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INNOVATIVE RESOURCES IN SMALL RUMINANT HEALTH
By
Sarah Paluso
B.S. Gettysburg College, 2016
A THESIS
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Science
(in Animal Science)
The Graduate School
The University of Maine
May 2019
Advisory Committee:
Anne Lichtenwalner, Associate Professor of Animal and Veterinary Sciences, Advisor
Juan Romero, Assistant Professor of Animal Nutrition
Con Sullivan, Assistant Professor of Biology
Sally Molloy, Assistant Professor of Genomics
INNOVATIVE RESOURCES IN SMALL RUMINANT HEALTH
By Sarah Paluso
Thesis Advisor: Dr. Anne Lichtenwalner
An Abstract of the Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science (in Animal Science) May 2019
Caseous lymphadenitis (CL) is a chronic bacterial infection caused by Corynebacterium pseudotuberculosis (C. pseudoTB) that affects small ruminants. This disease has a worldwide prevalence and results in significant economic losses to the sheep and goat industries. Antibiotics have had limited success in treating CL, due to the difficulty of penetrating the dry, thick-walled abscesses that characterize this disease. Essential oils are complex bioactive compounds that have been increasingly explored as sources of antimicrobial activity. Due to the nature of these oils, tissue and wood penetration may be possible, enabling topical and environmental treatment (e.g.; disinfectants of farm surfaces, such as feeders or shearing equipment). The purpose of this project was to evaluate certain essential oil components (EOCs) with known antibacterial properties for their bactericidal effects on C. pseudoTB using a standard disk diffusion assay. The most effective inhibition of C. pseudoTB growth in vitro was due to β-citronellol, carvacrol, thymol, and trans-cinnamaldehyde. These EOCs were able to inhibit C. pseudoTB growth at concentrations as low as 10 mg/ml. Essential oils and their components can also have toxic effects on eukaryotic cells. For this reason, a cell viability assay on a line of mammalian fibroblast cells (Buffalo rat liver cells) was conducted to evaluate the cytotoxicity of the
EOCs that were most effective at inhibiting C. pseudoTB growth. Based on direct microscopic
observations, EOCs damaged mammalian cells; this was not reflected in the cell viability assay.
Therefore, further research is needed on the toxicity of these components before use in vivo.
ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Anne Lichtenwalner, for her guidance and encouragement throughout my time at the University of Maine. I would also like to thank Ann Bryant for her great amount of help with the preparation and execution of the experiments in the lab. I would also like to thank all of my committee members, Dr. Juan Romero, Dr. Con Sullivan, and Dr. Sally Molloy for their continued support during this project and for their review of my thesis. Lastly, I would like to thank
Northeast Sustainable Agriculture Research and Education (NESARE) for funding this project. This material is based upon work supported by the National Institute of Food and Agriculture, U.S.
Department of Agriculture, through the Northeast Sustainable Agriculture Research and Education program under subaward number GNE18-184.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... iii
LIST OF TABLES ...... viii
LIST OF FIGURES ...... ix
Chapter
1. BACKGROUND ...... 1
Prevalence and Economic Impact ...... 1
Caseous Lymphadenitis ...... 3
Transmission ...... 3
Infections in Other Species ...... 4
Zoonotic Potential ...... 5
Corynebacterium pseudotuberculosis ...... 6
Virulence Factors ...... 7
Pathogenesis ...... 8
Environmental Stability ...... 9
Vaccines ...... 10
Diagnostic Tests ...... 11
Treatments and Control ...... 12
Essential Oils ...... 13
β-pinene ...... 15
Cuminaldehyde ...... 16
Carvacrol ...... 16
iv
Thymol ...... 17
Trans-cinnamaldehyde ...... 18
Para-anisaldehyde ...... 18
Alpha-terpinene ...... 19
β-citronellol ...... 20
Terpinolene ...... 20
2. SUSCEPTIBILITY OF C. PSEUDOTUBERCULOSIS TO ESSENTIAL OIL COMPONENTS ...... 21
Introduction ...... 21
Materials and Methods ...... 23
Preparation of C. pseudotuberculosis ...... 23
Culturing C. pseudotuberculosis for Disk Diffusion Assays ...... 23
Disk Diffusion Assay Trial 1 ...... 24
Disk Diffusion Assay Trial 2 ...... 25
Results ...... 26
Identification of C. pseudotuberculosis ...... 26
Disk Diffusion Assay Trial 1 ...... 26
Disk Diffusion Assay Trial 2 ...... 28
Discussion ...... 32
Conclusions ...... 36
3. SUSCEPTIBILITY OF BUFFALO RAT LIVER CELLS TO ESSENTIAL OIL COMPONENTS ...... 37
Introduction ...... 37
Materials and Methods ...... 37
Cell Viability Assay Trial 1 ...... 37
Cell Viability Assay Trial 2 ...... 38
v
Results ...... 39
Cell Viability Assay Trial 1 ...... 39
Cell Viability Assay Trial 2 ...... 40
Discussion ...... 43
Cell Viability Assay Trial 1 ...... 43
Cell Viability Assay Trial 2 ...... 44
BIBLIOGRAPHY ...... 47
APPENDICES ...... 61
Appendix A. C. pseudotuberculosis Identification ...... 61
Appendix B. Essential Oil Component Solubility ...... 62
Solubility of Essential Oil Components in Ethanol Trial 1 ...... 62
Methods ...... 62
Results ...... 62
Solubility of Essential Oil Components in Ethanol Trial 2 ...... 63
Methods ...... 63
Results ...... 63
Solubility of Essential Oil Components in Ethanol and Tween...... 63
Methods ...... 63
Results ...... 64
Appendix C. Buffalo Rat Liver Cell Culture Methods ...... 65
Maintaining BRL Cell Culture ...... 65
Restarting Cryopreserved BRL Cell Culture ...... 65
Counting BRL Cells ...... 66
Cell Viability Assay Trial 1 ...... 67
vi
Cell Viability Assay Trial 2 ...... 67
BIOGRAPHY OF THE AUTHOR...... 69
vii
LIST OF TABLES
Table 2.1. Chemical and physical properties of EOCs ...... 22
Table 2.2. Zones of C. pseudoTB inhibition by essential oil components (EOCs) after
24-, 48-, and 72-hours (hr) incubation...... 27
Table A.1 Identification of cryopreserved C. pseudoTB cultures ...... 61
Table B.1. Table B.1 Visual assessment of EOC solubility in 5% ethanol and water
(2=dissolved well; 1=dissolved slightly; 0=did not dissolve) ...... 62
Table B.2. Table B.2 Visual assessment of EOC solubility in Trial 2 (2=dissolved well;
1=dissolved slightly; 0=did not dissolve) ...... 63
Table B.3. Visual assessment of EOC solubility (sol) in Ethanol and Tween (2=dissolved
well; 1=dissolved slightly; 0=did not dissolve) ...... 64
Table C.1. Layout of 96-well plate for BRL cell viability assay Trial 1 (shaded wells
indicate no treatment; BC=β-citronellol; CV=carvacrol) ...... 67
Table C.2. Layout of 96-well plate #1 for BRL cell viability assay Trial 2 (shaded wells
indicate no treatment; BC=β-citronellol; CV=carvacrol) ...... 67
Table C.3. Layout of 96-well plate #2 for BRL cell viability assay Trial 2 (shaded wells
indicate no treatment; CU=cuminaldehyde; TH=thymol) ...... 67
Table C.4. Layout of 96-well plate #3 for BRL cell viability assay Trial 2 (shaded wells
indicate no treatment; TC=trans-cinnamaldehyde) ...... 68
viii
LIST OF FIGURES
Figure 2.1. Developed API® Coryne test strip inoculated with ATCC strain of C. pseudoTB ...... 26
Figure 2.2. Zones of C. pseudoTB inhibition at final essential oil component
concentrations of 100 mg/ml...... 28
Figure 2.3. Zones of C. pesudoTB inhibition by β-citronellol after 24-, 48-, and 72-hours
(hr) incubation. Values are the mean of 3 replicates; error bars are the
standard deviations of the replicates ...... 29
Figure 2.4. Zones of C. pesudoTB inhibition by carvcacrol after 24-, 48-, and 72-hours (hr)
incubation. Values are the mean of 3 replicates; error bars are the standard
deviations of the replicates ...... 29
Figure 2.5. Zones of C. pesudoTB inhibition by cuminaldehyde after 24-, 48-, and 72-hours
(hr) incubation. Values are the mean of 3 replicates; error bars are the
standard deviations of the replicates ...... 30
Figure 2.6. Zones of C. pesudoTB inhibition by thymol after 24-, 48-, and 72-hours (hr)
incubation. Values are the mean of 3 replicates; error bars are the standard
deviations of the replicates ...... 30
Figure 2.7. Zones of C. pesudoTB inhibition by trans-cinnamaldehyde after 24-, 48-, and
72-hours (hr) incubation. Values are the mean of 3 replicates; error bars are
the standard deviations of the replicates ...... 31
Figure 2.8. Zones of C. pseudoTB inhibition at final essential oil component concentrations
of 10 mg/ml. Values are the mean of 3 replicates; error bars are the standard
deviations of the replicates ...... 31
Figure 3.1. Viability of BRL cells treated with EOCs and controls (higher absorbance
readings indicate more viable cells). Values are the mean of 3 replicates ...... 40
ix
Figure 3.2. Viability of BRL cells treated with EOCs (higher absorbance readings indicate
more viable cells). Values are the mean of 3 replicates; error bars are the
standard deviations of the replicates ...... 41
Figure 3.3. Viability of BRL cells treated with bleach and untreated (higher absorbance
readings indicate more viable cells). Values are the mean of 3 replicates; error
bars are the standard deviations of the replicates...... 41
Figure 3.4. BRL cells at treated with β-citronellol at final concentrations of A) 100 mg/ml,
B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification) ...... 42
Figure 3.5. BRL cells at treated with carvacrol at final concentrations of A) 100 mg/ml,
B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification) ...... 42
Figure 3.6. BRL cells at treated with cuminaldehyde at final concentrations of A) 100
mg/ml, B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification)...... 42
Figure 3.7. BRL cells at treated with thymol at final concentrations of A) 100 mg/ml, B)
50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification) ...... 42
Figure 3.8. BRL cells at treated with trans-cinnamaldehyde at final concentrations of A)
100 mg/ml, B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification) ...... 43
Figure 3.9. BRL cells treated with 10% bleach for 24 hours from A) Plate 1, B) Plate 2,
and C) Plate 3 from viability assay Trial 2 (10x magnification) ...... 43
Figure 3.10. BRL cells from A) Plate 1, B) Plate 2, and C) Plate 3 from viability assay Trial
2 (10x magnification) ...... 43
Figure C.1 Confluent BRL cells (10x magnification) ...... 65
x
CHAPTER 1
BACKGROUND
Prevalence and Economic Impact
Caseous lymphadenitis (CL) is a chronic disease caused by the pathogenic bacterium
Corynebacterium pseudotuberculosis (C. pseudoTB) that affects small ruminants worldwide. CL is present in nearly all countries with farmed ruminants and its effects on the sheep and goat industries have been reported since the early 1900s. It has been identified in North and South America, Europe, Asia,
Australia, Africa, and in the Middle East (Baird & Fontaine, 2007). From 1996 to 2004, 64 countries reported having animals with CL to the World Animal Health Organization (OIE), however this is likely underestimated as CL is not considered a reportable disease in all countries (OIE, 2009; Abebe et al.,
2015).
External abscesses caused by CL infection can decrease the value of the animal hide as well as the amount of wool production. An Australian study estimated a 4.1-6.6% loss of wool in the first year of
CL infection (Paton et al., 1994). The disease can be fatal when internal abscesses, often occurring in the lungs and abdominal cavity, rupture and release bacterial toxins. In countries with a large sheep industry such as Australia, the economic losses from CL were estimated at $15 million per year (Paton et al.,
1988; Paton, 1993). Approximately half of this economic loss is due to the need for trimming and downgrading of carcasses displaying CL lesions, which can take up to 60-70% of a meat inspector’s time
(Paton, 2002). The other half is due to lower wool production, particularly in sheep with external abscesses (Paton et al., 1988). Western Australia reported CL prevalence rates of approximately 50-60% in the 1970s and 1980s, however these rates decreased by the early 2000s to 20-30% (Batey, 1986a;
Middleton et al., 1991).
Small ruminants make up an important sector of agriculture in low income and developing countries. Goats are of particular economic importance to developing countries in tropical and dry areas
1 where it can be difficult to maintain other species of ruminant livestock. Due to goats’ reliability, fast breeding rate, and relatively low nutritional requirements, many farmers in these areas rely on goat milk and meat production as a main source of income (Escareño et al., 2013). In 2017, countries designated as “least developed countries” produced 1.28 million tons of goat meat and 5.27 tons of goat milk; these were valued at $2.62 billion and $982 million respectively (FAOSTAT, 2017). These countries also produced 1.05 million tons of sheep meat and 2.14 million tons of sheep milk, valued at $1.43 billion and $812 million respectively (FAOSTAT, 2017). Economic losses due to CL can be most detrimental to these countries where resources to control the disease are most limited. CL is a major factor in organ/carcass condemnation from sheep and goats in many African countries including South Africa,
Tanzania, Kenya, Mali, and Nigeria (Abebe et al., 2015). In Nigeria and Ethiopia where goats contribute a significant amount to the agricultural economy, CL prevalence has been estimated at 50% and 27.5%, respectively (Abebe et al., 2015).
Sheep and goat farms also represent an important sector of U.S. agriculture. As of January 1,
2018, there were an estimated 5.23 million sheep and 2.62 million goats in the U.S. (USDA NASS, 2018).
In 2017, the United States produced 76.9 thousand tons of sheep and goat meat and 25.8 thousand tons of goat milk (FAOSTAT, 2017). Sheep infected with CL exhibit a decrease in wool production of approximately 5%, resulting in a loss of about $500,000 annually (Paton et al., 1994). CL has historically been highly prevalent in the western United States and Canada with incidence rates reaching 42% and
50-94%, respectively (Stoops et al., 1984; Stanford et al., 1998). The national wool, mohair, and milk production of these animals was worth approximately 9.39 million dollars as of the 2012 USDA census; decreases in wool, milk, and meat production caused by CL may lead to substantial economic impacts for these farmers. In Maine, a 2012 USDA Sustainable Agriculture Research and Education (SARE)- funded study found that approximately 22% of the Maine sheep tested in the study were seropositive for CL exposure using the Synergistic Hemolysin Inhibition (SHI) assay. Additionally, 43% of the Maine
2 sheep farms tested had at least one animal that tested positive for CL (Lichtenwalner, 2014). Due to the highly contagious nature of the pathogen and the prevalence observed on Maine farms, it could be predicted that the total percentage of infected sheep has since increased, unless proper biosecurity measures were administered. However, CL is not currently a reportable animal disease in Maine (ME
DACF, 2011). Locally, nationally, and internationally, CL may substantially impact the sheep and goat industry.
Caseous Lymphadenitis
In small ruminants, CL results in the formation of pyogranulomatous lesions. These are often expressed externally in the form of dry “caseous” abscesses within the superficial lymph nodes (Valli and
Parry, 1993). CL can also occur internally, primarily in the mediastinal lymph nodes or lung parenchyma, either independently or together with external lesions. This form of the disease can also affect other organs such as the liver, kidneys, and mammary glands. Less commonly, lesions also form in the heart, brain, spinal cord, testes, uterus, and joints (Valli and Parry, 1993).
Transmission
The most common route of CL infection is through the skin, when minor wounds or abrasions come into contact with infective material, either directly (trans-dermal contact) or indirectly (contact with contaminated fomites, such as contaminated shearing equipment). A respiratory route of infection has also been theorized, due to reports of some cases with an abundance of lesions concentrated in the lungs and airways (Stoops et al., 1984). However, other studies have indicated that these cases may be consistent with systemic infections that have spread through blood or lymph vessels (Brogden et al.,
1984; Nairn and Robertson, 1974). CL is usually introduced to a healthy flock by the addition of an infected animal (Ayers, 1977). It may also be introduced by human activity (contaminated equipment, clothing, etc.), although this is much less common (Baird and Fontaine, 2007).
3
The location of abscess formation is thought to be indicative of the route of infection due to the differences between sheep and goat infection. Sheep normally acquire infection through minor skin abrasions resulting from sheering, ear tagging, and castration. Goats are thought to acquire the disease orally or through skin on the face and head as this is consistent with common goat behaviors such as mutual grooming, head butting, and mouthing (Baird and Fontaine, 2007). Therefore, the visceral form of the disease is more common in sheep, whereas the external form of the disease, especially around the head and neck, is more common in goats (Baird and Fontaine, 2007).
Aside from minor skin wounds, other risk factors for acquiring CL include the breed and age of the animal (Baird and Fontaine, 2007; Pepin et al., 1994a). There are also several risk factors associated with farm environment such as the presence of wooden feeders or fencing that may harbor the bacterium and infect animals through the oral cavity or via splinters (Augustine and Renshaw, 1986).
Sheep dipping (immersing sheep in vats of antiparasitic solutions following shearing) has also been identified as a common source of CL transmission, although this practice is no longer very common
(Paton et al., 2002; Nairn and Robertson, 1974).
Insect vectors have been suggested as an important route of transmission for infections in horses, cattle, and buffalo, but not small ruminants (Barba et al., 2015; Spier et al., 2004; Braverman et al., 1999; Ghoneim et al., 2001). This discrepancy is likely due to the differences in the biovars that infect these animals.
Infections in Other Species
C. pseudoTB is pathogenic to many other mammalian species, however the resulting disease depends on the host species and geographical location. In horses, this organism can cause several different disorders that likely vary due to secondary factors based on geographic distribution such as seasonality, environmental conditions, and arthropod vectors (Baird and Fontaine, 2007). These disorders include ulcerative lymphangitis, folliculitis and furunculosis (equine contagious acne, Canadian
4 horsepox, equine contagious pustular dermatitis), and deep-seated abscessation (pigeon fever,
Wyoming strangles, false strangles) (Baird and Fontaine, 2007). Less commonly, C. pseudoTB infection has also resulted in abortion and mastitis in mares (Poonacha & Donahue, 1995; Addo et al., 1974).
Cattle can also be infected by C. pseudoTB, although not as commonly. C. pseudoTB infections of dairy cattle have resulted in abscesses (both subcutaneous and within the lymph nodes associated with the respiratory tract), mastitis, and ulcerative dermatitis. These effects on dairy cattle are mostly confined to Israel, but some reports have come from other countries as well (Yeruham et al., 1997; Baird and Fontaine, 2007).
C. pseudoTB also infects various species of farmed camelids, such as llamas and alpacas, in North and South America. It causes lymphadenitis and subcutaneous abscesses similar to CL of sheep and goats (Anderson et al., 2004). In buffalo, C. pseudoTB infections cause oedematous skin disease which is characterized by swellings of the skin particularly on the limbs, abdomen, and dewlap (Selim, 2001).
Natural C. pseudoTB infections have also been reported in farmed pig and in non-domestic goat populations (Oliveira et al., 2014; Colom-Cadena et al., 2014; Varela-Castro et al., 2017). Experimental infections have been induced in various laboratory animals including mice, rabbits, and guinea pigs
(Jolly, 1965).
Zoonotic Potential
Caseous lymphadenitis is considered a zoonotic disease. Although it is not very common, humans can acquire a form of lymphadenitis when infected with C. pseudoTB. The first reported case of human lymphadenitis was in a 37-year-old man from Panama in 1966. This man did not have any pre- disposing factors that may have made him susceptible to the disease, such as pre-existing illness, immunosuppression, or direct animal contact (Lopez et al., 1966). Many of the human cases reported since then have been in generally healthy people that were occupationally exposed to sheep on a regular basis (Peel et al., 1997). A review of human lymphadenitis cases in Australia found that the site
5 of infected lymph nodes was generally associated with the patient’s occupation; axillary lymphadenitis is thought to have occurred due to exposure of the hands and arms, and was commonly observed in sheep shearers, abattoir workers, and butchers. In contrast, farm workers often displayed inguinal lymphadenitis, thought to be caused by environmental exposure of the feet and legs (Peel et al., 1997).
C. pseudoTB is also considered a public health threat due to its ability to produce the diphtheria toxin after lysogenization with a Corynebacterium diphtheria phage (Maximescu et al., 1974). Because of this, many countries require the identification of toxigenic C. pseudoTB to be reported to disease control agencies. Currently, there has only been one reported case of toxigenic C. pseudoTB isolated from a human, but no source of infection was listed (Wagner et al., 2010).
Corynebacterium pseudotuberculosis
The genus Corynebacterium is classified along with Mycobacterium, Nocardia, and Rhodococcus
(CMNR). Most members of the CMNR group are characterized by a layer of mycolic acids surrounding the peptidoglycan layer, which is somewhat functionally equivalent to the outer membrane of Gram- negative bacteria. C. pseudoTB is a non-spore forming, non-motile, facultative intracellular bacterium.
Stained smears of C. pseudoTB show Gram-positive coccibacilli or bacilli in palisade or “Chinese letter” arrangements (Baird and Fontaine, 2007). There is also a coccoid form of the bacteria, although it is less common (Chirino-Z´arraga et al., 2006).
C. pseudoTB is divided into two biovars: biovar ovis (biovar I) and biovar equi (biovar II). Biovar ovis is responsible for infections of small ruminants and does not reduce nitrate to nitrite, while biovar equi is responsible for infections of horses and cattle and is capable of reducing nitrate to nitrite (Songer et al., 1988). Nitrate reduction may not be the only criterium for this classification, as nitrate-negative strains of C. pseudoTB have also been isolated from horses and cattle and cross-infection between small ruminants and these animals has never been reported (Connor et al., 2000; Yeruham et al., 1997;
6
Oreiby, 2014). Genetic analyses have suggested that the two biovars are undergoing anagenesis in which biovar equi is evolving into biovar ovis (Oliveira et al., 2016).
Virulence Factors
One of the main virulence factors of C. pseudoTB is the exotoxin phospholipase D (PLD). PLD is a permeability factor that catalyzes the hydrolysis of ester bonds in sphingomyelins of mammalian cell membranes (Bernheimer et al., 1980; Pepin et al., 1994a). PLD is crucial to the virulence of C. pseudoTB and the progression of CL. C. pseudoTB isolates with experimentally-inactivated PLD are unable to cause typical CL abscesses (Hodgeson et al., 1999). PLD contributes to the pathogenesis of C. pseudoTB through several different mechanisms. The increased permeability of endothelial cells leads to leakage of plasma from the bloodstream into the lymphatic system, which could assist the bacteria in migrating throughout the host (Batey, 1986b). PLD also causes dermonecrosis and has cytotoxic effects on host leukocytes (Muckle and Gyles, 1982; Tashjian and Campbell, 1983). It may also interfere with ovine neutrophil chemotaxis during early stages of infection (Yozwiak and Songer, 1993).
Another major virulence factor of C. pseudoTB is the mycolic acid coat surrounding the cell wall.
Mycolic acids are long fatty acids commonly found in the cell walls of Actinomycetes. The mycolic acids of C. pseudoTB have been shown to have a lethal effect on caprine and murine macrophages and had negative effects on glycolytic activity, membrane integrity, and viability of these cells (Hard, 1975). In addition to its cytotoxic properties, the waxy mycolic acid coat is thought to provide the bacteria with mechanical and potentially biochemical protection, allowing it to survive for extended periods of time in the environment and within mammalian host cells (Baird and Fontaine, 2007).
Virulence factors of C. pseudoTB continue to be an important area of research in terms of control and prevention of CL. The C. pseudoTB genome contains seven pathogenicity islands which harbor several virulence factor genes including for fimbrial subunits, adhesion factors, iron uptake, and secreted toxins. These pathogenicity islands indicate lateral gene transfer which increases the
7 adaptability of this pathogen to different environments (Ruiz et al., 2011). Serial passage of a C. pseudoTB strain in a murine model increased the virulence of that strain, further indicating the adaptability of the pathogen to its host environment (Silva et al., 2017).
Pathogenesis
Following infection, C. pseudoTB migrates to the local drainage lymph node. At this stage (1-4 days post-infection), neutrophils are recruited to both the site of initial infection and the local drainage lymph node to which the bacteria have migrated. During the second phase of infection (5-10 days post- infection), pyogranulomas begin to develop following the recruitment of macrophages and lymphocytes.
The final stage is characterized by the maturation and stabilization of the pyogranulomas (Pepin et al.,
1997). These pyogranulomas consist of a necrotic center surrounded by a layer of macrophages, with an adjacent layer of lymphocytes; the entire nodular mass is encapsulated by thick fibrous tissue.
Heterogeneity in the types of macrophages composing these lesions has been observed, suggesting that the histological composition of pyogranulomas can differ based on the immune status of the host (Pepin et al., 1992). Additionally, CD4+ T cells are the predominant lymphocytes making up the immature lesions of the second phase of infection, but CD8+ T cells become more prominent in mature lesions.
This shift in cellular composition of the pyogranulomas may correspond to either the persistence or elimination of the infection by the host (Pepin et al., 1994b).
Once ingested, C. pseudoTB can survive for over 48 hours within host macrophages (Stefanska et al., 2010). The mycolic acid cell coat is thought to be the reason C. pseudoTB is able to avoid degradation by the hydrolytic enzymes of the phagolysosome. More specifically, some ovine macrophages are unable to produce nitric oxide when infected with C. pseudoTB, which could be due to the mycolic acids or other bacterial antigens that are able to attenuate nitric oxide production (Bogdan et al., 1997). There is some controversy as to whether or not C. pseudoTB can replicate within host macrophages. Some studies have found increases, while others found no significant increase in the number of C. pseudoTB
8 isolated from macrophages at different time points post infection (McKean et al., 2005; Stefanska et al.,
2010). The number of viable phagocytes decreases over time when infected with C. pseudoTB in vivo, indicating that the bacteria has cytotoxic effects on these cells. Although some closely related intracellular bacteria such as Mycobacterium tuberculosis have been shown to induce apoptosis of infected macrophages, studies suggest that macrophages infected with C. pseudoTB do not experience apoptosis or autophagy (Lee et al., 2006; Stefanska et al., 2010). Instead, several studies suggest a necrotic effect of C. pseudoTB on host macrophages indicated by degeneration of various membrane components (Tashjian and Campbell, 1983; Hard, 1972; Hard 1975).
Environmental Stability
One of the major difficulties in managing CL is the environmental stability of the pathogen.
When CL abscesses rupture, many viable bacteria (approximately 106 to 5x107 per gram) are released onto the adjacent skin and hair, as well as into the environment (Brown and Olander, 1987). The bacteria can then survive in the environment for extended periods of time. When mixed with fomites such as wood shavings, hay, and feces, C. pseudoTB has been able to survive on inanimate surfaces
(plastic, wood, or steel) for up to 55 days (Augustine and Renshaw, 1986). These inanimate surfaces, including shearing or handling equipment, are likely come into direct contact with, and therefore could infect, many animals over these prolonged periods. In cool and damp conditions, such as bedding or soil, the bacteria can survive for six months or longer (Batey, 1986b). Common chemical disinfectants such as calcium hypochlorite, formalin, and cresol are generally successful in killing C. pseudoTB on inanimate surfaces, provided that they are exposed long enough to eliminate any organic material left behind
(Ismail and Hamid, 1972).
9
Vaccines
Due to the difficulty of controlling CL once it is present on a farm, a great deal of effort has gone into finding an effective and efficient vaccine against the disease. Bacterin, toxoid, combined, and live CL vaccines have been developed and a few are commercially available in some countries. There is a commercially available vaccine for sheep and a conditionally licensed vaccine for goats 3 months of age or older in the U.S. (LeCuyer et al., 2016). The vaccine used for sheep in the U.S. contains a combination of C. pseudoTB bacterin and toxoid as well as clostridial toxoids (Caseous D-TTM; Colorado Serum Co.,
Denver, CO, USA). It has been shown to offer some protection to sheep that were experimentally infected with the disease and also to reduce the number of internal and external CL abscesses
(Piontkowski and Shivvers, 1998). A similar vaccine called GlanvacTM made up of formalin-killed C. pseudoTB and clostridial toxoids is available in Australia. This vaccine was reported to significantly reduce the number of lesions in experimentally-infected goats; however, one study claimed that the PLD toxoid offered greater protection when not combined with other clostridial components (Brown et al.,
1986; Anderson and Nairn, 1984). Most of the experimental and available vaccines for CL contain chemically or genetically inactivated PLD, as this is one of the main virulence factors of the pathogen.
However, PLD does not stimulate cell-mediated immunity and therefore may be best utilized as part of a combined vaccine (Hodgson et al., 1999). Numerous studies have aimed to identify additional C. pseudoTB virulence factors that may be used as novel vaccine targets (Santana-Jorge et al., 2016;
Seyffert et al., 2014; Silva et al., 2018; Silva et al., 2013).
A major issue with available CL vaccines is they interfere with accurate diagnostic testing.
Vaccinated animals will test positive for CL in serologic tests, which tend to be based on detection of antibodies to PLD or other major antigens. Therefore, it is recommended that animals only be vaccinated if CL is already be present on the farm. Available vaccines also require yearly boosters, which can be costly and impractical for some farmers.
10
Diagnostic Tests
Accurate diagnostic tests are crucial in preventing the introduction of CL into a previously uninfected flock. Characteristic CL abscesses, composed of light green, slightly dry material, are fairly indicative of the presence of the disease, although there are other pyogenic organisms that can result in abscess formation. In some cases, detection can be as simple as isolating and identifying the bacteria from superficial abscesses. However, bacteriological diagnosis can be difficult in older chronic abscesses due to a lack of viable bacteria (Baird and Fontaine, 2007). C. pseudoTB can also be overgrown by other common skin bacteria such as Staphylococci and Streptococci on culture media and in ruptured abscesses, resulting in a false negative diagnosis (Brown et al., 1987). Once isolated, the organism can be identified through biochemical profiling kits such as the Analytical Profile Index (API) Coryne kit
(bioMerieux, UK). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) is also a highly specific method of identifying Corynebacterium species (Konrad et al., 2010). Although the isolation and characterization of C. pseudoTB from abscesses is the most reliable method of CL diagnosis, accurate serologic diagnostic tests are very important due to the number of cases where no external abscesses are present. Animals may harbor the organism in the absence of active or visible abscesses, so serologic diagnosis, which documents exposure to C. pseudoTB, is considered a prudent management practice.
A commonly used diagnostic test for CL in sheep and goats is the Synergistic Hemolysin
Inhibition (SHI) assay, which is a serological assay that detects antibodies for the C. pseudoTB exotoxin.
This assay was originally developed for the detection of C. pseudoTB in horses but was found to also be successful in small ruminants (Knight, 1977; Brown et al., 1985). Enzyme-linked immunosorbent assays
(ELISAs) have also been widely used in the detection of subclinical cases of CL, utilizing various antigen preparations including immunoglobulin, recombinant exotoxin, cell supernatant, and cell wall antigens
(Binns et al., 2007; Menzies et al., 1994; Dercksen et al., 2000; Kaba et al., 2001; Maki et al., 1985).
Although they are currently the most commonly utilized diagnostic test for CL, ELISAs are still not
11 entirely accurate in detecting the disease. They have typically displayed higher sensitivity in goats compared to sheep due to differences in serologic responses (Dercksen et al., 2000; Hoelzle et al., 2013).
Additionally, ELISAs based solely on PLD have been found to be insufficient in detecting all infected animals, especially sheep, suggesting that utilizing a combination of immunodominant C. pseudoTB antigens may result in more accurate detection (Hoelzle et al., 2013). Other serological diagnostic methods that have been developed for CL include indirect hemagglutination, complement fixation, immunodiffusion, and PCR (Shigidi, 1979; Burrell, 1980; Pacheco et al., 2007).
One of the major issues with serologic diagnostic tests is their lack of specificity. They may be unable to distinguish between C. pseudoTB antigens versus those of closely related bacteria, or C. pseudoTB antibodies acquired due to natural exposure versus those due to vaccination or to absorption of maternal antibodies (Binns et al, 2007; Komala et al., 2008; Ellis et al., 1991). The sensitivity of these tests is also problematic as they can vary depending on the immune status of the animal (recent exposure may result in false negatives while the host’s immune system is responding to the pathogen), route and amount of bacterial exposure, and infection stage (Pepin et al., 1993; Binns et al., 2007;
Komala et al., 2008). Therefore, after establishing a biosecure environment so that no new infections are expected, repeated serologic tests (at least two negatives, two to four weeks apart initially) and throughout the lifetime of the animal are recommended to confirm CL-negative status.
Treatments and Control
In cases of CL that are detected via external abscesses, a common treatment method is to drain the abscesses and clean the area with iodine solution (de Sá Guimarães et al., 2011). This procedure has to be done very carefully so as to not contaminate the environment or surgical equipment with infective material that could be transferred to other animals. The issue with this form of treatment is it has to be able to kill all of the bacteria to prevent recurrence of the disease. This is often not possible especially in cases where internal lesions are present. Internal abscesses are inaccessible and infected animals may
12 be able to spread C. pseudoTB via respiratory secretions if pulmonary abscesses rupture. Therefore, the most effective way to eradicate CL from a farm is to cull the infected animals, an action that, itself, can incur large economic losses to the farm.
Although C. pseudoTB is susceptible to several antibiotics in vitro, the intracellular nature of the pathogen as well as the biofilm formation that occurs during natural infection significantly decreases the effect of antibiotic drugs in vivo (Muckle and Gyles, 1982; Olson et al., 2002). The thick encapsulation of the caseous lesions also makes it difficult for antibiotics to penetrate (Baird and Fontaine, 2007). In vivo, a combination of rifamycin and oxytetracycline has had some success in treating CL. However, organisms can develop resistance to rifamycin quite quickly, so this treatment should only be considered when absolutely necessary (Senturk and Temizel, 2006). Additionally, rifampin is not currently approved for use in food producing animals in the United State and Canada, as it may have carcinogenic properties
(Giguere et al., 2013). The low efficacy and lack of food safety, along with the high cost of these treatments make them impractical for controlling the disease.
The chronic nature of this disease, the ability of the organism to escape detection, the inability of vaccines to effectively prevent infection, and the lack of effective and available antibiotics to treat the disease are strong reasons to find alternative, readily available means for preventing and treating CL.
Essential Oils
Essential oils (EOs) are volatile bioactive compounds that are formed as secondary metabolites of various aromatic plants (Bakkali et al., 2008). In plants, these oils offer natural protection as antibacterials, antivirals, antifungals and insecticides (Bakkali et al., 2008). Their medicinal properties have been explored by humans for hundreds of years. Essential oils are complex mixtures, usually made up of 20-60 different components (Bakkali et al., 2008). The concentrations of these components vary within and between different types of EOs and determine their biological activity. This variation can be due to extraction method, climate, soil composition, plant organ, age, and vegetative cycle stage
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(Masotti et al., 2003; Angioni et al., 2006). Most EO components are monoterpenes or monoterpenoids and the rest are aromatic or aliphatic (Bakkali et al., 2008).
Many EO components are characterized by their hydrophobicity, lipophilicity, and high ratios of octanol to water which make them able to dissolve and permeate many common polymers (Nostro et al., 2013). These properties also allow EO components to penetrate bacterial cell walls and cytoplasmic membranes, altering their integrity and resulting in the leakage of important cellular material such as
ATP, ions, and nucleic acids (Helander et al., 1998; Lambert et al., 2001; Ultee et al., 1999). Altering the integrity of bacterial cell membranes is the proposed antibacterial mechanism of many EO components.
However, there is a great deal of variation in EO components in terms of their strength as antibacterial agents against different types of bacteria. Some additional properties of these components have been described, but this is still a current area of research.
Essential oils can also have cytotoxic effects on eukaryotic cells, generally targeting the inner cell membranes and organelles (Richter and Schlegel, 1993). As in bacteria, the EO components alter the permeability of eukaryotic inner cell membranes, resulting in leakage of important ions and proteins.
The increased permeability of mitochondrial membranes can cause cell death by necrosis or apoptosis
(Yoon et al., 2000; Armstrong, 2006).
Antibiotic resistant bacteria pose a major threat to public health worldwide. The number of multi-drug resistant (MDR) bacteria has increased with the frequent use of antibiotic drugs in both hospital and community settings (WHO, 2014). Following this trend of opposition to antibiotics, the FDA has recently begun enforcing stricter regulation of antibiotics in food-producing animals. The Veterinary
Feed Directive (VFD) enacted in 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 (FDA, 2015).
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In an attempt to combat growing numbers of antibiotic resistant bacteria, researchers have been seeking alternative forms of treatment for bacterial infections. Due to their known antimicrobial properties, essential oils and their components are an appealing natural alternative to synthesized antibiotic drugs.
Some EO components have shown success in inhibiting even MDR bacteria such as
Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB). Drug resistance is becoming increasingly problematic to the treatment of TB. In 2017, 30% of reported TB cases tested positive for resistance to rifampin, and approximately 180,000 cases were classified as multidrug- resistant or extensively drug-resistant (WHO, 2018). One study evaluated the minimum inhibitory concentrations (MIC) of 25 essential oil constituents against M. tuberculosis. The ten EO components with the lowest MICs were cinnamic acid, p-anisaldehyde, β-pinene, carvacrol, cinnamaldehyde, β- citronellol, cuminaldehyde, α-terpinene, terpinolene, and thymol (Andrade-Ochoa et al., 2015). The antitubercular activities of EOs containing β-pinene, cuminaldehyde, and cinnamaldehyde have also been shown in other studies (Elhassan et al., 2016; Andrade-Ochoa et al., 2013). Some terpenes have also been shown to enhance the activity of traditional tuberculostatic drugs possibly due to their ability to interfere with the Mycobacterium efflux pumps (Sieniawska et al., 2017). Due to the similarities between C. pseudoTB and M. tuberculosis, similar effects of EO components might be expected.
β-pinene
β-pinene is a monoterpenoid associated with several EOs including pine (Pinus), lavender
(Lavandula), rosemary (Rosmarinus), and turpentine (Syncarpia). EOs with high concentrations of β- pinene have been shown to inhibit the growth of both Gram-positive and Gram-negative bacteria (Soni et al., 2016). β-pinene has a synergistic effect when combined with certain antibiotic drugs and was able to lower these drugs’ minimum inhibitory concentration (MIC) against Methicillin-resistant
Staphylococcus aureus (MRSA) (da Silva et al., 2012). β-pinene can also significantly reduce biofilm
15 formation of Candida albicans; biofilm formation is a factor that enhances pathogen growth (da Silva et al., 2012). β-pinene can, however, cause extreme damage to the lungs and respiratory system if inhaled or swallowed and significant irritation when in contact with skin. β-pinene exhibited cytotoxic effects on murine macrophages at concentrations higher than 0.125 mg/mL (da Silva et al., 2012).
Cuminaldehyde
Cuminaldehyde is a monoterpenoid found in certain types of cumin (Cuminum), cinnamon
(Cinnamomum), and caraway (Carum) essential oils. Cuminaldehyde and cumin essential oil have antibacterial effects on several pathogenic Gram-positive and Gram-negative bacteria, including some
MDR strains (Sheikh et al., 2010; Belal et al., 2017; Naveed et al., 2013). Cuminaldehyde has also demonstrated antifungal, anticancer, antidiabetic, and antiinflammatory properties (Ebada, 2017). This
EO component induces apoptosis in several different cancer cell lines, however it was nontoxic to PC12 cells (Ebada, 2017; Morshedi et al., 2015). Cuminaldehyde can cause acute toxicity if ingested at full strength and may cause minor skin irritation (NCBI, 2019).
Carvacrol
Carvacrol is a phenolic monoterpenoid found in various essential oils including thyme (Thymus), oregano (Origanum), wild bergamot (Citrus), pepperwort (Lepidium), and more. Carvacrol has demonstrated antibacterial activity against several Gram-negative and Gram-positive strains of bacteria, although it may have stronger antibacterial effects on Gram-positive bacteria (Ait-Ouazzou et al. 2013).
It has also demonstrated antifungal, antioxidant, and anticancer activities (Sharifi-Rad et al., 2018). The antimicrobial activity of carvacrol has been proposed to occur through several different mechanisms.
The hydrophobicity of this compound as well as the presence of a free phenolic hydroxyl group appear to be essential components for carvacrol’s ability to inhibit bacterial growth (Nostro & Papalia, 2012;
Ben Arfa et al., 2006). Like other terpenes, its hydrophobicity is likely responsible for permeation and
16 depolarization of the bacterial membrane (Xu et al., 2008). It may also deplete bacterial ATP by reducing
ATP synthesis or increasing ATP hydrolysis (Nostro and Papalia, 2012; Ultee et al., 1999). Carvacrol has also exhibited the ability to inhibit bacterial cell growth in biofilms, specifically in the initial phase of biofilm formation. Carvacrol can interfere with the initial adherence of Pseudomonas aeruginosa, a
Gram-negative bacterium with a high propensity for developing biofilms. It is suspected that this ability is due to the hydrophilicity of the compound, allowing it to diffuse through the polar polysaccharide matrix that make up these structures (Soumya et al., 2011). In eukaryotic cells, carvacrol may alter the integrity of inner cell membranes and organelles (Bakkali et al., 2008). The toxicity of carvacrol has been tested on several cell lines. In the intestinal cell line Caco-2, carvacrol exhibited genotoxic effects on purine bases at a concentration of 460 µM (69.10 µg/ml) (Llana‐Ruiz‐Cabello et al., 2014a). Carvacrol also reduced Caco-2 cellular viability at concentrations of 500 µM (75.11 µg/ml) or higher and caused morphological changes related to apoptosis at concentrations of 230 µM (34.55 µg/ml) and higher
(Llana-Ruiz-Cabello et al., 2014b). In rats, the median lethal dose of carvacrol was 810 mg/kg when administered orally, but ten-fold lower (80 mg/ml) when administered intravenously, indicating that the method of delivery was crucial to toxicity (Suntres et al., 2015). However, no mutagenic or genotoxic effects were detected in Chinese hamster lung fibroblasts, human hepatocytes, or human lymphocytes in vitro (Llana‐Ruiz‐Cabello et al., 2014a; Maisanaba et al., 2015).
Thymol
Thymol is a phenolic monoterpenoid found primarily in thyme (Thymus), basil (Ocimum), oregano (Origanum), and bergamot (Citrus) EOs. It is an isomer of carvacrol and therefore exhibits similar antibacterial mechanisms and can inhibit many of the same bacteria. Thymol and carvacrol have been used in conjunction as animal feed supplements in several studies. As dietary supplements, these
EO components have been reported to decrease intestinal inflammation and oxidative stress in weaning piglets and to increase growth and immune responses in broiler chickens (Wei et al., 2017; Hashemipour
17 et al., 2013). Thymol does not appear to have genotoxic effects on mammalian cells (Maisanaba et al.,
2015; Llana-Ruiz-Cabello et al., 2014a). In Caco-2 cells, thymol had no effect on cellular viability at concentrations up to 250 µM (37.56 µg/ml), but induced morphologic changes related to apoptosis at concentrations of 125 µM (18.78 µg/ml) and higher (Llana-Ruiz-Cabello et al., 2014b).
Trans-cinnamaldehyde
Trans-cinnamaldehyde is an aromatic phenylpropanoid and is the main constituent of cinnamon tree bark. It can also be found in other Cinnamomum species such as camphor and cassia. Cinnamomum
EOs have long been used in alternative medicine practices and have recently been found to have antidiabetic properties (Hassan et al., 2012). Due to its hydrophobicity, trans-cinnamaldehyde can inhibit bacterial growth by altering the fluidity of bacterial cell membranes (Trinh et al., 2015). Other antibacterial mechanisms of this EO component include inhibition of bacterial cell division by binding the FtsZ protein, ATPase inhibition, and disruption of carbohydrate, amino acid, and lipid metabolism
(Domadia et al., 2007; Amalaradjou and Venkitanarayanan, 2011a). Cinnamaldehyde has been shown to inhibit E. coli biofilms, potentially by interfering with the swimming motility of the bacteria (Niu and
Gilbert, 2004). It can also inhibit biofilm formation by downregulating genes associated with quorum sensing (Amalaradjou and Venkitanarayanan, 2011b). A study conducted on cinnamaldehyde’s ability to inhibit Helicobacter pylori found that this pathogen did not develop resistance to the EO component even after ten passages grown at sub-inhibitory concentrations (Ali et al., 2005). Exposure to trans- cinnamaldehyde can cause irritation of the skin, eyes, upper respiratory tract, and mucous membranes and can be toxic in large doses (NCBI, 2019).
Para-anisaldehyde
Para-anisaldehyde is an aromatic benzaldehyde that can be found in American cranberry
(Viburnum), anise (Pimpinella), fennel (Foeniculum), and vanilla (Vanilla) EOs. Staphylococcus aureus
18 cells exposed to p-anisaldehyde showed loss of membrane integrity and cytoplasmic leakage (Shi et al.,
2017). This EO component also has a synergistic effect when combined with the antibiotic, nisin, to inhibit the growth of S. aureus and Listeria monocytogenes (Shi et al., 2017; Chen et al., 2016). In addition to its antibacterial properties, p-anisaldehyde also has strong fungicidal and acaricidal properties (Shreaz et al., 2011; Lee, 2004). Cytotoxic effects of p-anisaldehyde have also been reported in vitro. P-anisaldehyde has been shown to decrease pyruvate/malate-mediated respiration of mitochondria from mouse liver cells but has low cytotoxicity on H9c2 rat cardiac myoblasts (Wolf et al.,
1982; Shreaz et al., 2011). Additionally, fennel oil containing 7.7% p-anisaldehyde has low cytotoxicity against HeLa, Caco-2, MCF-7, CCRF-CEM, and CEM/ADR5000 cells (Sharopov et al., 2017). The dermal toxicity of p-anisaldehyde is fairly low, but the acute inhalation toxicity is high (NCBI, 2019).
Alpha-terpinene
Alpha-terpinene is a monoterpene that can be found in essential oils from the genera Citrus,
Eucalyptus, and Juniperus, as well as cardamom (Elettaria), marjoram (Origanum marjorana), and tea tree (Melaleuca alternifolia) EOs. Few studies have analyzed the antibacterial activity of α-terpinene alone, however several studies have focused on the uses of tea tree oil. This EO has shown to have antibacterial activity against several species of bacteria as well as antifungal, antiviral, and other medicinal properties (Carson et al., 2006). α-terpinene has in vitro trypanocidal activity against
Trypanosoma evansi and in vivo it increased the longevity of mice infected with this parasite (Baldissera et al., 2016). It has antioxidant properties but easily forms skin allergens via autoxidation upon air exposure (Rudback et al., 2012). This EO component also has an indirect role in small ruminant agriculture; the normal diet of dairy goats of Mediterranean rangelands contains several kinds of terpenes, one of which is α-terpinene. These terpenes are degraded by rumen bacteria at different rates depending on their structure (Malecky et al., 2012). Inhalation or ingestion of concentrated α-terpinene is extremely harmful and potentially fatal (NCBI, 2019).
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β-citronellol
β-citronellol is a monoterpenoid found in rose (Rosa) and Pelargonium essential oils. It currently has several pharmacological uses as an anticonvulsant, antihyperalgesic, an orofacial antinociceptive
(Santos et al., 2019). It is used commercially as a biochemical pesticide due to its low toxicity and ability to attract mites (Ju-Hyun et al., 2008). Citronellol has a polar hydroxy group which interferes with cell membrane fluidity and protein conformation, giving it stronger antimicrobial properties (Mulyaningsih et al., 2011). It has been shown to inhibit several Gram-positive and Gram-negative species of bacteria including MRSA (Kotan et al., 2007; Mulyaningsih et al., 2011). Citronellol was found to reduce in vitro swarming behavior and to inhibit haemolysin activity, thus potentially reducing the virulence of Proteus mirabilis (Echeverrigaray et al., 2008). Citronellol also inhibited biofilm growth in several bacterial species including S. aureus, E. coli, and L. monocytogenes (Millezi et al., 2012; de Oliveira et al., 2010).
Low doses of citronellol cause minor skin irritation in both humans and animals. High doses administered to the skin or in food resulted in death of some laboratory animals (USEPA, 2009).
Terpinolene
Terpinolene is a monoterpene found in EOs from several genera of plants including Citrus,
Mentha, Juniperus, and Myristica. Few studies have been conducted on the antibacterial properties of terpinolene alone. This EO component is known for its antioxidant properties and may inhibit certain genes involved in cancer progression (Turkez et al., 2015; Okumura et al., 2012). Terpinolene is non- cytotoxic to human lymphocytes but has a sedative effect after being inhaled by mice (Turkez et al.,
2015; Ito and Ito, 2013).
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CHAPTER 2
SUSCEPTIBILITY OF C. PSEUDOTUBERCULOSIS TO ESSENTIAL OIL COMPONENTS
Introduction
Corynebacterium pseudotuberculosis is a member of the CMNR group of bacteria which includes
Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus. C. pseudoTB is the etiologic agent of caseous lymphadenitis (CL), a highly contagious and chronic infection that affects small ruminants characterized by “caseous” abscesses of the external or internal lymph nodes (Valli and Parry, 1993). C. pseudoTB is highly stable both in the host and in the surrounding environment. The thick-walled abscesses make antibiotic treatment of CL very difficult because the bacteria are protected by layers of fibrotic tissue (Baird and Fontaine, 2007). When these abscesses rupture, the bacteria are released into the environment where they may get the chance to infect a new host. In cool and damp conditions, such as bedding or soil, C. pseudoTB can survive for over six months (Batey, 1986b). Due to the difficulty of treating CL and eradicating the bacteria from a farm, an alternative method of treatment is needed to control the disease.
Essential oils are complex bioactive chemicals that are derived from various species of aromatic plants. Essential oils and their components have demonstrated antimicrobial properties on a wide range of bacteria, including some that are resistant to typical antibiotic drugs (Bakkali et al., 2008). Several essential oil components (EOCs) have been reported to inhibit the in vitro growth of Mycobacterium tuberculosis (Andrade-Ochoa et al., 2015). Due to the similarities between C. pseudoTB and M. tuberculosis, similar effects of EO components might be expected. The purpose of this study was to determine the minimum inhibitory concentrations (MICs) of nine EOCs against C. pseudotuberculosis in vitro (Table 2.1). The EOCs that were chosen (β-citronellol, β-pinene, carvacrol, cuminaldehyde, p- anisaldehyde, thymol, trans-cinnamaldehyde, α-terpinene, and terpinolene) have all previously been reported to inhibit M. tuberculosis growth (Andrade-Ochoa et al., 2015).
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Table 2.1 Chemical and physical properties of essential oil components (EOCs)
EOC Formula Physical Chemical Structure (TCI America State product code)
Thymol (M0410) C10H14O Solid
Carvacrol C10H14O Liquid (C0026)
Trans- C9H8O Liquid cinnamalehyde (C0352) p-anisaldehyde C8H8O2 Liquid (A0480)
β-pinene C10H16 Liquid (P0441)
α-terpinene C10H16 Liquid (M0317)
Terpinolene C10H16 Liquid (T0817)
β-citronellol C10H20O Liquid (C0370)
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Cuminaldehyde C10H12O Liquid (I0168)
Materials and Methods
Preparation of C. pseudotuberculosis
Six cryopreserved Corynebacterium isolates (Table A.1) which had been previously identified as
C. pseudotuberculosis based on phenotypic assessment (BiologTM) were acquired from the University of
Maine Veterinary Diagnostic Lab; identification was verified using API® Coryne (bioMérieux). For each cryopreserved C. pseudoTB culture, a cryobead was transferred to a microfuge tube containing 1 ml brain heart infusion (BHI) broth (BBLTM) using sterile technique. The tubes were vortexed briefly and incubated at 37° C for 24 hours. The broth cultures were streaked for isolation with 10 µl loops onto blood agar (BA) plates (Northeast Laboratories) and incubated at 37° C in 5% CO2 for 48 hours. A colony from each plate was picked and added to a microfuge tube containing 300 µl sterile DI water, and vortexed to mix an inoculum. A new BA plate was flooded with each inoculum, then incubated at 37° C for 48 hours. API® Suspension Medium was then inoculated with each C. pseudoTB culture. C. pseudoTB suspensions and reagents were then added to the API® Coryne test strip according to API® procedure
(bioMérieux).
Culturing C. pseudotuberculosis for Disk Diffusion Assays
A cryobead was transferred from a cryopreserved tube of C. pseudoTB (ATCC® 19410) to a microfuge tube containing 1 ml BHI broth using sterile forceps. The microfuge tube was vortexed briefly and incubated at 37° C. After 24-hours incubation, the C. pseudoTB culture was streaked on a BA plate for isolation. The plate was incubated at 37° C in 5% CO2 for 48 hours, then a representative colony was picked and Gram stained for assessment of morphologic identification to detect possible contamination.
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The plate was then stored at 4° C. Five hundred µl of the 24-hour C. pseudoTB culture was transferred to each of two 50 ml centrifuge tubes containing 30 ml of sterile BHI broth; a third tube was left blank as a control. These tubes were incubated at 37° C in air, while shaking at 130 cycles/minute. After 48 hours of incubation, 10 µl from each tube was picked with a sterile loop and Gram stained for assessment of morphologic identification to detect possible contamination. The inocula were then measured for percent transmittance (T) with a spectrophotometer. Tubes were vigorously vortexed in between measurements as C. pseudoTB forms clumps in broth culture. One of the C. pseudoTB tubes was chosen to inoculate the BA or BHI plates for the disk diffusion assays, with preference given to the tube with transmittance closest to 80%. The chosen inoculum was diluted to 80% T with sterile BHI broth. To determine how many colony forming units (CFUs) were being added to the plates, a count was performed on a C. pseudoTB culture in BHI broth at 80% T. This culture was serially diluted 10-fold to
-4 10 . One hundred µl of this dilution was spread on three BHI plates and incubated at 37° C in 5% CO2 for
48 hours. Colony forming units were counted in triplicate on each plate. The averages of the counts were used to calculated CFU/ml at 80% T which was found to be approximately 5.63 x 106 CFU/ml BHI broth.
Disk Diffusion Assay Trial 1
Essential oil components (EOCs) were obtained from TCI America. The solubilities of EOCs in ethanol, Tween-80, and DI water were evaluated to determine optimal concentrations needed
(Appendix B). Cuminaldehyde was mixed 1:1 (v/w) with Tween 80, then diluted in sterile DI water to create final cuminaldehyde concentrations of 200, 100, and 10 mg/ml. Carvacrol, β-pinene, α-terpinene, terpinolene, and β-citronellol were each mixed 1:1 (v/w) with Tween 80, then diluted in sterile DI water to final EOC concentrations of 100, 10, and 1 mg/ml solution. Trans-cinnamaldehyde and p-anisaldehyde were mixed 1:1 with ethanol (v/v), then mixed 1:1 with Tween 80 (v/w), then diluted in sterile DI water to final EOC concentrations of 100, 10, and 1 mg/ml solution. Thymol was mixed 1:1 with ethanol (w/v).
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This solution was mixed 1:1 with Tween-80 (v/w) and diluted in sterile DI water to final thymol concentrations of 100, 10, and 1 mg/ml. BHI agar plates (DibcoTM) were inoculated with 100 µl C. pseudoTB culture at approximately 5.63 x 106 CFU/ml BHI broth. A control plate was inoculated with 100
µl BHI broth. Additional controls were gentamicin (40 mg/ml DI water), ethanol (100 µl/ml DI water),
Tween 80 (200 mg/ml DI water) and DI water. One 6 mm paper disk (BBLTM) was placed in the center of each BHI plate with sterile forceps. Twenty-seven µl of the EOC dilutions or controls (gentamicin, ethanol, Tween 80, DI water) were pipetted onto the disks. The plates were incubated at 37° C in 5% CO2 and the zones of inhibition (diameter of the zone with no bacterial growth including the disk) were measured at 24-, 48-, and 72-hours incubation.
Disk Diffusion Assay Trial 2
Based on the results of Trial 1, β-citronellol, carvacrol, cuminaldehyde, thymol, and trans- cinnamaldehyde were mixed in 1:1:2 (v/v/w) solutions with ethanol and Tween 80. The β-citronellol, thymol, and trans-cinnamaldehyde solutions were then diluted in sterile DI water to final EOC concentrations of 100, 50, 10, and 1 mg/ml. The carvacrol and cuminaldehyde solutions were diluted in sterile DI water to final EOC concentrations of 100, 70, 40, and 10 mg/ml. BHI agar plates were inoculated with 100 µl C. pseudoTB culture at approximately 5.63 x 106 CFU/ml BHI broth. A control plate was inoculated with 100 µl BHI broth. One 6 mm paper disk was placed in the center of each BHI plate with sterile forceps. Twenty-seven µl of the EOC dilutions or controls (gentamicin, ethanol, Tween
80, DI water) were pipetted onto the disks in triplicate. The plates were incubated at 37° C in 5% CO2 and the zones of inhibition (diameter of the zone with no bacterial growth including the disk) were measured at 24-, 48-, and 72-hours incubation.
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Results
Preparation of C. pseudotuberculosis
All six API test strips had identical results and were identified as C. pseudotuberculosis when compared to the API database (Figure 2.1).
Figure 2.1 Developed API® Coryne test strip inoculated with ATCC strain of C. pseudoTB
Disk Diffusion Assay Trial 1
β-pinene, α-terpinene, and terpinolene had minimal (< 10 mm) or no (6 mm) zones of inhibition at any of the concentrations tested (Table 2.2). 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 (Table 2.2). β-citronellol and trans-cinnamaldehyde had substantial zones of inhibition (≥ 10 mm) at final concentrations of 10 mg/ml and greater at all time points (Table 2.2). Zones of inhibition on EOC- treated plates generally decreased in size over time (Table 2.2). At the highest concentration tested, β- citronellol, carvacrol, thymol, and trans-cinnamaldehyde had the largest zones of inhibition (Figure 2.1).
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.
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Table 2.2 Zones of C. pseudoTB inhibition by essential oil components (EOCs) after 24-, 48-, and 72-hours (hr) incubation.
EO Component Concentration Zone of inhibition (mm) (µl/ml) 24 hr 48 hr 72 hr Cuminaldehyde 200 42 13 11 100 18 11 10 10 8 6 6 Carvacrol 100 35 23 13 10 6 6 6 1 8 6 6 β-pinene 100 10 6 6 10 6 6 6 1 9 6 6 α-terpinene 100 11 6 6 10 9 6 6 1 6 6 6 Terpinolene 100 6 6 6 10 6 6 6 1 6 6 6 β-citronellol 100 26 21 16 10 12 10 10 1 6 6 6 Thymol 100 38 32 32 10 10 9 9 1 9 8 8 Trans- 100 62 62 49 cinnamaldehyde 10 26 11 11 1 6 6 6 p-anisaldehyde 100 15 10 10 10 10 6 6 1 6 6 6
27
70 60 50 40 30 20
10 Zoneof inhibition (mm) 0 24 48 72 Time (hours)
Cuminaldehyde Carvacrol β-pinene α-terpinene Terpinolene β-citronellol Thymol Trans-cinnamaldehyde p-anisaldehyde
Figure 2.2 Zones of C. pseudoTB inhibition at final essential oil component concentrations of 100 mg/ml.
Disk Diffusion Assay Trial 2
β-citronellol had substantial zones of inhibition (≥ 10 mm) at final concentrations of 10 mg/ml or greater at all time points (Figure 2.3). It has no zones of inhibition (6 mm) at a final concentration of 1 mg/ml (Figure 2.3). Carvacrol had substantial zones of inhibition (≥ 10 mm) at final concentrations of 10 mg/ml or higher at all time points (Figure 2.4). Cuminaldehyde had substantial zones of inhibition (≥ 10 mm) at final concentrations of 100 mg/ml and 40 mg/ml but not at 70 mg/ml or 10 mg/ml (Figure 2.5).
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 (Figure 2.6). It had minimal (< 10 mm) or no (6 mm) zones of inhibition at a final concentration of 1 mg/ml at all time points and 10 mg/ml at 72-hours incubation (Figure 2.5). Trans-cinnamaldehyde had substantial zones of inhibition (≥ 10 mm) at final concentrations of 10 mg/ml or greater at all time points (Figure 2.7). It had minimal (> 10 mm) or no (6 mm) zones of inhibition at a final concentration of 1 mg/ml (Figure 2.7). Zones of inhibition on EOC- treated plates generally decreased in size over time (Figure 2.8). 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.
28
Figure 2.3 Zones of C. pesudoTB inhibition by β-citronellol after 24-, 48-, and 72-hours (hr) incubation. Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
Figure 2.4 Zones of C. pesudoTB inhibition by carvacrol after 24-, 48-, and 72-hours (hr) incubation. Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
29
Figure 2.5 Zones of C. pesudoTB inhibition by cuminaldehyde after 24-, 48-, and 72-hours (hr) incubation. Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
Figure 2.6 Zones of C. pesudoTB inhibition by thymol after 24-, 48-, and 72-hours (hr) incubation. Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
30
Figure 2.7 Zones of C. pesudoTB inhibition by trans-cinnamaldehyde after 24-, 48-, and 72-hours (hr) incubation. Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
Figure 2.8 Zones of C. pseudoTB inhibition at final essential oil component concentrations of 10 mg/ml. Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
31
Discussion
β-citronellol, carvacrol, thymol, and trans-cinnamaldehyde were best able to inhibit C. pseudoTB growth in vitro of the nine EOCs tested. Based on the disk diffusion assay, MICs of β-citronellol, carvacrol, thymol, and trans-cinnamaldehyde are each 10 mg/ml. Based on the size of the zones of inhibition at this concentration it appears that trans-cinnamaldehyde has the strongest inhibitory effect on C. pseudoTB growth, followed by thymol, carvacrol, and β-citronellol. Although cuminaldehyde was able to inhibit C. pseudoTB growth at a final concentration of 40 mg/ml, it did not have zones of inhibition at a final concentration of 70 mg/ml. Cuminaldehyde would have to be retested at these concentrations to determine the MIC.
The EOCs used in these disk diffusion assays were chosen based on their effective inhibition of
M. tuberculosis in a previous study (Andrade-Ochoa et al., 2015). However, the MICs from that study were much lower than those found against C. pseudoTB. Thymol, carvacrol, cinnamaldehyde, β- citronellol, and cuminaldehyde were reported to have MICs against M. tuberculosis at final concentrations of 0.78, 2.02, 3.12, 6.25, and 10.41 µg/ml, respectively (Andrade-Ochoa et al., 2015). The antibiotic controls Isoniazid and Rifampicin had fairly comparable antitubercular effects to thymol, with
MICs of 0.26 µg/ml and 0.60 µg/ml, respectively (Andrade-Ochoa et al., 2015). Additionally, the EOCs that were unable to inhibit the growth of C. pseudoTB (p-anisaldehyde, β-pinene, terpinolene, and α- terpinene) at concentrations of 100 mg/ml or lower were reported to inhibit the growth of M. tuberculosis at relatively low final concentrations of 4.16, 10.41, 8.33, and 10.41 µg/ml, respectively
(Andrade-Ochoa et al., 2015). These MICs against M. tuberculosis were determined using an Alamar Blue bactericidal assay in which a color change in the media indicated the presence of live bacteria by measuring their cellular respiration. The viability of M. tuberculosis was measured after 7 days of incubation with the EOCs in 96-well plates (Andrade-Ochoa et al., 2015). However, there appears to be some variability in the Alamar Blue bactericidal assays. Another study evaluated the antitubercular
32 activity of carvacrol and thymol using the same assay and the same strain of M. tuberculosis and reported MICs of 41.6 µM (6.25 µg/ml) and 332.85 µM (50.00 µg/ml), respectively (Alokam et al., 2014).
This study also reported that the antibiotic controls Isoniazid and Rifampicin were relatively much more effective at inhibiting M. tuberculosis in this assay, with MICs of 0.66 µM (0.09 µg/ml) and 0.23 µM (0.19
µg/ml), respectively (Alokam et al., 2014). The antitubercular activities of these EOCs is attributed to their lipophilic nature, allowing them to easily interact with and permeate the lipophilic mycobacterial cell wall (Andrade-Ochoa et al., 2015). The cell wall of C. pseudoTB is also lipophilic due to the presence of mycolic acids and would therefore likely be affected similarly by EOCs. In this study, the MICs against
C. pseudoTB, versus M. tuberculosis, may appear to be higher due to differences in bactericidal assay techniques utilized. In liquid media, EOCs may have more contact with the bacteria, allowing them to inhibit their growth more effectively (Van de Vel, et al., 2017).
Disk diffusion assays have been used to evaluate the MICs of EOCs against other strains of bacteria. In a disk diffusion assay, cinnamaldehyde was reported to have MICs of 2 µg/ml and 1 µg/ml against H. pylori and E. coli, respectively (Ali et al., 2005). These relatively low MICs could be due to the differences in bacterial species being tested (Van de Vel et al., 2017). However, another difference in this study was that dimethyl sulfoxide (DMSO) was used as an emulsifying agent to dilute the EOCs that were being tested rather than Tween-80 (Ali et al., 2005). The type of emulsifier or solvent used to dilute EOCs has been shown to have an effect on the antimicrobial activity of those EOCs (Van de Vel et al., 2017). One study reported that EOCs emulsified with different types of gums (fenugreek gum, xanthan gum, Arabic gum and yellow mustard gum) were more effective at inhibiting Salmonella typhimurium compared to when they were emulsified with Tween-80 (Si et al., 2006). Thymol was reported to be less effective at inhibiting S. typhimurium growth in vitro when it was mixed with higher concentrations (0.100 µl/l) of Tween-80 as opposed to lower concentrations (0.0125 µl/l) (Juven et al.,
1994). Several other studies have found that using Tween-80 as an emulsifying agent can decrease the
33 antimicrobial effects of EOCs (Ma et al., 2016; Nielsen et al., 2016). These effects may be due to surfactant reduction the diameter of nutrient particles, leading to higher nutrient availability, or acting as a nutrient source itself (Nielsen et al., 2016). Therefore, it is possible that the use of Tween-80 as an emulsifying agent in our disk diffusion assays increased the MICs of the EOCs that were tested. However, the effects of Tween-80 on the antimicrobial function of EOCs may be more substantial in broth dilution assays as opposed to disk diffusion assays. The antifungal activity of several EOCs, including carvacrol and cinnamaldehyde, was reported to decrease with the addition of 0.5% Tween-80 when using a broth dilution method, but remained unchanged when used in an agar dilution method (Inouye et al., 2001).
In this study, the Tween control plate shown minimal or no zones of inhibition, indicating that it did not have an effect on the MICs. However, the effects that Tween-80 may have on the activity of EOCs should be considered in their future applications.
A notable observation from these disk diffusion assays was the decrease in size of the zones of inhibition over time. Short-term availability for bioactivity is a common problem with essential oils and their components due to their volatile nature (Wattanasatcha et al., 2012). Many EOCs can be chemically unstable under certain conditions, often related to heat, light, or oxygen exposure (Turek and
Stintzing, 2012). These factors can cause chemical reactions that may alter the structure of the EOCs, resulting in a change of function or bioactivity (Turek and Stintzing, 2012). To address this issue, several studies have attempted to increase the sustainability of essential oils through a variety of encapsulation methods. One study found that thymol nanoparticles encapsulated with sodium caseinate and chitosan hydrochloride were more effective at inhibiting the growth of Gram-positive bacterium than non- encapsulated thymol (Zhang et al., 2014). This is supported in another study which found encapsulated and non-encapsulated thymol nanoparticles had comparable MICs and MBCs against S. aureus, E. coli, and P. aeruginosa (Wattanasatcha et al., 2012). Porous starch microspheres have also been tested as an encapsulation method of essential oils and their components, with the end product being a free-flowing
34 powder of microspheres approximately the size of pollen grains (Glenn et al., 2010). Although encapsulation of the EOCs that inhibit C. pseudoTB may increase the longevity of their bioactive properties, it would increase the cost, potentially making them an impractical option for some farmers.
Another factor of the decreased EOC bioactivity to consider is the surface on which the component is inhibiting bacterial growth. On BHI media, C. pseudoTB likely has a better chance of overcoming antibacterial effects of the EOCs than on surfaces that are not nutrient-rich. One possible utilization of these EOCs in the control of CL is as a disinfectant of commonly contaminated farm surfaces such as feeders, fencing, and shearing equipment. Due to their hydrophobicity and high ratios of octanol to water, EOCs are able to dissolve and permeate many common polymers, which may help them penetrate the wooden surfaces commonly found on farms (Nostro et al., 2013). Although C. pseudoTB is able to survive on these surfaces for extended periods of time, the bacteria may be more susceptible to bactericidal effects of EOCs on these surfaces when nutrients are not readily available
(Augustine and Renshaw, 1986). The bactericidal effects of EOCs are also dependent on pH. At low pH levels, some EOCs are more hydrophobic, allowing them to more easily dissolve into bacterial membranes and therefore increasing antibacterial activity (Juven et al., 1994). This effect has been demonstrated with carvacrol, thymol, and cinnamaldehyde, which were reported to have higher MICs against Brachyspira hyodysenteriae when grown in media at a pH of 7.5 compared to a pH of 6.0 (Vande
Maele et al., 2015). The pH of the BHI agar used in this experiment was approximately 7.4 (BDTM). The pH of wood is typically between 4.0 and 6.0, at which the EOCs may be more effective at inhibiting C. pseudoTB growth (Perma-Chink®, 2014). To validate this hypothesis, further research could be done to evaluate the MICs of these components against C. pseudoTB on different surfaces.
The C. pseudoTB bacteria used in this experiment were exposed to EOCs during the exponential phase of growth. EOCs can have different antimicrobial effects on bacteria depending on the growth phase the bacteria are in when EOC exposure occurs. One study reported that L. monocytogenes was
35 resistant to the effects of the EOC, S-carvone, when in the stationary phase of its growth (Karatzas et al.,
2000). The authors hypothesized that this change in susceptibility was likely associated with the physiological changes the bacteria experiences when entering the stationary phase, such as decreased membrane fluidity and expression of stress-related proteins (Karatzas et al., 2000). Additionally, EOCs themselves can have effects on the growth phase of certain bacteria. Some EOCs, including carvacrol, cinnamaldehyde, and thymol, were reported to prolong the lag phase and reduce the exponential growth rate and final densities of Bacillus cereus growth in carrot broth (Valero and Giner, 2006). For these reasons, it is important to consider what phase C. pseudoTB may be in when utilizing these EOCs in vivo. If the bacteria have reached the stationary phase of their growth, either in a CL abscess or on a farm surface, these EOCs may not be as effective as antimicrobial agents.
Conclusions
β-citronellol, carvacrol, thymol, and trans-cinnamaldehyde were able to inhibit C. pseudoTB growth in vitro at final concentrations of 10 mg/ml or greater. However, the MICs of EOCs 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. pseudoTB in small ruminants. The major limitations of the application of these EOCs 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.
36
CHAPTER 3
SUSCEPTIBILITY OF BUFFALO RAT LIVER CELLS TO ESSENTIAL OIL COMPONENTS
Introduction
Essential oils are complex bioactive compounds derived from various aromatic plants. Many of the components that make up these oils have strong antimicrobial properties against a wide range of bacteria. For this reason, essential oil components (EOCs) are often used and studied as an alternative form of antimicrobials (Bakkali et al., 2008). However, in addition to their antimicrobial properties, some
EOCs can also have cytotoxic effects on mammalian cells, specifically targeting inner membranes and organelles (Bakkali et al., 2008). In order to use EOCs as antimicrobials, their cytotoxicity against various mammalian cells need to be evaluated at the concentrations that inhibit bacterial growth. The purpose of this study was to test the cytotoxicity against mammalian fibroblast cells of five EOCs (β-citronellol, carvacrol, cuminaldehyde, thymol, and trans-cinnamaldehyde) that have been shown inhibit the growth of Corynebacterium pseudotuberculosis at concentrations of 100 mg/ml or lower.
Materials and Methods
Cell Viability Assay Trial 1
Buffalo Rat (Rattus norvegicus) liver (BRL) fibroblast cells were obtained from ATCC® CRL-
1442TM. Essential oil components (EOCs) were obtained from TCI America. BRL cells were grown in
Eagle’s Minimal Essential Medium (EMEM) with L-glutamine (ATTC® 30-2003) and 10% fetal bovine serum (FBS) (ThermoFisher Scientific, cat. # R92157) according to the specifications in Appendix C. BRL cells were counted (Appendix C) and diluted to a concentration of 5 x 104 cells/ml with EMEM + 10%
FBS. One hundred µl of the cell suspension was pipetted into each well of a 96-well plate, equivalent to
5,000 cells/well (CellTiter 96® Assay Protocol, Promega, 2012). The plate was incubated overnight at 37°
C in 5% CO2. EOCs (β-citronellol and carvacrol) were mixed in a 1:1 (v/v) solution with ethanol (EtOH)
37
(Sigma-Aldrich), then this solution was mixed 1:1 (v/w) with Tween 80 (ThermoFisher Scientific).
EOC/EtOH/Tween solutions were diluted in EMEM + 10% FBS to final EOC concentrations of 200, 100, and 10 mg/ml. The overnight culture of BRL cells was observed and imaged with an inverted microscope at 10x. The media was removed with a multi-channel pipet and replaced with 100 µl/well PBS
(phosphate-buffered saline; Sigma-Aldrich) to wash the cells twice. Fifty µl EMEM + 10% FBS were added to each of the wells. An additional 50 µl was added to the outer and untreated wells of the plate. Fifty µl of EOC dilutions in EMEM + 10% FBS were added to the wells as shown in Appendix C (Table C.1). The plate was incubated in a humidified incubator at 37° C in 5% CO2 for 24 hours. The wells were imaged after 24-hours incubation with an inverted microscope at 10x magnification. Twenty µl CellTiter 96®
AQueous One Solution Reagent, containing a tetrazolium compound and an electron coupling reagent, was added to the EOC-treated wells and the control wells. The plate was returned to the humidified, 37°
C, 5% CO2 incubator. Absorbance was read at 490 nm on a spectrophotometer after 1-, 2.25-, 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.
Cell Viability Assay Trial 2
BRL cells were counted using the BRL cell counting procedure (Appendix C) and diluted to a concentration of 5 x 104 cells/ml with EMEM + 10% FBS. One hundred µl of the cell suspension were pipetted into three 96-well plates (5,000 cells/well) as shown in Appendix C (Tables C.2, C.3, C.4). One hundred µl sterile DI water was pipetted into the outer and untreated wells. The plates were incubated overnight in a humidified incubator at 37° C in 5% CO2. EOCs (β-citronellol, carvacrol, cuminaldehyde, thymol, and trans-cinnamaldehyde) were diluted to final concentrations of 200, 100, and 20 mg/ml with
EMEM + 10% FBS in a total volume of 300 µl. Bleach was diluted to 200 µl/ml in EMEM + 10% FBS in a total volume of 600 µl. The overnight cultures of BRL cells were imaged with an inverted microscope at
38
10x. The media was removed with a multi-channel pipet from the wells containing cells and replaced with 100 µl/well PBS to wash the cells twice. Fifty µl EMEM + 10% FBS were added to each of the wells.
An additional 50 µl was added to the untreated cells as a control. Fifty µl of EOC or bleach dilutions were added to the wells as shown in Appendix C (Tables C.2, C.3, C.4). The plates were incubated in a humidified incubator at 37° C in 5% CO2 while shaking at 130 cycles/minute for 24 hours. The wells were imaged after 24-hours incubation with an inverted microscope at 10x. The media was removed from the wells and replaced with 100 µl EMEM + 10% FBS. Twenty µl CellTiter 96® AQueous One Solution Reagent was added to the EOC-treated wells and the control wells. The plate was returned to the humidified, 37°
C, 5% CO2 incubator. 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 (CellTiter 96® Assay Protocol, Promega, 2012).
Results
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 3.1).
39
2.5
2
1.5
1
0.5 Absorbance 490 at nm
0 100 mg/ml 50 mg/ml 10 mg/ml Controls Treatment/Concentration
B-citronellol Carvacrol Bleach Ethanol Tween Ethanol/Tween No treatment
Figure 3.1 Viability of BRL cells treated with EOCs and controls (higher absorbance readings indicate more viable cells). Values are the mean of 3 replicates.
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 3.2 and 3.3). 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 3.2). There was a large amount of variability in the replicates for some of the treatments (Figures 3.2 and 3.3).
All of the EOCs affected BRL cell morphology based on visual assessment with an inverted microscope. These effects were more detectable at higher concentrations of EOCs (Figures 3.4, 3.5, 3.6,
40
3.7, and 3.8). Bleach did not appear to have a strong effect on BRL cell morphology (Figure 3.9).
Untreated BRL cells appeared to have experienced some damage and cell death (Figure 3.10).
1.4
1.2
1
0.8
0.6
0.4
Absorbance 490 at nm 0.2
0 100 50 10 Concentration (mg/ml)
β-citronellol Carvacrol Cuminaldehyde Thymol Trans-cinnamaldehyde
Figure 3.2 Viability of BRL cells treated with EOCs (higher absorbance readings indicate more viable cells). Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
0.9 0.8 0.7 0.6 0.5 0.4 0.3
0.2 Absorbance 490 at nm 0.1 0 1 2 3 Plate #
Bleach No treatment
Figure 3.3 Viability of BRL cells treated with bleach and untreated (higher absorbance readings indicate more viable cells). Values are the mean of 3 replicates; error bars are the standard deviations of the replicates.
41
A B C
Figure 3.4 BRL cells at treated with β-citronellol at final concentrations of A) 100 mg/ml, B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification)
A B C
Figure 3.5 BRL cells treated with carvacrol at final concentrations of A) 100 mg/ml, B) 50 mg/ml, and C) 10 mg/ml carvacrol for 24 hours (10x magnification)
A B C
Figure 3.6 BRL cells treated with cuminaldehyde at final concentrations of A) 100 mg/ml, B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification)
A B C
Figure 3.7 BRL cells at treated with thymol at final concentrations of A) 100 mg/ml, B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification)
42
A B C
Figure 3.8 BRL cells at treated with trans-cinnamaldehyde at final concentrations of A) 100 mg/ml, B) 50 mg/ml, and C) 10 mg/ml for 24 hours (10x magnification)
A B C
Figure 3.9 BRL cells treated with 10% bleach for 24 hours from A) Plate 1, B) Plate 2, and C) Plate 3 from viability assay Trial 2 (10x magnification)
A B C
Figure 3.10 BRL cells from A) Plate 1, B) Plate 2, and C) Plate 3 from viability assay Trial 2 (10x magnification)
Discussion
Cell Viability Assay Trial 1
No conclusions could be drawn from the EOC-treated wells because the amount of ethanol and
Tween that the components were diluted in had cytotoxic effects on the BRL cells. The number of viable cells did not vary consistently with varying concentrations of EOCs. This may be because of the differing
43 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
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 (Figure
3.2). However, the images taken of these wells after treatment do not support this finding (Figure 3.5 and 3.8). Visual assessment of the BRL cells treated with EOCs 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
(Figures 3.5, 3.7, and 3.8). These may be the EOCs themselves as they were unable to dissolve in the cell culture media, or they could be due to interactions between the EOCs and the media. Some EOCs are known to interact and bind with proteins in media. For example, thymol has been reported to bind with the protein bovine serum albumin in media (Veldhuizen et al., 2007). These interactions may interfere with the colorimetric reading of the cell viability assay or they may alter the cytotoxic/antimicrobial properties of the EOC (Veldhuizen et al., 2007). 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 EOCs, even after replacing the treated media, may have had an unintended effect on the color change of the media that is measured.
Trans-cinnamaldehyde and carvacrol both had higher absorbance readings at higher concentrations, even though no viable cells were detected with microscopy. However, several other studies have evaluated the cytotoxicity of EOCs using similar cell viability assays. One study assessed the
44 cytotoxicity of carvacrol and thymol against Caco-2 cells using an MTS assay and reported that thymol had no effect on cellular viability at concentrations up to 250 µM (37.56 µg/ml) and that carvacrol reduced cellular viability at concentrations of 500 µM (75.11 µg/ml) and higher. However, through morphologic assessments, the researchers were able to detect cellular damage at lower concentrations when compared to the MTS assay (Llana-Ruiz-Cabello et al., 2014b). Several other studies have assessed the cytotoxicity of EOCs with MTT-based assays. MTT assays have been reported to be successful for evaluation of the cytotoxicity of carvacrol, cuminaldehyde, terpinolene, and p-anisaldehyde on a variety of mammalian cell lines (Bimpzok et al., 2008; Morshedi et al., 2015; Turkez et al., 2015; Shreaz et al.,
2011; Sharopov et al., 2017). MTT assays are very similar to MTS assays in that they both measure the reduction of tetrazolium salts by viable cells. One of the main differences between these assays is that the byproduct of the MTS assay is soluble formazan whereas the byproduct of the MTT assay is insoluble formazan, although this difference should not have an effect on their ability to evaluate cytotoxicity of
EOCs. Overall, it can be concluded that β-citronellol, carvacrol, cuminaldehyde, thymol, and trans- cinnamaldehyde 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 EOCs 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. In piglets, a
1:1 mixture of carvacrol and thymol was used as a dietary supplement at a concentration of 100 mg/kg without altering the biomarkers of the intestinal barrier (Wei et al., 2017). In broiler chickens, a 1:1 mixture of carvacrol and thymol was used at concentrations as high as 200 mg/kg as a dietary supplement and was found to increase body weight and improve immune function of the birds without toxic side effects (Hashemipour et al., 2013). These findings suggest low toxicity of carvacrol and thymol
45 when administered orally. However, oral administration of EOCs 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 EOCs would have enough bactericidal activity to kill or inhibit the growth of C. pseudoTB, when and if they migrated to the source of the abscess. For these reasons, dermal administration of the EOCs to external CL abscesses may be a more viable approach. Many EOCs 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 EOCs on mammalian skin cells is needed before they can be used in vivo as antibacterial agents for C. pseudoTB.
46
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APPENDIX A: C. PSEUDOTUBERCULOSIS IDENTIFICATION
C. pseudotuberculosis is a BSL-2 organism. Therefore, all work with C. pseudoTB was done in the
BSC with proper personal protective equipment (lab coat, double gloves, eye shield, and mask).
Table A.1 Identification of cryopreserved C. pseudoTB cultures
Identity Date cyropreserved ATCC 19410 9/13/13 Farm #1 (goat pus) 4/2/14 Acc: 10241 (goat) 12/7/08 Acc: 16784-1 (from abscess) 4/2/18 Acc: 16787 (from abscess) 3/5/18 Acc: 10616 2009
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APPENDIX B: ESSENTIAL OIL COMPONENT SOLUBILITY
Solubility of EOCs in Ethanol: Trial 1
Methods
Five hundred µl of liquid essential oil components (EOCs) (α-terpinene, β-citronellol, β-pinene, carvacrol, cuminaldehyde, p-anisaldehyde, terpinolene, trans-cinnamaldehyde) or 0.5 g of solid EOCs
(thymol, trans-cinnamic acid) were added to 500 µl ethanol (EtOH) and vortexed well to mix. Fifty µl of
EOC/EtOH solutions were added to 950 µl sterile DI water and vortexed well to mix. The solubility of the
EO components in 5% EtOH and water were visually assessed.
Results
β-pinene, α-terpinene, terpinolene, and cuminaldehyde in 5% ethanol and water created a homogenous solution. Thymol, carvacrol, and β-citronellol in 5% ethanol and water dissolved slightly with some oily bubbles or swirls remaining. Trans-cinnamaldehyde, p-anisaldehyde, and trans-cinnamic acid did not dissolve in 5% ethanol and water and sedimented at the bottom of the tube (Table B.1).
Table B.1 Visual assessment of EOC solubility in 5% ethanol and water (2=dissolved well; 1=dissolved slightly; 0=did not dissolve)
EO component Solubility Thymol 1 Carvacrol 1 Trans-cinnamalehyde 0 p-anisaldehyde 0 β-pinene 2 α-terpinene 2 Trans-cinnamic acid 0 Terpinolene 2 β-citronellol 1 Cuminaldehyde 2
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Solubility of EOCs in Ethanol: Trial 2
Methods
EOCs were mixed in a 1:1 solution with ethanol (w/v) and vortexed well to mix. Fifty µl of
EOC/EtOH solutions were added to 950 µl sterile DI water and vortexed well to mix. Final solubility of
EOCs in EtOH and water was visually assessed.
Results
Thymol, carvacrol, β-pinene, α-terpinene, terpinolene, and β-citronellol in equal parts ethanol
(w/v) and water created a homogenous solution. Cuminaldehyde dissolved slightly in the EtOH and water, but many oily bubbles remained. Trans-cinnamaldehyde and p-anisaldehyde dissolved in the ethanol, but sedimented at the bottom of the tube when mixed with water. Trans-cinnamic acid did not dissolve in the EtOH or EtOH/water (Table B.2).
Table B.2 Visual assessment of EOC solubility in Trial 2 (2=dissolved well; 1=dissolved slightly; 0=did not dissolve)
EO compound Solubility Thymol 2 Carvacrol 2 Trans-cinnamalehyde 0 p-anisaldehyde 0 β-pinene 2 α-terpinene 2 Trans-cinnamic acid 0 Terpinolene 2 β-citronellol 2 Cuminaldehyde 1
Solubility of EOCs in Ethanol and Tween
Methods
Five hundred µl of liquid EOCs (trans-cinnamaldehyde, p-anisaldehyde) or 0.5 g of solid EOC components (thymol, trans-cinnamic acid) were added to 500 µl ethanol and vortexed to mix. Five hundred µl EOC/EtOH solutions were added to 0.5 g Tween 80 and vortexed well to mix. Four hundred
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µl of the EOC/EtOH/Tween solutions were added to 600 µl DI water and vortexed to mix. Solubility of
EOC components in ethanol, Tween, and water was visually assessed.
Results
Thymol, trans-cinnamaldehyde, and p-anisaldehyde formed homogenous solutions when mixed with ethanol, Tween, and water. Trans-cinnamic acid formed a solid layer when added to ethanol and therefore could not be mixed with Tween and water (Table B.3).
Table B.3 Visual assessment of EOC solubility (sol) in Ethanol and Tween (2=dissolved well; 1=dissolved slightly; 0=did not dissolve)
EO component Sol. in EtOH Sol. in EtOH/Tween Sol. in EtOH/Tween/water Thymol 2 2 2 Trans-cinnamic acid 0 Not tested Not tested Trans-cinnamaldhyde 2 2 2 p-anisaldehyde 2 2 2
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APPENDIX C: BUFFALO RAT LIVER CELL CULTURE
Maintaining BRL Cells
Buffalo Rat (Rattus norvegicus) liver (BRL) fibroblast cells were obtained from ATCC® CRL-
1442TM. BRL cells were grown in Eagle’s Minimal Essential Medium (EMEM) + 10% fetal bovine serum
(FBS) in a humidified, 37° C, 5% CO2 incubator. Media was replaced every 48 hours and cells were passaged (1:3 to 1:6) when confluent (Figure B.1). All cell culture media, PBS, and trypsin-EDTA aliquots were warmed in a 37° C water bath prior to use. Cell culture work was done in the BSC with the exception of microscopy and centrifugation.
Figure C.1 Confluent BRL cells (10x magnification)
Restart Cryopreserved BRL Cell Culture
To start the BRL cell culture, a cryovial of BRL cells was thawed in a 37° C water bath and transferred to a 15 ml centrifuge tube. Eight ml 37° C EMEM + 10% FBS were added slowly to the tube containing cells. The cells were centrifuged at 200g for 5 minutes and the supernatant was removed with a 10 ml pipet. Five ml EMEM + 10% FBS at 37° C was added to the cells, pipetted to mix, and transferred to a 25 cm2 cell culture flask. The flask was capped loosely and incubated in a humidified incubator at 37° C in 5% CO2. Media was replaced after 24 hours incubation.
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When BRL cells were confluent in the 25 cm2 flask, they were transferred to a 75 cm2 flask. Cell culture media was removed from 25 cm2 flask with a 10 ml pipet. Cells were washed with 2.5 ml PBS.
PBS was removed and 1 ml trypsin-EDTA was added to the flask and rocked gently to cover the cells. The flask was incubated at 37° C in 5% CO2 until cells had rounded and lifted off (approximately 3 minutes).
Four ml EMEM + 10% FBS was added to the flask and pipetted to mix. The cell suspension was then transferred to a 15 ml centrifuge tube and centrifuged at 130g for 5 minutes. The supernatant was removed and 5 ml EMEM + 10% FBS was added and pipetted to mix. Four ml of the cell suspension was added to a 75 cm2 flask containing 12 ml EMEM + 10% FBS. The flask was capped loosely and incubated in a humidified incubator at 37° C in 5% CO2.
Counting BRL Cells
Cell culture media was removed from the 75 cm2 flask with a 25 ml pipet. Cells were washed with 5 ml PBS. PBS was discarded and 3 ml trypsin-EDTA was added to the flask and rocked gently to cover the cells. The flask was incubated at 37° C in 5% CO2 (humidified) until the cells had rounded and lifted off (approximately 3 minutes). Eight ml EMEM + 10% FBS was added to the flask and pipetted to mix. The cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 130g for 5 minutes. The supernatant was removed with a 10 ml pipet and 5 ml EMEM + 10% FBS was added and pipetted to mix. One half ml of cell suspension was transferred to a 1.5 ml microfuge tube. The cells were vortexed briefly and 50 µl was transferred to a new microfuge tube. To detect viable cells, 50 µl
0.4% trypan blue solution was added to the cell suspension and vortexed briefly. Ten µl of this cell suspension was added to a hemocytometer. The number of viable (not stained) and non-viable (stained) cells was counted under a dissecting microscope at 100x magnification. The average number of live, dead, and total cells per square was calculated and these averages were used to calculate cells/ml using the equation c = n * 104 * 2, where “c” is the concentrations of cells per ml and “n” is the average number of cells per square of the hemocytometer.
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Cell Viability Assay Trial 1
Table C.1 Layout of 96-well plate for BRL cell viability assay Trial 1 (shaded wells indicate no treatment; BC=β-citronellol; CV=carvacrol)
1 2 3 4 5 6 7 8 9 10 11 12 A B BC BC CV Bleach Tween No trt C 100 10 50 100 200 D mg/ml mg/ml mg/ml µl/ml µl/ml E BC CV CV EtOH EtOH F 50 100 10 100 (100 µl/ml) G mg/ml mg/ml mg/ml µl/ml Tween (200 µl/ml) H
Cell Viability Assay Trial 2
Table C.2 Layout of 96-well plate #1 for BRL cell viability assay Trial 2 (shaded wells indicate no treatment; BC=β-citronellol; CV=carvacrol)
1 2 3 4 5 6 7 8 9 10 11 12 A B BC BC CV Bleach C 100 10 50 100 D µl/ml µl/ml µl/ml µl/ml E BC CV CV No F 50 100 10 trt G µl/ml µl/ml µl/ml H
Table C.3 Layout of 96-well plate #2 for BRL cell viability assay Trial 2 (shaded wells indicate no treatment; CU=cuminaldehyde; TH=thymol)
1 2 3 4 5 6 7 8 9 10 11 12 A B CU CU TH Bleach C 100 10 50 100 D µl/ml µl/ml µl/ml µl/ml E CU TH TH No F 50 100 10 trt G µl/ml µl/ml µl/ml H
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Table C.4 Layout of 96-well plate #3 for BRL cell viability assay Trial 2 (shaded wells indicate no treatment; TC=trans-cinnamaldehyde)
1 2 3 4 5 6 7 8 9 10 11 12 A B TC TC 10 No C 100 µl/ml trt D µl/ml E TC 50 Bleach F µl/ml 100 G µl/ml H
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BIOGRAPHY OF THE AUTHOR
Sarah Paluso was born in Windham, Maine on September 1, 1994. She was raised in Windham,
Maine and graduated from Windham High School in 2012. She attended Gettysburg College and graduated in 2016 with a Bachelor’s degree in Biology. She returned to Maine and entered the Animal and Veterinary Sciences graduate program at The University of Maine in the fall of 2017. Sarah is a candidate for the Master of Science degree in Animal and Veterinary Sciences from the University of
Maine in May 2019.
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