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Efficacy of Tea Tree Oil in the Treatment of Equine Streptothricosis
Accepted Manuscript Efficacy of Tea Tree Oil in the Treatment of Equine Streptothricosis Callan C. Frye, Di Bei, Jacquelyn E. Parman, Jessica Jones, Adam J. Houlihan, Amanda Rumore PII:
S0737-0806(18)30803-7
DOI:
https://doi.org/10.1016/j.jevs.2019.05.011
Reference:
YJEVS 2746
To appear in:
Journal of Equine Veterinary Science
Received Date: 2 February 2019 Revised Date:
9 May 2019
Accepted Date: 10 May 2019
Please cite this article as: Frye CC, Bei D, Parman JE, Jones J, Houlihan AJ, Rumore A, Efficacy of Tea Tree Oil in the Treatment of Equine Streptothricosis, Journal of Equine Veterinary Science (2019), doi: https://doi.org/10.1016/j.jevs.2019.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Efficacy of Tea Tree Oil in the Treatment of Equine Streptothricosis
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Callan C. Fryea, Di Beia, Jacquelyn E. Parmana, Jessica Jonesa, Adam J. Houlihana, Amanda
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Rumorea*
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24503, USA
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Department of Biological Sciences, Randolph College, 2500 Rivermont Avenue, Lynchburg, VA
6 ABSTRACT
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Streptothricosis is a dermatitis characterized by matted tufts of hair and coalescing, pustular
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crusts that affects many livestock species, including horses. It results from cutaneous infection
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by the actinobacterium Dermatophilus congolensis. For economic reasons, the ailment is often
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treated with commercially available over-the-counter (OTC) products or home remedies rather
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than prescribed medications. This study aimed to determine the efficacy of tea tree oil (TTO), an
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essential oil of Melaleuca alternifolia, as an OTC treatment for streptothricosis. Bacteria were
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isolated from presumptive streptothricosis lesions on horses at a farm in Forest, Virginia. These
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isolates were microbiologically and genetically confirmed to be D. congolensis. The
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antimicrobial activity of TTO against D. congolensis isolates was determined by minimum
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inhibitory concentration and disc diffusion assays and compared to three OTC products
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advertised specifically for the treatment of “rain rot,” a colloquial term for streptothricosis. A
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1% TTO solution (v/v, in baby oil) and the three selected OTC products were applied to equine
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streptothricosis lesions to evaluate in vivo resolution of the lesions. Tea tree oil exhibited
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antimicrobial behavior against D.congolensis in vitro and produce marked improvement of
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streptothricosis lesions in vivo. These results have implications for development of tea tree oil as
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a possible treatment for streptothricosis.
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Keywords
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Streptothricosis; rain rot; Dermatophilus congolensis; Melaleuca alternifolia; tea tree oil.
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*Corresponding author: E-mail: [email protected]; Postal address: 2500 Rivermont
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Avenue, Lynchburg, VA 24503, USA
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1. Introduction Streptothricosis, otherwise known as dermatophilosis and numerous colloquialisms (e.g. rain rot, rain scald), is a common dermatitis that manifests as purulent, coalescing crusts and
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matted hair on many animal species, particularly domesticated ruminants and horses [1]. The
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pustular process entraps hair shafts, forming thick, adherent mats of encrusted hair. This skin
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condition is caused by an infection of the opportunistic pathogen Dermatophilus congolensis, a
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gram-positive, microaerophilic actinobacterium [2]. D. congolensis was long believed to exist
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primarily in soil, and its ability to survive in tropical soil samples would seem to support this [3],
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but attempts to isolate the organism from soil surrounding affected animals have been
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unsuccessful [4]. It is alternatively hypothesized that D. congolensis may asymptomatically
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colonize the skin and hair of livestock and cause infectious disease under certain environmental
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conditions [5]. Prolonged exposure to high humidity, heat, and continuous rainfall facilitates
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proliferation of the microorganism and epidermal penetration by branching filaments [2].
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Consequently, livestock in tropical, subtropical, and temperate areas with humid climates and
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distinct rainy seasons are more often afflicted by streptothricosis [1,6]. In addition to these
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exogenous risk factors, a genetic basis for streptothricosis susceptibility has been identified in
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certain bovine breeds [7,8]. However, a comparable genetic basis in equids has yet to be
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identified.
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Acute streptothricosis usually self-resolves within 14-21 days if infected horses are
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sheltered to prevent further wetting of the coat [5,9]. Chronic infection is characterized by
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alopecia and keratinized material collecting around thick, exudative crusts of serocellular or
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hemorrhagic origin [6,10]. These conditions are associated with lameness, loss of performance
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[9,11], secondary cutaneous infection by Staphylococcus, Streptococcus, and Corynebacterium
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species [12], mortality [5], and spontaneous abortion [13]. Streptothricosis also diminishes the
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economic value affected livestock [14]. Treatment for minor cases of equine streptothricosis typically includes topical medicated
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shampoo, such as those containing chlorhexidine, and keeping the area dry until the lesions self-
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resolve. Systemic antibiotic treatment is reserved for severe cases [15]. D.congolensis is
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susceptible to several antibiotics [6,16] and long-acting oxytetracycline is highly effective in
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sheep and cattle [17]. However, antibiotics may be relatively expensive and can disrupt the
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normal gut microflora of the animal [18,19]. Additionally, systemic antibiotic use in horses does
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not always result in full resolution of the infection [10], and is therefore typically paired with a
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topical treatment [5]. Due to these obstacles, streptothricosis often goes untreated [11] or is
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addressed using empirical remedies developed by owners and handlers.
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Tea tree oil (TTO), an essential oil of the Melaleuca alternifolia tree, effectively inhibits a variety of bacterial species, even at low concentrations [20], and possesses a natural capacity to
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penetrate dermal layers [21]. Despite this, the efficacy of TTO against D. congolensis is
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unconfirmed. Its performance as an effective treatment for “rain rot” is only supported
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anecdotally by horse owners and handlers who claim that topically applying an amalgam of tea
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tree oil and a gel (e.g. Aloe vera gel) to streptothricosis lesions advances recovery. Essential oil
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from thyme (Thymus vulgaris) contains several common components with TTO and is an
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effective inhibitor of D. congolensis in vitro, but to our knowledge, no other studies have
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evaluated the use of an essential oils in the treatment of streptothricosis [22].
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We sought to evaluate the efficacy of TTO in the treatment of equine streptothricosis.
The aims of the present paper were:
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To use minimum inhibitory concentration and disc diffusion assays to assess the
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in vitro antimicrobial activity of tea tree oil against D.congolensis as compared to
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OTC treatments advertised for the treatment of “rain rot.”
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To evaluate in vivo effectiveness of a 1% (v/v, in baby oil) tea tree oil solution in
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the treatment of confirmed D.congolensis streptothricosis lesions.
Conclusion: In this study, tea tree oil was clinically effective for treatment of D.congolensis
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streptothricosis.
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2. Materials and Methods
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2.1 Study Design
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Four horses of various breeds from Brook Hill Farm Retirement Center for Horses in Forest, Virginia (USA) were used for this study. Each horse had visible, spontaneously occurring
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streptothricosis lesions in at least three separate regions of the body. All horses were between the
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ages of 16 and 27 years and did not receive treatment for streptothricosis for 30 days prior to the
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experiment. Horses were housed in a larger group of approximately forty horses on 50 acres of
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grass pasture. Three of the horses were under regular, light work but none were stabled at any
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point during the study. Diet was primarily grass and hay; however additional grain and
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supplements for individual horses was not controlled. Climatic conditions for the duration of the
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study (late May – early July) was hot and humid (avg. high 28°C, 72% humidity). This work was
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approved by the Randolph College Institutional Animal Care and Use Committee (IACUC).
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A D. congolensis stock strain (ATCC 14637) was acquired from the American Type Culture Collection and used as a control (stock) organism for all experimental work. Stock
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cultures were maintained on brain heart infusion agar (Difco™, Becton, Dickinson and Company,
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Franklin Lakes, NJ) at 37°C.
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Clinical isolates were acquired from spontaneously occurring streptothricosis lesions at
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various locations from each horse (Figure 1) by combining methods previously described [23,24].
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Briefly, sterile forceps were used to remove so-called ‘paintbrush lesions,’ which were identified
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as thick, adherent tufts of encrusted hair that resemble the head of a dried paintbrush when
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removed [9]. Lesion samples were then placed in sterile microcentrifuge tubes for transport to
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the laboratory where 1 mL of sterile water was added. The tubes were incubated for 15 minutes
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in a 35ºC, 5% CO2 environment to release zoospores. Isolated colonies were obtained by using a
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sterile inoculating loop to streak one loopful of the uppermost liquid from each tube onto brain-
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heart infusion agar (BHI) (Difco™, Becton, Dickinson and Company, Franklin Lakes, NJ)
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supplemented with 5% defibrinated sheep’s blood and 1000 IU/mL polymyxin B (Sigma-Aldrich,
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St. Louis, MO). Plates were incubated for 48 hours at 35ºC in a 5% CO2 atmosphere. The
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resulting colonies were inspected for phenotypic characteristics consistent with D. congolensis
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(beta-hemolytic, yellow, hard colonies with an irregular margin that adhere to the agar and have
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a depressed periphery), and presumptive D.congolensis isolates were cultured in BHI broth at
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37°C. Both the stock strain (ATCC 14637) and clinical isolates were tested for catalase and
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urease activity and a Gram stain and microscopic examination of cell morphology was
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performed to positively confirm identity. Isolates that were catalase and urease positive, gram-
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positive, and coccoid or filamentous with transverse septa were phenotypically confirmed to be
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D. congolensis.
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2.3 Genetic Identification
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DNA was isolated from the stock strain and clinical isolates using the DNeasy Kit (Qiagen, Germantown, MD) per manufacturer instructions. Polymerase chain reaction (PCR)
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was performed using primers specific to the D. congolensis agc gene: forward 5’-
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CTTCAGCAGAAAATTCACCA-3’ and reverse 5’-CGTACATTCCCGGAATCTTC-3’
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(Integrated DNA Technologies, Skokie, IL), which yield an expected 438 bp product [25]. Each
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PCR reaction contained: 250 ng of template DNA, 1 µM of each primer, and 1x GoTaq Green
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Master Mix (Promega, Madison, WI) in a total volume of 25 µl. The following temperature
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profile was used for PCR amplification: initial denaturation at 94ºC for 2 minutes followed by 40
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cycles of amplification (94ºC for 30 seconds, 55ºC for 30 seconds, and 72ºC for 30 seconds) and
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a final extension of 72ºC for 60 seconds. The PCR products were subjected to electrophoresis for
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60 minutes on a 1% agarose gel stained with GelRed ® (Biotium, Freemont, CA). The products
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were visualized under UV light using the Gel Doc system (Bio-Rad, Hercules, CA). Presence of
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a 438 bp band indicated that the isolates were D. congolensis.
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2.4 Sterility of TTO and OTC Products
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Pharmaceutical-grade tea tree oil (TTO) and three OTC products (hereafter called “P1”,
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“P2”, and “P3”) were purchased from tack or pet supply stores. Active and inactive ingredients
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of each OTC topical treatment, as listed on the product label, are shown in Table 1. The sterility
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of TTO and the OTC products was assessed by performing isolation streaks of each on BHI agar
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plates and incubating for 48 hours at 37ºC.
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2.5 Disk Diffusion Assay
D. congolensis stock strain (ATCC 14637) and the clinical isolates were grown in BHI
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broth for 24 hours 35ºC in 5% CO2 and 100 µL was uniformly spread on the surface of BHI agar
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plates. Sterile blotting paper disks (10 mm diam.) were saturated with 5 µL of TTO or undiluted
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OTC product (P1, P2, and P3). Paper disks were left at room temperature for ten minutes to
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allow complete absorption of the liquid and disks was placed onto the surface of the inoculated
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BHI agar using sterile forceps. Plates were incubated for 72 hours at 35ºC in 5% CO2 and the
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diameter of the zone of clearing around each disk was measured. The disk diffusion assay was
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performed in triplicate for each treatment.
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2.6 Minimum Inhibitory Concentration Assay The minimum inhibitory concentrations (MIC) of each treatment against both the D.congolensis stock strain (ATCC 14637) and the clinical isolates was determined using the
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method described by O’Bryan et al. [26]. Two-fold serial dilutions were done in a BHI broth
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containing 0.15% agar. All tubes were incubated at 35ºC for 72 hours and the MIC was
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determined as the lowest concentration able to inhibit visible bacterial growth.
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2.7 In Vivo Evaluation of Effectiveness of Tea Tree Oil
A 1% TTO solution (v/v, in baby oil) and P1, P2 and P3 were tested in vivo; the active
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and inactive ingredients of each OTC product are listed in Table 1. Each treatment was applied
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to three separate and isolated regions of streptothricosis lesions on three different horses. All
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regions had been positively identified to contain D. congolensis. OTC products were applied
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according to instructions provided on the product label. The 1% (v/v) TTO solution was
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aseptically prepared in store-bought baby oil (containing mineral oil and fragrance only) and
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liberally applied using a spray-nozzle bottle. This concentration of tea tree oil was chosen in
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compliance with commercial safety standards [27]. Baby oil was selected as the carrying
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substance for TTO because in vitro testing had confirmed it possessed no antimicrobial activity
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against D. congolensis (data not shown). Each treatment was applied to the same infected area
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once daily for eight consecutive days. Photographs were taken in natural light on days 1 and 9
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using a Canon EOS Rebel T5 digital single-lens reflex (DSLR) camera fitted with a standard EF-
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S 18-55mm f/3.5-5.6 IS II lens.
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A point-scale assessment was developed based on published criterion of physical
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identification of streptothricosis and with veterinary consultation (see Supplementary Figure 1).
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The severity of each localized streptothricosis infection was evaluated by a licensed veterinarian,
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blinded to the treatment type, prior to application of any treatment (day 1) and again 24hr
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following the final application (day 9).
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Statistical analysis was completed using GraphPad Prism® Version 7 software. All data are expressed as mean +/- standard deviation (SD) of independent triplicates unless otherwise
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noted. A one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test was
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performed for multiple comparisons across treatment groups. The level of significance is
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indicated with single asterisk (*) for p ≤ 0.05.
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3. Results
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3.1 Positive Identification of D. congolensis from Suspected Streptothricosis Lesions
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Four clinical isolates and the stock strain (ATCC 14637) formed colonies on BHI agar supplemented with sheep’s blood and polymyxin that were morphologically consistent with D.
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congolensis (irregularly-shaped, yellow pigmented, colonies exhibiting a cakey, crumb-like
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appearances). All were gram-positive, urease positive, and β-hemolytic; however, only the stock
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strain and one of the clinical isolates were catalase positive (Table 2). Based on its complete
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consistency with the D. congolensis stock strain (ATCC 14637), this clinical isolate was retained
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for further testing (Figure 2).
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3.2 D.congolensis Clinical Isolate Confirmed by Genetic Analysis The D.congolensis clinical isolate and stock strain (ATCC 14637) were subcultured in BHI broth and genetically analyzed for the presence of the agc gene, which encodes an alkaline
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ceramidase protein and is a specific genetic marker of D. congolensis [28]. Genomic DNA was
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extracted from the clinical isolate and stock strain and polymerase chain reaction (PCR) was
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conducted using gene specific primers for the agc gene. Gel electrophoresis of the PCR products
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confirmed amplification of the 438 bp agc gene fragment, thus providing confirmation that the
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clinical isolate was D.congolensis (Figure 2).
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3.3 Treatment Sterility Confirmed
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Sterility of the 100% tea tree oil (TTO) and the three OTC products (P1, P2, P3) was
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determined by spreading each on BHI agar, incubating at 37°C, and examining for bacterial
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growth after 24 hours. The TTO and all three products (P1, P2, P3) were sterile (Table 1) and
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used for further in vitro and in vivo testing. Store bought baby oil (listed ingredients: mineral oil
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and fragrance) used as the emulsifier in preparation of the 1% (v/v) tea tree oil solution for in
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vivo application was also negative for microbial contaminants (data not shown).
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3.4 Tea Tree Oil Inhibits Growth of D. congolensis In Vitro
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Disk diffusion assays using TTO produced 20 ± 3.70 mm diameter zones of clearing on D.congolensis (ATCC 14637) lawns (Table 3). P1 and P2 also produced zones of clearing: 19 ±
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4.88 mm and 34 ± 13.6 mm, respectively. TTO, P1, and P2 all inhibited growth of D.congolensis
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(ATCC 14637) compared to the sterile water control (p <.0001). P3 did not produce zones of
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clearing (Table 3).
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Minimum inhibitory concentrations (MICs) were determined for TTO, P1, P2, and P3 using D.congolensis (ATCC 14637) in BHI broth containing 0.15% agar. P1 and P3 showed no
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inhibition at the concentrations tested. The MIC for TTO was 7.25% ± 3.56% and the MIC for
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P2 was 3.48% ± 1.04% (Table 4). The ANOVA within-group comparison was p < 0.0001.
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3.5 Tea Tree Oil Effectively Resolves Streptothricosis Lesions In Vivo Equine streptothricosis lesions were evaluated before and after treatment by a licensed
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veterinarian using the Streptothricosis Score Chart (Supplementary Figure 1). Both 1% TTO (v/v,
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in baby oil) and P1 significantly improved the overall severity of the streptothricosis lesions
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(Figure 3a), while P2 and P3 did not produce significant changes in the severity of
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streptothricosis lesions during the eight-day trial period. Representative pre- and post-treatment
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images of the improvement seen using the 1% TTO solution (v/v, in baby oil) is shown in Figure
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3b.
231 Figure 4 shows changes in pre- and post-treatment lesions. One percent TTO (v/v, in
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baby oil) produced significant improvement of coat condition and reduction in erythema, while
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P1 showed resolution of lesion advancement, a reduction in excoriation, and improvement of
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barrier quality. These changes were significant between groups as determined with a one-way
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ANOVA and Tukey post-hoc testing (p < 0.05).
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4. Discussion While typically self-resolving, horse owners often treat streptothricosis with commercially available topical treatments. More severe or chronic cases may warrant the use of
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systemic antibiotics. In several reported instances, these antibiotic regimens saw the resolution of
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D. congolensis infection within 10 days [6,11]. However, these treatments have deleterious
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effects on equine gut microflora [18,19] and lower cure rates than topical treatments [10]. In the
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current study, a topically-applied 1% tea tree oil solution (v/v, in baby oil) allowed resolution of
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streptothricosis lesions within 8 days.
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Interestingly, of the three OTC products tested during these experiments, the product that demonstrated the most significant improvement on the Streptothricosis Score Chart during the
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experiment, P1, contained a proprietary blend of essential oils, including tea tree oil.
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Improvement of afflicted areas was marked and may signal a synergistic effect of the three
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essential oils in the product. P1 also showed inhibitory effects in vitro via the disk diffusion
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assay, but a minimum inhibitory concentration (MIC) could not be established. This was likely
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due to the relatively low initial concentration of the three essential oils in the product, thus any
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dilution conducted as part of the MIC experiments may have brought them below the threshold
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for inhibition.
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The other two products tested, both of which were sulfur-based, did not show
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improvement on the Streptothricosis Score Chart during the 8 day experiment when applied as
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described in the Materials & Methods section. P2, which contained a proprietary blend of alkyl
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sulfonate and alkyl sulfate, significantly inhibited D.congolensis in vitro, but did not improve
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lesions scores on the Streptothricosis Score Chart in vivo. P3, which lists sulfur as the only active
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ingredient, did not inhibit D.congolensis in vitro or show in vivo improvement of streptothricosis
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lesions as per the Streptothricosis Score Chart. While sulfur-based topical treatments are
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effective against other actinomycetes [29], it is possible this formulation was not specific enough
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for the causative agent of streptothricosis, D. congolensis. In vivo results for P2 and P3 may
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have been different if the products were applied more frequently than once per day or for more
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than eight days. Moreover, P2 was supplied with a foaming applicator which made it difficult to
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apply the solution as liberally as the other treatments.
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While the popularity of essential oils is rising, the use of TTO-based treatments is not without concern. Oral ingestion of TTO has been associated with loss of consciousness,
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disorientation, and rashes in humans [21,27,30]. Dermal application of undiluted TTO to three
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cats with shaved but intact skin resulted in neurotoxicity and potentially exacerbated underlying
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health conditions in one cat, leading to its death [31]. Slight dermal irritation has also been
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observed in response to repeated topical applications of 25% TTO (v/v, in paraffin oil) to shaved
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rabbits, though this concentration far exceeded the 1% TTO concentration recommended by the
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European Cosmetic Toiletry and Perfumery Association [21,27,32]. No adverse reactions,
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dermal or otherwise, were seen on the part of the participating horses in this study. While a 1%
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solution of TTO is supported as safe and effective for equine application, there is a concern that
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horse-owners may mistakenly apply undiluted TTO to streptothricosis lesions, as higher
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concentrations (≥ 5% TTO) may cause epidermal irritation and possible damage to other tissues
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[33]. For this same reason, it is also critical TTO is not applied to or near mucosal membranes,
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including the eyes, nose, or mouth. Thus, proper application of TTO requires prior knowledge of
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how to safely prepare and apply the compound.
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cattle and sheep. Bovine streptothricosis is a serious economic detriment in Central and Western
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Africa [1], so TTO may allow treatment of bovine streptothricosis in areas with lower access to
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regular veterinary care. In this study, we compared the efficacies of tea tree oil and three OTC
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products as treatments against equine streptothricosis. Tea tree oil is an easily accessed,
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relatively inexpensive, and potent compound with antimicrobial properties. Our research shows
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it is effective against Dermatophilus congolensis, the etiological agent of streptothricosis. We
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hope that, while our research was the first to explore tea tree oil as an effective and practical
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treatment for equine streptothricosis, other studies will seek to investigate its safety and efficacy
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in other affected species.
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While this research focused on tea tree oil, other essential oils may produce similar antimicrobial effects [20]. Additional research to evaluate the synergistic effect of tea tree oil
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with other essential oils might yield useful results, as some studies have found particular
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combinations of essential oils exert greater antimicrobial activity than the component essential
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oils in isolation [34]. Future research into the synergistic effects of essential oil combinations
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may lead to additional promising findings regarding the antimicrobial benefits of these emerging
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treatments.
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4.1 Conclusion
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In vitro testing revealed that tea tree oil produced large zones of clearing on disk
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diffusion plates and had a low minimum inhibitory concentration against the etiological agent of
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streptothricosis. In vivo testing demonstrated that a 1% (v/v, in baby oil) tea tree oil solution can
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ameliorate confirmed cases of streptothricosis in horses. Both in vitro and in vivo results support
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tea tree oil as an effective agent against D. congolensis, the causative agent of streptothricosis,
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and its ability to advance lesion resolution.
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5. Conflict of Interest AR is a regular volunteer and former donor to Brook Hill Farm Retirement Center for Horses. All other authors declare no competing interests.
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329 6. Funding
This work was supported by Randolph College, Lynchburg, VA; and the Virginia
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Foundation for Independent Colleges (VFIC), Richmond, VA. These sources of funding had no
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role in the design of this study; the collection, analysis, or interpretation of data; writing the
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article; nor the decision to submit this article for publication.
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335 7. Author Contributions
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Callan Frye: Investigation, Formal Analysis, Writing – Original Draft, Writing – Review &
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Editing, Visualization. Di Bei: Investigation, Formal Analysis. Jacquelyn Parman:
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Investigation, Formal Analysis. Jessica Jones: Investigation, Formal Analysis. Adam
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Houlihan: Conceptualization, Methodology, Writing – Review & Editing, Supervision, Project
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Administration, Funding Acquisition. Amanda Rumore: Conceptualization, Methodology,
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Formal Analysis, Visualization, Writing – Review & Editing, Supervision, Project
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Administration, Funding Acquisition
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8. Acknowledgments
The authors thank Jo Anne Miller, Tracy Russler, and all Brook Hill Retirement Center
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for Horses staff and volunteers for providing assistance and subjects for use in this study. They
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also thank Catherine Khoo and Aaron Whalen for laboratory support services.
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9. References
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[1] Zaria LT. Dermatophilus congolensis infection (dermatophilosis) in animals and man! An update. Comp Immun Microbiol Infect Dis 1993;16:179–222. [2] Martinez D. Chapter 2.4.9. Dermatophilosis. OIE ITerrestrial Manuali, 2008. [3] Martinez D, Prior P. Survival of Dermatophilus congolensis in tropical clay soils submitted to different water potentials. Vet Microbiol 1991;29:135–45. doi:10.1016/03781135(91)90121-U. [4] Pal M. Prevalence in India of Dermatophilus congolensis infection in clinical specimens from animals and humans. Rev Sci Tech OIE 1995;14:857–63. doi:10.20506/rst.14.3.882. [5] Scott DW. Chapter 7 - Skin diseases. In: Divers TJ, Peek SF, editors. Rebhuns Dis. Dairy Cattle. 2nd ed., Elsevier; 2008, p. 299–301. [6] Searcy GP, Hulland TJ. Dermatophilus dermatitis (streptothricosis) in Ontario. 1. Clinical observations. Can Vet J 1968;9:7–15. [7] Maillard J-C, Chantal I, Berthier D, Thevenon S, Sidibe I, Razafindraibe H. Molecular immunogenetics in susceptibility to bovine dermatophilosis. Ann N Y Acad Sci 2002;969:92–6. doi:10.1111/j.1749-6632.2002.tb04357.x. [8] Maillard JC, Martinez D, Bensaid A. An amino acid sequence coded by the exon 2 of the BoLA DRB3 gene associated with a BoLA class I specificity constitutes a likely genetic marker of resistance to dermatophilosis in Brahman zebu cattle of Martinique (FWI). Ann N Y Acad Sci 1996;791:185–97. doi:10.1111/j.1749-6632.1996.tb53525.x. [9] Overview of dermatophilosis. Merck Vet Man 2018. https://www.merckvetmanual.com/integumentary-system/dermatophilosis/overview-ofdermatophilosis (accessed June 8, 2018). [10] Awad WS, Nadra-Elwgoud, Abdou MI, El-Sayed AA. Diagnosis and treatment of bovine, ovine and equine dermatophilosis. J Appl Sci Res 2008;4:367–74. [11] Pascoe RR. Further observations on dermatophilus infections in horses. Aust Vet J 1972;48:32–4. doi:10.1111/j.1751-0813.1972.tb02207.x. [12] Elizabeth A Mauldin, Jeanine Peters-Kennedy. Integumentary System. In: Grant Maxie, editor. Jubb Kennedy Palmers Pathol. Domest. Anim., vol. 1. 6th ed., Saunders Ltd.; 2015, p. 509–736. [13] Sebastian MM, Giles RC, Donahu JM, Sells SF, Fallon L, Vickers ML. Dermatophilus congolensis-associated placentitis, funisitis and abortion in a horse. Transbound Emerg Dis 2008;55:183–5. doi:10.1111/j.1865-1682.2007.00981.x. [14] Yeruham I, Elad D, Perl S. Economic aspects of outbreaks of dermatophilosis in firstcalving cows in nine herds of dairy cattle in Israel. Vet Rec 2000;146:695–8. doi:10.1136/vr.146.24.695. [15] Scott D, Miller W. Equine Dermatology. 2nd ed. Saunders Elsevier; 2010. [16] Abu-Samra MT. A study of the ultrastructure and the life cycle of Dermatophilus congolensis. Zentralblatt Für Veterinärmedizin Reihe B 1979;26:110–24. doi:10.1111/j.1439-0450.1979.tb00799.x. [17] Overview of Dermatophilosis - Integumentary System. Merck Vet Man n.d. https://www.merckvetmanual.com/integumentary-system/dermatophilosis/overview-ofdermatophilosis (accessed June 8, 2018).
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[18] Costa MC, Stämpfli HR, Arroyo LG, Allen-Vercoe E, Gomes RG, Weese J. Changes in the equine fecal microbiota associated with the use of systemic antimicrobial drugs. BMC Vet Res 2015;11:19. doi:10.1186/s12917-015-0335-7. [19] Harlow BE, Lawrence LM, Flythe MD. Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: responses to antibiotic challenge. Vet Microbiol 2013;166:225–32. doi:10.1016/j.vetmic.2013.05.003. [20] Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 1999;86:985–90. doi:10.1046/j.1365-2672.1999.00780.x. [21] Carson CF, Riley TV. Antimicrobial activity of the essential oil of Melaleuca alternifolia. Lett Appl Microbiol 1993;16:49–55. doi:10.1111/j.1472-765X.1993.tb00340.x. [22] Yardley A. A preliminary study investigating the effect of the application of some essential oils on the in vitro proliferation of Dermatophilus congolensis. Int J Aromather 2004;14:129–35. doi:10.1016/j.ijat.2004.06.001. [23] Haalstra RT. Isolation of Dermatophiliis congolensis from skin lesions in the diagnosis of streptothricosis. Vet Rec 1965;77:824–5. [24] Burd EM, Juzych LA, Rudrik JT, Habib F. Pustular Dermatitis Caused by Dermatophilus congolensis. J Clin Microbiol 2007;45:1655–8. doi:10.1128/JCM.00327-07. [25] García A, Martínez R, Benitez-Medina JM, Risco D, García WL, Rey J, et al. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis. J Vet Sci 2013;14:491–4. doi:10.4142/jvs.2013.14.4.491. [26] O’Bryan CA, Crandall PG, Chalova VI, Ricke SC. Orange essential oils antimicrobial activities against Salmonella spp. J Food Sci 2008;73:M264–7. doi:10.1111/j.17503841.2008.00790.x. [27] Scientific Committee on Consumer Products (SCCP). Opinion on tea tree oil 2008. [28] Garcı́a-Sánchez A, Cerrato R, Larrasa J, Ambrose NC, Parra A, Alonso JM, et al. Identification of an alkaline ceramidase gene from Dermatophilus congolensis. Vet Microbiol 2004;99:67–74. doi:10.1016/j.vetmic.2003.10.028. [29] Gupta AK, Nicol K. The use of sulfur in dermatology. J Drugs Dermatol JDD 2004;3:427– 31. [30] Carson CF, Riley TV. Safety, efficacy and provenance of tea tree (Melaleuca alternifolia) oil. Contact Dermatitis 2001;45:65–7. doi:10.1034/j.1600-0536.2001.045002065.x. [31] Bischoff K, Guale F. Australian Tea Tree (Melaleuca Alternifolia) Oil Poisoning in Three Purebred Cats. J Vet Diagn Invest 1998;10:208–10. doi:10.1177/104063879801000223. [32] Beckmann B, Ippen H. Teebaum-Öl. Dermatosen 1998:120–4. [33] Sigma-Aldrich. Safety data sheet: tea tree oil 2015. [34] Gutierrez J, Barry-Ryan C, Bourke P. The antimicrobial efficacy of plant essential oil combinations and interactions with food ingredients. Int J Food Microbiol 2008;124:91–7. doi:10.1016/j.ijfoodmicro.2008.02.028.
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Table 1: List of products tested and sterility results Microbial b
P3
Sulfur
a
Purified water
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Blend of Alkyl Sulfonate, Alkyl Sulfate (18%)
Petroleum distillates, Zinc Stearate, Cade Oil, Glycerin
—
—
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Product Active Ingredients Inactive Ingredients: Growth a TTO Volatile essential oil from Melaleuca Baby Oil alternifolia, mostly terpene — hydrocarbons P1 Blend of Lavender Oil, Eucalyptus Canola Oil — Oil, Tea Tree Oil (3.20%)
Tea Tree Oil (TTO) was prepared as a 1% solution in baby oil for in vivo application.
b
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Products were aseptically cultured on BHI agar at 37°C for 3 days and examined for microbial growth (+ / -)
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Table 2: Presumptive laboratory identification of D.congolensis stock strain and clinical isolates Clinical Isolates 1
2
Catalase
+
+
—
Urease
+
+
+
—
+
+
β-hemolysis β-hemolysis β-hemolysis β-hemolysis β-hemolysis +
+
+
+
+
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Blood Hemolysis
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Characteristic
Stock Strain (ATCC 14637)
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Table 3: Antimicrobial activity of TTO and tested products against D.congolensis by disk diffusion methoda
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Zone Diameter (mm)b M
SD
p-value
TTO
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±3.70
<0.0001
P1
19
±4.88
<0.0001
P2
34
±13.6
<0.0001
P3
NID
NID
a
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Product
—
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zones of clearing quantified by average of four perpendicular measurements of diameter of clear area to the nearest millimeter Abbreviations: TTO, tea tree oil; NID, no inhibition detected; M, mean; SD, standard deviation
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Table 4: Minimum inhibitory concentration (MIC) of products a tested against D.congolensis a
MIC [% v/v]
TTO
7.24
3.56
P1
NID
—
P2
3.48
1.04
P3
NID
—
p-value
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SD
<0.0001
—
<0.0001
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M
Product
a
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Abbreviations: NID, no inhibition detected; M, mean; SD, standard deviation
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438
Figure Legends
439 Figure 1: Representative images of equine streptothricosis observed on a test subject. (A)
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anterior and posterior view of afflicted distal limb (B) ‘paintbrush’ lesion removed from afflicted
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region.
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Figure 2: Microbiological and genetic confirmation of D.congolensis isolation. (A)
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D.congolensis (ATCC 14637) stock strain and (B) clinical isolate grown on BHI agar at 35°C
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and 5% CO2. C) PCR amplification of D.congolensis (ATCC 14637) stock strain (L1) and
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clinical isolate (L2) for the agc gene with identical bands between 400 and 500bp. MWM, 100bp
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molecular weight markers (Bioline, Memphis, TN); L1, Lane 1; L2, Lane 2.
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Figure 3: Pre- and post-treatment assessment of overall streptothricosis severity. Horses were
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treated daily for 8 days and blindly evaluated on Day 1 and Day 9 by a licensed veterinarian. (A)
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overall score obtained pre- and post-treatment using the Streptothricosis Score Chart
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(Supplementary Figure 1). Each treatment was applied to three affected regions on three separate
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horses. (B) Representative images of streptrothricosis infection pre- and post-treatment with 1%
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tea tree oil (TTO) solution (v/v, in baby oil). Significant differences between groups was
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determined with one-way ANOVA and Tukey post-hoc testing (* p < 0.05).
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Figure 4: Pre- and post-treatment assessment of individual features of streptothricosis. Horses
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were treated daily for 8 days and blindly evaluated on Day 1 and Day 9 by a licensed
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veterinarian using the Streptothricosis Score Chart (Supplementary Figure 1). Shown are
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individual scores for (A) coat condition (B) lesion advancement (C) inflammation (localized) (D)
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erythema (localized reddening or heat) (E) excoriation (tissue destruction) (F) lichenification
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(thickening) and (G) barrier (breakdown). Significant change from pre- to post-treatment
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between groups was determined with one-way ANOVA and Tukey post-hoc testing (* p < 0.05).
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Highlights •
Limited evidence exists supporting the use of essential oils in the treatment of equine
•
Tea tree oil (TTO) shows antimicrobial activity towards D.congolensis, the causative agent of equine streptothricosis, in vitro
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A 1% tea tree oil (TTO) emulsion is effective at resolving streptothricosis lesions in vivo
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streptothricosis
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Ethical Statement I testify on behalf of all co-authors that our article submitted to Journal of Equine Veterinary Science:
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Title: Efficacy of Tea Tree Oil in Treatment of Equine Streptothricosis All authors: Callan Frye, Di Bei, Jacquelyn Parman, Jessica Jones, Adam Houlihan, and Amanda Rumore
Date: January 2, 2019
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Corresponding author’s signature:
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1) this material has not been published in whole or in part elsewhere; 2) the manuscript is not currently being considered for publication in another journal; 3) all authors have been personally and actively involved in substantive work leading to the manuscript, and will hold themselves jointly and individually responsible for its content. 4) this research was conducted under approval of the Randolph College Institutional Animal Care and Use Committee (IACUC)
S0737-0806(18)30803-7
DOI:
https://doi.org/10.1016/j.jevs.2019.05.011
Reference:
YJEVS 2746
To appear in:
Journal of Equine Veterinary Science
Received Date: 2 February 2019 Revised Date:
9 May 2019
Accepted Date: 10 May 2019
Please cite this article as: Frye CC, Bei D, Parman JE, Jones J, Houlihan AJ, Rumore A, Efficacy of Tea Tree Oil in the Treatment of Equine Streptothricosis, Journal of Equine Veterinary Science (2019), doi: https://doi.org/10.1016/j.jevs.2019.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Efficacy of Tea Tree Oil in the Treatment of Equine Streptothricosis
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Callan C. Fryea, Di Beia, Jacquelyn E. Parmana, Jessica Jonesa, Adam J. Houlihana, Amanda
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Rumorea*
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a
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24503, USA
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Department of Biological Sciences, Randolph College, 2500 Rivermont Avenue, Lynchburg, VA
6 ABSTRACT
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Streptothricosis is a dermatitis characterized by matted tufts of hair and coalescing, pustular
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crusts that affects many livestock species, including horses. It results from cutaneous infection
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by the actinobacterium Dermatophilus congolensis. For economic reasons, the ailment is often
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treated with commercially available over-the-counter (OTC) products or home remedies rather
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than prescribed medications. This study aimed to determine the efficacy of tea tree oil (TTO), an
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essential oil of Melaleuca alternifolia, as an OTC treatment for streptothricosis. Bacteria were
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isolated from presumptive streptothricosis lesions on horses at a farm in Forest, Virginia. These
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isolates were microbiologically and genetically confirmed to be D. congolensis. The
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antimicrobial activity of TTO against D. congolensis isolates was determined by minimum
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inhibitory concentration and disc diffusion assays and compared to three OTC products
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advertised specifically for the treatment of “rain rot,” a colloquial term for streptothricosis. A
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1% TTO solution (v/v, in baby oil) and the three selected OTC products were applied to equine
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streptothricosis lesions to evaluate in vivo resolution of the lesions. Tea tree oil exhibited
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antimicrobial behavior against D.congolensis in vitro and produce marked improvement of
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streptothricosis lesions in vivo. These results have implications for development of tea tree oil as
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a possible treatment for streptothricosis.
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Keywords
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Streptothricosis; rain rot; Dermatophilus congolensis; Melaleuca alternifolia; tea tree oil.
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*Corresponding author: E-mail: [email protected]; Postal address: 2500 Rivermont
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Avenue, Lynchburg, VA 24503, USA
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1. Introduction Streptothricosis, otherwise known as dermatophilosis and numerous colloquialisms (e.g. rain rot, rain scald), is a common dermatitis that manifests as purulent, coalescing crusts and
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matted hair on many animal species, particularly domesticated ruminants and horses [1]. The
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pustular process entraps hair shafts, forming thick, adherent mats of encrusted hair. This skin
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condition is caused by an infection of the opportunistic pathogen Dermatophilus congolensis, a
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gram-positive, microaerophilic actinobacterium [2]. D. congolensis was long believed to exist
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primarily in soil, and its ability to survive in tropical soil samples would seem to support this [3],
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but attempts to isolate the organism from soil surrounding affected animals have been
39
unsuccessful [4]. It is alternatively hypothesized that D. congolensis may asymptomatically
40
colonize the skin and hair of livestock and cause infectious disease under certain environmental
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conditions [5]. Prolonged exposure to high humidity, heat, and continuous rainfall facilitates
42
proliferation of the microorganism and epidermal penetration by branching filaments [2].
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Consequently, livestock in tropical, subtropical, and temperate areas with humid climates and
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distinct rainy seasons are more often afflicted by streptothricosis [1,6]. In addition to these
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exogenous risk factors, a genetic basis for streptothricosis susceptibility has been identified in
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certain bovine breeds [7,8]. However, a comparable genetic basis in equids has yet to be
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identified.
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Acute streptothricosis usually self-resolves within 14-21 days if infected horses are
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sheltered to prevent further wetting of the coat [5,9]. Chronic infection is characterized by
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alopecia and keratinized material collecting around thick, exudative crusts of serocellular or
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hemorrhagic origin [6,10]. These conditions are associated with lameness, loss of performance
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[9,11], secondary cutaneous infection by Staphylococcus, Streptococcus, and Corynebacterium
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species [12], mortality [5], and spontaneous abortion [13]. Streptothricosis also diminishes the
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economic value affected livestock [14]. Treatment for minor cases of equine streptothricosis typically includes topical medicated
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shampoo, such as those containing chlorhexidine, and keeping the area dry until the lesions self-
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resolve. Systemic antibiotic treatment is reserved for severe cases [15]. D.congolensis is
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susceptible to several antibiotics [6,16] and long-acting oxytetracycline is highly effective in
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sheep and cattle [17]. However, antibiotics may be relatively expensive and can disrupt the
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normal gut microflora of the animal [18,19]. Additionally, systemic antibiotic use in horses does
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not always result in full resolution of the infection [10], and is therefore typically paired with a
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topical treatment [5]. Due to these obstacles, streptothricosis often goes untreated [11] or is
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addressed using empirical remedies developed by owners and handlers.
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Tea tree oil (TTO), an essential oil of the Melaleuca alternifolia tree, effectively inhibits a variety of bacterial species, even at low concentrations [20], and possesses a natural capacity to
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penetrate dermal layers [21]. Despite this, the efficacy of TTO against D. congolensis is
67
unconfirmed. Its performance as an effective treatment for “rain rot” is only supported
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anecdotally by horse owners and handlers who claim that topically applying an amalgam of tea
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tree oil and a gel (e.g. Aloe vera gel) to streptothricosis lesions advances recovery. Essential oil
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from thyme (Thymus vulgaris) contains several common components with TTO and is an
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effective inhibitor of D. congolensis in vitro, but to our knowledge, no other studies have
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evaluated the use of an essential oils in the treatment of streptothricosis [22].
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We sought to evaluate the efficacy of TTO in the treatment of equine streptothricosis.
The aims of the present paper were:
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•
To use minimum inhibitory concentration and disc diffusion assays to assess the
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in vitro antimicrobial activity of tea tree oil against D.congolensis as compared to
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OTC treatments advertised for the treatment of “rain rot.”
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•
To evaluate in vivo effectiveness of a 1% (v/v, in baby oil) tea tree oil solution in
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the treatment of confirmed D.congolensis streptothricosis lesions.
Conclusion: In this study, tea tree oil was clinically effective for treatment of D.congolensis
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streptothricosis.
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2. Materials and Methods
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2.1 Study Design
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Four horses of various breeds from Brook Hill Farm Retirement Center for Horses in Forest, Virginia (USA) were used for this study. Each horse had visible, spontaneously occurring
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streptothricosis lesions in at least three separate regions of the body. All horses were between the
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ages of 16 and 27 years and did not receive treatment for streptothricosis for 30 days prior to the
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experiment. Horses were housed in a larger group of approximately forty horses on 50 acres of
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grass pasture. Three of the horses were under regular, light work but none were stabled at any
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point during the study. Diet was primarily grass and hay; however additional grain and
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supplements for individual horses was not controlled. Climatic conditions for the duration of the
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study (late May – early July) was hot and humid (avg. high 28°C, 72% humidity). This work was
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approved by the Randolph College Institutional Animal Care and Use Committee (IACUC).
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A D. congolensis stock strain (ATCC 14637) was acquired from the American Type Culture Collection and used as a control (stock) organism for all experimental work. Stock
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cultures were maintained on brain heart infusion agar (Difco™, Becton, Dickinson and Company,
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Franklin Lakes, NJ) at 37°C.
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Clinical isolates were acquired from spontaneously occurring streptothricosis lesions at
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various locations from each horse (Figure 1) by combining methods previously described [23,24].
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Briefly, sterile forceps were used to remove so-called ‘paintbrush lesions,’ which were identified
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as thick, adherent tufts of encrusted hair that resemble the head of a dried paintbrush when
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removed [9]. Lesion samples were then placed in sterile microcentrifuge tubes for transport to
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the laboratory where 1 mL of sterile water was added. The tubes were incubated for 15 minutes
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in a 35ºC, 5% CO2 environment to release zoospores. Isolated colonies were obtained by using a
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sterile inoculating loop to streak one loopful of the uppermost liquid from each tube onto brain-
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heart infusion agar (BHI) (Difco™, Becton, Dickinson and Company, Franklin Lakes, NJ)
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supplemented with 5% defibrinated sheep’s blood and 1000 IU/mL polymyxin B (Sigma-Aldrich,
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St. Louis, MO). Plates were incubated for 48 hours at 35ºC in a 5% CO2 atmosphere. The
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resulting colonies were inspected for phenotypic characteristics consistent with D. congolensis
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(beta-hemolytic, yellow, hard colonies with an irregular margin that adhere to the agar and have
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a depressed periphery), and presumptive D.congolensis isolates were cultured in BHI broth at
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37°C. Both the stock strain (ATCC 14637) and clinical isolates were tested for catalase and
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urease activity and a Gram stain and microscopic examination of cell morphology was
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performed to positively confirm identity. Isolates that were catalase and urease positive, gram-
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positive, and coccoid or filamentous with transverse septa were phenotypically confirmed to be
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D. congolensis.
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2.3 Genetic Identification
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DNA was isolated from the stock strain and clinical isolates using the DNeasy Kit (Qiagen, Germantown, MD) per manufacturer instructions. Polymerase chain reaction (PCR)
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was performed using primers specific to the D. congolensis agc gene: forward 5’-
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CTTCAGCAGAAAATTCACCA-3’ and reverse 5’-CGTACATTCCCGGAATCTTC-3’
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(Integrated DNA Technologies, Skokie, IL), which yield an expected 438 bp product [25]. Each
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PCR reaction contained: 250 ng of template DNA, 1 µM of each primer, and 1x GoTaq Green
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Master Mix (Promega, Madison, WI) in a total volume of 25 µl. The following temperature
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profile was used for PCR amplification: initial denaturation at 94ºC for 2 minutes followed by 40
129
cycles of amplification (94ºC for 30 seconds, 55ºC for 30 seconds, and 72ºC for 30 seconds) and
130
a final extension of 72ºC for 60 seconds. The PCR products were subjected to electrophoresis for
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60 minutes on a 1% agarose gel stained with GelRed ® (Biotium, Freemont, CA). The products
132
were visualized under UV light using the Gel Doc system (Bio-Rad, Hercules, CA). Presence of
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a 438 bp band indicated that the isolates were D. congolensis.
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2.4 Sterility of TTO and OTC Products
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Pharmaceutical-grade tea tree oil (TTO) and three OTC products (hereafter called “P1”,
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“P2”, and “P3”) were purchased from tack or pet supply stores. Active and inactive ingredients
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of each OTC topical treatment, as listed on the product label, are shown in Table 1. The sterility
139
of TTO and the OTC products was assessed by performing isolation streaks of each on BHI agar
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plates and incubating for 48 hours at 37ºC.
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2.5 Disk Diffusion Assay
D. congolensis stock strain (ATCC 14637) and the clinical isolates were grown in BHI
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broth for 24 hours 35ºC in 5% CO2 and 100 µL was uniformly spread on the surface of BHI agar
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plates. Sterile blotting paper disks (10 mm diam.) were saturated with 5 µL of TTO or undiluted
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OTC product (P1, P2, and P3). Paper disks were left at room temperature for ten minutes to
147
allow complete absorption of the liquid and disks was placed onto the surface of the inoculated
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BHI agar using sterile forceps. Plates were incubated for 72 hours at 35ºC in 5% CO2 and the
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diameter of the zone of clearing around each disk was measured. The disk diffusion assay was
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performed in triplicate for each treatment.
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2.6 Minimum Inhibitory Concentration Assay The minimum inhibitory concentrations (MIC) of each treatment against both the D.congolensis stock strain (ATCC 14637) and the clinical isolates was determined using the
154
method described by O’Bryan et al. [26]. Two-fold serial dilutions were done in a BHI broth
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containing 0.15% agar. All tubes were incubated at 35ºC for 72 hours and the MIC was
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determined as the lowest concentration able to inhibit visible bacterial growth.
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2.7 In Vivo Evaluation of Effectiveness of Tea Tree Oil
A 1% TTO solution (v/v, in baby oil) and P1, P2 and P3 were tested in vivo; the active
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and inactive ingredients of each OTC product are listed in Table 1. Each treatment was applied
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to three separate and isolated regions of streptothricosis lesions on three different horses. All
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regions had been positively identified to contain D. congolensis. OTC products were applied
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according to instructions provided on the product label. The 1% (v/v) TTO solution was
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aseptically prepared in store-bought baby oil (containing mineral oil and fragrance only) and
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liberally applied using a spray-nozzle bottle. This concentration of tea tree oil was chosen in
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compliance with commercial safety standards [27]. Baby oil was selected as the carrying
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substance for TTO because in vitro testing had confirmed it possessed no antimicrobial activity
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against D. congolensis (data not shown). Each treatment was applied to the same infected area
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once daily for eight consecutive days. Photographs were taken in natural light on days 1 and 9
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using a Canon EOS Rebel T5 digital single-lens reflex (DSLR) camera fitted with a standard EF-
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S 18-55mm f/3.5-5.6 IS II lens.
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A point-scale assessment was developed based on published criterion of physical
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identification of streptothricosis and with veterinary consultation (see Supplementary Figure 1).
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The severity of each localized streptothricosis infection was evaluated by a licensed veterinarian,
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blinded to the treatment type, prior to application of any treatment (day 1) and again 24hr
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following the final application (day 9).
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Statistical analysis was completed using GraphPad Prism® Version 7 software. All data are expressed as mean +/- standard deviation (SD) of independent triplicates unless otherwise
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noted. A one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test was
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performed for multiple comparisons across treatment groups. The level of significance is
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indicated with single asterisk (*) for p ≤ 0.05.
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3. Results
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3.1 Positive Identification of D. congolensis from Suspected Streptothricosis Lesions
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Four clinical isolates and the stock strain (ATCC 14637) formed colonies on BHI agar supplemented with sheep’s blood and polymyxin that were morphologically consistent with D.
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congolensis (irregularly-shaped, yellow pigmented, colonies exhibiting a cakey, crumb-like
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appearances). All were gram-positive, urease positive, and β-hemolytic; however, only the stock
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strain and one of the clinical isolates were catalase positive (Table 2). Based on its complete
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consistency with the D. congolensis stock strain (ATCC 14637), this clinical isolate was retained
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for further testing (Figure 2).
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3.2 D.congolensis Clinical Isolate Confirmed by Genetic Analysis The D.congolensis clinical isolate and stock strain (ATCC 14637) were subcultured in BHI broth and genetically analyzed for the presence of the agc gene, which encodes an alkaline
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ceramidase protein and is a specific genetic marker of D. congolensis [28]. Genomic DNA was
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extracted from the clinical isolate and stock strain and polymerase chain reaction (PCR) was
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conducted using gene specific primers for the agc gene. Gel electrophoresis of the PCR products
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confirmed amplification of the 438 bp agc gene fragment, thus providing confirmation that the
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clinical isolate was D.congolensis (Figure 2).
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3.3 Treatment Sterility Confirmed
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Sterility of the 100% tea tree oil (TTO) and the three OTC products (P1, P2, P3) was
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determined by spreading each on BHI agar, incubating at 37°C, and examining for bacterial
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growth after 24 hours. The TTO and all three products (P1, P2, P3) were sterile (Table 1) and
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used for further in vitro and in vivo testing. Store bought baby oil (listed ingredients: mineral oil
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and fragrance) used as the emulsifier in preparation of the 1% (v/v) tea tree oil solution for in
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vivo application was also negative for microbial contaminants (data not shown).
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3.4 Tea Tree Oil Inhibits Growth of D. congolensis In Vitro
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Disk diffusion assays using TTO produced 20 ± 3.70 mm diameter zones of clearing on D.congolensis (ATCC 14637) lawns (Table 3). P1 and P2 also produced zones of clearing: 19 ±
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4.88 mm and 34 ± 13.6 mm, respectively. TTO, P1, and P2 all inhibited growth of D.congolensis
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(ATCC 14637) compared to the sterile water control (p <.0001). P3 did not produce zones of
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clearing (Table 3).
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Minimum inhibitory concentrations (MICs) were determined for TTO, P1, P2, and P3 using D.congolensis (ATCC 14637) in BHI broth containing 0.15% agar. P1 and P3 showed no
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inhibition at the concentrations tested. The MIC for TTO was 7.25% ± 3.56% and the MIC for
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P2 was 3.48% ± 1.04% (Table 4). The ANOVA within-group comparison was p < 0.0001.
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3.5 Tea Tree Oil Effectively Resolves Streptothricosis Lesions In Vivo Equine streptothricosis lesions were evaluated before and after treatment by a licensed
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veterinarian using the Streptothricosis Score Chart (Supplementary Figure 1). Both 1% TTO (v/v,
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in baby oil) and P1 significantly improved the overall severity of the streptothricosis lesions
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(Figure 3a), while P2 and P3 did not produce significant changes in the severity of
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streptothricosis lesions during the eight-day trial period. Representative pre- and post-treatment
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images of the improvement seen using the 1% TTO solution (v/v, in baby oil) is shown in Figure
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3b.
231 Figure 4 shows changes in pre- and post-treatment lesions. One percent TTO (v/v, in
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baby oil) produced significant improvement of coat condition and reduction in erythema, while
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P1 showed resolution of lesion advancement, a reduction in excoriation, and improvement of
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barrier quality. These changes were significant between groups as determined with a one-way
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ANOVA and Tukey post-hoc testing (p < 0.05).
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4. Discussion While typically self-resolving, horse owners often treat streptothricosis with commercially available topical treatments. More severe or chronic cases may warrant the use of
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systemic antibiotics. In several reported instances, these antibiotic regimens saw the resolution of
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D. congolensis infection within 10 days [6,11]. However, these treatments have deleterious
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effects on equine gut microflora [18,19] and lower cure rates than topical treatments [10]. In the
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current study, a topically-applied 1% tea tree oil solution (v/v, in baby oil) allowed resolution of
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streptothricosis lesions within 8 days.
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Interestingly, of the three OTC products tested during these experiments, the product that demonstrated the most significant improvement on the Streptothricosis Score Chart during the
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experiment, P1, contained a proprietary blend of essential oils, including tea tree oil.
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Improvement of afflicted areas was marked and may signal a synergistic effect of the three
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essential oils in the product. P1 also showed inhibitory effects in vitro via the disk diffusion
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assay, but a minimum inhibitory concentration (MIC) could not be established. This was likely
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due to the relatively low initial concentration of the three essential oils in the product, thus any
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dilution conducted as part of the MIC experiments may have brought them below the threshold
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for inhibition.
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The other two products tested, both of which were sulfur-based, did not show
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improvement on the Streptothricosis Score Chart during the 8 day experiment when applied as
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described in the Materials & Methods section. P2, which contained a proprietary blend of alkyl
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sulfonate and alkyl sulfate, significantly inhibited D.congolensis in vitro, but did not improve
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lesions scores on the Streptothricosis Score Chart in vivo. P3, which lists sulfur as the only active
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ingredient, did not inhibit D.congolensis in vitro or show in vivo improvement of streptothricosis
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lesions as per the Streptothricosis Score Chart. While sulfur-based topical treatments are
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effective against other actinomycetes [29], it is possible this formulation was not specific enough
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for the causative agent of streptothricosis, D. congolensis. In vivo results for P2 and P3 may
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have been different if the products were applied more frequently than once per day or for more
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than eight days. Moreover, P2 was supplied with a foaming applicator which made it difficult to
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apply the solution as liberally as the other treatments.
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While the popularity of essential oils is rising, the use of TTO-based treatments is not without concern. Oral ingestion of TTO has been associated with loss of consciousness,
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disorientation, and rashes in humans [21,27,30]. Dermal application of undiluted TTO to three
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cats with shaved but intact skin resulted in neurotoxicity and potentially exacerbated underlying
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health conditions in one cat, leading to its death [31]. Slight dermal irritation has also been
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observed in response to repeated topical applications of 25% TTO (v/v, in paraffin oil) to shaved
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rabbits, though this concentration far exceeded the 1% TTO concentration recommended by the
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European Cosmetic Toiletry and Perfumery Association [21,27,32]. No adverse reactions,
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dermal or otherwise, were seen on the part of the participating horses in this study. While a 1%
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solution of TTO is supported as safe and effective for equine application, there is a concern that
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horse-owners may mistakenly apply undiluted TTO to streptothricosis lesions, as higher
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concentrations (≥ 5% TTO) may cause epidermal irritation and possible damage to other tissues
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[33]. For this same reason, it is also critical TTO is not applied to or near mucosal membranes,
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including the eyes, nose, or mouth. Thus, proper application of TTO requires prior knowledge of
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how to safely prepare and apply the compound.
299 As previously stated, streptothricosis affects an assortment of animal species, especially
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cattle and sheep. Bovine streptothricosis is a serious economic detriment in Central and Western
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Africa [1], so TTO may allow treatment of bovine streptothricosis in areas with lower access to
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regular veterinary care. In this study, we compared the efficacies of tea tree oil and three OTC
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products as treatments against equine streptothricosis. Tea tree oil is an easily accessed,
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relatively inexpensive, and potent compound with antimicrobial properties. Our research shows
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it is effective against Dermatophilus congolensis, the etiological agent of streptothricosis. We
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hope that, while our research was the first to explore tea tree oil as an effective and practical
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treatment for equine streptothricosis, other studies will seek to investigate its safety and efficacy
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in other affected species.
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While this research focused on tea tree oil, other essential oils may produce similar antimicrobial effects [20]. Additional research to evaluate the synergistic effect of tea tree oil
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with other essential oils might yield useful results, as some studies have found particular
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combinations of essential oils exert greater antimicrobial activity than the component essential
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oils in isolation [34]. Future research into the synergistic effects of essential oil combinations
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may lead to additional promising findings regarding the antimicrobial benefits of these emerging
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treatments.
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4.1 Conclusion
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In vitro testing revealed that tea tree oil produced large zones of clearing on disk
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diffusion plates and had a low minimum inhibitory concentration against the etiological agent of
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streptothricosis. In vivo testing demonstrated that a 1% (v/v, in baby oil) tea tree oil solution can
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ameliorate confirmed cases of streptothricosis in horses. Both in vitro and in vivo results support
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tea tree oil as an effective agent against D. congolensis, the causative agent of streptothricosis,
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and its ability to advance lesion resolution.
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326 327 328
5. Conflict of Interest AR is a regular volunteer and former donor to Brook Hill Farm Retirement Center for Horses. All other authors declare no competing interests.
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329 6. Funding
This work was supported by Randolph College, Lynchburg, VA; and the Virginia
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Foundation for Independent Colleges (VFIC), Richmond, VA. These sources of funding had no
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role in the design of this study; the collection, analysis, or interpretation of data; writing the
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article; nor the decision to submit this article for publication.
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335 7. Author Contributions
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Callan Frye: Investigation, Formal Analysis, Writing – Original Draft, Writing – Review &
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Editing, Visualization. Di Bei: Investigation, Formal Analysis. Jacquelyn Parman:
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Investigation, Formal Analysis. Jessica Jones: Investigation, Formal Analysis. Adam
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Houlihan: Conceptualization, Methodology, Writing – Review & Editing, Supervision, Project
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Administration, Funding Acquisition. Amanda Rumore: Conceptualization, Methodology,
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Formal Analysis, Visualization, Writing – Review & Editing, Supervision, Project
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Administration, Funding Acquisition
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8. Acknowledgments
The authors thank Jo Anne Miller, Tracy Russler, and all Brook Hill Retirement Center
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for Horses staff and volunteers for providing assistance and subjects for use in this study. They
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also thank Catherine Khoo and Aaron Whalen for laboratory support services.
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9. References
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[1] Zaria LT. Dermatophilus congolensis infection (dermatophilosis) in animals and man! An update. Comp Immun Microbiol Infect Dis 1993;16:179–222. [2] Martinez D. Chapter 2.4.9. Dermatophilosis. OIE ITerrestrial Manuali, 2008. [3] Martinez D, Prior P. Survival of Dermatophilus congolensis in tropical clay soils submitted to different water potentials. Vet Microbiol 1991;29:135–45. doi:10.1016/03781135(91)90121-U. [4] Pal M. Prevalence in India of Dermatophilus congolensis infection in clinical specimens from animals and humans. Rev Sci Tech OIE 1995;14:857–63. doi:10.20506/rst.14.3.882. [5] Scott DW. Chapter 7 - Skin diseases. In: Divers TJ, Peek SF, editors. Rebhuns Dis. Dairy Cattle. 2nd ed., Elsevier; 2008, p. 299–301. [6] Searcy GP, Hulland TJ. Dermatophilus dermatitis (streptothricosis) in Ontario. 1. Clinical observations. Can Vet J 1968;9:7–15. [7] Maillard J-C, Chantal I, Berthier D, Thevenon S, Sidibe I, Razafindraibe H. Molecular immunogenetics in susceptibility to bovine dermatophilosis. Ann N Y Acad Sci 2002;969:92–6. doi:10.1111/j.1749-6632.2002.tb04357.x. [8] Maillard JC, Martinez D, Bensaid A. An amino acid sequence coded by the exon 2 of the BoLA DRB3 gene associated with a BoLA class I specificity constitutes a likely genetic marker of resistance to dermatophilosis in Brahman zebu cattle of Martinique (FWI). Ann N Y Acad Sci 1996;791:185–97. doi:10.1111/j.1749-6632.1996.tb53525.x. [9] Overview of dermatophilosis. Merck Vet Man 2018. https://www.merckvetmanual.com/integumentary-system/dermatophilosis/overview-ofdermatophilosis (accessed June 8, 2018). [10] Awad WS, Nadra-Elwgoud, Abdou MI, El-Sayed AA. Diagnosis and treatment of bovine, ovine and equine dermatophilosis. J Appl Sci Res 2008;4:367–74. [11] Pascoe RR. Further observations on dermatophilus infections in horses. Aust Vet J 1972;48:32–4. doi:10.1111/j.1751-0813.1972.tb02207.x. [12] Elizabeth A Mauldin, Jeanine Peters-Kennedy. Integumentary System. In: Grant Maxie, editor. Jubb Kennedy Palmers Pathol. Domest. Anim., vol. 1. 6th ed., Saunders Ltd.; 2015, p. 509–736. [13] Sebastian MM, Giles RC, Donahu JM, Sells SF, Fallon L, Vickers ML. Dermatophilus congolensis-associated placentitis, funisitis and abortion in a horse. Transbound Emerg Dis 2008;55:183–5. doi:10.1111/j.1865-1682.2007.00981.x. [14] Yeruham I, Elad D, Perl S. Economic aspects of outbreaks of dermatophilosis in firstcalving cows in nine herds of dairy cattle in Israel. Vet Rec 2000;146:695–8. doi:10.1136/vr.146.24.695. [15] Scott D, Miller W. Equine Dermatology. 2nd ed. Saunders Elsevier; 2010. [16] Abu-Samra MT. A study of the ultrastructure and the life cycle of Dermatophilus congolensis. Zentralblatt Für Veterinärmedizin Reihe B 1979;26:110–24. doi:10.1111/j.1439-0450.1979.tb00799.x. [17] Overview of Dermatophilosis - Integumentary System. Merck Vet Man n.d. https://www.merckvetmanual.com/integumentary-system/dermatophilosis/overview-ofdermatophilosis (accessed June 8, 2018).
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[18] Costa MC, Stämpfli HR, Arroyo LG, Allen-Vercoe E, Gomes RG, Weese J. Changes in the equine fecal microbiota associated with the use of systemic antimicrobial drugs. BMC Vet Res 2015;11:19. doi:10.1186/s12917-015-0335-7. [19] Harlow BE, Lawrence LM, Flythe MD. Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: responses to antibiotic challenge. Vet Microbiol 2013;166:225–32. doi:10.1016/j.vetmic.2013.05.003. [20] Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 1999;86:985–90. doi:10.1046/j.1365-2672.1999.00780.x. [21] Carson CF, Riley TV. Antimicrobial activity of the essential oil of Melaleuca alternifolia. Lett Appl Microbiol 1993;16:49–55. doi:10.1111/j.1472-765X.1993.tb00340.x. [22] Yardley A. A preliminary study investigating the effect of the application of some essential oils on the in vitro proliferation of Dermatophilus congolensis. Int J Aromather 2004;14:129–35. doi:10.1016/j.ijat.2004.06.001. [23] Haalstra RT. Isolation of Dermatophiliis congolensis from skin lesions in the diagnosis of streptothricosis. Vet Rec 1965;77:824–5. [24] Burd EM, Juzych LA, Rudrik JT, Habib F. Pustular Dermatitis Caused by Dermatophilus congolensis. J Clin Microbiol 2007;45:1655–8. doi:10.1128/JCM.00327-07. [25] García A, Martínez R, Benitez-Medina JM, Risco D, García WL, Rey J, et al. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis. J Vet Sci 2013;14:491–4. doi:10.4142/jvs.2013.14.4.491. [26] O’Bryan CA, Crandall PG, Chalova VI, Ricke SC. Orange essential oils antimicrobial activities against Salmonella spp. J Food Sci 2008;73:M264–7. doi:10.1111/j.17503841.2008.00790.x. [27] Scientific Committee on Consumer Products (SCCP). Opinion on tea tree oil 2008. [28] Garcı́a-Sánchez A, Cerrato R, Larrasa J, Ambrose NC, Parra A, Alonso JM, et al. Identification of an alkaline ceramidase gene from Dermatophilus congolensis. Vet Microbiol 2004;99:67–74. doi:10.1016/j.vetmic.2003.10.028. [29] Gupta AK, Nicol K. The use of sulfur in dermatology. J Drugs Dermatol JDD 2004;3:427– 31. [30] Carson CF, Riley TV. Safety, efficacy and provenance of tea tree (Melaleuca alternifolia) oil. Contact Dermatitis 2001;45:65–7. doi:10.1034/j.1600-0536.2001.045002065.x. [31] Bischoff K, Guale F. Australian Tea Tree (Melaleuca Alternifolia) Oil Poisoning in Three Purebred Cats. J Vet Diagn Invest 1998;10:208–10. doi:10.1177/104063879801000223. [32] Beckmann B, Ippen H. Teebaum-Öl. Dermatosen 1998:120–4. [33] Sigma-Aldrich. Safety data sheet: tea tree oil 2015. [34] Gutierrez J, Barry-Ryan C, Bourke P. The antimicrobial efficacy of plant essential oil combinations and interactions with food ingredients. Int J Food Microbiol 2008;124:91–7. doi:10.1016/j.ijfoodmicro.2008.02.028.
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Table 1: List of products tested and sterility results Microbial b
P3
Sulfur
a
Purified water
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Blend of Alkyl Sulfonate, Alkyl Sulfate (18%)
Petroleum distillates, Zinc Stearate, Cade Oil, Glycerin
—
—
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Product Active Ingredients Inactive Ingredients: Growth a TTO Volatile essential oil from Melaleuca Baby Oil alternifolia, mostly terpene — hydrocarbons P1 Blend of Lavender Oil, Eucalyptus Canola Oil — Oil, Tea Tree Oil (3.20%)
Tea Tree Oil (TTO) was prepared as a 1% solution in baby oil for in vivo application.
b
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Products were aseptically cultured on BHI agar at 37°C for 3 days and examined for microbial growth (+ / -)
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Table 2: Presumptive laboratory identification of D.congolensis stock strain and clinical isolates Clinical Isolates 1
2
Catalase
+
+
—
Urease
+
+
+
—
+
+
β-hemolysis β-hemolysis β-hemolysis β-hemolysis β-hemolysis +
+
+
+
+
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Characteristic
Stock Strain (ATCC 14637)
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Table 3: Antimicrobial activity of TTO and tested products against D.congolensis by disk diffusion methoda
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Zone Diameter (mm)b M
SD
p-value
TTO
20
±3.70
<0.0001
P1
19
±4.88
<0.0001
P2
34
±13.6
<0.0001
P3
NID
NID
a
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Product
—
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zones of clearing quantified by average of four perpendicular measurements of diameter of clear area to the nearest millimeter Abbreviations: TTO, tea tree oil; NID, no inhibition detected; M, mean; SD, standard deviation
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Table 4: Minimum inhibitory concentration (MIC) of products a tested against D.congolensis a
MIC [% v/v]
TTO
7.24
3.56
P1
NID
—
P2
3.48
1.04
P3
NID
—
p-value
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SD
<0.0001
—
<0.0001
SC
M
Product
a
—
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Abbreviations: NID, no inhibition detected; M, mean; SD, standard deviation
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438
Figure Legends
439 Figure 1: Representative images of equine streptothricosis observed on a test subject. (A)
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anterior and posterior view of afflicted distal limb (B) ‘paintbrush’ lesion removed from afflicted
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region.
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Figure 2: Microbiological and genetic confirmation of D.congolensis isolation. (A)
445
D.congolensis (ATCC 14637) stock strain and (B) clinical isolate grown on BHI agar at 35°C
446
and 5% CO2. C) PCR amplification of D.congolensis (ATCC 14637) stock strain (L1) and
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clinical isolate (L2) for the agc gene with identical bands between 400 and 500bp. MWM, 100bp
448
molecular weight markers (Bioline, Memphis, TN); L1, Lane 1; L2, Lane 2.
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Figure 3: Pre- and post-treatment assessment of overall streptothricosis severity. Horses were
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treated daily for 8 days and blindly evaluated on Day 1 and Day 9 by a licensed veterinarian. (A)
452
overall score obtained pre- and post-treatment using the Streptothricosis Score Chart
453
(Supplementary Figure 1). Each treatment was applied to three affected regions on three separate
454
horses. (B) Representative images of streptrothricosis infection pre- and post-treatment with 1%
455
tea tree oil (TTO) solution (v/v, in baby oil). Significant differences between groups was
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determined with one-way ANOVA and Tukey post-hoc testing (* p < 0.05).
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Figure 4: Pre- and post-treatment assessment of individual features of streptothricosis. Horses
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were treated daily for 8 days and blindly evaluated on Day 1 and Day 9 by a licensed
460
veterinarian using the Streptothricosis Score Chart (Supplementary Figure 1). Shown are
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individual scores for (A) coat condition (B) lesion advancement (C) inflammation (localized) (D)
462
erythema (localized reddening or heat) (E) excoriation (tissue destruction) (F) lichenification
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(thickening) and (G) barrier (breakdown). Significant change from pre- to post-treatment
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between groups was determined with one-way ANOVA and Tukey post-hoc testing (* p < 0.05).
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FIGURE 2
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FIGURE 3 29
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Highlights •
Limited evidence exists supporting the use of essential oils in the treatment of equine
•
Tea tree oil (TTO) shows antimicrobial activity towards D.congolensis, the causative agent of equine streptothricosis, in vitro
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A 1% tea tree oil (TTO) emulsion is effective at resolving streptothricosis lesions in vivo
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streptothricosis
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Ethical Statement I testify on behalf of all co-authors that our article submitted to Journal of Equine Veterinary Science:
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Title: Efficacy of Tea Tree Oil in Treatment of Equine Streptothricosis All authors: Callan Frye, Di Bei, Jacquelyn Parman, Jessica Jones, Adam Houlihan, and Amanda Rumore
Date: January 2, 2019
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Corresponding author’s signature:
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1) this material has not been published in whole or in part elsewhere; 2) the manuscript is not currently being considered for publication in another journal; 3) all authors have been personally and actively involved in substantive work leading to the manuscript, and will hold themselves jointly and individually responsible for its content. 4) this research was conducted under approval of the Randolph College Institutional Animal Care and Use Committee (IACUC)