of ruvA mutant and parental strain 5A18NP1 after 14 h treatment with the indicated doses of mitomycin C in BSK II medium. Colony counts on duplicate or triplicate plates were utilized to determine the concentration of surviving B. burgdorferi. Results shown are for representative experiments from three independent studies for each treatment.

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Mitomycin C causes DNA damage by crosslinking complementary DNA strands at CpG sequences and is a commonly used reagent to examine the cellular response to DNA damage. To determine the sensitivity of the ruvA mutants to mitomycin C we assessed the viability of the ruvA mutant T11P01A11 and the parental strain 5A18NP1 after 14 hours exposure to increasing doses of mitomycin C. Treated cultures were plated and the number of colonies visible after 2-3 weeks incubation was used to assess spirochete survival. Figure 5B shows the survival of B. burgdorferi colonies after exposure to varied concentrations of mitomycin C. We observed no differences in survival between the ruvA mutant and the parental strain 5A18NP1.

Discussion Top

We conducted a genetic screen for trans-acting factors involved in vlsE antigenic variation in B. burgdorferi. Of the seven genes examined in this screen, four exhibited diverse PCR-RFLP patterns of vlsE, indicating that the inactivated genes are not centrally involved in vlsE recombination in C3H/HeN mice. Mutant MG065 in BB0098 (annotated as a recombination and DNA strand exchange inhibitor and as mutS-II in other organisms) was not recovered from inoculated mice, and thus could not be evaluated for vlsE variation. Two of the remaining transposon mutants, which had disruptions in the B. burgdorferi orthologs of the Holliday junction helicase genes ruvA and ruvB, had reduced vlsE recombination. The only other mutant that exhibited reduced infectivity and PCR-RFLP diversity was a mutS mutant, T05P01C02. A second mutS mutant, T10P01D06, had an insertion site near the end of the gene (insertion ratio 0.97); this clone had normal infectivity and RFLP results, indicating that a functional product is likely produced. The possible role of mutS in vlsE recombination will be investigated further in a separate study.

The predicted proteins encoded by the B. burgdorferi BB0023 and BB0022 open reading frames have a high degree of homology with the Holliday junction helicase proteins RuvA and RuvB, respectively, of other organisms. In Escherichia coli, the 22-kDa protein RuvA specifically targets RuvB to the Holliday junctions [36],[37], and the combined action of RuvAB results in branch migration during homologous recombination [38]. The 19-kDa RuvC resolvase cleaves the Holliday junction and is required for resolution of Holliday junctions in E. coli [39]. No homolog of RuvC has been identified in Lyme disease Borrelia, although RuvC orthologs have been identified in other spirochetes including the relapsing fever organism B. turicatae, T. pallidum, T. denticola and L. interrogans. Aravind et al. has proposed that a family of predicted LE exonucleases may substitute for RuvC in B. burgdorferi [40]. In addition to their role in recombination, E.coli ruv mutants exhibit defects in DNA repair such as increased sensitivity to UV light and mitomycin C [41],[42].

The infectivity phenotypes of the ruvA mutant T11P01A01 and the ruvB mutant T03TC051 resemble those of B. burgdorferi strains that lack lp28-1 [14],[15],[17],[18] or the vls locus [19], although some differences exist. As shown in Tables 2 and 3, the ruvA and ruvB mutants exhibited lower culture positivity than the parental strain 5A18NP1 in immunocompetent C3H/HeN mice. The ruvA mutant culture positivity pattern was the same as the lp28-1^– clone 5A8 on day 14 post inoculation in C3H/HeN mice, with only joint specimens yielding positive cultures (Table 3); however, 7/20 cultures in diverse tissues were positive for the ruvA mutant at day 28 post inoculation, as compared to a lack of positive cultures in the 5A8-inoculated animals. The ability of some ruvA^– organisms to survive is most likely due to the ability to express VlsE and to undergo limited vlsE recombination. The ruvA mutant and the lp28-1^– clone 5A8 is able to infect all tissues in C3H/scid mice, as has been observed previously with lp28-1^– strains and clones in which the vls locus was deleted from lp28-1 by telomere-mediated plasmid truncation [19]. Thus mutation of ruvA results in reduced ability to survive in the presence of the adaptive immune system, perhaps due to antibody responses against invariant or limited variation forms of VlsE.

We also tested the ability of B. burgdorferi carrying ruvA or ruvB mutations to survive in ticks. B. burgdorferi were readily recovered from both fed and unfed ticks suggesting that the ruvA and ruvB mutations do not abrogate the ability of B. burgdorferi to survive in ticks. However, the mutant spirochetes did not establish infection in C3H/HeN mice following tick inoculation (Table 4). These data suggest that the lack of detectable organisms at day 28 post mouse inoculation (Table 4) was due to the effects of ruvA and ruvB mutations on survival in mice rather than poor replication in ticks or transmission from ticks to mammals. The failure to recover organisms in cultures derived from mouse inoculation via ticks may be due to the relatively low number of organisms delivered during tick inoculation as compared to the 10⁵ Borrelia per mouse used in the needle inoculation studies.

The reduced ability of the ruvA mutant T11P01A01 to undergo vlsE recombination is illustrated in Figures 3, 4, and S3. Figure 3 shows that vlsE variants were not detected on days 7 and 14 in C3H/HeN mice inoculated with the ruvA mutant; while vlsE variants are predominant on day 28, only one or two variant clones are detected in each mouse (Figures 4 and 5). Remarkably, no vlsE variants were detected at any time point following infection of C3H/scid mice with the ruvA mutant, whereas all of the clones examined at 28 days post inoculation were variants when C3H/scid mice were inoculated with the parental 5A18NP1 strain. Combining the reduced culture positivity results with the decreased vlsE variant diversity observed with the ruvA mutant, the following is evident: 1) inactivation of ruvA results in reduction of vlsE recombination rates, resulting in only a few variants in C3H/HeN mice and the lack of detected variants in C3H/scid mice; and 2) in C3H/HeN mice, the few clones that have undergone vlsE recombination are able to survive after the anti-VlsE response is activated, whereas nearly all of the parental clones are eliminated by day 28 post inoculation.

The types of recombination events observed in the ruvA mutant were also quite different than those observed in wild-type B. burgdorferi. The average length of observed recombination events was an order of magnitude larger than in wild type clones. Moreover, most of the predicted recombination events observed in the ruvA mutant also appeared to represent intermittent recombination events, with regions of recombination interrupted by stretches of the unchanged, parental vlsE sequence (Figure 5). Finally, vls2, vls4, and vls5 were the only silent cassettes utilized in the ruvA mutant. The ruvB mutant also exhibited a limited repertoire of silent cassette usage (cassettes 2, 3, 4, 5, and 8). Taken together, these results may indicate that the recombination process is different in the ruvA mutant than in wild-type organisms; i.e., the observed changes could involve a different, underlying recombination mechanism other than the as yet cryptic, vlsE-specific process that appears to be induced during mammalian infection.

We found that sensitivity of B. burgdorferi to UV radiation and mitomycin C exposure was not affected by ruvA mutation. In other bacteria such as E. coli, deficiencies in RuvA, RuvB, or RuvC result in increased sensitivity to DNA-damaging agents, including UV irradiation and mitomycin C [43]. UV irradiation causes intra-strand thymine dimers in DNA, and the damage is repaired by RecA-mediated SOS response [44]. Mitomycin C exposure induces inter-strand cross-link in DNA, and an extended SOS response is needed to repair the damaged DNA [45]. Induction of genes encoding DNA repair enzymes during the SOS response is one of the most important DNA repair system in bacteria [46]. However, the B. burgdorferi genome does not encode a homolog of the SOS response repressor LexA, and SOS boxes (LexA binding sites in promoter regions) are not present upstream of recA and other genes that comprise the SOS regulon [47]. Also, expression of recA is not induced following UV exposure [48]. Thus B. burgdorferi appears to lack an SOS regulon. As an obligate parasite, B. burgdorferi is never exposed directly to UV light, and thus may have lost its ability to upregulate genes involved in the repair of DNA damage caused by radiation and other agents.

Thus far, our attempts to restore full infectivity and vlsE recombination through complementation with ruvA in the shuttle vector pKFSS1 have been unsuccessful. In addition, we have also tried using constructs containing ruvAruvB and ruvAruvBqueApfpB, in case the transposon insertion in ruvA has some polar effect on the expression of downstream genes. A 264-bp region upstream of ruvA was used in all of these constructs; it is presumed to contain the promoter, although the region lacks a strong consensus promoter sequence (e.g. -35 and -10 sequences with the proper spacing). BB0024, the gene upstream of ruvA, is oriented in the opposite direction, indicating that ruvA is the first gene in the operon. Dresser et al. [49] have also been unsuccessful in trans complementing ruvA and ruvB mutants obtained by allelic exchange. The ruvA and ruvB mutants obtained by our laboratory and by Dresser et al. [49] were derived independently by different means (transposon mutagenesis and gene disruption by allelic exchange), yet have nearly identical phenotypes. Thus it is likely that the lack of complementation observed in these studies is due to technical complications, as has been commonly observed in genetic studies involving infectious B. burgdorferi. We will continue our efforts to complement the ruvA and ruvB mutants in an attempt to fulfill the ‘molecular Koch’s postulates’ on the roles of these genes in infectivity in immunocompetent mice and in vlsE recombination.

The current study along with those by Liveris et al. [31] and Dresser et al. [32] provide some insight into the mechanism of recombination in the vls antigenic variation system. The central role of RuvAB indicates that heteroduplex formation with branch migration is important in this process. The lack of a requirement for RecA [31],[32] is surprising. RecA provides two important functions in other microorganisms: ATP-dependent formation of RecA-single-stranded DNA nucleoprotein filaments that facilitate the homologous base pairing during heteroduplex formation [50]; and cleavage of LexA to activate the SOS response through its activity as a co-protease when bound to single-stranded DNA. As indicated above, B. burgdorferi lacks a recognizable LexA ortholog, so the latter function is likely to be unimportant (although co-protease activity is present in B. burgdorferi RecA). Liveris et al. [31] state that the apparent lack of effect of recA mutation on vlsE recombination indicates that homologous recombination mechanisms are not involved in this process. However, ‘templated’ vlsE sequence changes (those involving incorporation of silent cassette sequences) consistently occur at the same position as the optimal alignment between the silent cassette and vlsE; i.e., the observed vls silent cassette/vlsE recombinations are always homologous, and never nonhomologous [11]. vlsE recombination is induced during mammalian infection; it is possible that factor(s) that bind specifically to vls sequences and promote strand invasion and heteroduplex formation may be expressed under these conditions and hence fulfill a RecA-like (but site-specific) role. Such factor(s) have not been identified to date. Once a heteroduplex is formed, RuvAB may promote the migration of the heteroduplex branch point, thus extending the region of strand exchange. In a set of 126 vlsE variants that appeared to contain a single recombination event [11], the putative minimal recombination events ranged from 1 nt to 349 nt, with a median value of less than 5 nt. This result indicates that the strand replacement process is often interrupted quite soon after its initiation. The observed range of sequence identity between donor and recipient sequences flanking the region of sequence change was from 0 nt to 88 nt, with (remarkably) as little as 6 nt on one side or the other [11]. It is currently unknown whether the role of the RuvAB migrase is to promote heteroduplex formation in these regions of sequence identity (thus nucleating the strand exchange event), or whether it is also able to ‘drive’ the strand pairing through regions of sequence differences.

Gene conversion has been described as the predominant mechanism in other bacterial and protozoal antigenic variation systems, including those of relapsing fever Borrelia (e.g. B. hermsii), Neisseria gonorrhoeae, Anaplasma marginale, Trypanosoma cruzi, and Babesia bovis [51],[52],[53],[54]. Lyme disease and relapsing fever Borrelia are closely related, and VlsE and the variable large protein (Vlp) antigenic variation proteins have some sequence similarity. However, in B. hermsii the gene conversion events result from homologous recombination within well-demarcated upstream homology sequences (UHS) and downstream homology sequences (DHS), resulting in replacement of nearly the entire expression site gene with a silent gene sequence [53],[55]; therefore it is different from the vls system both in terms of mechanism and gene conversion outcome. The N. gonorrhoeae pilE system, the A. marginale MSP2, and the B. bovis VESA1a systems each involve segmental gene conversion events utilizing multiple silent gene copies to produce highly variable pilin or surface protein sequences. The pilE system is dependent upon RecA and other recombination system proteins that are not required for vlsE recombination, as described in detail by Dresser et al. [32]. In A. marginale, the gene conversion event is almost always ‘anchored’ in a conserved region flanking the hypervariable region [56]. The mechanisms of B. bovis VESA1a recombination are not well characterized, but are likely to involve eukaryote-specific elements that will differ considerably from those present in the prokaryote B. burgdorferi. Thus, based on current knowledge, the vls system may represent a unique gene conversion process that is mechanistically dissimilar to other known antigenic variation systems.

Materials and Methods Top

Ethics statement

All procedures involving mice conducted at the University of Texas Health Science Center at Houston were reviewed and approved by the Animal Welfare Committee of that institution. All mouse studies conducted at the Tulane National Primate Research Center were reviewed and approved by its Institutional Animal Care and Use Committee (IACUC).

Bacterial strains and growth medium

The transformable, infectious Borrelia burgdorferi B31 clone 5A18NP1 was used for generation of all mutants. 5A18NP1 is a genetically modified clone in which plasmids lp28-4 and lp56 are missing and BBE02, encoding a putative restriction-modification enzyme, has been disrupted [33]. Borrelia burgdorferi B31 clone 5A8, containing all plasmids except lp28-1, was isolated previously from the low-passage strain B31 [15],[57] and was used as negative control in mouse inoculation experiments. All strains used in this study had undergone no more than two subcultures since clone isolation prior to infectivity studies. B. burgdorferi were grown at 34°C in 3% CO₂ in Barbour-Stoenner-Kelly II (BSK-II) medium supplemented with appropriate antibiotics as described previously [58]. The in vitro growth rates of the ruvA and ruvB mutants and the parental strain 5A18 NP1 were determined by daily quantitation of organisms in triplicate cultures by dark-field microscopy. E. coli TOP10, a DH5α^TM-derived strain obtained from Invitrogen Corporation (Carlsbad, CA, USA), was used for the preparation of plasmids for electroporation into B. burgdorferi.

Inactivation of DNA recombination and repair genes

Genes putatively involved DNA recombination and repair were inactivated by transposon mediated mutagenesis as part of a transposon signature tagged mutagenesis (STM) study on-going in the laboratory. This study will be described in detail in another publication (Lin, T., L. Gao, C. Zhang, E. Odeh, and Norris, S.J., manuscript in preparation). Briefly, twelve independent mutant libraries, each having a unique 7 bp sequence tag, were created using modified versions of the suicide plasmid pMarGentKan. This plasmid was graciously provided by Dr. P. E. Stewart (Rocky Mountain Laboratories, National Institutes of Health, Hamilton, MN) and is a modified version of pMarGent [59] in which a kanamycin resistance cassette (flaB::aph1) was inserted in the ‘backbone’ of the vector, outside the himar1-based transposable element and a flgB:aacC1 gene is inserted within the transposable element (P. E. Stewart, unpublished data). After insertion of unique sequence tags into pMarGentKan, 5 µg of each plasmid was electroporated into B. burgdorferi B31 using a modification of previously described methods [59],[60]. The transformants were incubated in BSK-II medium without antibiotics for recovery overnight hours and plated in solid BSK-II media with 200 µg/ml of kanamycin and 40 µg/ml of gentamicin as described previously [57]. Colonies were selected and cultured in liquid BSK-II medium until mid-log phase prior to addition of 15% (v/v) glycerol and storage at -70°C. The transposon insertion site was determined by restriction digestion of the Borrelia genomic DNA, plasmid rescue in E. coli, and sequencing as described previously [59]. Properties of the transposon mutants selected for this study are shown in Table 1.

Determination of plasmid content

The plasmid profiles of DNA recombination and repair mutants were determined by a microtiter plate-based PCR method as previously described [15] or by a multiplex PCR scheme followed by detection using Luminex® FlexMAP^TM technology. The Luminex® procedure will be described in detail in another article (S. J. Norris, J. K. Howell, E. Odeh, T. Lin, and D. G. Edmondson, manuscript in preparation). Briefly, multiplex PCR reactions were performed to amplify plasmid-specific regions. The resultant PCR reactions were treated with exonuclease I and shrimp alkaline phosphatase (Exo/SAP, U. S. Biologicals) to remove excess nucleotides and primers, and then subjected to allele specific primer extension (ASPE) in the presence of biotin-dCTP using primers specific for individual plasmid products. The 5′ end of the ASPE primers contains an xTAG® universal tag sequence and the 3′ end of the ASPE primers contains the plasmid-specific sequences. The 5′ universal tag sequence was hybridized to the complementary anti-tag sequence coupled to a particular xMAP® bead set and biotin labeled DNA was detected with PhycoLink® Streptavidin-R-Phycoerythrin (SAPE). Detection and analysis was carried out using the Luminex® 200^TM System (Luminex Corporation, Austin, TX), and samples were scored as plasmid positive or negative based on the median fluorescence intensity values for each probe set.

Mouse infection studies

Clones with transposon insertions in DNA recombination and repair genes were tested individually for infectivity in 4-week-old C3H/HeNHsd (wild type) by needle inoculation (Table 1). Mutant clones, the parental strain 5A18NP1, and clone 5A8 were cultured to mid-log phase, and groups of 4-5 mice were inoculated with 1×10⁵ organisms subcutaneously at the base of the tail as described previously [57]. Skin biopsies were collected aseptically at day 4, 7, 14 post inoculation. The mice were sacrificed at day 28 post inoculation, and skin, tibiotarsal joint, heart, and urinary bladder were collected. The tissue specimens were cultured in 6 ml BSK II medium containing kanamycin and gentamicin at 34°C in 3% CO₂. The cultures were checked for spirochetes by dark-field microscopy at 2 and 4 weeks, and positive cultures were diluted and subsurface plated in 0.7% solid BSKII -agarose medium to isolate individual colonies. For selected mutants, similar studies were performed in 5-week-old, female C3H.C-Prkdĉscid/ICRSmnHsd SCID mice (Harlan, Indianapolis, IN). For clarity, this mouse strain is referred to as C3H/scid in this article. Anti-VlsE responses were assessed by ELISA as described previously [61].

Tick inoculation studies

B. burgdorferi mutant and wild-type strains were grown in BSK-II medium that was supplemented with 6% rabbit serum (Pel-Freez Biologicals) and 45.4 µg/ml rifampicin, 193 µg/ml phosphomycin, 0.25 µg/ml amphotericin, 200 µg/ml kanamycin (all from Sigma-Aldrich). Gentamicin (Gibco) at 50 µg/ml was added to the mutant strain cultures only. Ixodes scapularis nymphal ticks from the Tulane National Primate Research Center tick colony were capillary fed with culture medium that contained the ruvA mutant, ruvB mutant, or the 5A18NP1 wild type B. burgdorferi, each at a concentration of about 9×10⁷ cells/ml. Capillary feeding was performed by a procedure described previously [20]. Ticks were then allowed to rest for 21 to 25 days in a humidified environment at 22°C. Ten flat (unfed) nymphs from each group were surface disinfected and crushed in 30 µl of sterile PBS. Half of this volume was cultured in 5 ml of BSK II medium, supplemented with antibiotics as above, for a total of 12 weeks, and monitored weekly after the second week. . The remaining half was distributed on microscope slides for quantitation of spirochetes (see below).

The remaining flat ticks were placed on mice. Three 8-10 week-old C3H/HeN female mice were used for each B. burgdorferi strain, and each mouse received 10-12 nymphs. Engorged (fed) ticks (about 75% of the initial number) were collected within 6 days, and cleaned, crushed, and cultured or prepared for DFA as described above. All of the mice were euthanized 4 weeks after the ticks had dropped off, and heart, bladder, one ear, and one tibiotarsal joint were collected from each animal. In each case, half of the organ sample was snap frozen in liquid nitrogen and stored and the other half was placed in culture for 8 weeks.

Immunofluorescence

A direct fluorescent antibody (DFA) assay was performed to evaluate survival of each B. burgdorferi strain in ticks as described previously [62]. Briefly, tick smears from flat and fed ticks (see above) were air-dried on glass slides, acetone fixed and stored at -20°C until DFA examination. Slides were incubated with 40 µl of a 1:10 dilution of fluorescein-labeled anti-Borrelia species antibody (Kirkegaard & Perry Laboratories (Gaithersburg, MD)) for 30 minutes at 37°C. Following incubation, the slides were washed in phosphate-buffered saline and examined by fluorescence microscopy (magnification, 600X). For unfed ticks and for each spirochetal strain, spirochetes in 10 microscope fields were counted in each of 10 ticks per strain. For fed ticks, between 5 and 10 fields were counted in 24-26 ticks per strain. The mean number of spirochetes per field and the standard deviations are presented in Table 4. Statistical significance (p<0.05) was assessed by ANOVA.

RFLP analysis and sequencing of amplified vlsE cassette regions

As a screen for vlsE sequence variation, cultures obtained from mouse infections were subjected to vlsE cassette region amplification followed by RFLP analysis. Five µl of unpurified culture was used as the DNA template. The vlsE expression cassette was amplified by using primers 4066 and 4120 [6] and the Phusion^TM High-Fidelity DNA Polymerase (Finnzymes, Inc. Woburn, Massachusetts, USA). PCR reactions were performed in volumes of 50 µl containing 10 µl of 5× HF buffer with 7.5 mM MgCl₂, 1 µl of 10 mM dNTPs, 1 µl of 25 mM primers, and 1 U of Phusion^TM High-Fidelity DNA Polymerase. PCR reactions were performed in a Eppendorf Mastercycler® thermocycler (Foster City, California, USA), using the following conditions; 98°C for 2 min followed by 30 cycles of 1) denaturation at 98°C for 10 sec, 2) annealing at 61°C for 20 sec, and 3) extension at 72°C for 45 sec and a final extension at 72°C for 10 min. For RFLP analysis, 10 µl of PCR product was digested with 2.5 U of the restriction endonuclease HphI (New England BioLabs, Ipswich, MA) for 2 hrs at 37°C in 10×NEBuffer 4. The digests were separated in a 2% (w/v) agarose gel at a 100V constant voltage for 1.5 hrs in the presence of ethidium bromide. The Hi-Lo^TM DNA marker (Bionexus, Oakland, CA) was used as a molecular size marker. Gels were imaged with UV light illumination.

For sequence determination, the PCR products from individual clones were amplified as described above, purified using the Qiaquick® PCR purification kit (Qiagen), and sequenced on both strands at the High-Throughput Genomics Unit (Department of Genome Sciences, University of Washington, Seattle), using the same primers used for PCR amplification. Each DNA sequence was compared with their corresponding chromatographs and the parental vlsE sequence to verify the quality and accuracy of the sequence data.

vlsE sequence analysis

The sequence of the vlsE cassette region of clone 5A18NP1 (GenBank GQ369288) is identical to that of B. burgdorferi B31-5A3, from which the vlsE sequence (U76405) was derived initially [6]. All B. burgdorferi clones and the GenBank numbers corresponding to their vlsE cassette region sequences are listed in Table S3. For simplicity, the clone names refer to all clones resulting from T11P01A01 infection as ruvA1 and all T03TC051 derivatives as ruvB1. All clones resulting from infection with the parental clone are given the prefix 5A18NP1. The clone names (e.g. RuvA1SD14M1S2) are in the following format: infecting strain name; S indicates C3H/scid mice (all others are C3H/HeN mice); D7, D14 or D28 = 7, 14 or 28 days post inoculation; mouse number (M1, M2, etc.); S, E, J, H, B = skin, ear, tibiotarsal joint, heart, and bladder, respectively; and the clone number from that tissue. The clone numbers and their GenBank accession numbers are also provided at the website http://www.uth.tmc.edu/pathology/borreli​a/ .

vlsE sequence analysis of variants was performed as described previously [11]. The output of this Excel®/Visual Basic-based program is a color-coded map of possible vlsE recombination events, as shown in Figure 4 and Figure S2. The minimum and maximum deduced regions of recombination were determined by analyzing the longest contiguous recombination event in the variant sequence. The relationships among clones with different vlsE sequences were displayed graphically as phylogenetic trees. Phylogenetic trees were constructed using the online phylogenetic program Phylogeny.fr [63], based on the aligned parental and variant vlsE sequences.

Sensitivity to UV radiation and mitomycin C

The ruvA mutant T11P01A01 and the parental strain 5A18NP1 were tested for UV sensitivity using a modification of the method described by Miller, et al. Cultures were grown to a density of 1×10⁷ organisms/ml. The spirochetes were centrifuged and resuspended in phosphate buffered saline (PBS) at a concentration of 1×10⁸ organisms/ml, and 0.1 ml of the suspensions was rapidly aliquoted into 24-well plates. The bacteria were irradiated with UV light (254 nm) using a Spectrolinker XL-1000 UV cross-linker (Spectronics, Westbury, N.Y.) at doses of 0, 3.0, 4.5, 6.0, 7.5 and 9.0 mJ/cm². The UV treated bacteria were protected from room light, diluted in BSK-II and cultured in BSK-II plates in duplicate or triplicate, at a concentration of 100 organisms per plate. The colonies were counted after 2 weeks incubation.

For determination of mitomycin C sensitivity, fresh cultures of Borrelia were grown to mid-log phase and diluted to a density of 1×10⁷ organisms/ml. Mitomycin C (Sigma-Aldrich, St. Louis, MO) was added to 1 ml cultures at final concentrations of 0, 15, 30, 60, 120, 240, 480, 960, 2000, and 4000 ng/ml, and the cultures were incubated at 34°C in 3% CO₂. The concentration of motile spirochetes was determined by dark-field microscopy at 2, 4, 6, 8, 12, 16, and 24 hours. At 14 hours, the cultures were serially diluted in BSK-II to obtain concentrations 1000 cells/ml, and 0.1 ml portions of each dilution were cultured in BSKII plates with antibiotics as described above. Each culture was plated in duplicate and incubated 7-10 days at 34°C. Mitomycin C sensitivity was measured by counting the surviving colonies. Untreated cells grown in an identical manner served as controls.

RT-PCR analysis of ruvA operon

RNA was isolated from B. burgdorferi using RNA-Bee® (Tel-Test, Inc., Friendswood, Texas). Borrelia cultures were grown to a concentration of approximately 5×10⁷ organisms/ml. Spirochetes from 1 ml of culture were sedimented by centrifugation and resuspended in 1 ml of RNA-Bee®, and RNA isolation was carried out as per manufacturer’s instructions. The resulting RNA was quantitated by UV spectroscopy, checked visually on an agarose gel, and then treated with DNase I (RNase-free, New England Biolabs) following the manufacturer’s suggested procedure. Reverse transcription was performed using 200 ng of RNA and primers specific to the ruvA operon following the two-step protocol found in the Enhanced Avian HS RT-PCR kit from Sigma-Aldrich. Subsequent PCR reactions used two µl of the resultant cDNA, 25 µM of specific primers and 2X PCR mix from New England Biolabs. Primer sequences and reaction conditions are shown in Table S2.

Supporting Information Top

Figure S1.

Gene arrangement and transcription patterns of the ruvAB locus in the parental clone 5A18NP1 and the ruvA mutant T11P01A01. (A) The ruvAB locus, with presumed cotranscribed genes queA and pfpB. The location of the transposon insertion site in the ruvA mutant T11P01A01 is shown. (B) RT-PCR analysis of transcription of the ruvAB locus of 5A18NP1 and T11P01A01, using the primer pairs shown in (A). RT = with reverse transcriptase, no RT = without reverse transcriptase.

(0.26 MB PDF)

Figure S2.

Detailed analysis of the possible recombination events in the vlsE variant clonotypes isolated 28 days post inoculation of C3H/HeN mice with the ruvA mutant T11P01A01 and the ruvB mutant T03TC051. The method of analysis is described in detail in Ref. [11]. Briefly, the horizontal colored bars represent regions of each silent cassette (vls2 to vls16, top to bottom) that may have contributed to sequence changes found in the variant clone. Dark regions in each bar correspond to the regions of sequence changes, whereas the lighter portion of each bar represents the maximal possible region of that silent cassette that could have been exchanged into vlsE to produce the observed sequence change. The locations and silent cassette sources of the most likely recombination events are marked by a red dashed box.

(0.65 MB PDF)

Figure S3.

Reduced vlsE sequence diversity generated following inoculation of C3H/HeN mice with the ruvB mutant T03TC051. Clones were isolated from three C3H/HeN mice 28 days post inoculation with 10⁵ T03TC051. The vlsE cassette region sequences of each clone were optimally aligned and then analyzed for sequence diversity using a phylogenetic tree program. The groups of clones isolated from each mouse and their tissue source are indicated. Trees are rooted with the 5A18NP1 parental vlsE sequence.

(0.08 MB PDF)

Table S1.

The B. burgdorferi ruvA mutant T11P01A01 and the ruvB mutant T03TC051 exhibit decreased vlsE sequence variation during infection of C3H/HeN and C3H/SCID mice.

(0.01 MB PDF)

Table S2.

Primers used in RT-PCR of the ruvAB locus.

(0.01 MB PDF)

Table S3.

B. burgdorferi isolates generated in this study.

(0.43 MB PDF)

Acknowledgments Top

We thank D. Scott Samuels for providing the shuttle vector pKFSS1, Jerrilyn K.Howell for helpful suggestions and assistance with the animal studies, and Douglas J. Botkin for advice on vlsE sequence analysis. We also thank Ashley R. Dresser, Pierre-Olivier Hardy, and George Chaconas for sharing their results prior to publication.

Author Contributions Top

Conceived and designed the experiments: TL LG DGE MTP SJN. Performed the experiments: TL LG DGE MBJ. Analyzed the data: TL LG DGE MBJ MTP SJN. Contributed reagents/materials/analysis tools: TL LG SJN. Wrote the paper: TL DGE MTP SJN.

References Top

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While it is not unusual for a person to respond to a particular, single treatment, most often what seems to be an incorrigible health problem must be tackled from many different sources at once, say, nutritional, medical, Candidiasis treatment, allergy treatment, chelation therapy, and so on, depending upon the person and the problem.

The unlucky invasion of Borrelia burgdorferi, the spiral-shaped microbe injected by at least one species of tick, Ixodes scapularis, seems to present the unwitting victim with arthritic symptoms that also may require more than one approach for its solution. Thankfully, if diagnosed early enough, antibiotics can easily wipe out the invading population, and bring about swift remission. The antibiotics, of course, should be heavily accompanied with Lactobaccilus acididophilus1, so that while treating the Lyme Disease, we do not also unwittingly bring about a fungal infection of Candidiasis.

There are about 1,200 cases of Lyme Disease reported across the United States each year; there were 1,282 cases reported in 19934.

The disease remains concentrated along the coastal plain of the Northeast and mid-Atlantic region, in the upper Midwest, and along the Pacific coast, although the disease has been reported in 32 states.

In a Science News report, researchers at the University of Connecticut Health Center in Farmington and the Yale-New Haven Hospital examined 70 children diagnosed with Lyme Arthritis Disease and found that only 53% actually harbored the Lyme-causing bacterium Borrelia burgdorferi. The remaining 47% had been misdiagnosed.4

There is some hysteria regarding the incidence of this disease, possibly due to extensive adverse publicity. One thousand two hundred and eighty-two cases out of perhaps several hundred thousand with tick bites is not exactly a national emergency, although for a percentage of those afflicted, the disease can be rather significant and even catastrophic.

Lyme disease has the following symptoms: begins with reddened area that doesn’t itch, resulting from tick bite, but expands over time, measuring several inches across; clearing of bite area begins in center resembling a bull’s-eye; flu-like symptoms: chills, fever, fatigue, joint and muscle pain; may develop a rash which disappears in a few days; may have tingling and numbness; non-symmetrical joint problems; other symptoms may also occur; sometimes sensitivity to light, stiff neck, headache, sleepiness, mood changes and memory loss; swelling and aching joints for months or years at a time; and vague, migrating musculature pains.

The characteristics of Lyme Disease were first laid out in 1975 when two mothers were told that their children had Juvenile Rheumatoid Arthritis. The name “Juvenile” does not distinguish it’s clinical pattern from that of “Adult” Rheumatoid Arthritis, but merely tells the parent that this horrible, crippling disease occurred in their child, a fact that most parents already know. What is new is the diagnoses of “Rheumatoid Arthritis.”

These two mothers soon learned that many other children and adults in their geographical region were afflicted with the same symptoms, and since Rheumatoid Arthritis does not seem to cluster in a regional geography (with some exceptions), Dr. Stephen E. Malawista of Yale University, among others, began to look for a source of this apparently new disease. Dr. Malawista discovered that many of his patients suffered from a range of symptoms among which might be those that resembled Rheumatoid Arthritis.

The cause of Lyme Disease was determined to be a microbe transmitted by a tick, in this first instance, from the species Ixodes capularis. Since this tick was common in the grasses and woods near Lyme, Connecticut, the cluster of symptoms obtained the name “Lyme Disease.”

As Dr. Willy Burgdorfer, who worked for Rocky Mountain Laboratories in Hamilton, MT, identified the damaging microbe, the bacteria was named Borrelia burgdorferi, which is a spiral-shaped bacterium similar in shape to the spirochete, Treponema pallidum, which causes syphilis.

Since this initial set of discoveries, it’s clear that similar diseases have existed in Australia, Africa, Europe and Asia. It also appears in every one of the states in the United States, but seems to be particularly common in northern California, Minnesota and the northeast.

Infection by Borrelia burgdorferi occurs chiefly in the spring, summer or early fall, because of the life cycle of the Ixodes scapularis tick.

Three Stages of Borrelia burgdorferi

There are three stages to the life cycle of the tick, and at each stage they have a favorite host, although they will attach themselves to a range of animals, including the human species.

The larva from Ixodes scapularis emerges in the summer from eggs deposited in the spring, and attaches itself to a small vertebrate such as a white-footed mouse, where it imbibes its first meal. If this mouse is infected with Borrelia burgdorferi spirochetes, the larva feeding on the mouse’s blood will also become infected.

Later, the larva molts into a nymph, and during the spring and summer (usually mid-May through July) this nymph takes a second meal. If the larva was infected, it may very well pass Borrelia burgdorferi onto its second host. This nymph is now about the size of a small seed, say, a poppy seed, and is responsible for most human infections.

The nymph molts again, and by October is the size of a larger seed, like an apple’s. Again this tick feeds, at least by winter or spring, and they also mate to produce eggs that begin the cycle all over again. Usually ticks do their mating on white-tailed deer, which is why they are referred to as a “deer tick”.

In some regions of the United States, between 15 and 30 percent of the Ixodes scapularis nymph and adult ticks are infected with Borrelia burgdorferi — some 50 percent of adult ticks are infected. The adult ticks are more likely to infect humans because they have had more opportunity, throughout their life-cycle, to do so.

About 1 to 3 percent of adults who are bitten by the infected tick contract Lyme disease, meaning that a high percentage of those infected are able to master the infection.

The tick attaches itself to the skin of its host, where it takes its meal of blood. At this time Borrelia burgdorferi begins to multiply in the gut of the tick, whence it crosses into the tick’s circulation system, migrating to the salivary glands and passing with the tick’s saliva through the host’s skin.

A tick must be attached to its host for 36 to 48 hours before an infectious dose of Borrelia burgdorferi is transmitted. This is fortunate, because most folks who are bitten by a tick will find it prior to the infectious event.

Lyme Disease Symptoms

Those infected by the bacteria Borrelia burgdorferi usually have a set of characteristic symptoms:

Stage I Symptoms

About 60% will notice a round rash called an erythema chronicum migrans (ECM), as doctors like to have a nice, neat name for everything they observe.
Three days to a month later there will be a redness at or near the site of the tick bite.
The reddened area does not itch or hurt, but it will expand over time until it may measure several inches across.
There is a clearing that begins in the center, as the rash enlarges, resembling a bull-eye. Some may acquire the rash, but fail to see these characteristics because of the bite’s location.
The rash may disappear within weeks or even days.
Days or weeks later, a variety of other early symptoms affecting many areas of the body appears, and these symptoms are thought to be from the spread of the spirochete to many different tissues through the blood stream. The symptoms will include flulike symptoms, such as chills, fever, fatigue, joint and muscle pains, and loss of appetite.
Stage II Symptoms

Weeks to months later, about 10% of those afflicted will experience transient heart dysfunction. There will be varying degrees of heart blockage. Neurologic abnormalities include headaches, profound fatigue, meningitis, cranial nerve problems (neuropathies), including fascia palsies, and sensory and motor nerve problems.

Cardiac problems occur with 5 to 10 percent of those infected, if they have been untreated. Usually this condition is not noticed by the infected person, but can be detected by a physician. The heart irregularities persist for but a week to 10 days and probably will not require the use of a pacemaker.

Early symptoms may also include mild musculoskeletal disturbances, where patients complain of vague, migrating pain without swelling in muscles, tendons or joints. The jaw, the temporomandibular joint, may be affected. These symptoms, too, will decrease in weeks to months.

However, in about a half a year after the initial infection, 50% of those infected (without treatment) will suffer episodes of obvious arthritis, including the symptoms of swelling and discomfort in one or more joints, but often the knee.

Stage III Symptoms

Ten percent of those who reach the “arthritic” point will go on to suffer chronic Lyme Arthritis. These patients will find joints swelling for months at a time, or certain joints will become enlarged and achy for a year or more.

In these latter stages, joints, the central nervous system and the skin may be involved. Arthritis can develop from a few weeks to several years after Stage I. Sixty percent suffer at least one episode of arthritis if untreated. Usually the joint arthritis is but one-sided, and migration of the joint pain may prefer the larger joints, especially the knees.

Attacks may last for weeks or months, although they may also become less frequent over time and eventually disappear, leaving about 10% with damaged joints.

Sometimes neurological problems also appear, in about 20 percent of untreated patients, including Bell’s palsy. Bell’s palsy is one of our listed “Rheumatoid Diseases,” a collagen tissue disease, and so there must be more than one causation for that affliction2.

Other neurological afflictions include meningitis (sensitivity to light, stiff neck, headache), encephalitis (sleepiness, mood changes, memory loss), and radiculoneuropathy, where the roots of nerves that stem from the spinal cord to the periphery of some level of the body becomes irritated. These regions may be painful, tingle, or even go numb.

In traditional Rheumatoid Arthritis, a joint affected on one side of the body will also have a matching joint affected on the other side of the body. This is not true for Lyme Arthritis where only one joint may be affected on one side of the body.

Although the skin, heart, joints and nervous system are usually targeted, as the Borrelia burgdorferi bacteria can invade any system in the body, every organ or system in the body can also produce its own variation of symptoms. This ability to invade all human systems, too, is a similarity to the syphilis spirochete.

Traditional and Untraditional Treatments

Thankfully, if diagnosed early enough, antibiotics can easily wipe out the invading population, and bring about swift remission. (The antibiotics, of course, should be heavily accompanied with Lactobaccilus acididophilus1 so that while treating the Lyme Disease, we do not also unwittingly bring about a fungal infection of candidiasis.)

The key to solving Lyme Arthritis is early diagnoses and antibiotic treatment. Early diagnoses can be difficult, especially when the characteristic rash is not present. Since flulike symptoms can arise from many different sources, as described in Dr. Paul Pybus’ The Herxheimer Effect3, it becomes most difficult for a physician to make an early diagnoses. The patient’s history, especially their recent exposure to woods, ticks, and bites, and especially noting the characteristic bulls-eye lesions on the skin, all are most important for early diagnosis.

Although a definitive test for Borrelia burgodrferi bacteria is possible, the test is presently a low-yield procedure. A direct examination of body fluids and tissues is not recommended because there will be so few organisms found. There are no blood tests that can make an early diagnoses of Lyme Arthritis within the time length required for an early diagnosis, although surely someone, somewhere is working to develop such an early test, probably based on DNA of the microorganism. The study of body serums, serology, using indirect immunofluorescent assay or enzyme-linked immunoabsorbant assay has a slow antibody response and is positive in but 50% of Stage I infections, and should antibiotics be used, the test is often aborted.

Since Lyme Arthritis is potentially disabling, extreme vigilance must be taken by those who traverse woods and grasses, but overall, it may not cause serious problems for more than 10 percent of those who have received Borrelia burgdorferi through a tick bite.

Many who think they have Lyme Arthritis actually suffer from other forms of disease states, but among those who are found among the 10 percent seriously affected, there seems to be no good solution to the problem, because, after the early stage of the disease, antibiotics seem to be ineffective.

The primary problems with traditional treatments consists of the following: (1) Inability to diagnose the disease early without specific noting of the bulls-eye lesions, or having at hand an accurate, clear, case history. This delay affects treatment response by use of antibiotics, and often also causes over-extended usage of antibiotics; (2) Over-extended usage of antibiotics increases overgrowth of organisms-of-opportunity in the intestinal tract, such as Candida albicans, which condition also creates additional disease states, including some that mimic various arthritides forms, and also increases food allergies over time; (3) Many treated cases linger with pain, increasing systemic damage, and lessened vigor over many years, often ending up damaged organs and joints.

There is hope, however.

Alternative Treatments

Anti-Amoebic (Anti-Microbial) Treatment

Gus J. Prosch, M.D., Jr. of Birmingham, Alabama suggests a trial of the Rheumatoid Arthritis or Rheumatoid Disease treatment protocol as recommended by The Arthritis Fund/The Rheumatoid Disease Foundation: “I’ve seen Lyme Arthritis Disease clear up after using a course of anti-micro-organism drugs as recommended by Professor Roger Wyburn-Mason for Rheumatoid Diseases.

“Although Lyme Arthritis Disease, and other diseases such as Gout, Carpal Tunnel Syndrome, and Tendinitis are not supposed to be the same kind of diseases as Rheumatoid Arthritis, I’ve seen them all respond one time or another to the same treatment we use for Rheumatoid Arthritis.”19

In the case of Lyme Arthritis Disease, Dr. Prosch will give metronidazole (or one of the other 5-nitroimidazoles described) in heavier doses, for a longer period of time than recommended for Rheumatoid Disease.

Artificial Fever and Herbs

Agatha Thrash, M.D. and Calvin Thrash, M.D. write that “About one-third of patients with chronic infectious arthritis derive substantial benefits from fever treatments, one-third derive only moderate benefits, and one-third little or no help.

“In gonococcal arthritis, swelling and pain is often astonishingly helped. Patients suffering from [Osteoarthritis] receive temporary benefit, and the fever treatments may be used along with general arthritis treatment of diet and physical conditioning.”24

The Case of John Woodworth

Agatha M. Thrash, M.D., Uchee Pines Institute, Seale, Alabama was visited by a 54 year old Caucasian with Lyme Arthritis Disease. John had typical symptoms of pain in the joints, neurologic symptoms and specific rash beginning with a small, raised, red area and spreading concentrically outward with fading in the mid-portion.

Dr. Thrash administered a series of 15 fever treatments in which the mouth temperature was brought up to 1030 Fahrenheit as many times as possible during the 15 treatments, each day. John took five treatments, then skipped 2 days and repeated this schedule 3 times.

Dr. Thrash writes: “Once in a great while a person with Lyme Disease does not clear completely with the first series of 15 treatments and must take a second series. This was the case with John Woodworth. We waited 3 weeks between series to give the body a good chance to reset the immune system. Fever enhances the effectiveness of the immune system, but the body adapts to the fever and the response begins to weaken after about 5 days. For this reason we skip 2 days each week and rest 1 to 3 weeks between series.”

John also was given golden seal and echinacea, the first being anti-bacterial, and the second being a boost to the immune system. “The way to make it is by bringing a quart of water to a gentle simmer, adding 1 tablespoon of golden seal root powder and 1 heaping tablespoon of echinacea (chopped whole plant). It should be simmered for 20 to 25 minutes, cooled, strained, and drunk throughout the day. Make it up fresh every day.

“On the first day in chronic cases, and for 5 to 10 days during the acute phase, the patient should take 2 quarts of the tea daily. The patient should also take Nutri-bioticTM (grapefruit seed extract) obtainable from any health food store. Put 6 to 15 drops in a quart of water or tea and drink 2 quarts a day.”

Dr. Thrash says that “We have had several typical cases of Lyme Disease, complete with the tick bite and rash, which have been treated with a series of artificial fever treatments. None of them, including John Woodworth, has had further illness, as long as 2 years follow-up later.”18

Agatha Thrash, M.D., Calvin Thrash, M.D., Home Remedies, Thrash Publications, available from this foundation.

Homeopathic Remedy

According to Stephen Tobin, D.V.M.

Stephen Tobin, D.V.M.,31 is a veterinarian in a Lyme Arthritis Disease infested region. He’s treated many cases of Lyme Disease in dogs, cats and horses.

After trying a number of homeopathic remedies, he settled on Ledum (Genus Epidemicus) in a 200 or 1M potency, three times a day for three days. Dr. Tobin says, “Every animal treated this way has shown immediate improvement, whether they were only recently infected or have had the disease for years, treated or not with antibiotics. A number of pet owners, on seeing how well it cured their companions, took it themselves, with equally good results.

“As a preventative, I use the Borrelia burgdorferi nosode 60X, giving one dose daily for one week, then one dose per week for one month, then one dose every six months, the same way I administer all the nosodes I give in place of vaccinations.

“I have had only one failure in almost two hundred animals so treated. This is more effective than the vaccine for Lyme Disease used in dogs, which often has the effect of producing symptoms of Lyme Disease, including lameness, swollen joints, lethargy, inappetance (lack of appetite), kidney failure, and cruciate degeneration (cross-shaped as in the cruciate ligaments of the knee)

“I have seen no side effects from the nosode itself.”

According to Dr. Catherine Russell

Although rare in Mexico, Dr. Catherine Russell,30 Guadalajara, Mexico, successfully treats Lyme Arthritis Disease just as she would any other arthritis. She uses proteolytic enzymes, niacinamide 500 mg 3 X daily, and homeopathic symptomatic medicine according to individual symptoms and modalities. Sometimes she also uses herbs, especially stinging nettle which she picks and prepares herself, especially when a lot of uric acid is involved.

Venus Fly Trap Carnivora Treatment

Carnivora is an extract of the Venus Fly Trap plant (Dionaea muscipula) that was developed by the German oncologist (cancer specialist) Helmut Keller, M.D.

According to medical reporter Morton Walker, D.P.M.,22 “Since 1981, over 2,000 patients have been treated with Carnivora. Among them has been President Ronald Reagan who received the substance postoperatively following his operation for malignant polyps of the colon. The President took Carnivora drops for their healing and preventive powers against cancer recurrence.”

“Actor Yul Brynner also received dosages of Carnivora in injections and/or Carnivora drops.” Yul Brynner’s lung tumor’s were rapidly diminishing in size until he foolishly followed the recommendation of a New York City oncologist and failed to keep up with the remedy.”

“Carnivora has a proven 82 to 87% remission rate for most types of carcinoma when the patient’s immune system has not been compromised by conventional, allopathic chemotherapy or radiation therapy.”22

As Carnivora is a relatively new product, no one knows how many different organisms it can inhibit, or kill. In addition to its immune stimulating properties, the Carnivora extract “has been used successfully for the treatment of chronic diseases including most forms of cancer, neurodermitis, ulcerative colitis, Crohn’s disease, multiple sclerosis, all types of herpes infections, primary chronic polyarthritis, and almost any immune deficiency state,” and AIDS.

After it’s use, Harvey Bigelsen, M.D. was so impressed with the reduction of pleomorphic organisms in a patient’s blood that he ordered a supply of Carinvora for his own use.22 Pleomorphic organisms are micro-organisms that change form and function depending upon their surrounding environmental conditions.

Dorothea M. Linley, M.D.

Dorothea M. Linley, M.D. says, “In August 1986, I came down with a flu-like illness: pains in muscles, back, headache, fatigue and even my hair was painful. Fever and migrating arthritis followed by cardiac fibrillation which was controlled by oral magnesium. A blood test for Lyme disease was positive. . . .

“It settled in my knees, the left swelling so severely I needed crutches. I took 1 gram of tetracycline daily for 3 weeks and then 2 grams daily for three more weeks. Then my stomach rebelled and it was stopped.

“I was left with 50% reduced motion in both knees. Three months later I learned that the North (negative, south seeking) magnet would reduce swelling and pain. I applied a North pole magnet to both knees for 30 minutes twice a day5. In one week my knee flexion was doubled. In 6 months flexion was almost normal except for something that didn’t feel right in the middle of the joint.

“Two months later I experimented with germanium sesquioxide6. On a dose of 900 mg daily the right knee returned to normal in 3 days and after reducing the dose to 300 mg daily, the left knee took about 2 months to become normal. . . .

“I was 5 years without any symptoms.

“In May 1992 my left knee started swelling and a blood test was positive for Lyme. Whether this was a new case or a return of the first attack I do not know. What I did know was that I had no desire to take antibiotics again. I had just finished treatment for Candidiasis7.

“In mid-July I learned about Carnivora [Dionaea muscipula, Venus Fly Trap plant extract] from Morton Walker’s article in the Townsend Letter [for Doctors]8. I learned that Carnivora would kill bacteria, yeast, parasites and viruses without harming beneficial intestinal bacteria.

“I believe the Carnivora has killed the Lyme bacteria as well as Candida overgrowth. I lost 10 pounds, my skin is younger looking, my bowels are functioning normally for the first time in my life, my fingernails are hard and growing well, and my digestion is improved, needing fewer digestive aids.

“I hope my story will help those many patients suffering from chronic illness which may be due to Lyme. I was fortunate in that I did not take antibiotics until after a positive blood test. Those who treat symptoms clinically typical of Lyme before developing a positive blood test will never test positive and thus obscure the diagnosis. There are many people out there in this situation.”

Dorothea M. Linley, M.D. can be reached at 220 Banks Rd., Easton, Connecticut 06612.

Neurokinesiological Testing and Herbal Remedies of Louis Marx, M.D.

Louis Marx, M.D. Recommended Treatment

Louis J. Marx, M.D.11 combines neurokinesiology as a research and diagnostic tool with herbal programs.

A simple explanation of kinesiology given by Dr. Marx is that “if you touch a blocked acupuncture point while testing a muscle, that muscle will test weak. . . . Kinesiology is a technique for testing the integrity of the energy supply to an organ, as evidenced by a specific muscle. It uses the concept of organ-muscle linkage. . . . Any muscle can be used.

“Neurokinesiology differs from applied and other forms of kinesiology by not relying on the organ-muscle linkage . . . it tests directly through the nervous system . . . Dr. Calvin Alldredge has developed reflex points for the various known and unknown infections, hormonal system, nutritional status and toxic substance. By testing through the nervous system we are getting responses from the body’s innate intelligence.”

In Dr. Marx’s Neurokinesiological Testing and Herbal Remedies, writing on herbal remedies (tinctures or capsules), he says, ” Most programs last about three weeks. Usually, the patient experiences an improvement from a program within a day or two. However, during the third week after beginning a program, the patient may feel a decline in well-being and may develop new symptoms. It is very important to understand what is happening to avoid discouragement and loss of confidence in the treatment.

Insert Here are more books Icon: Louis J. Marx, M.D., Healing Dimensions of Herbal Medicine, available from this foundation; also Neurokinesiological Testing and Herbal Remedies; 3418 Loma Vista Road, Suite 1-A, Ventura, CA 93003

“What really happens is that the herbals have just about resolved those problems being treated, and the immune system no longer has to concern itself with those specific disorders. Therefore, the immune system starts attacking another group of infections, etc. This can happen a number of times with some patients. However, each group of new programs brings more energy and well-being than the prior programs. Expect this and explain it to the patient so they will not get discouraged.

“Sometimes a patient may present a specific complaint and testing identifies a specific factor as causing that complaint. However, after taking the program for that problem, the symptom remains. If you retest, you will identify another basis for the symptom which was not identified in your first examination. It is like the problem has layers to it, and as one layer is uncovered, a deeper one surfaces. Occasionally, you may need to treat two or three factors before there is a final resolution of the complaint. Usually the response is rapid enough and dramatic enough to leave little doubt about the benefit of the herbal programs.”

Dr. Marx, using neurokinesiological testing and herbal remedies, has developed a combination of herbal recommendations for almost every disease condition. These include, but are not limited to, viruses (assorted, intestinal, liver, herpes, wart, immune, life force), parasites, bacteria, Mycoplasma, rickettsia, yeast/fungi, onco genes (involved in cancer), and even the will to live.

Dr. Marx says, “Once you learn the system, you can apply it to any remedy. You can test your foods, supplements, drugs, etc.”

The drops can be put in water or juice, or taken directly into the mouth. The amount of alcohol ingested is negligible, and many reformed alcoholics tolerate the tincture well without inducing a craving for alcohol.

The capsules are best taken after meals. The capsules and tinctures do not need to be taken at the same times of day. All the tinctures or extracts can be taken together. If there are six bottles of liquids to be taken, they all can be put in a small amount of juice. Shake and drink followed by some extra juice to chase down the taste. It is actually better to squeeze the dropper top and squirt out one dropper full. It is more accurate than counting out the drops. That’s because some of the liquids are thicker, therefore, have larger drops.

If the extracts or tinctures are too thick, just add a few drops of water to the bottles.

Most herbal programs contain three bottles — one bottle of cut dried herbs in capsules. There are 70 caps to each bottle. The other two are one half ounce bottles of liquid extracts or tinctures. On each of these bottles are letters AO, RVO, VO, JRVO just before the program name. These letters stand for the extractants used in making the formula. A stands for alcohol, O stands for oil, R stands for rice water, V stands for vinegar, and J stands for fruit juice. T stands for tincture, and so (T)-(AO) stands for tincture of oil in an alcohol base. SPIRO- (L) would stand for the Lyme Spirochete.

Louis J. Marx, M.D. recommends that herbal remedies be ordered from Monastery of Herbs, PO Box 3123, Granada Hills, CA 91394; (800) 352-4372; (818) 360-4871. There are other good sources, but best one check through a health professional, as many herbs are either not what they are supposed to represent, or have been degraded throughout its cycle.

In the case of Lyme Arthritis Disease, Dr. Marx recommends the use of:

(T)-(AO)     SPIRO-(L) @ 30 drops/day for 1/2 Fld. Oz.

(T)-(RVO)     SPIRO-(L) @ 30 drops/day for 1/2 Fld. Oz.

Herbal Blend SPIRO-(L) @ 4 Capsules/day for 50 Caps.

The Case of Pearl Bennette Atkin, R.N., M.A., C.S.

Where Carnivora extract helped one person, it seemed to become a burden for another. Pearl Atkin, RN, MA, CS, Briarcliff Manor, New York, fought against Lyme disease, to win. Healer Atkin10 wrote to Townsend Letter for Doctors for advice in November 1992, saying:

In June of 1984 I attended a professional conference in the wooded town of Armonk, New York. Within a couple of weeks I had flu-like symptoms — aches and pains all over my body, especially a headachy feeling each afternoon, and discomfort in my coccyx area. After the summer cold the aches and pains subsided, but I continued to experience a ‘hypoglycemia-type’ of late afternoon problem, which I took care of with a fruit and nut snack, and sometimes with an ounce or two of vodka.

But even after that, I was still left with an aching coccyx, and at that point I decided my mattress had to be traded for a softer, more luxurious type — such as deep innerspring, or foamy and waterbed types. I had always thought that a waterbed was decadent — until that summer. When I first tried out a waterbed, I was able to get comfortable on it in a second — and my coccyx felt good in any position!

Then, after about six weeks of these unpleasant symptoms, as I was finally sleeping a little more comfortably at night, . . . an official from the conference that I had attended 8 weeks before called to ask how I felt, and asked if I had [any of about 40 symptoms] — and I’d experienced many of them. I had recently turned 50, and felt that [age] was the reason — now I knew differently — I had Lyme [Arthritis] Disease.

I was given the name of a Westchester Lyme [Arthritis Disease] specialist. I called him, then went for a consultation, and told him about my symptoms. Blood was drawn, and I was put on 7-10 days of tetracycline 240 mg 4 times a day. I was informed that, although I had not had the classic bull’s-eye — I had had a rashy welt initially on my forehead that I thought was poison ivy — two weeks later it had spread down to my neck and chin areas — I would know it was ‘Lyme’ if, after taking each tetracycline capsule I would have an exacerbation of the headachy, flu-like symptoms. That’s exactly what happened. I continued taking the capsules until the exacerbation experience stopped — and then for a few more days — which was a total treatment of about 10-12 days. Yet the blood work was negative.

During and for some time after the treatment period, I continued to be troubled, frequently, by the peculiar headaches, though I had hardly ever had headaches before. And these felt different from any I had experienced before. As they were miraculously cured, almost instantaneously, by a shot of vodka, it was my treatment of choice whenever they returned.

That was the summer of 1984. Very gradually, over many months, the headaches became less frequent and less intense. But two years later, in 1986, my headachy symptoms flared again, and I again went on tetracycline 500 mg 4 times a day for 7-10 days. It did the trick this second time also.

Two years later, in 1988, there was a third episode. I had the headachy feeling again, and the doctor gave me a choice of 7 days in the hospital with an intravenous (IV) drip of penicillin (which was the treatment of choice at that moment in time) — or I could try 7-10 days of tetracycline 500 mg again.

I opted for the oral treatment, and took more vodka every afternoon with my snack parties!

The headache receded to a low level that was tolerable and I went on about my life. [During these episodes, the first two measurements for Lyme antibodies (titers) were negative — but I understand that false negatives are common; then a third sample was taken, but that one was lost.]

But now I’m right in the middle of it again — my fourth serious episode. This past summer I went to Eastern Europe on a genealogical tour and when I returned home I had a protracted case of ‘jet lag.’ One day I noticed a rashy welt in my pubic/groin area, and the next day I had 1030 F temperature. I called my gynecologist — but he was on vacation. His ‘covering’ doctor called back, and we ran through the ‘could be’s — and came up with my fourth recurrence of Lyme Disease.

He prescribed 500 mg of tetracyline 4 times a day again for 7-10 days. The temperature and rash were gone immediately! When I called my gynecologist the next week, he said I should continue on the tetracycline for 1 month — that was the protocol for Lyme this year.

By the second week, however, the tetracycline was giving me nausea, queasiness, stomach and abdominal discomfort, and I was having headaches around the clock. So I called a doctor friend (an internist), and he changed me to Amoxicillin (straight penicillin 500 mg 3 times a day), for another 2 weeks.

That treatment gave me back my appetite, and took the worst of the headaches away. But what else is it doing to my poor system? I’ve been taking Lactibaccilus acidophilus1 to counteract the killing of the good intestinal microrganisms.

“. . . I’ve paid no attention to Lyme Disease therapies the last few years, because I’ve had other things to deal with personally and professionally that seemed more important than a ‘hypoglycemic headache’ but now my spotlight [is] on Lyme Disease, . . .”

On December 1993, Pearl Atkin12 described the great help she’d gotten, particularly from Lous Marx, M.D. of California. She wrote:

“I want to let you know how well I’ve been since you published my last ‘Letter [in Townsend Letter for Doctors and Patients] . . . regarding my plea for help with my fourth Lyme Disease attack. Each time I have had a Lyme infection I have been treated with massive doses of antibiotics, but have nevertheless been left with a pattern of recurrent headaches identical to those experienced during the initial acute Lyme episodes. This last time the headaches were so severe that they were disorienting as well as disabling. For several months I could not remember what it was like to have a clear-thinking and pain-free head.”

One of your subscribers, Dr. Dorothea Linley from Connecticut, contacted me with information about treatment that had been successful for her own chronic Lyme infection. She had been working with a nutritionist who advised Carnivora22 (Venus Fly Trap extract) as part of a program that also included many vitamin supplements and glandulars. After I began this program my headaches cleared up about 70%. But each time I opened [and began using] a new bottle of Carnivora I had a recurrence of devastating flu-like symptoms3 — high fever, and head and full body aches for 3 days. After three rounds of this (approximately 3 months) I stopped those treatments, as I decided to accept the 70% remission and live with it!

At this time a physician — Dr. Louis Marx11 — sent a copy of his herbal book to me, having earmarked the page on ‘Spirochetes; Lyme Disease.’ Reading this book, I was impressed with his work of the last 10 years on ‘designer herbals’ (with Dr. Clifford Alldridge), and called him to discuss my situation. He encouraged me to order the Spirochete-Lyme program — but told me to expect that it might not give me 100% relief, and also that I might need to do several more herbal programs, as one layer of disorder after another required attending to. . . .

“Within three days my headachy feeling that I have lived with for years was gone — and my head was clear as a bell! It was unbelievable, and extremely pleasurable.”

Pearl Bennette Atkin, RN, MA, CS can be reached at 85 Aspinwall Road, PO Box 950, Briarcliff Manor, New York 10510, telephone (914) 941-8926.

Nutritional and Immune Support

The Case of Sarah Statesmyer

Sarah Statesmyer, age 16, came to the office of Robin Ellen Leder, M.D.,29 in Bronx, New York, complaining of severe fatigue and episodes of debilitating joint pain, especially in her knees. Low energy made it extremely difficult for Sarah to do her work at a law firm on her bad days. She felt like she could barely walk. Her symptoms, she reported, had been ongoing since childhood.

Sarah’s parents thought her problem stemmed from a tick bite years earlier, but a blood test was only suggestive of Lyme Arthritis Disease, and was not conclusive, probably because of the time that had passed since Sarah’s first exposure to the Borrellia burdorfi organism. However, because of the described symptoms and probable history of Lyme Arthritis disease, this condition was considered to be the most likely cause of Sarah’s problem.

Dr. Leder discussed traditional, possibly long-term, antibiotic therapy with Sarah, and also the importance of supporting the immune system in chronically symptomatic Lyme Arthritis Disease patients.

Sarah and her family chose to begin treatment using the nutritional approach offered by Dr. Leder.

To help design a diet that would benefit Sarah, Dr. Leder began by having Sarah take a six hour glucose tolerance test and a special blood test for food allergies. She was found to be quite hypoglycemic (tendency to low blood sugar), and also to have sensitivities to a number of foods.

Sarah’s diet was changed and she was required to eat a minimum of five to six times per day, and every meal or snack was to include some form of protein. All foods that Sarah was allergic or sensitive to, according to her blood test, were eliminated from her diet.

Dr. Leder, according to her custom, also asked Sarah to eliminate other common foods that have a history of being allergenic, even though they were not on Sarah’s list, and, in addition, to cut out any other foods that had any remote history suggestive of allergy.

A broad spectrum of nutritional supplements and plenty of water completed Sarah’s program.

“With the help of an exceptionally supportive family, Sarah’s symptoms literally disappeared, her energy was restored to a level normal for a healthy young woman, and, during several months of follow-up, no further flare-ups of her joint pain occurred.”

Universal Oral Vaccination

When former Iowa Congressman Berkley Bedell13 testified before the U.S. Senate Health Appropriations Subcommittee Chaired by Senator Tom Harkin, also of Iowa, on June 24, 1993, he gave witness to a powerful and obviously safe method of solving Lyme Arthritis Disease. (See beginning of this chapter.)

He described a procedure whereby the killed bacteria, Borrelia burgdorferi, was injected above a cow’s udder, above the base of the teat, (where the antigen or allergen is sure to reach the cistern) prior to the birth of her calf. Colostrum — the cow’s first milk after the calf is born — is processed into whey — the liquid left after milk has been coagulated by the aid of a coagulating enzyme called rennet.

Congress Bedell also gave witness to the effects of an over-powerful, suppressive governmental organization that would prevent people from trying every 1-1/2 hours for a few weeks the whey of this milk to learn if their Lyme Arthritis Disease will disappear. He reports that the company that cured him “dares not sell such a medicine, because of FDA regulations.”21

Later the same farmer that cured Congressman Bedell of his Lyme Arthritis Disease prepared a homeopathic remedy which, Bedell reports, had 80-90% success in treating patients for whom conventional treatments had not been effective.

When specific personalities in the U.S. Department of Agriculture were shown in court to have falsified data regarding this patent — apparently as part of governmental suppression (although according to one source there may have been personality conflicts also) — it became only the second patent in U.S. history to receive by vote of U.S. Congress an additional 16 year lifetime protection.

On April 2, 1968, a patent number 3,376,198,23 “Method of Producing Antibodies in Milk,” was granted to William E. Petersen, St. Paul, MN and Berry Campbell, Monrovia, California, assigned to Collins Products, Inc., Waukon, Iowa. Other patents for additional discoveries have since been granted to Mary E. Collins and Robert A. Collins (Patent No. 4,402,938; September 6, 1983; Gregory B. Wilson and Gary V. Paddock (March 28, 1989); Robert A. Collins and Philip F. Weighner (Patent No. 4,843,065; June 27, 1989); Robert Collins K (Patent No. 5,102,669; April 7, 1992)

The original work on development of the cows’-milk vaccine was performed at the University of Minnesota, School of Biochemistry under the direction of the patent assignees. (Porter: Biological Abstracts 1953, p. 951, par. 10, 185). In August, 1951, Dr. Porter, then “working on his doctoral thesis, suggested the possibility of manufacturing antibodies in the cow’s udder by infusion of antigen into the udder of a lactating cow.” (Patent No. 3,376,198)

A spokesman for a group that prefers not to be identified, says that the protective element “seems to be a system of peptides that is produced by the cow. . . . Basic research beginning in the late sixties was directed to identify the active products (biological and chemical) in the whey product. This has proven very difficult and especially because the activity is not an antibody per se, but appears to be the action of a low molecular weight material.

“Several important activities can be found in the product that is produced by infusion of specific antigens into the udder (above the udder into the cistern) of a cow after collecting the colostrum and milk for the final product production. These are being researched.

Many people have used the product, and it seems not to matter whether the cow’s colostrum is used, made into whey, the cow’s milk is used, or a homeopathic remedy is prepared, whether or not pasteurized, whether or not lypholized, or pasteurized and lypholized — all are effective, although transfer factor, an additional protective substance, as described in one patent seems to be of higher yield in the colostrum.

Reminiscent of what has become the routine human use of dimethylsulfoxide (DMSO) or antibiotics restricted by law to veterinarians and those practicing husbandry, marked “Not For Human Use,” some dairy farmers purchase products for their animals’ disease protection, but use the products on themselves with success.

Those with access to a cow can purchase standardized antigens (killed) or allergens from biological supply sources which can be inoculated through the cow’s udder or into the base of the udder (into the cistern) at the proper time before calving. A variety or blend of organisms or substances — pollen, cat, dog, or cow hair if one is allergic, or specific antigens against a given disease condition — will result in a milk product that will cure and protect from an equally large and varied number of pathogenic organisms or allergens, respectively.

In homeopathic remedies produced by Beaumont Bio-Med, Waukon, Iowa, conditions aided are Rheumatism, Rheumatoid Arthritis, coughing, respiratory, sore throat, skin conditions, acne blemishes, upset stomach, cold and flu, diarrhea and impetigo.

See Universal Oral Vaccine, Anthony di Fabio, this foundation.

A few homeopathic remedies based on the described principles can be obtained from Beaumont Bio-Med, PO Box 6, Waukon, Iowa 52171; (800) 332-2249.

As a general principle, this method will vaccinate safely against any allergen or antigen — any substances which when introduced into the body create antibodies such as allergenic pollens, house dust, animal hairs, or micro-organism proteins.

According to Patent Number 3,376,198, antigenic protections can be developed against “bacteria, viruses, proteins, animal tissue, plant tissue, spermatozoa, rickettsia, metazoan parasites, mycotic molds, fungi, pollens, dust and similar substances. . . . exemplary antigens include: bacterial — Salmonella pullorum, Salmonella typhi, Salmonella paratyphi, Staphylococcus aureus, a Streptoccus agalactiae, g Streptococcus agalactiae, Staphylococcus albus, Staphylococcus pyogenes, E. Coli pneumococci, streptococci, and the like; viral — Influenza type A, fowl pox, turkey pox, herpes simplex and the like; protein — egg albumin and the like; tissue — blood and sperm.”

Protected, according to this and a later patent, were mice, cows, goats, chickens and pigs.

For allergy prevention, one can use a mixture of hair (cats, dogs, cattle), making a vaccine. (Many milk-producing farmers become allergic to cow’s hair.) Other allergens, such as pollens, can also be introduced, such that many other allergies can be beneficially affected.

It’s also good for chickenpox, cold sores, genital herpes, Cryptocides sporidium, and for anti-inflammatory conditions, as it is heavy with complement and anti-complement (C3B), substances that assist in the destruction of invasive organisms.

In work supported by the National Institutes of Health and by Philip Morris Cos.,27 “A modified version of a protein extracted from whey blocked the AIDS virus from infecting cells in the test tube,” according to Dr. Robert Neurath, head of the laboratory of Biochemical Virology at the Lindsley F. Kimball Research Institute of the New York Blood Center.

“Scientists modified a whey protein called beta-lactoglobulin to produce a substance called B69, which they discovered latched onto a protein structure called CD4 on the surface of cells.” This prevented AIDS virus from using CDS as an entryway into the cells.

Dr. Jeffrey Laurence, an AIDs researcher at Cornell Medical College in New York, cautioned that HIV can infect some cells without using the CD4 gateway.

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According to a source,23 “One North Dakota support group uses this substance for multiple sclerosis with beneficial results.”

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In a 1984 study reported in Medical Microbiology and Immunology32 IgA-rich cow colostrum containing anti-measles lactoglobulin resistant proteases was orally administered to patients with MS. Measles-positive antibody colostrum was orally administered every morning to 15 patients with multiple sclerosis at a daily dosage of 100 ml for 30 days. Similarly, measles-negative antibody control colostrum ( < 8)was orally administered to 5 patients. Of 7 anti-measles colostrum recipients, 5 patients improved and 2 remained unchanged. Of 5 negative (< 8) recipients, 2 patients remained unchanged and 3 worsened. These findings suggested the efficacy of orally administered anti-measles colostrum in improving the condition of MS patients (P < 0.05).

The Case of Dorothy Johnson

Dorothy Johnson,28 49 years-of-age, was diagnosed at the Mayo Clinic with multiple sclerosis, a slowly progressive Central Nervous System disease characterized by patches of demyelinated nerve tissue of the brain and spinal cord. Demyelination is the loss of insulative protective tissues that surround nerve tissue.

Dorothy suffered from varied and multiple neurological symptoms and signs, such as shakiness, numbness in legs, difficulty in climbing stairs, tingling in hands and feet, cramping of legs, and other symptoms.

Although multiple sclerosis may go into remission and then recur, often with greater severity, over a period of four years Dorothy became progressively worse, until she met Herb Saunders, a farmer who had been treating people for various conditions by the use of specially prepared colostrum from a cow.

Dorothy took 4 tablespoons of colostrum a day for two years, and gradually improved, until all of her symptoms disappeared.

When some symptoms did reappear after a period of time without the colostrum, her husband obtained three more bottles of specially prepared colostrum, and again the symptoms disappeared. She has continued without symptoms for several years.

The Case of Judith Toliver

Dorothy’s experience was repeated by that of Judith Toliver,28 26 years-of-age, who was wheel-chair bound. Her blood was injected into the cistern of a cow, and the prepared colostrum given to her in the same manner as that described for Dorothy. After one year of treatment, she was able to walk upstairs with a cane.

Other Diseases

Early work using the described principle for Rheumatoid Arthritis involved staphylococcus and streptococcus killed organisms injected as antigens into the cow’s cistern, the successful results thus strongly supporting the infectious nature of Rheumatoid Arthritis. As many forms of Rheumatoid Diseases and related diseases seem to have an infectious and/or allergenic component, such as Ankylosing Spondilitis, candidiasis, Crohn’s disease, Fibrositis, Fibromyalgia, food allergies, rhinitis, and so on, this form of protection may be all-inclusive, inexpensive, and all-important.

According to one spokesperson,23 “The homeopathic remedy derived from this process has been found useful for various forms of arthritis.”

One hundred gallons of milk is taken from an inoculated cow, casein and fat separated by ultra-centrifuge, and pasteurized. It is then lypholized — frozen — that is the water is taken out under cold temperature. The resulting powder can then be used sub-lingually, or made into homeopathic remedies, or any other reasonable means for introducing it into the human or animal body.

The suppression of safe, workable treatments continues: The Minnesota diary farmer, Herb Saunders, 66, who cured Congressman Bedell, was prosecuted in St. James, Minnesota by the state prosecuting attorney for practicing medicine without a license. Herb was selling bovine colostrum (“first milk”) as a potential cure for cancer. “Saunders would sell each patient a cow for $2,500, but keep the cow on his farm. He would inject a sample of each patient’s blood into the cow’s udder [cistern], and then sell the colostrum to the cow’s owner for $35 a bottle. Saunders told an undercover state agent who posed as a cancer patient that he would ‘cough out’ his cancer within months if he would take colostrum, [and] refrain from chemotherapy.

“After two weeks of [court] trial — the longest this small community had ever seen — the result was a hung jury. The 6-person jury voted 5-1 to convict, but the last holdout, a part-time social studies teacher, apparently couldn’t decide whether Saunders was practicing medicine without a license or offering an alternative type of care that is not medical practice.”26

Berkley Bedell provided $21,000 for Saunders’ defense.

“The Watonwan County attorney’s office stated that it plans to retry Saunders.” Herb Saunders was indeed tried a second time, resulting in a hung jury more pronounced than the first time.26

To make these kinds of obviously safe treatments available to all, avoiding great costs and suffering under ineffective traditional treatments, each person is advised to write to his/her U.S. Senators and Representatives in support of freedom-of-choice-in-medicine legislation, and also to support similar bills at each state level.

Vaccines

There has been considerable interest in developing a vaccine against Borrelia burgdorferi, and so far the research looks quite promising, but there are years of work ahead before such vaccines are to be declared both safe and effective. Rather than inoculate humans with appropriate vaccines, possibly mice in nature can be vaccinated, thus breaking the Lyme Disease cycle9.

To develop any kind of effective vaccine against Lyme Disease, however applied, will surely provide knowledge on how to protect folks from the invasions of the syphilis spirochete.

According to reports in the Journal of Longevity Research, Dr. Henry Jay Heimlich, who developed the Heimlich maneuver, and who also pioneered successful organ replacement using part of a patient’s stomach to construct an esophagus, is investigating (in China) the use of a curable malaria organism as a cure for AIDS and possibly also Lyme disease. Malaria is said to stimulate production of TNC (Tumor Necrosis Factor), Interleukin-1, and other regulators of cellular immunity which has been associated with Lyme Disease14.

Royal Raymond Rife Technology

Superb technology was developed by Royal Raymond Rife under sponsorship of Timken ball-bearing funding. According to 1930’s reports by physicians associated with the University of Southern California, cancer and other diseases were being cured.

After the FDA persecuted and destroyed Rife’s work, his technology languished until James E. Bare, D.C. retraced Rife’s path, using modern technology. Dr. Bare and others have now produced results that seem to match, and in some respects exceed, Rife’s work. As they cannot build or sell these devices without incurring the ire of FDA, they have made circuit diagrams, video tapes and internet data available to those who would like to build the device themselves.

According to internet information, using this newly developed Rife instrument, frequencies 432, 484, 610, 790, and 864 will kill off the bacteria that causes Lyme disease.

Information can be obtained from James E. Bare, D.C. (505) 268-4272; fax (505) 268-4064; Email: rifetech@rt66.co m

References

1. See Anthony di Fabio, Friendly Bacteria — Lactobacillus acidophilus & bifido bacterium, The Arthritis Trust of America/The Rheumatoid Disease Foundation, 7111 Sweetgum Road, Suite A, Fairview, Tn 37062-9384, 1989.

2. Anthony di Fabio, Rheumatoid Diseases Cured at Last, The Arthritis Fund/The Rheumatoid Disease Foundation, Op.Cit., 1985.

3. Dr. Paul K. Pybs, The Herxheimer Effect, The Arthritis Fund/The Rheumatoid Disease Foundation, Op.Cit., 1992.

4. “Picking Out the Lymes From the Lemons,” Townsend Letter for Doctors, 911 Tyler St., Port Townsend, WA. 98368-6541, May 1993, p. 408; reprint from Science News.

5. See, William H. Philpott, M.D., Magnetic Resonance Bio-Oxidative Therapy for Rheumatoid and Other Degenerative Diseases, The Arthritis Fund/The Rheumatoid Disease Foundation, Op.Cit., 1994.

6. See Anthony di Fabio, Germanium, The Arthritis Fund/The Rheumatoid Disease Foundation, Op.Cit., 1989.

7. See Gus J. Prosch, Jr., M.D., Candidiasis: Scourge of Arthritics, The Arthritis Fund/The Rheumatoid Disease Foundation, Op.Cit., 1994.

8. See Morton Walker, D.P.M., “The Carnivora Cure for Cancer, AIDS & Other Pathologies — Part II, Townsend Letter for Doctors, May 1992, #106, p. 329.

9. See Fred S. Kantor, “Disarming Lyme Disease,” Scientific American, 415 Madison Avenue, New York, NY 10017-1111, September 1994, p. 34.

10. Pearl Atkin, R.N., M.A., CS, “My Experience With Lyme Disease,” Townsend Letter for Doctors, Op.Cit., November 1992, p. 997.

11. Louis J. Marx, M.D., Healing Dimensions of Herbal Medicine, 3418 Loma Vista Road, Suite 1-A, Ventura, CA 93003, date unknown.

12. Pearl Atkin, R.N., M.A., CS, “Treatment of Lyme Disease,” Townsend Letter for Doctors, Op.Cit., December 1993, p. 1220.

13. Congressman Berkely Bedell, “Bedell Testifies Before U.S., Senate,” Townsend Letter for Doctors, Op.Cit., December 1993, p. 1229.

14. “Malaria Therapy: A Cure for Cancer and Aids?” Journal of Longevity Research, Vol.1/No.2, December 1994, p. 8.

15. Anthony di Fabio, “Lyme Disease: Arthritis by Infection,” The Art of Getting Well, The Arthritis Trust of America/The Rheumatoid Disease Foundation, 7111 Sweetgum Road, Suite A, Fairview, Tn 37062-9384, 1994.

16. Textbook of Internal Medicine, J.B. Lippincott Company, East Washington Square, Philadelphia, PA 19105, 1989.

17. Burton Goldberg Group, Alternative Medicine: The Definitive Guide, Future Medicine Publishing Co., Inc., 10124 18th St., Court E, Puyallup, WA 98371.

18. Personal communication from Agatha M. Thrash, M.D., November 2, 1995.

19. Personal communication from Gus J. Prosch, Jr., M.D., November 20, 1995.

20. Jwing-Ming Yang, Arthritis — The Chinese Way of Healing and Prevention, YMAA Publication Center, Yang’s Martial Arts Association (YMAA), 38 Hyde Park Avenue, Jamaica Plain, Massachusetts 02130, 1991.

21. Personal letter from Berkley Bedell July 18, 1994.

22. Morton Walker, D.P.M., “The Carnivora Cure for Cancer, AIDS & Other Pathologies — Part I & II, Townsend Letter for Doctors, 911 Tyler St., Port Townsend, WA, 98368-6541, #95, p. 412; #106, p. 324.

23. Personal interview with, and correspondence from a scientist who chooses not to be identified.

24. Agatha Thrash, M.D., Calvin Thrash, M.D., Home Remedies, Thrash Publications, Rt. 1, Box 273, Seale, Alabama 36875.

25. United States Patent 3376198.

26. “Minnesota Milk-Cure Case Ends with Mistrial,” Townsend Letter for Doctors, 911 Tyler St., Port Townsend, Washington, 98368-6541, August/September 1995, p. 81; from Minneapolis Star Tribune, 3/16/95.

27. Malcolm Ritter, “Whey Could Prevent HIV Infection,” Wisconsin State Journal, January 31, 1996, quoting the February Nature Medicine.

28. Personal interview with Dorothy Johnson.

29. Personal communication from Robin Ellen Leder, M.D.

30. Personal interview with Dr. Catherine Russell.

31. Stephen Tobin, D.V.M., “Lyme Disease,” Townsend Letter for Doctors, 911 Tyler St., Port Townsend, WA, 98368-6541, January 1993, p. 63.

32. T. Ebina, et. al., Med Microbiol Immunol, Springer-Verlag, 173:87-93, 1984.

Supplement to The Art of Getting Well
http://www.garynull.com/articles/whichArticle.php?article=209
 
responsible editor/writer Anthony di Fabio
 
contributed by:
The Arthritis Trust of America,
7111 Sweetgum Road, Suite A
Fairview, TN 37062-9384
www.arthritistrust.org
Sources are given in references.
Authors of contributions\quotations are alphabetically arranged; major author, if any, is underlined.
Dr. Clifford Aldridge, Pearl Bennette Atkin, RN, MA, CS, Congressman Berkley Bedell, Dr. Willy Burgdorfer, Dr. Dorothea M. Linley, M.D., Stephen E. Malawista

Copyright ©1994
All rights reserved by The Roger Wyburn-Mason and Jack M. Blount Foundation for the Eradication of Rheumatoid Disease AKA The Arthritis Trust of America

Discovery of Lyme Disease

Regards,
Linda

… from IMVA Medical News Commentaries list.

Yulia Tymoshenko, prime minister of Ukraine and a candidate in upcoming
national elections, visits a children’s hospital in western Ukraine.

Prime Minister Tymoshenko stated, “We cannot relax even for a moment because the World Health Organization (WHO) predicts two more waves of flu, including the bird flu, are expected in Ukraine. There is no alternative to vaccination. The entire world is going this way…” A day earlier she admitted she was not vaccinated and that she prefers “like all other people” plans to rely on garlic, onion and lemon as a way of preventing the flu. Commenting on what’s going on in Eastern Europe F. William Engdahl says, “The degree of fraud, deceit, official cover-up and outright criminal endangerment of the broad population by the current Swine Flu hysteria is seemingly without precedent.”

What I said a few weeks ago about the economy I repeat again in terms of what is happening in the Ukraine with the swine flu, a new genetic strain of it or something entirely new. President Yushchenko said: “People are dying. The epidemic is killing doctors.” Doctors in Ukraine’s western Chernovetsky region, near the centre of the flu outbreak, said lab tests showed at least some of the fatalities appeared to be caused by something dissimilar to both common flu and swine flu.

OFFICIAL NUMBERS of Sick Released 12th of November

1,253,558 SICK

65,615 HOSPITALIZED

239 DEAD

Reports of aerial spraying right before the plague hit will probably never see the light of day but if a person does not believe that there are people who would do such a thing they really are not familiar with the dark side of humanity. Dr. Gregory J. Moran, an associate professor in the department of emergency medicine in Los Angles says, “Plague may be rare today in the United States, but health care professionals should be aware of its potential as a weapon, because its pathogens are easily aerosolized and the symptoms are not likely to arouse suspicion until an epidemic is evident. If plague were to be used as a biological weapon, the pathogens would likely be spread via aerosol and inhaled, leading to the pneumonic form.” [1]

After weeks of silence in the western press the New York Times finally reported on 13th of November, “When patients began arriving in Dr. Vyacheslav Bonder’s intensive care unit two weeks ago, their lungs so saturated with blood that they could barely gasp, the only thing he could compare it to was a field hospital in wartime. As soon as he hooked one patient up to a ventilator, a second and third would appear in the doorway.”[2]

The virus causing the deaths is extremely
aggressive, it doesn’t first infect the throat (as
is common in flu), but strikes the lungs directly.
Dr. Viktor Bachinsky

The entire nation of Ukraine is in an absolute state of panic as a mystery illness rips through their country. The pandemic in the Ukraine is threatening  the lungs of flu victims with hemorrhage The first alarm came in August when a microbiologist calls into a radio show[3] run by a doctor with dire warnings about what was about to be deliberately released in the Ukraine and then is hunted down in L.A. by a SWAT squad and its all displayed on this bizarre video. Now just two months later the Sunday Express is reporting, “A doctor in Western Ukraine who did not want to be named, said:” We have carried out post mortems on two victims and found their lungs are as black as charcoal. They look like they have been burned. It’s terrifying.”[4]

Please note that this is the first IMVA Emergency Alert and see associated essay and our Natural Emergency Medicine protocol for suggested best medical responses. My advice to my beloved readers and to the world at large is to prepare for the worst, accept and use worst case scenario planning and take what is happening to heart with a fierce determination to protect ones family from what seems likely to come. And this means eating more than garlic, onion and lemon though this is a good medicinal combination to start with.

Special Note: The Ukraine and the rest of Eastern Europe are currently the weakest pegs in the world financial system, besides California and a host of other states. While they are celebrating on Wall Street certain areas of the world are facing collapse. Crisis spreads to Eastern Europe as Ukraine, Hungary and Serbia call IMF Ukraine, Hungary, and Serbia are all in emergency talks with the International Monetary Fund, raising fears that an exodus of foreign investors will set off a systemic crisis across Eastern Europe. A team of IMF trouble-shooters rushed to Kiev on Wednesday to draw up a possible standby loan to help Ukraine stabilize its bank after a panic run on deposits this month. Meanwhile banks in Sweden and The Baltic states are in the eye of the storm after leaked reports that Sweden is bracing for a full-blown economic and political “breakdown” in Latvia. The Svenska Dagbladet newspaper said Sweden’s finance minister Anders Borg had told banks secretly that Latvia’s political order was unraveling, advising them to prepare for the collapse of Latvia’s rescue talks.

Eastern Europe offers us a looking glass into the future that is happening in certain places today. Consider that accurate news is a precious commodity rarer then exceptional stones. We hardly hear in the mainstream about the crisis facing many States in America, how much less about Eastern Europe. The model presented by many thinkers is one where we see the Internationalist One World Government super elite taking advantage of and instigating crisis in both economic and health areas simultaneously. Kind of like doing a cannon ball on top of a man coming up for air; what we are seeing today in the Ukraine and the rest of Eastern Europe might be a harbinger of what is planned for the rest of us.

From Reuters Health Information http://www.medscape.com/viewarticle/703037

By Megan Rauscher

NEW YORK (Reuters Health) May 19 – Almonds have the potential to boost immune health and reduce inflammation, according to research reported Tuesday at the 109th annual meeting of the American Society for Microbiology in Philadelphia.

Almonds, study presenter Dr. Giuseppina Mandalari told Reuters Health, may have a “helpful impact on the treatment of infective and chronic diseases. Almond skins can modulate the complex cytokine network during an immunological response and positively act as novel antiviral agents.”

Dr. Mandalari, from the Institute of Food Research, Norwich, UK, and colleagues evaluated the effects of natural and blanched almond skins on the release of cytokines in peripheral blood mononuclear cells (PBMC) either infected or not infected with herpes simplex virus-2 (HSV-2).

The study was funded by the Almond Board of California.

The addition of natural almond skins to PBMC resulted in a “significant decrease in HSV-2 replication, whereas other extracts did not significantly influence the replication of the virus,” they report in a meeting abstract.

Almond skins also boosted production of several pro-inflammatory cytokines including interferon (IFN)-alpha, IFN-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-12 (IL-12). Moreover, IL-10 and IL-4, representative of Th2 responses, were also detected.

“It is well known that efficient elimination of a viral infection requires a pro-inflammatory host response and the development of type 1 immunity, characterized by activation of mononuclear cells and production of pro-inflammatory cytokines, the scientists explain.

“Our data suggest that almond skins may improve the immune surveillance of PBMC towards viral infection, both by triggering the Th1 and stimulating Th2 subset. This could represent a new strategy towards the Th1/Th2 balance,” they conclude.

“If the study can be repeated in humans, it offers promise for preventing chronic inflammation and boosting immune function,” Dr. Mandalari said.