Archive for the ‘research’ Category

Nearly 30% of Ticks on Italian Dogs Found to be infected with Tick-borne Pathogens

http://online.liebertpub.com/doi/10.1089/vbz.2017.2154

Vector-Borne and Zoonotic Diseases

Molecular Survey on Rickettsia spp., Anaplasma phagocytophilumBorrelia burgdorferi sensu lato, and Babesia spp. in Ixodes ricinus Ticks Infesting Dogs in Central Italy

Morganti Giulia, Gavaudan Stefano, Canonico Cristina, Ravagnan Silvia, Olivieri Emanuela, Diaferia Manuela, Marenzoni Maria Luisa, Antognoni Maria Teresa, Capelli Gioia, Silaghi Cornelia, and Veronesi Fabrizia. https://doi.org/10.1089/vbz.2017.2154

Online Ahead of Print: October 12, 2017

ABSTRACT

Dogs are a common feeding hosts for Ixodes ricinus and may act as reservoir hosts for zoonotic tick-borne pathogens (TBPs) and as carriers of infected ticks into human settings. The aim of this work was to evaluate the presence of several selected TBPs of significant public health concern by molecular methods in I. ricinus recovered from dogs living in urban and suburban settings in central Italy.

A total of 212 I. ricinus specimens were collected from the coat of domestic dogs. DNA was extracted from each specimen individually and tested for Rickettsia spp., Borrelia burgdorferi sensu lato, Babesia spp., and Anaplasma phagocytophilum, using real-time and conventional PCR protocols, followed by sequencing.

Sixty-one ticks (28.8%) tested positive for TBPs; 57 samples were infected by one pathogen, while four showed coinfections. Rickettsia spp. was detected in 39 specimens (18.4%), of which 32 were identified as Rickettsia monacensis and seven as Rickettsia helvetica. Twenty-two samples (10.4%) tested positive for A. phagocytophilum; Borrelia lusitaniae and Borrelia afzelii were detected in two specimens and one specimen, respectively. One tick (0.5%) was found to be positive for Babesia venatorum (EU1).

Our findings reveal the significant exposure of dogs to TBPs of public health concern and provide data on the role of dogs in the circulation of I. ricinus-borne pathogens in central Italy.

 

For more:  https://madisonarealymesupportgroup.com/2017/10/04/droplet-digital-pcr-shows-60-bb-infection-rate-in-ticks-and-over-50000-spirochetes-per-adult-tick/

https://madisonarealymesupportgroup.com/2016/11/05/infected-ticks-in-ontario/

https://madisonarealymesupportgroup.com/2017/08/17/of-birds-and-ticks/

https://madisonarealymesupportgroup.com/2017/09/20/night-at-the-blood-sucking-creature-museum/

https://madisonarealymesupportgroup.com/2017/09/19/tbis-in-australia/

Oregano, Cinnamon, and Clove Found to Have High Anti-Persister Activity for Bb

https://doi.org/10.3389/fmed.2017.00169

ORIGINAL RESEARCH ARTICLE

Front. Med., 11 October 2017 | https://doi.org/10.3389/fmed.2017.00169

Selective Essential Oils from Spice or Culinary Herbs Have High Activity against Stationary Phase and Biofilm Borrelia burgdorferi

imageJie Feng1imageShuo Zhang1imageWanliang Shi1imageNevena Zubcevik2,imageJudith Miklossy3 and imageYing Zhang1*
  • 1Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States
  • 2Department of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, Charlestown, MA, United States
  • 3International Alzheimer Research Centre, Prevention Alzheimer International Foundation, Martigny-Croix, Switzerland

Although the majority of patients with acute Lyme disease can be cured with the standard 2–4 week antibiotic treatment, about 10–20% of patients continue suffering from chronic symptoms described as posttreatment Lyme disease syndrome. While the cause for this is debated, one possibility is that persister bacteria are not killed by the current Lyme antibiotics and remain active in the system. It has been reported that essential oils have antimicrobial activities and some have been used by patients with persisting Lyme disease symptoms. However, the activity of essential oils against the causative agent Borrelia burgdorferi (B. burgdorferi) has not been well studied. Here, we evaluated the activity of 34 essential oils against B. burgdorferi stationary phase culture as a model for persister bacteria. We found that not all essential oils had activity against the B. burgdorferi stationary phase culture, with top five essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) at a low concentration of 0.25% showing high anti-persister activity that is more active than the known persister drug daptomycin. Interestingly, some highly active essential oils were found to have excellent anti-biofilm ability as shown by their ability to dissolve the aggregated biofilm-like structures. The top three hits, oregano, cinnamon bark, and clove bud completely eradicated all viable cells without any regrowth in subculture in fresh medium, whereas but not citronella and wintergreen did not have this effect. Carvacrol was found to be the most active ingredient of oregano oil showing excellent activity against B. burgdorferi stationary phase cells, while other ingredients of oregano oil p-cymene and α-terpinene had no apparent activity. Future studies are needed to characterize and optimize the active essential oils in drug combination studies in vitro and in vivo and to address their safety and pharmacokinetic properties before they can be considered as a novel treatment of persistent Lyme disease.

 Introduction

Lyme disease or Borreliosis is the most common vector borne illness in the United States with an estimated 300,000 cases per year (1). The illness is transmitted by a tick bite and in some endemic areas, where more than 40% of the ticks are infected with the causative agent of Borreliosis, Borrelia burgdorferi sensu lato complex species, which increases the risk of transmission to human host (2).

The difficulty in the clinical management of Borreliosis is that the current treatment regimen recommended for acute stage of illness of 21 days of Doxycycline (Dox) (3) leaves over 20% of patients with chronic symptoms that can last over 6 months (4). These chronic symptoms can be debilitating fatigue, muscular and joint pain, and cognitive and neurologic impairment. While we do not yet understand the full spectrum of etiologies, research evidence in animal studies illuminates that persistence of infection is one of them. The persistence of the organism after antibiotic treatment is seen in dogs (5), mice (67), monkeys (8), as well as humans (9), but viable organisms are very difficult to be cultured from the host after antibiotic treatment.

Once the disease has been acquired, it can spread from the skin to various secondary organs throughout the body, including heart, joints, peripheral and central nervous system (10). The early stage of the illness tends to be easier to cure, but it can become more difficult to treat when the disease has progressed to late stage (11). This further challenges conventional antibiotic monotherpy such as intravenous ceftriaxone, which has not been proven successful with a subset of patients presenting with complex chronic symptoms (12).

One of the reasons for this failure, clinically relatable, would be that the host is infected with organisms that are enriched in variant persister forms or the disease when not treated in early stage can progress allowing persisters to further develop (round bodies and biofilm-like microcolonies and larger aggregated biofilm structures). Analogous variant atypical persister forms can be found in stationary phase cultures and under stress conditions such as starvation and antibiotic exposures (1315). It is worth noting that the current antibiotics used to treat Lyme disease such as Dox, amoxicillin, and cefuroxime (CefU) are highly active against the growing spirochetal form of B. burgdorferi but have poor activity against the atypical persister forms (round bodies, microcolonies, and biofilm) enriched in stationary phase cultures (1417). These persister forms that are not killed by the current Lyme antibiotics may underlie the persistent symptoms in patients despite the standard antibiotic treatment.

To identify drugs that target the persister forms, we screened FDA-approved drug library and NCI compound libraries (1418) against stationary phase cultures enriched in round bodies and microcolonies as well as antibiotic-induced round body persisters (19). Using these models, we identified a range of drugs such as daptomycin (Dap), clofazimine, anthracycline antibiotics, and sulfa drugs, etc., which have good activity against the Borrelia persister forms. However, some of these persister-active agents are either very expensive, difficult to administer, and have to be given intravenously, or have significant side effects.

Essential oils are concentrated volatile liquids that are extracted from plants, most of which are used as spices and culinary herbs. It has been reported in the literature that essential oils have antimicrobial activities (20), and anecdotal patient reports from the internet suggest that some essential oils may improve symptoms of patients with persistent Lyme disease (http://essentialoiladvocate.info/2015/11/6-essential-oils-to-help-fight-lyme-disease.html/http://paulaquinlan.com/products/therapeutic-essential-oils/http://drericz.com/beating-lyme-disease-with-essential-oils/). However, only one study has been done, which assessed the activity of essential oils on B. burgdorferi, where it showed that volatile oil from Cistus creticus has growth inhibiting activity on growing B. burgdorferi (21). Because the current Lyme antibiotics (e.g., Dox, amoxicillin, CefU) are already very good at killing the log phase B. burgdorferi but have poor activity against stationary phase B. burgdorferi (141617). In addition, it is the dormant persister forms enriched in stationary phase cultures (often in variant morphological forms such as round bodies and microcolonies and biofilm) that may be involved in persistent infection that is not cured by the current Lyme antibiotics. Moreover, no study has been performed to evaluate the activity of essential oils on non-growing stationary phase B. burgdorferipersisters. Thus, the purpose of this study is to comprehensively evaluate the activity of essential oils for activity against the more difficult to kill persister forms of B. burgdorferi that are enriched in the stationary phase culture (22). To achieve this goal, we screened a panel of essential oils from common commercial sources for activities against B. burgdorferi stationary phase cells and found that not all essential oils have activity against B. burgdorferi, with oregano, cinnamon bark, and clove bud having among the highest anti-persister activity in vitro.

Materials and Methods

Strain, Media, and Culture Techniques

Low passaged (less than eight passages) B. burgdorferi strain B31 5A19 was kindly provided by Dr. Monica Embers (16). The B. burgdorferi B31 strain was grown in BSK-H medium (HiMedia Laboratories Pvt. Ltd.) and supplemented with 6% rabbit serum (Sigma-Aldrich, St. Louis, MO, USA). All culture medium was filter-sterilized by 0.2 µm filter. Cultures were incubated in sterile 50 ml conical tubes (BD Biosciences, CA, USA) in microaerophilic incubator (33°C, 5% CO2) without antibiotics. After incubation for 7 days, 1 ml stationary-phase B. burgdorferi culture (~107spirochetes per milliliter) was transferred into a 96-well plate for evaluation of potential anti-persister activity of essential oils (see below).

Essential Oils and Drugs

A panel of commercially available essential oils was purchased from Plant Therapy (ID, USA), Natural Acres (MO, USA), or Plant Guru (NJ, USA). Carvacrol, p-cymene, and α-terpinene were purchased from Sigma-Aldrich (USA). Essential oils were added to BSK-H medium or B. burgdorferi cultures to form aqueous emulsion suspensions by vigorous vortexing, followed immediately by serially diluting the essential oil suspensions to desired concentrations into B. burgdorferi cultures. Essential oils were also dissolved in organic solvent dimethyl sulfoxide (DMSO) at 20%, followed by dilution at 1:20 into 7-day-old stationary phase culture to 1% final concentration. To make further dilutions for evaluating anti-Borrelia activity, the 1% essential oils were further diluted with the stationary phase culture to achieve desired dilutions. Dox, CefU (Sigma-Aldrich, USA), and (Dap) (AK Scientific, Inc., USA) were dissolved in suitable solvents (2324) to form 5 mg/ml stock solutions. The antibiotic stocks were filter-sterilized by 0.2 µm filter and stored at −20°C.

Microscopy

The B. burgdorferi cultures were examined using BZ-X710 All-in-One fluorescence microscope (KEYENCE, Inc.). The SYBR Green I/PI viability assay was performed to assess the bacterial viability using the ratio of green/red fluorescence to determine the live:dead cell ratio, respectively, as described previously (1422). This residual cell viability reading was confirmed by analyzing three representative images of the bacterial culture using epifluorescence microscopy. BZ-X Analyzer and Image Pro-Plus software were used to quantitatively determine the fluorescence intensity.

Evaluation of Essential Oils for Their Activities against B. burgdorferi Stationary Phase Cultures

To evaluate the activity of essential oils, aliquots of the essential oils or drugs were added to 96-well plate containing 100 µL of the 7-day-old stationary phase B. burgdorferi culture to obtain the desired concentrations. In the primary essential oil screen, each essential oil was assayed in four concentrations, 1, 0.5, 0.25, and 0.125% (v/v) in 96-well plate. Dap, Dox, and CefU were used as control drugs at 40, 20, 10, and 5 µM, respectively, since this drug combination has been shown to completely eradicate B. burgdorferi persisters in our previous studies (1525). The active hits were further confirmed with lower 0.1 and 0.05% concentration; all tests were run in triplicate. All the plates were incubated at 33°C and 5% CO2 without shaking for 7 days when the residual viable cells remaining were measured using the SYBR Green I/PI viability assay and epifluorescence microscopy as described (1422).

Antibiotic Susceptibility Testing

To qualitatively determine the effect of essential oils in a high-throughput manner, 10 µl of each essential oil from the prediluted stock was added to 7-day-old stationary phase B. burgdorferi culture in the 96-well plate. Plates were sealed and placed in 33°C incubator for 7 days when the SYBR Green I/PI viability assay was used to assess the live and dead cells as described (14). Briefly, 10 µl of SYBR Green I (10,000× stock, Invitrogen) was mixed with 30 µl propidium iodide (PI, 20 mM, Sigma) into 1.0 ml of sterile dH2O. Then 10 µl staining mixture was added to each well and mixed thoroughly. The plates were incubated at room temperature in the dark for 15 min followed by plate reading at excitation wavelength at 485 nm and the fluorescence intensity at 535 nm (green emission) and 635 nm (red emission) in microplate reader (HTS 7000 plus Bio Assay Reader, PerkinElmer Inc., USA). With least-square fitting analysis, the regression equation and regression curve of the relationship between percentage of live and dead bacteria as shown in green/red fluorescence ratios was obtained. The regression equation was used to calculate the percentage of live cells in each well of the 96-well plate.

The standard microdilution method was used to determine the MIC of carvacrol, based on inhibition of visible growth of B. burgdorferi by microscopy. Carvacrol was added to B. burgdorferi cultures (1 × 104spirochetes per milliliters) to form aqueous suspension by vortex. The carvacrol suspension was twofold diluted from 0.5% (equivalent to 4.88 µg/ml) to 0.008% (equivalent to 0.08 µg/ml). All experiments were run in triplicate. B. burgdorferi culture was incubated in 96-well microplate at 33°C for 7 days. Cell proliferation was assessed using the SYBR Green I/PI assay and BZ-X710 All-in-One fluorescence microscope (KEYENCE, Inc.).

Subculture Studies to Assess Viability of the Essential Oil-Treated B. burgdorferi Organisms

A 7-day-old B. burgdorferi stationary phase culture (500 µl) was treated with essential oils or control drugs for 7 days in 1.5 ml Eppendorf tubes as described previously (15). After incubation at 33°C for 7 days without shaking, the cells were collected by centrifugation and rinsed with 1 ml fresh BSK-H medium followed by resuspension in 500 µl fresh BSK-H medium without antibiotics. Then 50 µl of cell suspension was transferred to 1 ml fresh BSK-H medium for subculture at 33°C for 20 days. Cell proliferation was assessed using SYBR Green I/PI assay and epifluorescence microscopy as described above.

Results

Evaluation of Essential Oils for Activity against Stationary Phase B. burgdorferi

We evaluated a panel of 34 essential oils at four different concentrations (1, 0.5, 0.25, and 0.125%) for activity against a 7-day-old B. burgdorferistationary phase culture in the 96-well plates with control drugs for 7 days. Consistent with our previous studies (1425), Dap included as a persister drug control was shown to have higher activity against the B. burgdorferi stationary phase culture than the currently used antibiotics such as Dox and CefU for treating Lyme disease (Table 1), with a dose-dependent increase in killing activity. We used 40 µM Dap (64.8 µg/ml) as a positive persister drug control because this is a clinically achievable concentration that could cause near complete clearance of B. burgdorferistationary phase cells while the current Lyme antibiotics could not (1415) (Figure 1). Five essential oils (bandit, oregano, clove bud, geranium bourbon, and cinnamon bark) at 1% concentration showed more activity against the stationary phase B. burgdorferi culture than 40 µM Dap with the plate reader SYBR green I/PI assay (Table 1). We found some essential oils have autofluorescence, which interfered with the SYBR Green I/PI plate reader assay; however, we were able to resolve this issue present in some samples by fluorescence microscopy. As we previously described (18), we directly calculated the green (live) cell ratio of microscope images using Image Pro-Plus software, which could eliminate the background autofluorescence. Using the SYBR Green I/PI assay and fluorescence microscopy, we additionally found 18 essential oils that showed more or similar activity against the stationary phase B. burgdorferi at 1% concentration compared to the 40 µM Dap, included as a positive persister drug control (14), which could eradicate all live cells as shown by red (dead) aggregated cells (Table 1; Figure 1A). At 0.5% concentration, seven essential oils (oregano, cinnamon bark, clove bud, citronella, wintergreen, geranium bourbon, and patchouli dark) were found to have higher or similar activity against the stationary phase B. burgdorferi than 40 µM Dap by fluorescence microscope counting after SYBR Green I/PI assay (Table 1; Figure 1B). However, bandit thieves oil, while having good activity at 1%, had significantly less activity at 0.5% and lower concentrations (Table 1). Among the effective hits, five essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) still showed better activity than 40 µM Dap at 0.25% concentration (Table 1; Figure 1C). Eventually, oregano, cinnamon bark, and clove bud were identified as the most active essential oils because of their remarkable activity even at the lowest concentration of 0.125%, which showed similar or better activity than 40 µM Dap (Table 1; Figure 1D).

TABLE 1
www.frontiersin.orgTable 1. Effect of essential oils on a 7-day-old stationary phase Borrelia Burgdorferi.a

FIGURE 1
www.frontiersin.orgFigure 1. Effect of essential oils on the viability of stationary phase Borrelia burgdorferi. A 7-day-old B. burgdorferistationary phase culture was treated with essential oils at different concentrations (v/v), 1% (A), 0.5% (B), 0.25% (C), and 0.125% (D) for 7 days followed by staining with SYBR Green I/PI viability assay and fluorescence microscopy. Daptomycin was included as a persister-active positive control drug at 40, 20, and 10 µM in panels (A–C), respectively.

To further compare the activity of these active essential oils and find whether they could eradicate stationary phase B. burgdorferi at lower concentrations, we evaluated six essential oils (oregano, cinnamon bark, clove bud, citronella, geranium bourbon, and wintergreen) at even lower concentrations at 0.1 and 0.05%. We noticed that oregano could not wipe out stationary phase B. burgdorferi at 0.05% concentration as shown by some residual green aggregated cells (Table 2; Figure 2), despite oregano showing strong activity sterilizing all the stationary phase B. burgdorfericells at and above 0.1% concentration (Tables 1 and 2).

TABLE 2
www.frontiersin.orgTable 2. Comparison of essential oil activity against stationary phase Borrelia burgdorferi at 0.1 and 0.05% (v/v).a

FIGURE 2
www.frontiersin.orgFigure 2. Effect of active essential oils or their ingredients on stationary phase Borrelia burgdorferi. A B. burgdorferi stationary phase culture (7 days old) was treated with 0.1% (A) or 0.05% (B) essential oils (labeled on the image) or the ingredients (carvacrol, α-terpinene, or p-cymene) of oregano for 7 days followed by staining with SYBR Green I/PI viability assay and fluorescence microscopy.

To address potential concern that the essential oils may not dissolve in culture medium well and may affect the above results, we diluted all the 34 essential oils in DMSO and found 19 could be dissolved and 15 could not be dissolved in DMSO (see Table 1, Superscript c). For essential oils that can be dissolved in DMSO, we simultaneously dissolved them in DMSO and also in aqueous culture medium at the same dilutions of 0.5, 0.1, 0.05% and evaluated their activity against the 7-day-old stationary phase B. burgdorferi. However, we found that there was no significant difference in their anti-borrelial activity when dissolved in DMSO or in aqueous culture medium (p = 0.138–0.975) (see Table S1 in Supplementary Material).

Carvacrol As a Highly Potent Active Ingredient of Oregano Oil against Stationary Phase B. burgdorferi

To identify active ingredients of the oregano essential oil, we tested three major constituents (26), carvacrol, p-cymene, and α-terpinene on the stationary phase B. burgdorferi. Interestingly, carvacrol showed similar high activity against B. burgdorferi as oregano essential oil either at 0.1% (6.5 µM) or 0.05% (3.2 µM) concentration (Table 2; Figure 2, h). Meanwhile, we also found that carvacrol was very active against replicating B. burgdorferi, as shown with a very low MIC of 0.16–0.31 µg/ml. By contrast, p-cymene and α-terpinene did not have activity against the stationary phase B. burgdorferi (Table 2; Figure 2, i,j). Thus, carvacrol could be one of the most active ingredients in oregano oil that kill stationary phase B. burgdorferi.

Subculture Studies to Evaluate the Activity of Essential Oils against Stationary Phase B. burgdorferi

To confirm the activity of the essential oils in killing stationary phase B. burgdorferi, we performed subculture studies in BSK-H medium as described previously (15). To validate the activity of these essential oils, samples of essential oil-treated cultures were subjected to subculture after removal of the drugs by washing followed by incubation in fresh BSK medium for 21 days. According to the essential oil drug exposure experiments (Table 2), we used subculture to further confirm whether the top six active essential oils (oregano, cinnamon bark, clove bud, citronella, geranium bourbon, and wintergreen) could eradicate the stationary phase B. burgdorferi cells at 0.1 or 0.05% concentration. At 0.1% concentration, the subculture results were consistent with the above drug exposure results, and no regrowth in samples of three top hits, oregano, cinnamon bark, and clove bud was observed (Figure 3A, b–d). However, citronella, geranium bourbon, and wintergreen could not completely kill the stationary phase B. burgdorferi with many spirochetes being visible after 21-day subculture (Figure 3A, e–g). Subculture also confirmed the activity of carvacrol by showing no spirochetal regrowth in the 0.1% carvacrol-treated samples. However, in the p-cymene and α-terpinene subculture samples, growth in 0.1% concentration samples was observed. At 0.05% concentration, we observed no spirochetal regrowth after 21-day subculture in the oregano and cinnamon bark-treated samples (Figure 3B, b,c), even though some very tiny aggregated microcolonies were found after treatment (Figure 2B, b,c). Although the clove bud showed better activity than the cinnamon bark at 0.05% concentration (Table 2), it could not sterilize the B. burgdorferi stationary phase culture, as they all had visible spirochetes growing after 21-day subculture (Figure 3B, c,d). Additionally, 0.05% citronella, geranium bourbon, and wintergreen could not kill all stationary phase B. burgdorferi since many viable spirochetes were observed in the 21-day subculture (Figure 3B, e–g). Remarkably, 0.05% carvacrol sterilized the B. burgdorferi stationary phase culture as shown by no regrowth after 21-day subculture (Figure 3B, h).

FIGURE 3
www.frontiersin.orgFigure 3. Subculture of Borrelia burgdorferi after treatment with essential oils. A B. burgdorferi stationary phase culture (7 days old) was treated with the indicated essential oils at 0.1% (A) or 0.05% (B) for 7 days followed by washing and resuspension in fresh BSK-H medium and subculture for 21 days. The viability of the subculture was examined by SYBR Green I/PI stain and fluorescence microscopy.

Discussion

Previous in vitro studies showed that certain essential oils have antibacterial activity against multidrug resistant Gram-negative clinical isolates (27). In this study, we tested 34 essential oils from different plants on non-growing stationary phase B. burgdorferi as a model of persister drug screens. We were able to identify 23 essential oils at 1% concentration that are more active than the control persister drug Dap (40 µM or 64.8 µg/ml) 3 of which, oregano, clove bud, and cinnamon bark, highlighted themselves as having a remarkable activity even at a very low concentration of 0.125% (Table 1). Among them, oregano and cinnamon bark essential oils demonstrated the best activity as shown by complete eradication of stationary phase B. burgdorferi even at 0.05% concentration. In a previous study, oregano oil was found to have antibacterial activity against Gram-positive and Gram-negative bacteria (26). Here, for the first time, we identified oregano oil as having a highly potent activity against stationary phase B. burgdorferi. We tested three major ingredients of oregano oil (carvacrol, p-cymene, and α-terpinene) on B. burgdorferi, and found carvacrol is the major active component, which showed similar activity as the complete oregano oil (Figures 2 and 3). We could not rule out the possibility that other components may also have activity against B. burgdorferi. We plan to use GC/MS to identify components of the active essential oils and test them on B. burgdorferi in the future.

In addition to the above findings, we noted that oregano oil can dramatically reduce the size of aggregated biofilm-like microcolonies compared to the antibiotic controls (Figure 1). After treatment with 0.25% oregano essential oil, only some dispersed tiny red aggregated cells were left in the culture (Figure 1C). Interestingly, we observed that amount and size of aggregated biofilm-like microcolonies of B. burgdorferi dramatically reduced with increasing concentrations of oregano oil, as aggregated biofilm-like structures vanished after treatment with 0.5 or 1% oregano essential oil. When we reduced the concentration of oregano essential oil to 0.05%, it could not eradicate stationary phase B. burgdorferi (residual viability 56%, Figure 2B, b) but the size of aggregated microcolonies decreased significantly. By contrast, Dap could kill the aggregated biofilm-like microcolonies of B. burgdorferi as shown by red aggregated microcolonies but could not break up the aggregated microcolonies even at the highest concentration of 40 µM (Figure 1A). It has been shown that carvacrol and other active compositions of oregano oil could disrupt microbial cell membranes (20). Future studies are needed to determine whether oregano oil and other active essential oils have similar membrane disruption activity and could destroy the aggregated biofilm-like structures of B. burgdorferi.

We also noted that some essential oils such as oregano and cinnamon bark had relatively high residual viability percentage (Table 2) at a low concentration of 0.05%, but the treated B. burgdorferi cells did not grow in the subculture experiment (Table 2; Figure 3B, b,c). We speculate that these essential oils could dissolve the dead B. burgdorferi cells presumably due to their high lipophilicity. The reduction of the number of dead red cells by the essential oil made the residual viability percentage increase, although the number of live cells obviously decreased as well (Figure 2A, b–d and Figure 2B, b,c). In addition, these essential oils may also permanently damage B. burgdorferi during the treatment, such that even in the fresh medium, the residual B. burgdorferi cells still could not regrow.

Meanwhile, we found that, at a high concentration (above 1%), lemongrass or oregano essential oil showed apparent high residual viability percentage by the SYBR Green I/PI plate assay, compared with the microscopy counting data (Table 1; Figure 1A). This may be caused by strong autofluorescence of these essential oils that severely interfere with the SYBR Green I/PI assay. We studied the emission spectra of lemongrass essential oil using Synergy H1 multi-mode reader and found lemongrass essential oil emits the strongest autofluorescence. The peak fluorescence of lemongrass essential oil is at 520 nm that overlaps with the green fluorescence of SYBR Green I dye (peak is at 535 nm). The strong autofluorescence caused the abnormal residual viability percentage (above 100% in Table 1) using SYBR Green I/PI plate assay. We also found that the oregano essential oil emits autofluorescence at 535 nm, which pushed the green/red fluorescence ratio higher than their true values (Table 1). We were able to solve this problem by using fluorescence microscopy as a more reliable measure to confirm the results of SYBR Green I/PI plate reader assay (1418).

Additionally, we found cinnamon bark and clove bud essential oils showed excellent activity against stationary phase B. burgdorferi. Cinnamon bark essential oil eradicated the stationary phase B. burgdorferi cells even at 0.05% concentration (Table 2) while clove bud essential oil showed sterilization at 0.1% or above concentration. Extractions of cinnamon bark and clove bud have been used as flavors for food processing. Based on this discovery, effective oral regimens with low side effect may be developed to fight against Lyme disease in future studies.

In a previous study, it has been found that volatile oil from C. creticusshowed growth inhibiting activity against growing B. burgdorferi in vitro(21), but its activity against stationary phase bacteria enriched in persisters was not evaluated. In this study, we tested six Citrus plants (Citrus bergamia, Citrus sinensis, Citrus limonum, Citrus aurantifolia, Citrus racemosa, Citrus reticulata) on the stationary phase B. burgdorferi culture and found bergamot (C. bergamia) had high activity (residual viability 12%) at 1% concentration. The other Citrus essential oils did not show good activity against B. burgdorferi compared with clinically used Dox, CefU, or Dap (Table 1).

Although we found several essential oils (oregano, cinnamon bark, clove bud) that have excellent activity against B. burgdorferi stationary phase cells in vitro (Table 1), the effective dose that will show equivalent activity in vivo is unknown at this time largely because the active ingredients in the active essential oils and the pharmacokinetic profile of the active ingredients are not all known. Future studies are needed to identify the active ingredients of the active essential oils and determine their effective dosage in vivo. Identification of active components or active component combinations from essential oils may help to eliminate the quality difference of natural products such that MICs and MBCs of the active compounds could be tested on growing bacteria in future studies. We were able to identify carvacrol as the most active ingredient in oregano essential oil, and its pharmacokinetics has been studied as a feed addition in pigs (28) and topical oil in cattle (29). In the rat model, the calculated LD50 of carvacrol is 471.2 mg/kg (30). We noticed that the 0.05% of carvacrol used here, which is equivalent to 0.48 µg/ml or 3.2 µM and completely eradicated B. burgdorferi stationary phase cells in subculture (Figure 3), is lower than the peak plasma concentration (3.65 µg/ml) in the swine study (28). Clinically, an antimicrobial agent that penetrates the blood–brain barrier as well as the persistent Borreliaorganisms do, would be an ideal candidate for further study (31). Oil of Oregano is a good candidate for this consideration given that its main active ingredient—carvacrol is a phenolic monoterpenoid that is fat-soluble and found in animal studies to have blood–brain barrier penetration (32). These findings favor the application of carvacrol in future treatment studies. Importantly, carvacrol seems to be more active than Dap, the most active persister drug against B. burgdorferi (1415). In this study, 0.1% carvacrol (6.4 µM) showed much higher activity (2% residual viability) than 5 µM Dap (45% residual viability) (Tables 1 and 2). In addition, 0.05% carvacrol (3.2 µM) could eradicate B. burgdorferistationary phase cells with no regrowth in subculture, by contrast, 10 µg/ml Dap (6.2 µM), could not completely kill B. burgdorferi stationary phase cells as shown by regrowth in subculture (15). Furthermore, we know that the drug’s activity against non-replicating bacteria is not always consistent with its activity against growing bacteria. With that in mind, we also tested carvacrol on the growing B. burgdorferi cells and found its MIC to be 0.16–0.31 µg/ml. This result showed that carvacrol is a good drug candidate active against not only stationary phase B. burgdorferi but also log phase replicating cells. For consideration in clinical applications, there is limited safety information on carvacrol and essential oils in humans. In mice, carvacrol has been given at 40 mg/kg daily for 20 days with no apparent toxicity (33). However, carvacrol and other active components of essential oils showed certain cytotoxicity (IC50 of carvacrol was 200–425 µM) (3435) on mammalian cells and genotoxic activity in vivo (10 mg/kg) (36). We found that few essential oils have been used in human studies. This is probably because these essential oils are natural products, which are mixtures of multiple active components, and little is known about their active ingredients or their mechanisms of antibacterial action. Thus, more work is needed to identify their active compounds of the effective essential oils. In addition, it is well known that some effective drugs identified in vitro may fail when tested in vivo. Thus, adequate animal studies are needed to confirm the safety and efficacy of the active essential oils in in vivosetting before human studies.

In summary, we found that essential oils had varying degrees of activity against stationary phase B. burgdorferi. The most active essential oils are oregano, cinnamon bark, and clove bud, which seem to have even higher activity than the persister drug Dap. A particularly interesting observation is that these highly active essential oils had remarkable biofilm-dissolving capability and completely eradicated all stationary phase cells with no regrowth in vitro. In addition, carvacrol was found to be the most active ingredient of oregano with high activity against B. burgdorferi stationary phase cells. Future studies are needed to test whether carvacrol could replace the persister drug Dap in drug combinations against more resistant biofilm-like structures and for treating persistent Borrelia infections in animal models and in patients.

Author Contributions

YZ and JM conceived the experiments; JF, WS, and SZ performed the experiments; JF, NZ, JM, and YZ analyzed the data; and JF, NZ, JM, and YZ wrote the paper.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer AN and handling editor declared their shared affiliations.

Acknowledgments

We acknowledge the support of this work by Global Lyme Alliance, Lyme Disease Association, NatCapLyme, and Steve Sim Fund. YZ was supported in part by NIH grants AI099512 and AI108535.

Supplementary Material

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fmed.2017.00169/full#supplementary-material.

Referenceshttps://doi.org/10.3389/fmed.2017.00169

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**Comment**

While this is potentially great news for patients, please remember this was all done in vitro (in a test tube), similarly to the work on Stevia.  Also, we have no idea what dosage would be effective or safe for human consumption.

More on Stevia:

https://madisonarealymesupportgroup.com/2015/11/19/stevia-and-bb/

https://madisonarealymesupportgroup.com/2017/08/11/stevia-clinical-trial-underway/

 

 

Bb in California Chipmunk and Squirrels

http://online.liebertpub.com/doi/abs/10.1089/vbz.2017.2134#utm_source=FastTrack&utm_medium=email&utm_campaign=vbz

Distribution and Diversity of Borrelia burgdorferi sensu lato Group Bacteria in Sciurids of California

Roy Austin N., Straub Mary H., Stephenson Nicole, Sholty Kathleen E., and Foley Janet. Vector-Borne and Zoonotic Diseases. October 2017, ahead of print. https://doi.org/10.1089/vbz.2017.2134

Online Ahead of Print: October 4, 2017

ABSTRACT

California has a remarkable diversity of squirrel and chipmunk species (sciurids), and five named and several unnamed genospecies in the Borrelia burgdorferi sensu lato group (BBSL) of bacteria as well, many of which utilize sciurids as reservoirs. We investigated the prevalence, spatial distribution, and diversity of BBSL in sciurids of California by literature search, PCR of 585 ear tissue samples from 15 sciurid species prospectively collected across 19 California counties, and DNA sequencing when possible. Seven publications documented BBSL infections in western gray squirrels (Sciurus griseus), fox squirrels (Sciurus niger), eastern gray squirrels (Sciurus carolinensis), Douglas squirrels (Tamiasciurus douglasii), and redwood chipmunks (Tamias ochrogenys) in northern California. Prospective sampling added new BBSL infection records for long-eared chipmunks (Tamias quadrimaculatus), Allen’s chipmunks (Tamias senex), and Siskiyou chipmunks (Tamias siskiyou). Infection was detected in the Mendocino, North Coast, West Sierra, and Central Valley regions of California.

The overall PCR prevalence was 9.4% (n = 585), and exceeded 40% (n = 84) in Mendocino and farther north along the Pacific coast. Redwood (40.7%, n = 81) and Siskiyou (22.2%, n = 18) chipmunks had the highest prevalence of BBSL infection. BBSL infections were associated with arboreal and semiarboreal sciurid species and species occurring in conifer forests. Western gray squirrels and Allen’s chipmunks in Humboldt County and redwood chipmunks in Mendocino County were infected with B. burgdorferi sensu stricto, while we identified Borrelia bissettiae in Douglas squirrels and Siskiyou chipmunks in Humboldt and Del Norte Counties. This indicates that further study of sciurids can aid in describing the ecology of BBSL in California.

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For more:  https://madisonarealymesupportgroup.com/2016/10/02/the-role-of-birds-in-tickborne-illness/

https://madisonarealymesupportgroup.com/2017/08/17/of-birds-and-ticks/

 

Rob Thomas on Marisol’s 14-Year MSIDS Battle: ‘It’s Like a Weird Alien Inhabited My Wife’

http://www.etonline.com/exclusive-rob-thomas-marisols-14-year-lyme-disease-battle-its-weird-alien-inhabited-my-wife-87787?

EXCLUSIVE: Rob Thomas on Marisol’s 14-Year Lyme Disease Battle: ‘It’s Like a Weird Alien Inhabited My Wife’

By 
rob_thomas_marisol_thomas_gla_101316_getty_images_614394704.474104268_highres.jpg
Photo: Global Lyme Alliance / Getty Images

Rocking out, side of stage, while her husband, Matchbox Twenty frontman Rob Thomas, performed in Chula Vista, California, Marisol Thomas may have appeared a gorgeous picture of health and happiness as she sang along to the band’s hit, “Disease.” But behind the smiles, the scene was a rare moment of reprieve from a debilitating 14-year fight against a disease which Rob says has transformed his perspective on life, robbed any thoughts of having more children and “inhabited” parts of his wife.

 “It’s crazy how normal something abnormal can become,” Rob tells ET, during a rare interview with Marisol to promote Global Lyme Alliance’s 3rd Annual New York Gala — where he will perform and Marisol is an honoree for her advocacy work for Lyme and other tick-borne diseases. “It’s like if one day you woke up and there was an alien living in your house and the first couple of weeks you’d be f**king freaked out, but after a while you’d name it and it would just be there. This sickness has been like this weird alien that’s moved into our life and inhabited my wife. Sometimes she’s just not herself. Watching the person you’re closest to become someone other than herself is so [hard].”
The couple has grappled with Marisol’s health battles for more than a decade, during which she visited countless specialists, desperate for accurate diagnosis and treatment. Showing symptoms of autoimmune disease, she was told she had Multiple Sclerosis one minute, Lupus the next and at one point, while lying in a hospital for days, she believed she had pancreatic cancer.Rob frustratingly recalls one specialist exploring the possibility of Lyme (a potentially fatal illness spread by ticks) years ago, but the common diagnostic test is inaccurate more than half the time.However, after undergoing surgery to remove a lesion from her brain in 2015, doctors suspected and tested for the illness, eventually diagnosing eight tick-borne diseases — including late stage Neurological Lyme Borreliosis, Babesiosis and Bartonella — all of which induce different symptoms and require various treatments and doctors. The infections are also believed to have triggered Hashimoto’s disease and Lupus-like symptoms, as well as Atypical Trigeminal Neuralgia, nicknamed the “suicide disease,” because it attacks facial nerves and “makes you wish for death.”

“After brain surgery, everyone went, ‘You’re on the road to recovery — you’re doing great,’” reflects Marisol, 41. “But that was actually the easiest part. The real battle began when we finally figured out what was going on with me.”

While Lyme can be cured with antibiotics if caught early, Marisol’s late-stage diagnosis and presence of multiple infections means it’s harder to manage. Her treatment is constantly tweaked according to which infection is flaring up most, and involves a mixture of pharmaceutical meds and holistic therapies, some of which are not easily obtainable due to being commercially unviable.

Once a driven “A-type,” who thrived in co-managing Rob’s career and passionately running their Sidewalk Angels Foundation, which assists animal rescue groups, she says Lyme has “stolen” her life and frequently traps her on the tour bus with symptoms including visual disturbance, numbness, seizures and constant pain. Being correctly diagnosed means she has better treatment, fewer seizures and more hours of feeling “semi-functional,” but the former model says there’s still a “long road ahead.”

“It’s like you’re standing still unable to participate as everything keeps going on without you, then you wake up the next day and the same thing happens,” she explains. “I don’t consider what I’m doing living. I’m existing … and fighting to hopefully one day live again. That’s a horrible place to be in and I have support to pursue it, but I know there are people who don’t. You become so sick of being sick and want nothing more than a moment away from it, so if I have a [good] moment I run with it. My mom and I call them happy moments and I do more in those hours than I ever would’ve before!”

Currently traveling with Rob on Matchbox Twenty’s “A Brief History of Everything” tour, such escapism has included visiting the California Mid-State Fair and hanging with actor pal Wilmer Valderrama.

Although the couple relish those precious periods of respite from Marisol’s physical symptoms, the mental toll of the illness is an ongoing battle, largely thanks to the “stigma” attached to Lyme, an illness which has increased 25-fold since national surveillance began in 1982, with more than 329,000 new cases each year in the U.S. alone. Marisol notes how Lyme sufferers often get accused of “exaggerating, faking or needing psychiatric help,” which has triggered some to consider or commit suicide.

Despite harsh comments and being accused of sharing her story for attention, she continues to advocate for tick-borne diseases and will be honored alongside menswear designer Joseph Abboud at Global Lyme Alliance’s Oct. 11 gala at Cipriani, 42nd Street, where fellow Lyme sufferers Yolanda Hadidand Aly Hilfiger are Honorary Event co-chairs. Rob, who performed at last year’s gala, will once again hit the stage alongside Chris Daughtry.

For Rob, watching his wife of almost 18 years somewhat vanish into a daily health battle has been a harrowing journey, which inspired powerful solo hits like 2009’s “Your Diamonds” and the poignant 2016 ballad “Pieces.”
The ongoing struggles have come with significant personal costs, like sacrificing family hopes, social lives and holidays.

Once contemplating having children together, Rob — who has a 19-year-old son, Maison, from a previous relationship — admits all thoughts of having more kids were long ago swallowed by illness. “For so long now, we’ve told ourselves that we never really wanted kids together that we don’t know if that’s even true,” he admits. “We’ve known for the last decade that because of Mari’s health, that was something we couldn’t do and you just resign yourself to the fact. If we can have Mari back healthy and she can have a normal life, then we don’t need a family to complete us because we have so much time to make up for.”

“The real personal cost has been [little things] like one of our dearest friends visiting us on the road last night and she couldn’t get off the bus to see him,” he continues. “She loves hanging with everyone, but she’s always one door away unable to be part of it. Or there’s holidays she has to let go by. I’ve never seen anybody love Christmas like this girl loves Christmas, so to watch it pass [is tough]. These little things make a year suddenly go by without us realizing.”

While Rob, 45, remains deeply dedicated to Marisol — halting his 2015 solo tour after learning she needed surgery — he admits the helplessness and guilt that comes with being her spouse can be arduous. Some relationships don’t survive the perils of the disease, with Hadid stating in her new book (Believe Me: My Battle With the Invisible Disability of Lyme Disease) that her sickness contributed to the collapse of her seven-year marriage to David Foster.

Rob often feels “survivor’s guilt and remorse,” for being healthy. “Often their day-to-day life makes you feel selfish just by doing normal things,” he says. “l’ll say, ‘Man, I had the worst show,’ then realize who I’m talking to and go, ‘I’m sorry, that’s the stupidest thing to say!’ You get perspective. I don’t think I have anything to complain about because I’ve seen how bad it can get for someone. The great thing about going to the Global Lyme Alliance benefit last year was seeing we’re not alone. People talk about their health struggles and what the family goes through and it’s normal to hear someone say, ‘I can’t tell you how much I thank my family for not just killing me!’ — and think, ‘I totally get that.’”

“For public record, I’m not going to kill my wife!” he adds. “But they spend so much time trying to get better that, as a caretaker, you pick up the slack on everything else in their life. I get tired and frustrated, then I see a glimpse of Mari and we talk, recognize it, then keep going.”

Rob adds that having Marisol on tour has been a blessing because he’s always nearby, while still able to fulfil his duties with Matchbox Twenty.

The GRAMMY-nominated group, whose debut album Yourself or Someone Like You turns 21 next month, have loved being reunited following guitarist Kyle Cook’s brief fallout with Rob and departure from the band. Rob says the group is now stronger than ever.

“I don’t know that we’ve ever gotten along as well as we are now,” he shares. “We hashed everything out and have taken it to another level, where we enjoy each other’s company on and off stage. We’re constantly in communication, and even today, on a day off, we’re writing stupid, little things to each other. I have a solo record I’ve almost finished writing and will put out next year, but because of this tour, we know after that, we’re going to record new music and want to tour again.”

And, when that tour rolls around, Marisol naturally hopes to be side of stage for as many shows as she can. “On my good days, I make sure I’m there — it’s my favorite thing!” she says. “We always kid how I’m a road chick from way back … music and touring has been a huge part of my life, so having moments where I can stand side of stage and see Rob do his thing and be better than ever is amazing. Those are my happy moments.”

See ’80s-singing icon and new Dancing With the Stars contestant Debbie Gibson talk about her fight against Lyme disease in the video below.

 

 

Remembering Dr. Masters the Rebel For Lyme Patients Who Took on the CDC Single-handedly

The following story, covered by Pamela Weintraub, in Cure Unknown: Inside the Lyme Epidemic, should be made into a movie.

Seriously.  A Movie.

Way back in 2003 Weintraub interviewed Dr. Masters of Cape Girardeau, Missouri, about Lyme disease in the South, though the “powers that be,” continued to deny it (and still do).  The interview is broken down into 4 sections below.  If you have the time, please read Weintrab’s version.  With journalistic flair and insight as a patient herself, she reveals the character of the doctor who never shied away from controversy.  Highlights below.

EdMastersSmall.211133811

Dr. Ed Masters  http://www.georgialymedisease.org/we_salute

https://www.psychologytoday.com/blog/emerging-diseases/200906/rebel-cause-the-incredible-dr-masters-part-1  Part 1:  Masters started recognizing Lyme in his own patients after preparing for an entire year for a talk on Lyme for a forestry meeting in 1988.  He dutifully reported his cases to the Missouri Department of Public Health, who completely ignored him.  Masters took pictures of every rash with the corresponding patient and bought a special freezer to store patient rash and blood samples.

https://www.psychologytoday.com/blog/emerging-diseases/200906/rebel-cause-the-incredible-dr-masters-part-ii  Part 2:  Understanding treatment failure, Masters treated acute patients with 3-4 weeks with much success and others for longer.  Masters worked with Missouri entomologist, Dorothy Fier, who found borrelia in 2% of sampled lone star ticks and who supported Masters’ Missouri Lyme.  Despite publicity and validation, the CDC insisted that the EM rash was NOT diagnostic for LD for Missouri patients due to the fact that neither Ixodes dammini nor Ixodes pacificus were found there.  Go here:  http://steveclarknd.com/wp-content/uploads/2013/11/The-Confounding-Debate-Over-Lyme-Disease-in-the-South-DiscoverMagazine.com_.pdf for a great article on how Andrew Spielman’s tick maps ruled Lyme Land like the iron curtain, and frankly still do, dictating where Lyme is and is not.  https://madisonarealymesupportgroup.com/2017/08/24/canine-maps-better-than-the-cdcs-in-predicting-lyme-disease/  (nothing’s changed)

https://www.psychologytoday.com/blog/emerging-diseases/200906/rebel-cause-the-incredible-dr-masters-part-iii  Part 3:  CDC researchers camped out in Masters’ office for 2 weeks and then took blood and biopsy samples back to Fort Collins.  Two years later Masters received a draft that asserted that Missouri rashes were different from real Lyme rashes despite expert dermatologists from all over the globe ruling that these rashes were identical.  Masters went over the charts with a fine tooth comb and found huge CDC errors.  Another contentious point was the arbitrary cut off date imposed by the CDC which did not pick up serious late-stage symptoms.  Another was the CDC’s rejection of many positive blood tests performed in its own lab, as well as other lab work showing “motile spirochetes” in nearly 5% of lone star nymphs.  Long story short, after numerous revisions, Masters could never sign onto the bastardized study.  The CDC had purposely tossed out data and manipulated the results.

https://www.psychologytoday.com/blog/emerging-diseases/200906/rebel-cause-the-incredible-dr-masters-part-iv-conclusion  Part 4:  The CDC essentially tried blackmailing Masters into signing off on the study before they would let him see the final draft.  Refusing the bait, he published a letter of objection in the Journal of Infectious Diseases as well as an article of his own in Missouri Medicine which showed Missouri patients met the CDC surveillance definition for LD and growing evidence that lone star ticks were infected with an unidentified spirochete causing identical symptoms of LD in patients.  When the CDC study came out they unbelievably attributed the rashes they labeled STARI to an allergy to tick saliva!  In the acknowledgment section of the paper where dozens of folks were thanked, they completely omitted the man who made it all possible – Dr. Ed Masters.

If this makes you want to hit something, unfortunately, it only gets worse.

Supporting Masters’ theory, James H. Oliver, Jr., Callaway later showed that mice from 5 southern states tested positive for Borrelia burgdorferi, the causative agent of LD and had just as many reactive antibodies as mice from Connecticut.  He also found a range of new Southern ticks transmitting a literal hodgepodge of borrelia as well as a unique Southern strain of Bb with unusual outer surface proteins that are undetectable on Northern blood tests.  

This was over 20 years ago and Southern authorities and the CDC still deny Lyme:  https://madisonarealymesupportgroup.com/2016/09/24/arkansas-kids-denied-lyme-treatment/   They also still cling to the contention that STARI is a rash-only illness unrelated to Lyme and that antibiotics should be used sparingly if at all.  Masters’ patients all improved dramatically with longer antibiotic treatment.

Left out in the links above but on page 185 in Weintraub’s book she reveals another fly in the ointment: The CDC kept changing the blood tests from year to year until samples that were positive were now negative.

Microbiologist Tom Grier’s “A Short Historical Perspective of Lyme Disease,”  https://www.lymeneteurope.org/forum/viewtopic.php?t=5133#p37209  reveals how Masters constantly had the state of Missouri on his tail and how yet even today the state singles out and persecutes Lyme doctors that deviate from the norm (CDC guidelines).  In Masters’ case the state of Missouri illegally seized and destroyed all the patient’s medical records, blood samples, and skin biopsies in Masters’ freezer.  What they didn’t know is that Masters had his patients keep duplicates in their own freezers.  Better yet, all the patients had signed a paper announcing that the office samples were property of the patients alone – the legal loophole proving that seizing and destroying patient samples was a violation of their rights and medical records.  

When patients demanded to have their samples returned, only then did the state stop harassing Dr. Masters.

By the way, this persecution has been happening non-stop in every state in the U.S.  My own LLMD spent $50K out of pocket to defend his practice.  Here is one such example:   https://madisonarealymesupportgroup.com/2017/06/24/llmd-daniel-cameron-disciplined-by-ny-medical-authorities/

At least in Wisconsin the state medical boards strategically do not typically title the doctor’s offense with the word “Lyme” as they know patients will come unhinged.  There’s a lot of us out here!  What they often do is sift through patient records with such a fine toothed comb that they will find a nit.  Any little nit.  It is this nit that forces the doctor to pay a hefty fine, have their license suspended, or even have to close their practice.  For an example of this type of witch hunting:  https://www.uppitywis.org/blogarticle/wisconsin-lyme-doctor-gets-reprieve  (Dr. Hoffman was killed in a car crash last year, and is horribly missed.) 

A person once told Masters,

“Masters, they were having a big old fine party, and you’re the turd in the punchbowl.  You spoiled it.”

We need more turds in the punchbowl.

For another great piece on Dr. Masters:  1992+Lyme+Times+V3N2+Fall  Lyme Times, 1992

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**Dedicated to Kathy White, who tirelessly advocates for patients in the South despite the fact it doesn’t exist there.”**                           http://www.mvlymecenter.org/2013/06/24/lyme-stories-kathy-white-kansas-city/

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Droplet Digital PCR Shows 60% Bb Infection Rate in Ticks and Over 50,000 Spirochetes Per Adult Tick

https://www.cambridge.org/core/journals/parasitology/article/validation-of-droplet-digital-pcr-for-the-detection-and-absolute-quantification-of-borrelia-dna-in-ixodes-scapularis-ticks/A47CEB12C767C573BEB7AD8BD8E3A252

Validation of droplet digital PCR for the detection and absolute quantification of Borrelia DNA in Ixodes scapularis ticks

Summary

We evaluated the QX200 Droplet Digital PCR (ddPCR™, Bio-Rad) system and protocols for the detection of the tick-borne pathogens Borrelia burgdorferi and Borrelia miyamotoi in Ixodes scapularis nymphs and adults collected from North Truro, Massachusetts. Preliminary screening by nested PCR determined positive infection levels of 60% for B. burgdorferi in these ticks. To investigate the utility of ddPCR as a screening tool and to calculate the absolute number of bacterial genome copies in an infected tick, we adapted previously reported TaqMan®-based qPCR assays for ddPCR. ddPCR proved to be a reliable means for detection and absolute quantification of control bacterial DNA with precision as low as ten spirochetes in an individual sample. Application of this method revealed the average carriage level of B. burgdorferi in infected I. scapularis nymphs to be 2291 spirochetes per nymph (range: 230–5268 spirochetes) and 51,179 spirochetes on average in infected adults (range: 5647–115,797). No ticks naturally infected with B. miyamotoi were detected. The ddPCR protocols were at least as sensitive to conventional qPCR assays but required fewer overall reactions and are potentially less subject to inhibition. Moreover, the approach can provide insight on carriage levels of parasites within vectors.

 

**Comment**

While this is a great start, there are many other pathogens to be concerned with besides Borrelia burgdorferi (Bb), the agent of Lyme Disease:  https://madisonarealymesupportgroup.com/2017/07/01/one-tick-bite-could-put-you-at-risk-for-at-least-6-different-diseases/, and there are many more besides the six mentioned in this article.  Bartonella has not been proven conclusively to be transmitted by ticks, but it is highly likely.  It is also a frequent coinfection and can be spread by:

Arthropod vectors including fleas and flea feces, biting flies such as sand flies and horn flies, the human body louse, mosquitoes, and ticks; through bites and scratches of reservoir hosts; and potentially from needles and syringes in the drug addicted. Needle stick transmission to veterinarians has been reported. There is documentation that cats have received it through blood transfusion. 3.2% of blood donors in Brazil were found to carry Bartonella in their blood. Bartonella DNA has been found in dust mites. Those with arthropod exposure have an increased risk, as well as those working and living with pets that have arthropod exposure. 28% of veterinarians tested positively for Bartonella compared with 0% of controls. About half of all cats may be infected with Bartonella – as high as 80% in feral cats and near 40% of domestic cats. In various studies dogs have close to a 50% rate as well. Evidence now suggests it may be transmitted congenitally from mother to child – potentially leading to birth defects.  https://madisonarealymesupportgroup.com/2016/01/03/bartonella-treatment/

There is much work yet to be done.

 

 

 

 

 

Treat the Infection, Psychiatric Symptoms Get Better

It has long been a belief of mine that pathogen involvement should be considered with psychiatric issues, particularly with Lyme/MSIDS.  I came to this conclusion after watching my husband battle with anxiety, depression, memory loss, rage, sudden crying spells, fear of abandonment, and other bizarre symptoms https://madisonarealymesupportgroup.com/2015/09/16/bizarre-symptoms-msids/ which were completely new and out of the blue, while we were in treatment for Lyme/MSIDS.  Upon further reading and talking to other patients, I became convinced.

The following article adds further credence to this idea:  http://www.ijporlonline.com/article/S0165-5876(16)30380-9/fulltext

While this study is not a randomized clinical trial, it reveals improvement of psychiatric symptoms when concurrent sinusitis was treated and resolved.  

Treatment was amoxicillin & clavulanic acid.  A few received  clindamycin or azithromycin, all of which are common antibiotics for Lyme/MSIDS and other infections.

The conclusion of the study stated that there is mounting evidence that inflammation and/or infection can affect neuropsychiatric health.

This has been reported before by a prominent LLMD.

https://madisonarealymesupportgroup.com/2015/10/18/psychiatric-lymemsids/

LLMD, Dr. Horowtiz, goes on record stating that antibiotics are effective in Schizophrenia. With irony he points out that the authors attribute the reason minocycline helped these patients is due to its ability to affect glutamate pathways in the CNS, blocking nitric oxide-induced neurotoxicity, and inflammation in the brain. He reminds them that minocycline is a tetracycline antibiotic that very well may be treating an infection. He also emphatically states that he has had several schizophrenic patients test positive for Bb, the agent of Lyme Disease. After taking doxycycline they improved significantly and with the help of their psychiatrist, were able to reduce and in some cases eliminate all of their antipsychotic medication. It is important to note that patients remained stable on antibiotics but their symptoms returned if they stopped treatment. He says a doctor should suspect MSIDS in psychiatric patients if they have a symptom complex that has good and bad days with associated fevers, sweats and chills, fatigue, migratory joint and muscle pain, migratory neuralgias with tingling, numbness and burning sensations, a stiff neck and headache, memory and concentration problems, sleep disorders with associated psychiatric symptoms.
Horowtiz also reports the work of psychiatrist Dr. Brian Fallon who has linked Lyme Disease to paranoia, thought disorders, delusions with psychosis, schizophrenia, with or without visual, auditory or olfactory hallucinations, depression, panic attacks and anxiety, obsessive compulsive disorder, anorexia, mood lability with violent outbursts, mania, personality changes, catatonia, dementia, atypical bipolar disorder, depersonalization/derealization, conversion disorders, somatization disorders, atypical psychoses, schizoaffective disorder and intermittent explosive disorders. In children and adolescents MSIDS can mimic Specific or Pervasive Developmental Delays, Attention-Deficit Disorder (Inattentive subtype), oppositional defiant disorder and mood disorders, obsessive compulsive disorder, anorexia, Tourette’s syndrome, and pseudo-psychotic disorders.

Minocycline has also been found to help MS:  https://madisonarealymesupportgroup.com/2017/06/04/minocycline-for-ms-and-much-more/

For patients with psychiatric issues, it’s important to rule out infections as players, unfortunately, that’s often nearly impossible for Lyme/MSIDS as the testing is so poor.  ALL doctors need to become more educated on tick borne illness and its ability to cause and/or exacerbate psychiatric and cognitive issues.  Hopefully, long gone are the days where Lyme/MSIDS is considered a mild dermatological disease with some joint involvement and fatigue.

This stuff can put you in the psych ward.

For more:  https://madisonarealymesupportgroup.com/2017/01/17/lymemsids-and-psychiatric-illness/

https://madisonarealymesupportgroup.com/2015/10/18/psychiatric-lymemsids/

https://madisonarealymesupportgroup.com/2017/06/20/suicide-lyme-and-associated-diseases/

https://madisonarealymesupportgroup.com/2017/09/19/three-things-for-parents-to-watch-for-regarding-tbis/