Final report for GNE18-184
- Caseous lymphadenitis (CL) is a highly contagious and chronic disease that effects sheep and goats and is caused by the bacteria Corynebacterium pseudotuberculosis. Due to the nature of this pathogen, CL is very difficult to treat with standard antibiotics. C. pseudotuberculosis is also highly resilient and able to survive in the environment for extended periods of time where it can be easily spread throughout a flock. This disease causes a decrease in production in these animals and therefore a loss of income for farmers. The purpose of this project was to find an alternative method of treatment and/or control of the disease by using essential oil components with known antimicrobial properties. If effective, these essential oils could be used as oral or topical antibiotics for affected small ruminants, or as disinfectants for contaminated farm surfaces.
- The efficacy of several essential oil components as antimicrobials against C. pseudotuberculosis was tested using a standard disk diffusion assay. The most effective of these components were then tested for cytotoxic effects against mammalian fibroblast cells using a cell viability assay to determine their safety for use in small ruminants.
- The essential oil components most effective at inhibiting the in vitro growth of C. pseudotuberculosis were β-citronellol, carvacrol, cuminaldehyde, thymol, and trans-cinnamaldehyde. However, their ability to inhibit C. pseudotuberculosis growth decreased over time, likely due to the volatility of the essential oil components. The results of the cell viability assays were inconclusive; essential oil components appeared to interfere with the cell viability measurements.
- The essential oil components tested in this study could potentially be used as alternative antimicrobial treatments or disinfectants against C. pseudotuberculosis in small ruminants. The major limitations of these components are their short-term bioactivity and their potential for cytotoxicity to mammalian cells. Further research is needed in these areas before these components could be used in vivo.
- To determine which essential oil components are most effective as antimicrobials against Corynebacterium pseudotuberculosis
- To determine the cytotoxic effects of select essential oil components on mammalian fibroblast cells
The purpose of this project was to find an alternative method of treatment and control of caseous lymphadenitis (CL) using constituents of essential oils with known antimicrobial properties. CL is a disease that affects sheep and goats across the country and worldwide. A 2012 SARE-funded study of Maine sheep found that approximately 22% of Maine sheep tested positive for CL exposure using the Synergistic Hemolysin Inhibition assay. Additionally, 43% of the Maine sheep farms tested had at least one animal that tested positive for CL. Due to the highly contagious nature of the pathogen and the prevalence observed on Maine farms, it may be that the percentage of infected sheep has increased since that study, unless proper biosecurity methods were implemented. External abscesses caused by CL infection can significantly decrease the value of the animal hide as well as the amount of wool production. The disease is occasionally fatal when internal abscesses, which may occur in the lungs and abdominal cavity, rupture and release bacterial toxins. Although vaccination may decrease abscess formation, there are currently no widely effective control and prevention measures for this disease. Thus, it is an ongoing challenge for the small ruminant industry. Recent studies have shown that essential oil components can have antimicrobial properties against even highly antibiotic resistant bacteria. The results of this project offer farmers new options for alternative CL treatment; these may be more readily available than conventional antibiotics and may also be less likely to induce antibiotic resistance on the farm.
Antimicrobial Activity Assay: The essential oil components evaluated for their antimicrobial activity against Corynebacterium pseudotuberculosis were drawn from a list including cinnamic acid, p-anisaldehyde, β-pinene, carvacrol, cinnamaldehyde, β-citronellol, cuminaldehyde, α-terpinene, terpinolene, and thymol. These compounds were chosen due to their reported ability to inhibit the in vitro growth of Mycobacterium tuberculosis, a closely related bacterium to C. pseudotuberculosis. (Andrade-Ochoa et al., 2015) Antimicrobial activity was evaluated by determining the minimum inhibitory concentration (MIC) values, or the lowest possible concentration of the essential oil component that prevents visible growth of the bacteria. The values were determined using an agar disk diffusion test, a standard procedure in the microbiology field for testing antimicrobial efficacy in vitro. The ATCC strain of C. pseudotuberculosis was grown in Brain Heart Infusion (BHI) broth and spread evenly on the surface of BHI agar plates. Differing concentrations (ranging from 100 to 1 mg/ml) of the essential oil components diluted in Tween (a mild, cell-friendly detergent), ethanol, and water were added to sterile 6 mm paper disks in the center of each plate. Several experiments were conducted to determine the optimal concentrations of Tween and ethanol needed to dissolve these components, which are highly insoluble in water. Plates with disks treated with gentamicin (a conventional antibiotic), water, Tween, and ethanol were included as controls. The plates were incubated at ideal growth conditions for C. pseudotuberculosis (37° C in 5% CO2). The zones of inhibition around each disk (area where the bacteria are unable to grow) were measured after 24, 48, and 72 hours of incubation. The essential oil components that were most effective at inhibiting C. pseudotuberculosis growth with this method were retested in triplicate at a narrower range of concentrations to determine the MICs of each.
Cytoxicity Assay: The essential oil components that were most effective at inhibiting C. pseudotuberculosis growth in the disk diffusion assay were also assessed for their cytotoxicity against mammalian fibroblast cells to determine if they would be safe to use on and/or around animals. Fibroblast cells were chosen because they are a common cell type that would likely come into contact with the EO components if they were being used as topical antimicrobials or disinfectants of farm surfaces. The cells used in this experiment were Buffalo Rat liver (BRL) cells. These cells were grown in Eagle’s Minimal Essential Medium (EMEM) + 10% fetal bovine serum (FBS) until confluent. They were then added to 96-well plates at a concentration of 5,000 cells/well and incubated overnight (37° C in 5% CO2). The EO components (β-citronellol, carvacrol, cuminaldehyde, thymol, and trans-cinnamaldehyde) were diluted in ethanol, Tween, and EMEM to final concentrations of 100, 50, and 10 mg/ml and added to the cells. Untreated cells as well as cell treated with 10% bleach and ethanol/Tween were included as controls. The plates were incubated overnight (37° C in 5% CO2). Then, the cell viability reagent CellTiter 96® AQueous One Solution Reagent (Promega) was added to the wells and incubated. Absorbance was read at 490 nm on a spectrophotometer after 1-, 2-, and 3-hours incubation. The amount of formazan product as measured at 490 nm is used as a directly proportional indicator of the number of viable cells.
We found that the diluents, ethanol and Tween, had an effect on the viability of the cells, so this experiment was repeated with the EO components diluted directly in the cell culture media. Due to the low solubility of these EO components, the plates treated with EO components were then incubated with shaking in an attempt to maximize contact between the EO components and the BRL cells.
Disk Diffusion Assay Trial 1
In the preliminary disk diffusion assay (9 EO components tested), β-pinene, α-terpinene, and terpinolene had minimal (<10 mm) or no (6 mm, which is the diameter of the disk) zones of inhibition at any of the concentrations tested. Cuminaldehyde, carvacrol, thymol, and p-anisaldehyde had substantial zones of inhibition (≥ 10 mm) at final concentrations of 100 mg/ml or greater at all time points. β-citronellol and trans-cinnamaldehyde had substantial zones of inhibition at final concentrations of 10 mg/ml and greater at all time points. Zones of inhibition on EO component-treated plates generally decreased in size over time (Table 1). At the highest concentration tested, β-citronellol, carvacrol, thymol, and trans-cinnamaldehyde had the largest zones of inhibition (Figure 1). The gentamicin control had a consistent zone of inhibition (~25 mm) at all time points. The negative controls (ethanol, Tween, sterile DI water) had no zones of inhibition. The blank BHI inoculated plate had no bacterial growth.
Disk Diffusion Assay Trial 2
Of the most effective EO components, β-citronellol had substantial zones of inhibition at final concentrations of 10 mg/ml or greater at all time points. It has no zones of inhibition at a final concentration of 1 mg/ml (Figure 2). Carvacrol had substantial zones of inhibition at final concentrations of 10 mg/ml or higher at all time points (Figure 3). Cuminaldehyde had substantial zones of inhibition at final concentrations of 100 mg/ml and 40 mg/ml but not at 70 mg/ml or 10 mg/ml (Figure 4). Thymol had substantial zones of inhibition at final concentrations of 50 mg/ml and higher at all time points and at 10 mg/ml up to 48-hours incubation. It had minimal or no zones of inhibition at a final concentration of 1 mg/ml at all time points and 10 mg/ml at 72-hours incubation (Figure 5). Trans-cinnamaldehyde had substantial zones of inhibition at final concentrations of 10 mg/ml or greater at all time points. It had minimal or no zones of inhibition at a final concentration of 1 mg/ml (Figure 6). Zones of inhibition on EO component-treated plates generally decreased in size over time (Figure 7). The zones of inhibition at 10 mg/mL were the same or slightly larger in Trial 2 as compared to Trial 1 (Table 1; Figure 7). The gentamicin control had a consistent zone of inhibition (~25 mm) at all time points. The negative controls (ethanol, Tween, sterile DI water) had no zones of inhibition. The blank BHI inoculated plate had no bacterial growth.
β-citronellol, carvacrol, thymol, and trans-cinnamaldehyde were able to inhibit C. pseudotuberculosis growth in vitro at final concentrations of 10 mg/ml or greater. These concentrations are much higher than those reported to inhibit the in vitro growth of the related bacteria, M. tuberculosis (Andrade-Ochoa et al., 2015). However, the MICs of EO components are dependent on several factors including the type of bactericidal assay or emulsifying agent used, the pH of the media, and the growth phase of the bacteria. These components could potentially be used as alternative antimicrobial treatments or disinfectants against C. pseudotuberculosis in small ruminants. The major limitations of the application of these EO components are their short-term bioactivity and their potential cytotoxic effects on mammalian cells. Further research is needed in these areas before these components could be used in vivo.
Cell Viability Assay Trial 1
β-citronellol and carvacrol both inhibited cell viability versus the untreated controls, though neither had a linear effect due to concentration used. As well, cell viability was higher in all EOC treatments compared to vehicle controls or bleach. The untreated wells had the highest number of viable cells (Figure 8).
No conclusions could be drawn from the EO component-treated wells because the amount of ethanol and Tween in which the components were diluted had cytotoxic effects on the BRL cells. The number of viable cells did not vary consistently with varying concentrations of EO components. This may be because of the differing amounts of ethanol and Tween needed to dilute the components. Lack of media may have also reduced cell viability in wells with higher concentrations; because all of the wells must have the same total volume, cells treated with higher concentrations have less cell culture media available.
Cell Viability Assay Trial 2
The absorbance measurements of BRL cells treated with all concentrations of β-citronellol, cuminaldehyde, and thymol, the lowest final concentration (10 mg/ml) of carvacrol and trans-cinnamaldehyde, and of bleach and no treatment were all within the range of 0.2 to 0.3, indicating that there was no difference in cell viability (Figures 9 and 10). Trans-cinnamaldehyde had the highest absorbance readings at final concentrations of 50 mg/ml and 100 mg/ml (0.786 and 1.243, respectively). The higher concentrations of carvacrol also had relatively high absorbance readings of 0.537 and 0.732 respectively (Figure 9). There was a large amount of variability in the replicates for some of the treatments (Figures 9 and 10).
All of the EO components affected BRL cell morphology based on visual assessment with an inverted microscope. These effects were more detectable at higher concentrations of EO components. Bleach did not appear to have a strong effect on BRL cell morphology. Untreated BRL cells appeared to have experienced some damage and cell death.
According to the absorbance readings, the treatments with the most viable BRL cells were the highest final concentrations (50 mg/ml and 100 mg/ml) of trans-cinnamaldehyde and carvacrol. However, the images taken of these wells after treatment do not support this finding. Visual assessment of the BRL cells treated with EO components indicate that all of the components had a cytotoxic effect on the cells, especially at higher concentrations. Visual assessment of wells treated with carvacrol, thymol, and trans-cinnamaldehyde also revealed relatively large amorphous aggregates. These may be the EO components themselves as they were unable to dissolve in the cell culture media, or they could be due to interactions between the components and the media; some EO components are known to interact and bind with proteins in media. These interactions may interfere with the colorimetric reading of the cell viability assay or they may alter the cytotoxic/antimicrobial properties of the EO component. The CellTiter 96® AQueous One Solution Cell Proliferation Assay is an MTS-based assay that measures the increase in color intensity that occurs when live cells reduce the MTS tetrazolium compound, generating a colored formazan product. The absorbance reading at 490 nm is directly proportional to the number of viable cells. The presence of the EO components, even after replacing the treated media, may have had an unintended effect on the color change of the media that is measured. Based on morphological assessment, it can be concluded that β-citronellol, carvacrol, cuminaldehyde, thymol, and trans-cinnamaldehyde may have cytotoxic effects on BRL cells in vitro at concentrations of 10 mg/ml and higher based on microscopy observations. However, a different cell viability assay may have to be used to quantify these cytotoxic effects.
Although these EO components appear to decrease viability of mammalian fibroblast cells, a few of them have already been used in vivo on both humans and animals without reported toxic side effects. Carvacrol and thymol have been used as a dietary supplement in food-producing animals without reported toxic effects (Wei et al., 2017; Hashemipour et al., 2013). These findings suggest low toxicity of carvacrol and thymol when administered orally. However, oral administration of EO components may not be effective as a method for treating CL due to the location and thick encapsulation of CL abscesses as well as the volatility of the EOCs. It is unlikely that low doses of these EO components would have enough bactericidal activity to kill or inhibit the growth of C. pseudotuberculosis, when and if they migrated to the source of the abscess. For these reasons, dermal administration of the EO components to external CL abscesses may be a more viable approach. Many EO components are reported to cause skin irritation, however most studies involving dermal exposure of laboratory animals to EOCs report the LD50 values rather than the amount of damage or irritation to the skin (PubChem, 2019). Further evaluations of the cytotoxicity of these EO components on mammalian skin cells is needed before they can be used in vivo as antibacterial agents for C. pseudotuberculosis.
The EO components β-citronellol, carvacrol, cuminaldehyde, thymol, and trans-cinnamaldehyde are effective at inhibiting the in vitro growth of C. pseudotuberculosis at relatively low concentrations. We were unable to draw definitive conclusions about the cytotoxicity of these components against mammalian fibroblast cells at the concentrations needed to inhibit bacterial growth. Further research is needed in this area before these EO components can be used in a farm setting. If these components are found to have low toxicity towards mammalian cells, they could potentially be used as topical antimicrobials of CL abscesses. EO components are able to permeate many different surfaces which may make them more effective than traditional antibiotics at penetrating the thick-walled abscesses that characterize CL. These components could also potentially be used as disinfectants of farm surfaces that commonly harbor C. pseudotuberculosis following the rupture of abscesses such as wooden feeders, fencing, or shearing equipment. Essential oils and their components may be less toxic to animals and to the environment than typical disinfectants used on farm surfaces. Several of the EO components used in this experiment including thymol and carvacrol are Generally Regarded As Safe (GRAS; grasdatabase.org) One of the potential issues of utilizing EO components on farms is their short-term bioactivity, which may make them a less sustainable option for disinfection.
Education & Outreach Activities and Participation Summary
A presentation was given at the 2018 Maine Sheep Conference and Annual Meeting in Bangor, Maine. The conference was attended by approximately 15 sheep producers of the Maine Sheep Breeders Association (MSBA) as well as several members of the University of Maine Cooperative Extension livestock specialist group. This presentation covered general information about caseous lymphadenitis (CL) such as disease prevalence, transmission, and prevention. We also discussed our current research using essential oil components as antimicrobials for Corynebacterium pseudotuberculosis, the causative agent of CL.
A poster presentation outlining the major findings of this project was presented at the Maine Sustainability and Water Conference in Augusta, Maine on March 28, 2019. This conference was attended by students, researchers, and professionals from a variety of backgrounds focused on sustainability.
The project results were also presented at the University of Maine Student Symposium in Bangor, Maine on April 10, 2019. This symposium was attended by UMaine students, faculty, and the general public.
Two reports are currently in preparation for submission to a peer-reviewed, open-access journal. These articles will discuss antimicrobial evaluations of the EO components against C. pseudotuberculosis, as well as a literature review in this area.
This project may contribute to future sustainability in the small ruminant industry. This works suggests that essential oil components may be just as, if not more, effective at inhibiting the growth of Corynebacterium pseudotuberculosis compared to typical antibiotic treatments. If essential oils and/or their components are found to be safe and efficient to use in the control of CL, it could reduce the need for disinfectant methods that are toxic to animals and the environment. It could also reduce the need for typical antibiotic treatments, which could be crucial in the face of rising numbers of antibiotic resistant bacteria. Additionally, the Veterinary Feed Directive (VFD; FDA, 2015) requires medically important antimicrobials in feed to be used only under the supervision of a licensed veterinarian and limits the “extra-label” use of antibiotics, which further narrows the choice of treatment for small ruminants and other livestock. Having an alternative to traditional antibiotic drugs could greatly help especially small-scale farmers maintain healthy flocks.
Through this project I have gained a better understanding for the struggles that many small-scale farmers face in controlling diseases such as CL. This project helped me to improve my presentation skills including manuscript and poster preparation, public speaking, and data presentation and analysis. Through this process, I mentored an undergraduate student in the lab, Cassie Miller, as she conducted her senior capstone project on a related research topic. I have also learned many different laboratory techniques which will be valuable as I hope to continue to do research on the control and prevention of infectious diseases in livestock and other farm animals. This work has informed the project of a current NSF REU student working in Dr. Anne Lichtenwalner’s lab.