Intramuscular Injection of Plant Derived Antimicrobials: a Potenial Use Against M.Bovis in Cattle

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Master's Theses University of Connecticut Graduate School

12-2-2019

Intramuscular Injection of Plant Derived Antimicrobials: A Potenial Use Against M.Bovis in Cattle

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This work is brought to you for free and open access by the University of Connecticut Graduate School at OpenCommons@UConn. It has been accepted for inclusion in Master's Theses by an authorized administrator of OpenCommons@UConn. For more information, please contact [email protected]. Intramuscular Injection of Plant Derived Antimicrobials: A Potenial Use Against M.Bovis in Cattle

Elizabeth Joy Johnson

B.S. Biology, Elmira College, 2017

A Masters Thesis

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Masters of Science

at the

University of Connecticut

2019

Elizabeth Joy Johnson

2019

APPROVAL PAGE

Masters of Science

Intramuscular Injection of Plant Derived Antimicrobials: A Potenial Use Against M.Bovis in Cattle

Presented by

Elizabeth Joy Johnson, B.S.

Major Advisor……………………………………………………………….. Dr. Xiuchun (Cindy) Tian

Associate Advisor………………………………………………………………. Dr. Mary Anne Roshni Amalaradjou

Associate Advisor………………………………………………………………. Dr. Guillermo Risatti

University of Connecticut 2019

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Table of contents Approval page……………………………………………………………………………………………………………………… i Table of contents………………………………………………………………………………………………………………… ii List of figures………………………………………………………………………………………………………………………. iv List of tables………………………………………………………………………………………………………………………… iv List of abbreviations……………………………………………………………………………………………………………. v Chapter I: Mycoplasma I. Mycoplasma General information…………………………………………………………………………… 1 II. Morphology: Physical and Chemical………………………………………………………………………… 1 III. Mycoplasma membranes, transport systems, and metabolism……………………………….. 2 IV. Mycoplasma bovis General information…………………………………………………………………… 7 V. Prevalence……………………………………………………………………………………………………………….. 9 VI. Acquiring infection…………………………………………………………………………………………………… 10 VII. Laboratory diagnosis………………………………………………………………………………………………… 11 VIII. Antibiotic resistance…………………………………………………………………………………………………. 12 IX. Mycoplasma Genome……………………………………………………………………………………………….. 14 X. Virulence factors……………………………………………………………………………………………………….. 16 XI. Mycoplasma bovis immune function…………………………………………………………………………. 22 XII. Environmental survival………………………………………………………………………………………………. 28 XIII. Bovine Respiratory airway…………………………………………………………………………………………. 29 XIV. Mucociliary apparatus……………………………………………………………………………………………….. 31 XV. Pathogenesis and localization of antigen…………………………………………………………………… 34 XVI. Co-infection……………………………………………………………………………………………………………….. 34 XVII. Mycoplasma bovis arthritis……………………………………………………………………………………….. 35 XVIII. Mycoplasma bovis pneumonia………………………………………………………………………………….. 35 XIX. Mycoplasma bovis mastitis………………………………………………………………………………………... 39 Chapter 2: Essential Oils- Plant derived antimicrobials I. Background………………………………………………………………………………………………………………… 46 II. Historical use of essential oils…………………………………………………………………………………….. 48 III. Composition of essential oils……………………………………………………………………………………… 50 IV. Testing essential oils………………………………………………………………………………………………….. 51 V. The growth medium………………………………………………………………………………………………….. 53 VI. Tests of antibacterial activity of essential oils in food systems………………………………….. 54 VII. Antioxidant and anti-inflammatory activities of essential oils…………………………………… 57 VIII. Essential oils on ruminal function……………………………………………………………………………… 62

IX. Mode of antibacterial action…………………………………………………………………………………….. 69 a. Carvacrol…………………………………………………………………………………………………….. 72 b. Eugenol………………………………………………………………………………………………………. 74 c. Trans Cinnamaldehyde……………………………………………………………………………….. 74 X. Susceptibility of Gram-negative and Gram-positive organisms……………………………….. 77 XI. Synergism and antagonism between components of essential oils…………………………. 81 XII. Toxicity……………………………………………………………………………………………………………………. 83 a. Liver…………………………………………………………………………………………………………….. 90 b. Kidney…………………………………………………………………………………………………………. 93 XIII. Safety Data……………………………………………………………………………………………………………… 95 XIV. Neurological effects of essential oils……………………………………………………………………….. 96 XV. Part I and II References…………………………………………………………………………………………… 107 Chapter 3: Effects of intramuscular injected plant-derived antimicrobials in the mouse model i. Effects of intramuscular injected plant-derived antimicrobials in the mouse model…. 139 a. Abstract………………………………………………………………………………………………………… 140 b. Introduction………………………………………………………………………………………………….. 141 c. Methods……………………………………………………………………………………………………….. 143 d. Results………………………………………………………………………………………………………….. 148 e. Discussion…………………………………………………………………………………………………….. 154 f. Conclusion……………………………………………………………………………………………………. 159 ii. References………………………………………………………………………………………………………………. 161 Chapter 4: VigorSential I. University support……………………………………………………………………………………………………. 164 II. Executive Summary………………………………………………………………………………………………….. 167 III. Company description………………………………………………………………………………………………. 168 IV. Products and Services………………………………………………………………………………………………. 168 V. Markey analysis……………………………………………………………………………………………………….. 170 VI. Strategy of implementation…………………………………………………………………………………….. 171 VII. Financial plan and projections/ Organization and management team……………………… 172

Lists of Figures Title Page

Chapter III

Figure 1 Animal treatment and observation schemes. Mice arrived on Day -2 144 and were sexed. The mice then had a 48h acclimation time. On the morning of Day 0 the mice were weighed and injected. Mice were observed hourly for 2 h, and twice daily post-injection until Day 4 when mice were weighed, euthanized for dissection. Red vertical bars indicate the times of observation.

Figure 2 The averaged foot/leg scores (means+SD; upper panel) and number 151 of mice with scores of >0 (lower panel) in controls and TC treated mice. H and D: hour and day post-injection. Scoring system: 0=normal; 1=closed digits while walking well; 2=closed digits, no grip; 3=closed digits, no grip, dragging leg, foot flipped.

Figure 3 The averaged foot/leg scores (means+SD; upper panel) and number 152 of mice with scores of >0 (lower panel) in controls and CAR treated mice. H and D: hour and day post-injection. Scoring system: 0=normal; 1=closed digits while walking well; 2=closed digits, no grip; 3

Lists of Tables Title Page

Chapter III

Table 1 Levels of PDA treatments. DMSO was used as the vehicle control. 145

Table 2 Scoring systems for eating/drinking, physical activities, physical 146 appearance, and foot/leg function/appearance

Table 3 Mortality from CAR treatments (dead/total). No mortality was found 148 in the corresponding DMSO controls. Table 4 The averaged behavior scores and the number of animals receiving a 149 score of >0 during the first 12 h of TC and CAR treatments Table 5 Weights (g) and relative weights (%) of the liver and kidney in 153 control and PDA treated mice.

List of Abbreviations

BRD Bovine Respiratory Disease LPS Lipopolysaccharide PBP Periplasmaic binding proteins TCA Tricarboxylic acid cycle VSP Variable Surface Lipoprotein MALP Macrophage activating lipoprotein E2 Prostaglandin estrogen NO Nitric oxide TACA Serum antioxidant capacity Mb-LIP M.bovis lympho-inhibitory peptide PIMs Pulmonary intravascular macrophage ASL Air-surface liquid TAP Tracheal antimicrobial peptide LAP Lingual antimicrobial peptide SP-A Surfactant proteins A SP-D Surfactant proteins D UBRD Undifferentiated bovine respiratory disease EO Essential Oils MLC Minimal lethal concentration ICAM Intracellular adhesion molecule VCAM Vascular cell adhesion molecule LTA Lipoteichoic acid PPARs Peroxisome Proliferator Activated Receptor TC Transcinnamaldehyde OM Outer membrane HHP High Hydrostatic Pressure MIC Minimum inhibitory concentration Ip Intraperitoneal Iv Intravenous PDAs Plant Derived Antimicrobials

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Chapter I: Mycoplasma

Mycoplasma General Information Mycoplasmas are among the smallest free-living microorganisms, comprising over 100 species. The class Mollicutes was founded in 1956 by Edward and Freundt. The class Mollicutes is now used to classify organisms of the pleuropneumonia group and the order of Mollicutes.

Mollicutes are part of the Gram-positive lineage originally evolving from Clostridium-like bacteria via gene deletion. Most Mycoplasmas are facultative anaerobes, have a tri-layered cell membrane and cannot form a cell wall. Due to the lack of a cell wall, they have a pleomorphic shape, and antibiotic resistance. One family, Mycoplasmataceae, is composed of two generas,

Mycoplasma and Acholeplasma. Mycoplasma bovis (M. bovis) was initially called M.agalactiae var. (later subsp) bovis because of both clinical and biochemical similarities to M.agalactiae, the cause of contagious agalactia in small ruminants (Nicholas, 1998).

Many species of Mycoplasma are important veterinary pathogens that cause Epizooties list A diseases. It is generally believed that Mycoplasmas have a secondary role in infections by exacerbating pre-existing conditions. However, Mycoplasma bovis has been shown in recent years to have a primary role in infections such as bovine pleuropneumonia, Bovine Respiratory

Disease (BRD), arthritis, and mastitis in cattle (McAuliffe, 2004).

Morphology: Physical and Chemical

Typical mycoplasmal colonies have well-defined edges with an average diameter of

100µm. However, variations between 10-600µm have been seen. The colony has a dense center and a translucent periphery. Early microscopics described finding colonies below the agar surface, which was later proven true by doing a cross section of the agar plate. This growth

is possible because as fluid around a single mycoplasma dries, it is brought into the inside of the agar gel via capillary action. Once inside the gel, the bacteria begins to multiply and spread between the fibrils of the agar itself, resulting in a flat and thin periphery on the surface.

However, there are many variations of this growth. If two cells are close together, the resulting colony has two centers and a confluent periphery. When colonies are crowed, the nutrients are deficient resulting in overall smaller colonies, but the same characteristic shape occurs.

Vacuolization occurs in aging colonies. Mycoplasmas can absorb large amounts of lipid. The absorbed liquid becomes associated with the organism, forming the lace-like vacuolization pattern in the periphery. Lipid accumulation that results in this lace-like appearance is caused by lysis of the cells.

Based on dry weight, mycoplasmas are composed of 40-60% protein. When whole cells hydrolyze, at least 17 different amino acids are produced. Upon performing a starch-gel and polyacrylamide-gel electrophoresis, a lot of variation in protein bands among different species have been observed. Carbohydrates make up less then 0.1% of the cell based on dry weight

Total lipid content by dry weight is between 8-20%, with about half of the lipids being unsaponifiable lipids, glycolipids, and glycerides. The other half is exclusively polar phospholipids.

Mycoplasma membranes, transport systems, and metabolism

Mycoplasma cells contain a trilaminar membrane that surround the cytoplasm, which is packed with ribosomes, fibrillar DNA, one or more electron dense areas, and the occasional empty vesicle. The cytoplasmic membrane is about 5nm thick and is bound on either side by

3nm thick electron dense areas (overall membrane about 11nm). The outer layer is denser than the inner electron absorbing layer. In terms of the nuclear content, fibrils are about 3nm thick and are not contained within a membrane. Their cytoplasmic membrane is composed of lipids including cholesterol but has a high total protein content up to 50% by mass (Dermina et al.,

2009).

The lipoproteins of Gram-positive bacteria are anchored in the outer ‘leaflet’ of the plasma membrane whereas in Gram-negative bacteria they are anchored in the inner ‘leaflet’ of the outer membrane or in the outer ‘leaflet’ of the inner membrane, depending on cellular function. The presence of large amounts of lipopolysaccharide (LPS) in the outer membrane is a distinctive property of Gram-negative bacteria. However, the plasma membrane of some mycoplasma species also contains lipoglycans, which, although distinct from LPS, shares a repetitive oligosaccharide structure. Additionally, in mycoplasmas, some lipoproteins are variable antigens, with the ability to undergo both phase and sequence variation. Sequence variability based on the presence of repetitive motifs is, therefore, another property shared by

LPS and lipoproteins, along with their role in modulating the activity of the host immune system. The four main categories of known lipoprotein functions are structural (murein lipoproteins), transport (substrate- binding proteins of ABC transporters in mycoplasmas and

Gram-positive bacteria), adhesion (in mycoplasmas) and enzymatic (Sutcliffe and Russell, 1995;

Braun and Wu, 1994; Hayashi and Wu, 1990).

Two main types of lipoproteins variation have been described in mycoplasmas: one involves varying the number of tandem repeats at the carboxy terminus, thereby varying the lipoproteins size and the other involves turning lipoprotein synthesis on or off, a phase

variation. The phase variation of a given antigen can also effect, by differential masking, the epitope accessibility of another surface antigen, thereby increasing the possible variation

(Theiss et al., 1996). The size and phase variation of lipoproteins occurs at high frequency, and thus a given population of mycoplasmas cells always comprises variants differing in their lipoprotein repertoire (Rosengarten and Yogev, 1996).

Lipid peroxidation of biomembranes impacts the overall structural integrity and function of the membrane by decreasing the amount of polyunsaturated fatty acids, inhibiting membrane formation, and protein conformation. Oxidizing thiols that may affect enzyme activities in the membrane, decreasing lipid fluidity in the membrane, and liberating breakdown products from the site of lipid peroxidation (Ames, 1993).

Many genes encoding mycoplasma lipoproteins, such as Mbov_0395 and

MBOVPG45_0550, appear to be within operons encoding ABC transporters. Mbov_0395 and

MBOVPG45_0550 may be associated with the acquisition of nutrients for transport into the cell

(Browning et al., 2011). Some of these lipoproteins can also be targets for growth inhibitory antibody, as seen with the M. hyopneimoniae surface lipoprotein P65, which is a lipolytic enzyme (Schmidt et al., 2004). Mycoplasmas can stimulate and suppress lymphocytes in a non- specific polyclonal manner, and their cell components can modulate the activities of monocyte/macrophages and natural killer cells, triggering the production of many up-regulating and down regulating cytokines and chemokines (Razin et al., 1998). Mbov_0585

(MBOVPG45_0316; MMB_0536) encodes a predicted membrane adenosine triphosphatase and may serve as a coenzyme in intracellular energy transfer. Another interesting role that mycoplasma membrane proteins may play is in pulmonary surfactant disruptions. Both

surfactant protein A and inducible nitric oxide synthase are thought to be important in pulmonary defense against respiratory pathogens such as mycoplasmas (Hickman-Davis et al.,

2007).

In Gram-negative bacteria, some substrates (such as maltose and maltodextrins, histidine, oligopeptides, phosphate, etc.) are concentrated into the cell by high affinity transport systems which include several proteins and a common organization (Ames, 1986;

Shuman, 1987). A central component of these systems is a binding protein which is located in the periplasm between the outer and inner membranes. These water soluble periplasmic binding proteins (PBP) present high affinity for their specific substrates. Their concentration in the periplasm can reach very high values (1mM), which increases the availability of bound substrate in the inner membrane vicinity. Two proteins of the complex are hydrophobic and are intrinsic inner membrane proteins. Depending on the system there are also one or two hydrophilic proteins in the complex; they are believed to be bound to the inner face of the cytoplasmic membrane. These hydrophilic proteins belong to a family of homologous proteins associated with various systems for trans-membrane transport of very different substrates in a variety of organisms (Gilson et al., 1984; Higgins et al., 1986; Henderson and Maiden, 1987).

These proteins bind ATP and are believed to provide energy for transport through the membrane.

The proximity of the PBP to the cytoplasmic membrane is believed to be critical for the functioning of these high affinity transport systems. Examination of the NH2 terminal sequences of the malX and amiA gene products gives a possible clue to this question. MalX and AmiA, in

Gram-positive bacteria, and p37 in mycoplasma, are the functional equivalent of periplasmic

binding proteins involved in transport in Gram-negative bacteria. The NH2 terminal parts of the malE and malX gene products represent signal peptides that are exported through the cytoplasmic membrane. In MalE, the first 26 residues are cleaved upon export (Duplay et al.). In the case of MalX, the region of the potential cleavage site belongs to a well-defined category.

MalX protein is a lipoprotein. Lipoproteins are also exported through the cytoplasmic membrane, but the NH2 terminal cysteine is transformed into a lipo-amino acid. This lipophilic modification is through to be responsible for the membrane anchorage of some exported proteins (Nielsen and Lampen, 1982). The membrane attachment of MalX is thus likely to occur through the same mechanism. The proteins itself would be exposed to the outside face of the membrane in a water-soluble form.

Mycoplasma bovis lacks the tricarboxylic acid cycle (TCA), and their relatively small genome size limits their metabolic activities, which makes them largely dependent on the host for amino acids, nucleic acid precursors, and lipids. Its main energy compounds for growth include lactate and pyruvate since it is unable to ferment glucose or hydrolase arginine (Khan,

2005). Byproducts of mycoplasma metabolism include hydrogen superoxide and superoxide radicals. Upon infection, lymphocyte production is stimulated. However, antiphagocytic activity, immunosuppressive, and auto-immune phenomena have been observed because it has been noted that M. bovis can form biofilms and utilize macrophage machinery for its own use.

Mycoplasma membrane lipoproteins and certain lipids have been found to induce cytokine secretions (Nicolet, 1996). It is thought that the interaction of mycoplasmas with macrophages and monocytes induces the production of proinflammatory cytokines, interleukins, and interferon gamma.

Mycoplasma bovis General Information Mycoplasma bovis was first isolated from a severe mastitis case in the United States in

1961 (Hale et al., 1962) and the primary cause of respiratory disease in 1976 (Gourlay et al.,

1976). However, infections have been reported throughout the world before 1961 and continue to do so. In the United Kingdom, respiratory diseases are believed to affect 1.9 million cattle yearly, with at least a quarter to a third with M. bovis as the primary pathogen. In the United

States, $108 million is spent on diseases caused by M. bovis, with infection rates of up to 70% of a herd (McAuliffe, 2004). It is important to note that these estimates are likely underestimated due to poor identification and public knowledge.

Clinical signs of M. bovis pneumonia are indistinguishable from other causes of pneumonia. Clinical signs are anorexia, depression, lethargy, fever, hyperpnea, respiratory distress, and weight loss. In chronic cases more specific clinical signs such as lameness and chronic failure to respond to antibiotics occurs. However, even in these cases without testing specifically for M. bovis, the primary pathogen remains unknown. One of the best distinguishing features is the presence of caseonecrotic bronchopneumonia lesions that are generally exclusively associated with M. bovis infection.

The three main types of lesions are caseonecrotic bronchopneumonia, bronchopneumonia with foci of coagulation necrosis, and suppurative bronchopneumonia with abscessation. These lesions most often affect the cranial and middle lobes of the lung with severe cases affecting over 80% of the entire lung. In severe cases, the unaffected tissue is usually only a thin band of unaffected lung in the dorsocaudal aspect of the caudal lobes.

Nodules of caseous necrosis are seen in all affected areas with a large variation in the size of

the nodules. The nodules are typically circular, white, dry, crumbly, and bulging from the pleural or cut surfaces of the lung (Rodrigues et al., 1996; Shahriar et al., 2002; Khodakaram-

Tafti and Lopez, 2004; Gagea et al., 2006). Caseonecrotic lesions in experimental cases less than two weeks have not been noted.

Upon further histological investigation, chronic bronchointerstitial pneumonia is characterized by thickening of the bronchial wall due to excess lymphocytes (peribronchiolar lymphatic cuffings) and dense accumulation of lymphocytes around blood vessels (perivascular lymphocytic cuffings). This is commonly referred to as “cuffing pneumonia.” Purulent bronchiolitis, the accumulation of neutrophils and macrophages within alveolar lumens, epithelialization of alveolar septae, and in severe cases the complete or partial collapse of the affected lung (atelectasis) are also seen.

Other conditions such as meningitis, otitis media, ketato-conjunctivitis, and vaginitis and/or abortions are less frequent outcomes of M. bovis infections. In experimental cases of M. bovis, reproductive tract problems have been noted in both males and females, but specific causative evidence is hard to develop because of numerous associated factors. A 2005 report reported M. bovis isolation from the brain of calves that did not show neurological signs (Ayling,

2005 and Nicholas, 2008). In a different specific case from 2008, M. bovis was isolated from large spheroidal fibrinous lesion in the heart (Ayling, 2005 and Nicholas, 2008). These two separate reports are interesting in how they bring up possible other conditions related to M. bovis. However, these also appear to be very isolated occurrences because no other reports have been noted.

Prevalence

M. bovis infection has been reported in the United States, Canada, and most European countries. Prevalence of seropositive herds range from 20% to 100%, depending on the study and geographical region (Grand et al., 2002; Tenk et al., 2004; Ghadersohi et al., 2005). Calves with high serum antibody titers to M. bovis are at an increased risk (relative risk 1.7), although many calves with high titers do not have chronic pneumonia (Pollock et al., 2007).

Calves less than 4 months of age are at the highest risk of respiratory disease primarily caused by M. bovis (Stipkovits et al., 2000). Bennett and Jaspert, 1977, suggest that M. bovis prevalence peaks between 1 and 4 months of age, but specific time points are not known because of geographical differences. In healthy calves that have not been recently transported or introduced to new cattle, the prevalence of M. bovis is between 0% and 7% (ter Laak et al.,

1992; Grand et al., 2002; Thomas et al., 2002; Hirose et al., 2003; Rifatbegovic et al., 2007;

Wigins et al., 2007).

M. bovis prevalence is greater in feedlots and cattle that have been recently transported or co-mingled, even if no signs of infection are noticed. In one healthy herd of beef cattle, initial prevalence was between 40% and 60% when they initially arrived at a feed lot. However, by day

12 the prevalence of M. bovis increased to nearly 100% (Allen et al., 1991, 1992). In another study, 55% of previously seronegative calves for M. bovis became seropositive during the first seven weeks post-feedlot arrival (Tschopp et al., 2001). M.bovis has been isolated from 79% of seemingly healthy veal calves bronchoalveolar lavage fluid (Arcangioloi et al., 2007). Only 2% of nasal swabs came back M. bovis positive in calves that arrived in stocker and backgrounding

operations ten days prior (Wiggins et al., 2007). Out of the total calves that died within two months upon feedlot arrival, M. bovis was isolated from 46% of lungs. Interestingly in these calves, no gross or histological evidence of inflammation was seen (Gagea et al., 2006).

Acquiring infection

Introduction of M. bovis typically occurs via undiagnosed subclinical mycoplasma carriers. In these cattle, M. bovis is shed through nasal discharge without ever showing clinical signs of illness. Most subclinical cases are identified at necropsy or slaughter when cranioventral areas of the lungs are red-blue, firm, and ooze purulent materials on cut sections.

M.bovis can be acquired via ingesting infected milk and close contact with infected calves

(Pfutzner, 1990). There is still a lot of controversy over the incubation period of M. bovis, because the onset of clinical signs is not specific toward M. bovis caused diseases. In 1996, a naturally infected cohort of calves developed respiratory disease two weeks after contact with an infected herd (Adegboye et al., 1996). In experimental settings, respiratory disease and arthritis are seen around 8-10 post infection (Stipkovits et al., 2000). However, symptoms as early as 2-6 days have been recorded (Pfuzner, 1990).

Due to the common occurrence of pneumonia symptoms, several morbid calves are not initially identified. However, at slaughter, many of these unidentified cattle have detectable lung lesions accompanied by decreased weight gain. In 1996, Wittum et al. reported that 72% of 469 steers had at least one lung lesion. However, of this same group, only 35% had been treated for BRD during their life. Another study in 2018 found that 65% of animals that were never treated for BRD contained lung lesions. This information indicates frequent subclinical or

not easily diagnosed conditions. In comparison, 30% of animals that were treated more than once were affected by a moderate to severe bronchopneumonia, which highlights the ineffectiveness of current antibiotics (Caucci, 2018).

There are some experimental reports of cattle inoculated with M. bovis in which no symptoms appeared (Rodriguez et al., 1996). However, there are other experimentally inoculated cattle that exhibited fever, lethargy, lameness, and increased amount of joint fluid.

Some reports also include symptoms such as nasal discharge, cough, tachypnea, and loss of appetite (Gourlay et al., 1976; Rodriguez et al., 1996). Seronegative calves were introduced to a group of calves with endemic M. bovis pneumonia. The introduced calves remained seronegative for at least 29 to 35 days, and antibody titers did not develop until days 59 to 63

(Nagatomo et al., 1996).

Laboratory diagnosis

In vitro isolation of M. bovis can be extremely challenging because of the discrepancies between experimentally induced cases and natural cases, disease resistance differences depending on the strain, and lack of simple correlation between antibody titers and disease resistance. Diagnosis of M. bovis, in either individual animal or herd culture, is typically done via fluorescent antibody test (FA-test) and/or serology along with polymerase chain reactions

(PCR). Paired serum samples are frequently used to detect acute infections because antibody titers occur 10-14 days after initial infection, depending on the strain. Most misdiagnoses are the result of limited information regarding M. bovis incubation time. In experimental infections, the incubation time for mastitis and pneumonia caused by M. bovis are less than one week, and

one week, respectively. With some of the initial M. bovis studies in 1996, an incubation time of two to six days was reported (Pfutzner and Sachse, 1996). However, in 2011 Punyapomwithaya et al. found the average incubation time for a case of mastitis to be 13.6 days. The varying outcomes in incubation time are most likely caused by different bacteria phenotypes, depending on the strain found in a certain herd. Much of the information regarding virulence is unknown, but it is generally believed that each herd contains a slightly different variation of M. bovis.

Antibiotic resistance

The food industry is a primary user of antibiotics, and in many industrialized countries, the total amount of antibiotic used for food production exceeded the amount used in human medicine, and is thus, a significant factor in exposing bacteria to antibiotics (Levy, 1992).

Antibiotic resistance occurs when bacteria exchange antibiotic resistance genes. M. bovis is resistant to common antibiotics because it lacks a bacterial cell wall, making treatment difficult.

Typical bacterial cell walls are composed of a murein/peptidoglycan layer. Common antibiotics, such as beta-lactams (penicillin group), glycopeptides, and the clycoserine family have mode of actions that inhibit peptidoglycan synthesis, thus killing the bacteria. Since M. bovis lacks this layer, these common antibiotics are ineffective against it. The antibiotics that are regularly used to treat M. bovis infections are macrolides, modified macrolides, tetracyclines, aminoglycosides, chloramphenicols, pleuromutilins, and fluroquinolones, which all target specific bacteria biochemical pathways.

Macrolides are bacteriostatic and inhibit essential protein biosynthesis, via selective binding to bacterial ribosomal RNA. They stimulate the dissociation of peptidyl-tRNA from the ribosome during translocation (Menninger & Otto, 1982). Tulathromycin is rapidly distributed to the respiratory tract, eliminated slowly, and accumulated in neutrophils and macrophages

(Villarino et al., 2014). In cattle experimentally inoculated with M. bovis, tulathromycin treated calves displayed lower lung lesion proportion, lower mortality, fewer days with depressed demeanor, and higher body weight 14 days post-treatment than control calves (Bartram et al.,

2016).

Tetracyclines gain access into the bacterial cell by passive diffusion through hydrophilic pores in the outer cell membrane and then through the inner cytoplasmic membrane by an energy-dependent active transport (Chopra et al., 1992; Levy, 1992a). ‘Typical tetracyclines,’ which include tetracyclines, chloretracylcine, minocycline, and doxycycline, prevent bacteria growth by reversibly binding to the ribosomes and inhibiting protein synthesis; they are bacteriostatic agents which inhibit rather than kill in vitro (Chopra et al., 1992). These tetracyclines bind to the bacterial 30S ribosomal subunit and prevent attachment of aminoacyl- tRNA to the ribosomal receptor site in a reversible fashion. Binding of the tetracycline may cause distortion of the three-dimensional structure of the ribosome, which could then lead to changes in the accessibility of bases within the 16S rRNA (Chopra et al., 1992). Another possibility is that the binding of the tetracycline may block the entry of aminoacyl-tRNAs into the ribosome (Taylor et al., 1996). The important proteins in the 30S subunit with high-affinity binding of tetracyclines include S3, S7, S14, and S19, with evidence that tetracycline binds directly to S7 (Buck et al., 1990). The 16S rRNA, the regions of nucleotide 891 through

nucleotide 1054, may be involved in the antibiotic binding of tetracycline. Nucleotide 1052-

1054 have been suggested as the most likely to be associated with inhibition of protein synthesis (Chopra et al., 1992). ‘Atypical tetracyclines’ include chelocardin, anhydrotetracycline,

6-thiatetra-cycline and anhydrochlorotetraccline (Chopra, 1994; Oliva et al., 1992). These are bactericidal and interfere with membrane permeability resulting in cell damage which leads to cell lysis and death in vitro (Chopra, 1994; Oliva et al., 1992). There is no direct association between these atypical tetracyclines and the inhibition of protein synthesis by binding to the ribosomes (Oliva et al., 1992).

The numerous variations in M. bovis structure have also made vaccine development difficult. In Europe, no vaccines are approved that specifically prevent M. bovis, and in the

United States, there are some commercial vaccines available. However, vaccine effectiveness depends very much on the herd. It is possible, particularly for large farms, to have custom vaccines made using the specific strain of M. bovis present. This method is not frequently used, however, because it is expensive and every season, due to rapid alterations within the bacteria, a new custom vaccine would have to be made.

Mycoplasma Genome

The genome of M. bovis is small, with 1080bp, and a low G+C ratio of 27.8 to 32.9%.

DNA content ranges from 1.5% to 7%, whereas RNA content ranges from 3% to 17%, based on dry weight. This wide range is because the content will be the highest during early logarithmic growth and then decline very rapidly. Variations in measured amount can also be a result of cell damage during experimental processing. The chromosome is circular, and when under

replication, two Y forms are seen, similar to E. coli. The size of the chromosome is around 1000 x106 daltons, allowing for more than a thousand cistrons. Ribosomes are about 14nm in diameter and sedimentation coefficients of 72S is seen and when diluted with water results in three fractions of 70s, 49s, and 32s.

On either end of the cells, structurally dense blebs are seen. These areas do not contain nucleic acids, are not protein, and possibly contain lipid. The blebs have a dense elliptical outer plate that is adjacent to the cytoplasmic membrane along with fine threads that connect the flat plate to another elliptical plate. The area between the threads does not stain and is hypothesized to be microtubules. Behind the bleb, there is a large portion of protein, with unknown function. In the two ends of the cells, ribosomes are not seen. However, bleb regions are only seen in actively dividing cells.

Weise et al., (2011) were the first to publish a genome sequence of M. bovis, for the type strain PG45, which was initially isolated in 1961 from a severe case of mastitis in a cow.

The genome is 1,003,404bp in length, is 29.3% G+C and is predicted to encode 826 ORDFs.

Subsequently, the complete genome sequences of strain Hubei-1 (Li et al., 2011), which was isolated from a case of severe respiratory disease in Hubei province in China, and of strain

HB0801, which was isolated from lung lesions in a cow from Yingcheng in Hubei (Qi et al.,

2012). Comparison of their genomes revealed that HB0801 and Hubei-1 had a large inversion

(of 580kb), associated with two mobile genetic elements, ISMbov3 and ICEB-2, compared to

PG45. The HB0801 genome is 43,581 bp longer than that of Hubei-1, but 11,701 bp shorter than PG45. The differences between PG45 and HB0801 were mostly associated with insertion sequence, encode putative lipoproteins, and membrane proteins. The differences between

Hubei-1 and HB0801 are seen in nine distinct insertions in HB0801, many of which contained predicted lipoprotein and membrane protein genes. A surprising finding was the absence of the

Variable surface lipoproteins (vsp) gene cluster in Hubei-1, which is found in both PG45 and

HB0801. The adjacent xerC gene, which encodes the integrase-recombinase that mediates site specific inversion in the vsp gene family, thus generating phase variation in Vsp expression, was present. Vsps have been identified in all strains of M. bovis that have been characterized (Wise et al., 2011; Qi et al., 2012), suggesting that the absence of this cluster in Hubei-1 may be a result of deletion during culture of possibly an artefact of genome assembly.

Virulence factors

Variable surface lipoproteins (Vsp) are a family of immunodominant surface antigens that contribute to phenotype variability of the bacteria and the most known virulence factor of

M. bovis. They allow the bacteria to adhere to host cells and evade the immune system by binding antibodies. In M. bovis, there are five Vsp proteins thus far: Vsp A, Vsp B, VspC, Vsp F, and VspO (Sachse et al., 2000). These variations are responsible for the differing virulence amongst strains. Most common Vsp expression are seen with type B, O, and F (McAuliffe et al.,

2004). However, the exact correlation between Vsp expression and genotype has not been concluded.

It is known that Vsp are encoded by at least 13 VSP genes. In all of these genes, the N- terminus is conserved and works to anchor the proteins into the bacterial membrane. Overall

VSP proteins have multiple domains with large repeated segments of 6-87 amino acids (Sachse et al., 2000). However, the specific number of domains, the length of each repetitive sequence,

along with the prevalence of proline, glutamic acid, and lysine resides is highly variable. The variability among these characteristics is presumably important in immunogenicity, ligand binding, and cytoadhesion but specific links have not been found.

In the vsp gene locus itself, there has been numerous recombination events which may be important in variations amongst species isolates (Poumarat et al., 1999). Examples of such recombination events include adding and deleting of repeat sequences along with site specific

DNA inversions. Specifically, the addition or deletion of repeat sequences changes the size and antigenic variability of the encoded protein. However, site specific DNA inversions are important in phase variations and possibly lead to the expression of novel chimeric Vsp proteins. In 1996, Le Grand and colleagues found that the presence of antibodies specific to Vsp proteins influences which Vsp proteins the bacteria express with some difference in the ability to modulate an immune response.

A 48kDa membrane lipoprotein (P48) of M. bovis was discovered by Robino et al. It is homologous to the macrophage activating lipoprotein (MALP) of M. fermentans which is widely distributed in bacteria (Robino et al., 2005). It appears to be structurally and antigenically conserved in M. bovis strains and has the potential for use as a specific serological marker of infection. A paralogue encoding a hypothetical 68ka protein (P68) has been identified in M. bovis PG45 clonal variant 6. P68 shares the conserved selective lipoprotein-associated motifs of the MALP-related lipoproteins (Lysnyanksy et al., 2008). The P68 gene was only detected in some strains of M. bovis, all of which also contained the P48 gene. Thus far M. bovis is the only mycoplasma species found to contain multiple homologues of MALP.

Immunogenic protein pMB67 is produced during infection and is recognized by antibodies. This protein also has a high rate of phase and size variation that was shown in vitro

(Berens et al., 1996). Based on competitive adherence assays, P26 antigen is a 32 kDa hydrophilic protein that is important in cell adhesion. Treatment with neuraminidase highlighted the importance of sialyl acid receptors and adhesion interactions (Sachse et al.,

2006).

The Hsp60 gene in M. bovis has been shown to cause heat shock responses in natural infections (Scherm et al., 2002). However, there is very little information about this process and if it plays a role in bacteria protection or virulence.

Virulence related factors that contribute to the attenuation of highly passaged M. bovis strains, all the proteins of M.bovis were analyzed using VFDB. Eleven critical genes that likely contribute to the attenuation of highly passaged M.bovis strains were identified (Mbov_0722,

Mbov_0723, Mbov_0482, Mbov_0565, Mbov_0155, Mbov_0581, Mbov_0742, Mbov_0299,

Mbov_0212, Mbov_0797, Mbov_0567.

Mbov_0722 and Mbov_0723 are of high importance. They encode ascorbate-specific

PTS enzyme IIB and IIA components, respectively, which function in ascorbate and aldarate metabolism pathways by involving the PTS, the major carbohydrate transport system in bacteria, and they are responsible for the conversion of L-ascorbate into L-ascorbate-6 phosphate (Postma et al., 1993). The final product of this pathway is D-xylulose-5 phosphate, which is an intermediate in the pentose phosphate pathway. The primary purpose of this pathway is to generate a reducing equivalent of NADH that can be used in reductive

biosynthesis reactions, while the production of the pentose phosphate pathway intermediates ribose 5-phosphate and erythrose 4-phosphate are used to synthesize nucleotides and nucleic acids, and aromatic amino acids, respectively. This sugar has a role in gene expression, mainly by promoting the ChREBP transcription factor in the well-fed state (Iizuka and Horikawa, 2008).

The enolase encoded by Mbov_0482 is considered to be a virulence- related factor, and it participates in 7 metabolic pathways and interacts with other proteins as described previously. It catalyzes the reversible conversion of 2- phosphoglycerate into phosphoenolpyruvate. This reaction is present in the glycolysis/gluconeogenesis pathways of

M. bovis. Prokaryotic α-enolase may contribute to pathophysiological processes (Pancholi,

2001). A surface associated enolase is an adherence by binding plasminogen (Song et al., 2012).

The immunogenicity of enolase has also been observed in Mycoplasma synoviae (Berci et al.,

2008) and Mycoplasma capricoum subsp. Capripneuminiae (Zhao et al., 2012). Hence, enolase might be a significant protein that contributes to the virulence of M. bovis. However, two SNPs were not detected inside the enolase-encoding gene but were located approximately 300bp upstream of the gene, suggesting that both SNPs might cause some change in the promoter region.

L-lactate dehydrogenase encoded by Mbov_0565 is another protein that is important for metabolism and as a virulence- related factor. It interconverts L-lactate to pyruvate.

Additionally, this enzyme works in many metabolic pathways, including glycolysis/gluconeogenesis, cysteine, and methionine metabolism, pyruvate metabolism, and propanoate metabolism. Hence, this lactate dehydrogenase plays significant roles in a variety of

metabolic process. Moreover, L-lactate dehydrogenase was shown to be surface expressed and to interact with plasminogen (Grundel et al., 2015).

Pyruvate kinase (Mbov_0155) is also a very active protein in metabolism and energy production. It catalyzes the conversion of phosphoenolpyruvate to pyruvate. Then, mycoplasmas generate ATP via a protein-translocating ATP synthase by oxidizing organic acids

(pyruvate and lactate) to acetate and CO2. Moreover, the pyruvate kinase present in the genome of Mycoplasma suis is proposed to be required for the conversion of all NDPs and dNDPs to NTPs and dNTPs, respectively (Pollack et al., 2002). Hence, this protein is very important for energy production in Mycoplasma species and the SNP in the pyruvate kinase- encoding gene may lead to the attenuation of M. bovis.

The different molecular function of transketolase (Mbov_0212) include metal ion binding and transferase activity, and it is a key enzyme in the non-oxidative branch of the pentose phosphate pathway that transfers a two carbon glycolaldehyde unit from a ketose donor to aldose-acceptor sugar (Jores et al., 2009). Moreover, transketolase is an immunodominant membrane protein that may be sed as a biomarker for the serological diagnosis of contagious agalactia caused by M. mycoides subsp. Capri (Corona et al., 2013).

Hence, this protein may be involved in the immunogenicity and virulence of M. bovis.

ABC transporter proteins have different functions, including ATP binding, ATPase activity, and catalyzing the transmembrane movement of substances, such as importing sugars, amino acids, peptides, metal ions, and phosphates, and effluxing toxins, drugs, and proteins

(Higgins et al., 1986). The Mbov_0742 gene, which contained a SNP only in strain P180, encodes

a glycerol ABC transporter protein. The Mbov_0581 gene, which had an indel only in strain

P180, encodes multiple sugar ABC transporter protein. Both genes are considered to encode virulence related factors in the VFDB. In addition, the Mbov_0134 and Mbov_0018 genes encoding ABC proteins responsible for the uptake of simple sugar and spermidine/putrescine were found to have SNPs in all three attenuated strains.

NADH dehydrogenase (Mbov_0299) is considered to be a virulence- related factor. It generates energy by transferring electrons from NADH (oxidation) to quinine. NADH and

NADPH are possibly essential for the growth of M. suis (Guimaraes et al., 2011). In

Mycobacterium tuberculosis, a mutant lacking NuoG, a subunit of the type 1 NADH dehydrogenase complex, exhibited attenuated growth in vivo (Blomgran et al., 2012). Hence, this gene mutation might be significantly related to the further attenuation of M. bovis strain

P180.

Phosphate acetyltransferase (PTA) (Mbov_0567) catalyzes the formation of acetyl phosphate and acetyl CoA, which are used in many metabolic pathways. For example, taurine and hypotaurine metabolism, pyruvate metabolism, propanoate metabolism, and methane metabolism. Acetyl phosphate regulates various cellular processes, including cell division, outer membrane protein expression, osmoregulation, and biofilm development. Moreover, acetyl phosphate is required for the activation of the Rrp2-RpoN-RpoS pathway, which serves as a global signal in bacterial pathogenesis by activating virulent genes (Xu et al., 2010). Because of a

SNP in Mbov_0567, a proline residue was converted to threonine, resulting in polarity change that might be significant in virulence attenuation.

Membrane proteins influence cell shape, cell division, motility, and adhesion to host cells, and they are thought to be integrally involved in the pathogenesis of mycoplasmas. As is known, adhesion and invasion are generally considered to be virulence- associated processes.

M.bovis can adhere to and invade epithelial cells and immune cells. Membrane lipoproteins, such as Vsps, enolase, and Vpmax, play a significant role in the adhesion of M. bovis to host cells (Burki et al., 2015). Five genes encoding membrane proteins were shown to have SNPs, and four of them were predicted to encode secretory proteins with signal peptides, including

Mbov_0393, Mbov_0525, Mbov_0579, and Mbov_0797. Among them, Mbov_0797 encodes a

Vsp, and it was predicted to be a virulence- related factor, Mbov_0579 was predicted to encode the ADP- ribosyltransferase CDTa, which contains functional domains of the community- acquired respiratory distress syndrome toxin of M. pneumoniae (Kannan et al., 2014).

Mycoplasma bovis Immune function

Infusion of lysed M. bovis into the udders of cows resulted in severe, acute mastitis characterized by an eosinophilic response (Mosher et al., 1968). Infusion of 0.9, 9.0, and 18.0 mg of polysaccharide toxin into the udders of cows resulted in increased white cell counts in the milk of, respectively, 10, 100, and 175 times that of controls infused with PBS. Rectal temperature also increased. Physical examination of the inflamed quarters showed large, firm masses, and the milk obtained from these quarters was thick and ropy. Histologic examination of the inflamed tissue revealed many eosinophils and polymorphonuclear leukocytes in the alveoli, symptoms characteristic of mycoplasma mastitis caused by M. bovis (Mosher et al.,

1968). Intradermal injection of 0.1mg of the polysaccharide toxin into guinea pigs resulted in red swelling reactions measuring approximately 1.0cm in diameter approximately 2 hours after

inoculation. Maximum reactions were usually reached by 6 to 8 hours and subsided completely within 24 hrs. Histologic examination of the reaction sites showed edema with large influxes of eosinophils. Being a polysaccharide, it also evokes an eosinophilic response that is not observed in bovine mastitis caused by Staphylococcus, Streptococcus, or other bacterial agents. Since some dextrans activate complement by the alternative pathway (Pillemer et al., 1955), the finding that M. bovis toxin activates complement suggests a mechanism by which it may cause an inflammatory reaction. Activated complement is known to increase the vascular permeability of small blood vessels, which may account for the edema, and is chemotactic for leukocytes (Eisen, 1974), which may cause the accumulation of eosinophils at the reaction site.

The increase in vascular permeability caused by this toxin may also play a role in clinical cases of bovine mastitis by allowing for the widespread dissemination of the organism throughout the mammary gland.

Some species of mycoplasmas are believed to produce hydrogen peroxide. The hydrogen peroxide reacts with iron producing hydroxyl radicles which cause lipid peroxidation and oxidative damage to cell membranes. The amount of hydrogen peroxide produced appears to be dependent on the strain of M. bovis (Khan et al., 2005). When reactive oxygen metabolites are generated in an amount in which they exceed the body’s ability to safely detoxify them via antioxidant mechanisms, oxidative stress occurs. Oxidative stress then contributes to the onset of periparturient disorders in dairy cattle (Miller, 1993). Normal metabolism produces hydroxyl radicals, which are mutagenic in the body. Lipid peroxidation also naturally gives rise to mutagenic lipid epoxides, lipid hydroperoxides, and peroxyl radicals.

Ames et al. (1993) demonstrated in vitro that leukocytes and macrophages could inhibit

proliferation of varying lymphocyte subtypes. Proliferation is inhibited via the production of reactive oxygen intermediates, as well as prostaglandin estrogen (E2) and nitric oxide (NO).

Chirase et al. proposed in 2003, that increased stress on steers through marketing and transportation to the feedlot attributes to oxidative stress in the cattle overall. This increase in oxidative stress reduces antioxidant defense capacity of the animal, increases total lipid peroxidation overall resulting in increased susceptibility of cattle to BRD upon arrival in the feedlot. Serum total antioxidant capacity (TACA) is used to measure the reductant capacity/capability of the body. It was found that overall TACA decreased after transport inferring an overall decrease in the antioxidant defense system.

Gardner et al. took this finding further and found that nutritional stress is prevalent in calves that have been transported long distances particularly when moving from auction to feedlot. Some antioxidants are nutrients that can become depleted during marketing and transportation. There is a positive correlation between pre-transit and post-transit antioxidant capacity. This infers that calves with decreased antioxidant capacity after marketing most likely arrive at the feedlot with severely decreased antioxidant capacity (Sies, 1992). A decrease in antioxidant concentrations could cause a decreased ability of the calves to detoxify reactive oxygen species, therefore weakening their immune system.

Mycoplasma bovis is capable of adhering to neutrophils and embryonic bovine lung cells, capable of invading the respiratory epithelium. Once M. bovis invades the tracheal epithelium, it attaches to host receptors on airway cells promoting initial colonization of host tissues (Thomas, 1987). Partial binding inhibition (reduction by 36%) occurred using partially

trypsinized M. bovis cells (Thomas, 2003). This highlights the possible importance of trypsin- resistant proteins for M. bovis adherence.

Once inside the cells, the immune mechanisms is not well known. The role of the innate immune system in the lung remain largely unknown. M. bovis adheres to neutrophils, therefore, inhibiting oxidative burst, chemiluminescence, and degranulation in affected cells

(Finch and Howard, 1990; Thomas et al., 1991). The impaired neutrophils negatively affect the function of the rest of the immune system. However there is probably little direct effect on mycoplasmal infection propagation.

It is assumed that an effective antibody response might block M. bovis attachment, opsonize M. bovis, or activate complement-mediated killing. Once opsonized the classical pathway is activated in the presence of either IgG1 or IgG2 leading to dose dependent killing of

M. bovis. In this experiment, IgG1 was more effective at killing M. bovis than IgG2 (Howard,

1981). In naturally infected calves M. bovis specific IgM and IgG are abundantly found in serum.

However, in nasal secretions and bronchoalveolar fluid only low levels of M. bovis specific IgM and IgG are seen with an abundance of IgA (Boothby et al., 1983). In experimentally infected cattle CD8+ T cells were activated with lower response from CD4+ and γδ T cells based off lymphocyte blastogenesis and overall enhanced expression of IL-2 receptor α (CD25) (Vanden

Bush and Rosenbusch, 2003). This result suggests a Th2 predominant response.

The variations in Vsp expression on the M. bovis surface are proposed to play a large role in its evasion of the humoral immune response. When M. bovis bacteria with serum containing M. bovis antibody the expression of Vsp on M. bovis surface was altered. Reduced

expression of Pmb67 also occurred (Le Grand et al., 1996). M. bovis can suppress in vitro proliferation of lymphocytes to mitogens (Finch and Howard, 1990; Thomas et al., 1990). It also promotes apoptosis of lymphocytes in vitro (Vanden Bush and Rosenbusch, 2002). Inhibiton of mitgen-induced lymphocyte function is controlled via a Vsp-L (M. bovis lympho-inhibitory peptide (Mb-LIP)) (Vanden Bush and Rosenbusch, 2004).

During mycoplasma infections, cell-surface lipoproteins are the preferential target of the humoral immune response. P35 lipoproteins from Mycoplasma penetrans is the antigen recognized preferentially by the host immune system (Grau et al., 1995; Neyrolles et al., 1999).

Additionally, the lipoproteins of M.gallisepticum have been shown to be the most active immunogens during experimental chicken infections (Jan et al., 1995) and similar results have been obtained with mycoplasmas that infect cattle, including M.mycoides subsp. Mycoides SC

(el Abdo et al., 1998) and M .bovis. The greater immunogenicity of lipoproteins compared with other mycoplasma antigens is probably caused by their surface exposure and the presence of the amino-terminal lipoylated structure.

The term bacterial ‘modulin’ has been proposed to describe molecular moieties that induce cytokine synthesis with pathological consequences (Henderson et al., 1996).

Mycoplasmas produce at least three classes of modulins: lipoproteins and lipids (Razin et al.,

1998; Rottem and Naot, 1998) an in some strains a polypeptide superantigen (Cole et al., 1996).

Many studies have shown that mycoplasma lipoproteins stimulate monocytes and induce the secretion of pro-inflammatory cytokines, interleukin 6, and IL-1 (Razin et al., 1998;

Rawadi and Roman-Roman, 1996). Comparison of these effects of intact lipoproteins with those

of proteinase-K-treated lipoproteins reveals that the lipoylated amino terminus is responsible for the modulation activity (Brenner et al., 1997).

Human Toll-like receptor 2 (TLR-2) is the macrophage cell-surface receptors for LPS and lipoproteins (Brightbill et al., 1999; Aliprantis et al., 1999; Qureshi et al., 1999). LPS signaling via human TLR requires the LPS-binding protein (LBP) and is increased by soluble CD14 provided from the serum or co-expressed as a membrane-bound form in TLR-positive cells (Kirschning et al., 1998; Yang et al., 1998). LBP is thought to transport LPS monomers to host cells from LPS aggregates. After activation of TLR by LPS, the signal is transmitted across the plasma membrane, resulting in the activation of nuclear factor κβ (NF- κβ) and the regulation of responsive genes.

The synergy between lipoproteins and LPS in vivo in the induction of lethal shock and the production of pro-inflammatory cytokines suggests that lipoproteins and LPS activate macrophages in different ways (Zhange et al., 1998), perhaps as a result of interactions with different members of the TLR family.

Genetic mechanisms of lipoproteins variation in mycoplasmas includes: variation in the repertoire of lipoproteins expressed by mycoplasmas has been observed in vivo (Talkington et al., 1989; Levisohn et al., 1995); surface antigen variants of M. bovis and M. hominis can be selected in vitro by the addition of antibodies to the culture medium (Le Grand et al., 1996;

Jensen et al., 1995); the accessibility of antibodies to a mycoplasma colony depends on the side of the antigen (the number of carboxy-terminal repeats) (Levisohn et al., 1995). Elongated

surface lipoproteins protect mycoplasma cells from growth-inhibiting antibodies (Citti et al.,

1997).

If the variation of lipoproteins is a means of evading the host immune response, then the repertoire of expressed lipoproteins should vary during chronic infection. This is true in some mycoplasma species. It is often found that the expression of virulence factors and proteins involved in cell tropism is subject to extensive variations (Deitsch et al., 1997). Some experimental data have indicated that the variation of mycoplasma surface lipoproteins could be associated with changes in haemadsorptions. Surface variation could be considered a more general mechanism that allows the microorganism to cope with a fluctuating environment. This is also supported by the fact that surface variation also occurs in microorganisms that are not exposed to the host immune system (Deitsch et al., 1997).

Environmental survival

M. bovis can survive in the environment for several days when protected from sunlight.

Viable M. bovis has been found at 4˚C in sponges or milk for 2 months. In water viable M. bovis has been found for 2 months but with greater sensitivity to higher temperatures, 1-2 weeks survival at 20˚C and 1 week at 37˚C (Pfuzner, 1984). In other material M. bovis has been found on manure for 37 days, cotton for 18 days, straw for 13 days, and wood/stainless steel for 1-2 days (Ruffo et al., 1969).

The formation of biofilms is when inactive bacteria attach to a substrate, or each other, and then often become surrounded by an extracellular polysaccharide matrix. By forming biofilms, many types of bacteria can increase their resistance to host defense mechanisms and

become more resistant to environmental stress. For bacteria, environmental stress includes resistance to antimicrobials, antibiotics, and phagocytes. In general, biofilms are typically associated with heat resistance, protection from desiccation, and antimicrobial resistance.

Specifically, for M. bovis, the formation of biofilms is strain dependent with a strong correlation to the combination of Vsp genes being expressed. Strains expressing VspF typically have poor biofilm formation whereas strains expressing VspO or VspB typically have very durable biofilms (Caswell and Archambault, 2008; McAuiffe, 2006). It is believed that M. bovis biofilms release phagocytic enzymes that can further damage the surrounding tissue, therefore increasing host infection. Formation of these biofilms also enhances M. bovis’ ability to cause chronic infections because of their ability to periodically release planktonic cells. In experimental studies, M. bovis biofilms were formed as early as 20 hours post inoculation on glass coverslips.

Bovine Respiratory airway

The tracheobronchial tree in cattle is relatively long. This increases the amount of dead space volume when comparing to other animals. With increased dead space the amount of fresh oxygen that is delivered to the lung is affected, increasing the chance of alveolar hypoventilation because of particle obstruction. The increased dead space allows for a slightly larger increased surface area for particle deposition and an overall increase in the frequency of inhaled vapors and particulate matter (Ackermann, 2010).

The bovine lung contains interlobular septae with limited interdependence, increased resistance and decreased compliance. Collateral ventilation is reduced due to the lack of

broncho-alveolar communication (channels of Lambert), alveolar pores (pores of Kohn), and intrabronchiolar connections (channels of Martin). The lack of collateral ventilation promotes atelectasis, and these areas of the lung remain consolidated therefore lacking functional gas exchange. The regions of atelectasis are under hypoxic vasoconstriction prompting arterioles to shunt blood flow away from these areas to areas of the lung with better gas exchange

(Ackermann, 2010).

In lungs of calves after experimental respiratory infection with a clonal variant of M. bovis type strain PG45 an acute inflammatory response develops within 48hr post infection and that variation in expression of M. bovis Vsp antigens occurs in vivo. In cattle, both alveolar macrophages (Ams) and pulmonary intravascular macrophages (PIMs) are recognized by the

CD68 antibody used in the present study (Singh et al., 2004). Lung macrophages are known to produce several pro-inflammatory cytokines (TNF-α, IL-8, and IL-18) (Delclaux and Azoulay,

2003). TNF-α is thought to be a key mediator in eliciting the recruitment of neutrophils to the lung in mand and cattle as well (Zhang et al., 2000; Singh et al. 2004). In vitro studies showed that bovine Ams produce TNF-α after infection with M. bovis strain PG45 (Jungi et al., 1996).

Following respiratory infection with M. bovis, a specific immune response resulting in the production of antibodies to M. bovis Vsp antigens occur (Brank et al., 1999). Furthermore, a local pulmonary immune response develops in inflamed lungs which is characterized by hyperplasia of BALT also known as “cuffing pneumonia.” Positive immunohistochemical staining reactions for Vsp antigens by applying AR techniques was achieved for two of the three aAbs

(1A1, 1E5) tested. Negative immunohistochemical staining reaction with mAb I2 with specificity for the non-Vsp membrane surface antigen pMB67 of M. bovis is presumably due to the

destruction of antigenic epitopes recognized by this antibody because of formation fixation and processing techniques used for embedding of lung tissue samples. Vsp antigens were widely distributed within the lungs of the infected calves of this study and were already present at day two post infection within the cytoplasm of numerous macrophages and in other compartments of the lung (Buchneau et al., 2010).

Mucociliary apparatus

Submucosal glands and goblet cells are responsible for generating the air-surface liquid

(ASL) lining of the upper respiratory tract and pulmonary airway. This layer provides protection against inhaled particulate matter, aerosols, vapors, and microbials pathogens. ASL can be further broken down into two layers; a periciliary sol layer and a gel/mucus layer. The mucus layer is toward the airway lumen. The periciliary sol layer is found close to the apical cell surface. Here ciliary beat occurs because this layer is less viscous compared to the gel layer. The gel layer is formed of mucin glycoproteins and proteoglycans that are secreted by goblet cells and submucosal glands. Chronic inflammatory conditions increase the amount of the gel layer present (Ackermann, 2010). Dehydration enhances the viscosity of the ASL. Dehydration is frequently caused by aggregates of DNA and filamentous action that has accumulated from degraded neutrophils, leukocytes, necrotic epithelial cells, and bacterial biofilms (Ackermann,

2010).

The ASL is very active in the innate immune response. Epithelial cells secrete chloride ions causing water accumulation within the periciliary sol layer. When sodium is removed from the layer via the action of submucosal glands and serous cells allowing water to be reabsorbed.

The periciliary sol layer is also important in maintaining a pH that is slightly more acidic than blood (Ackermann, 2010).

In the ASL there are many molecules that are important in antimicrobial, inflammation, immunomodulation, as well as wound healing. Sodium and chloride levels regulate the osmotic gradient allowing for proper protein, enzyme, and peptide gradient. Lactoferrin binds and sequesters iron from microbial agents and lysozyme disrupts bacterial membranes. Defensins, cathelicidins, and other large proteins have been specifically identified in cattle (Ackermann,

2010).

In cattle, there are three beta-defensins including tracheal antimicrobial peptide (TAP), lingual antimicrobial peptide (LAP) and enteric antimicrobial peptide. The respiratory epithelial produce TAP and LAP, with TAP with greater expression levels. TAP and LAP form pores in membrane bacteria and enveloped viruses resulting in lysis. Beta defensins also trigger histamine release from mast cells enhancing acute inflammation, trigger the adaptive immune response via chemotactic interaction with dendritic cells, and induce wound healing with their mitogenic function in epithelial cells. Alpha defensins are produced by neutrophils and are only present after neutrophil recruitment and degranulation. Neutrophils also produce cathelicidin antimicrobial peptides that can also be released in low amounts by lung epithelial (Ackermann,

2010).

Particulate matter and pathogens that are inhaled and enter the deep lung are engulfed by alveolar macrophages. Upon activation macrophages, release cytokines, chemokines, and other intermediates that stimulate an immune response. Type 1 pneumocytes, line the alveoli,

are covered by a surfactant layer. This layer is composed of phosphatidylcholine and other phospholipids that help maintain proper surface tension, preventing alveolar collapse. Type II cells in the airway secrete surfactant. Intracellularly, surfactant proteins B and C are associated with surfactant and maintain surfactant folding/structure until release. Surfactant proteins A

(SP-A) and D (SP-D) are released by psuedostriated ciliated cells and have a strong antimicrobial and immunomodulatory role. SP-A and SP-D both have a carbohydrate recognition domain

(CRD) that binds the mannose residue inactive microbial agents. Once bound, the complex is taken up by alveolar macrophages. Once SP-A is released, it can activate macrophages, enhancing macrophage uptake and therefore the killing of microbial pathogens. SP-A can be taken up by the pulmonary lymphatic drainage system, via alveolar lumen liquid, entering the blood (Ackermann, 2010).

Since mycoplasmas lack a cell wall their cell membranes constituents are able to establish direct contact with target cell surface receptors. The ability of mAb Mb4F6 to significantly reduce M. bovis adherence indicated involvement of the corresponding antigen, a

26 kDa protein (P26), in adhesion processes. Interestingly, the different levels of expression of

P26 in strains 120 and 454 as seen in whole-cell protein patterns and immunoblots coincide with their differing adherence rates under identical conditions (equal numbers of mycoplasma cells per well, same density of EBL cell monolayer, practically equal H3 incorporation rates). The distinction in P26 expression could also partly explain that adherence of strain 454 was more inhibited in relative terms than that of strain 120 by equal amounts of the mAb. EBL cells could contain some of the same surface receptors for M. bovis cells as mature bovine lung.

Pathogenesis and localization of antigen

Immunohistochemistry studies have shown that caseonecrotic lesions contain M. bovis antigen throughout the foci with strong localization at the periphery in both the cytoplasm of macrophages and free in the caseonecrotic material itself. M. bovis antigen was also found in the bronchiolar lumen within eosinophilic cellular debris and between as well as within leukocytes (Thomas et al., 1986; Rodriguez et al., 1996; Khodakaram-Tafti and Lopez, 2004;

Gagea et al., 2006). In fibrinosuppurative bronchopneumonia with foci of coagulation necrosis,

M. bovis antigen is seen primarily at the periphery of foci along with the cytoplasm of necrotic neutrophils and macrophages. M. bovis antigen was occasionally seen within nonnecrotic neutrophils and free within edematous alveoli (Khodakaram-Tafti and Lopez, 2004; Gagea et al.,

2006).

The cytoplasm of macrophages in natural cases often contains M. bovis antigen

(Rodriguez et al., 1996; Kodakaram-Tafti and Lopez, 2004; Gagea et al., 2006) along with inside mononuclear cells in lymphoid follicles (Kodakaram-Tafti and Lopez, 2004). It is still unclear if the identified M. bovis bacteria have been phagocytosed and therefore killed by the macrophages or if the bacteria used the macrophage as a way to invade the immune response.

Co-infection

Most cases of M. bovis diseases also involve other bacteria. This interaction complicates identifying the specific role of M. bovis during infection. I one study M. bovis was the role pathogen isolated from the lungs of 34% of cases of fatal pneumonia (Byrne et al., 2001). Many cases are also infected with M. haemolytica, Pasteurella multocida, A. pyogenes or Mycoplasma

arginine (Byrne et al., 2001). Co-infection of M. bovis and M. haemolytica results in increased severity of clinical disease, especially when M. bovis predominates first. (Houghton and

Gourlay, 1983)

Mycoplasma bovis Arthritis

In arthritis cases caused primarily by M. bovis, it is believed infection is a result of pulmonary infection because arthritis is commonly associated with pneumonia in feedlot cattle with signs and symptoms of both occurring together (Gagea et al., 2006). In calves experimentally infected intranasally with M. bovis 14% to 66% develop arthritis with M. bovis present in affected joints (Gourlay et al., 1976; Thomas et al., 1986; Stipkovits et al., 2005).

Mycoplasma bovis Pneumonia

During a BRD outbreak in France, M. bovis was found in 8 out of 9 feedlots and in 60-

100% of tested cattle in each feedlot (Arcangioli et al., 2007). Two types of pneumonia are associated with M. bovis infections: contagious bovine pleuropneumonia and enzootic pneumonia in calves (calf pneumonia). Contagious bovine pleuropneumonia infection is frequently caused by Mycoplasma mycoides subspecies mycoides and is classified as a foreign animal disease. However, in calf pneumonia, mycoplasma species work in association with other pathogenic bacteria, causing primary and/or concurrent viral and/or bacterial infections.

Commonly associated bacteria include M. dispar, M. bovirhinus, M. bovigenitalium, Ureaplasma diversum. These associated bacteria are commonly found as normal flora of the upper respiratory tract. Pneumonia develops after the bacteria are introduced into the lower respiratory tract via impairment of mucociliary clearance and/or immune defense.

M. bovis adheres to and invades epithelial cells of the trachea and bronchioles.

Experimentally infected tracheal explants showed M. bovis antigen on the epithelial cell surface as well as in between epithelial cells (Thomas et al., 1987). The entry allows systemic infection via the blood. It is believed that adhesion is partly mediated by the Vsp along with P26 (Sachse et al., 1993, 1996). The adhesion appears to be temperature dependent, is disseminated via trypsin, and strength depends on M. bovis strain (Sachse et al., 1996; Thomas et al., 2003,

2005). Due to the strong variation between strains, it is believed that in some strains evasion across the epithelium occurs within 7 days of infection. However this is proposed to not occur in chronic stages (Thomas et al., 1986).

After intranasal and intratracheal challenge, M. bovis from the blood can be recovered via culture up to 9 days post infection (Thomas et al., 1986). In chronic infections, M. bovis has been recovered from the spleen, liver, and kidney 21 days after infection (Stipkovits et al.,

2005). M. bovis antigen has been isolated from macrophages that originated in hepatocyte cytoplasm, bile ductules, renal tubules, and occasionally in axons of facial nerves (Maeda et al.,

2003).

In vitro endothelial cells are susceptible to M. bovis infection. In these experiments there is not report of M. bovis antigen localization in vascular walls, limiting the experimental purpose of the study (Lu and Rosenbusch, 2004). After induced surface expression of adhesion molecular VCAM-1 and several cytokines, infected endothelial cells became activated.

However, with this, there was little necrosis of infected endothelial cells (Lu and Rosenbusch,

2004).

Bovine Respiratory Disease is estimated to account for 70- 80% of total morbidity. Total mortality is estimated at 40- 50% with the majority of cases occurring within the first 45 days upon arrival in the feedlot with M. bovis as a primary pathogen. Calves affected by BRD have lower average daily gains. In 1996, Smith and colleagues found that, during a 28-day receiving period, calves that became sick had an average daily gain that was 0.23 kg less than healthy calves. However, it is important to note that the pathogens present were not identified.

In a 2013 study of 3,519 white veal calves in Northern Belgium from 10 commercial veal farms identified M. bovis as the most prevalent bacteria. Of six identified pathogens, M. Bovis was identified in all 10 farms and affected 79.6% of all cattle. Calves that had been affected once by BRD had a reduction of 8.2kg and a reduction of 9.2kg if diarrhea occurred. Calves that were affected multiple times with BRD had an overall poorer carcass quality, primarily seen with undesirable red meat color at slaughter (Pardon, 2013).

In a study of 305 beef calves, 26.6% were positive for M. bovis on day 0 of arrival. By day

10, 62.7% were positive, and by day 42, 98.2% were positive for M. bovis. Calves that were M. bovis negative on day 0 gained more weight than calves that were culture positive. Conversely, calves that were negative on day 42 gained significantly more weight compared to M. bovis positive calves. Calves that were M. bovis negative at day 10 gained more weight per day compared to positive calves. This trend continued throughout the 42 days (Hanzlicek, 2011).

Cole et al. found that calves treated for BRD gained 0.12kg/d less for up to 56 days post- arrival. However, by day 149, the average daily gain was the same as the control due to compensatory gain. Hutcheson and Cole investigated compensatory gain further specifically

looking after the first month, found that although some compensatory gains occurred the cattle were still more susceptible to disease and lower overall production. Holland et al. reported that an additional 20 days on feed were necessary for heifers that were treated three times for BRD in order for them to reach similar body weights as untreated/healthy animals.

Longer transport distance for arriving calves is associated with increased morbidity presumably because of increased stress. Sanderson proposed a 10% increase in initial respiratory disease morbidity risk for each 160km (100miles) increase in transport (Sanderson,

2008). There have been associations between the pattern of calf sales at auction markets, time of year, and weather on the prevalence of shipping fever. Calves entering feedlot in November were at a 2-8 times greater risk of shipping fever than calves that were entering feedlots from

September or December. The risk of shipping fever was very low for calves that arrived at the feedlot during the first two weeks of September. However, the risk roughly doubled every two weeks until it reached its peak in November (Ribble, 1995).

Cattle that have been treated for undifferentiated bovine respiratory disease (UBRD) have been recorded to weigh less than healthy calves and to gain significantly less weight than control calves. Bateman et al. determined that cattle with UBRD gained 0.06kg/day less than untreated/healthy penmates. This correlates to affected animals being 11kg lighter after 180 days. It has been shown that out of 360 cases of UBRD, 5-28.9% of treated animals relapsed at least once. Of those that relapsed, 41% were treated again on or before day 5 post-arrival, and

17% of cattle that did not relapse were treated in the same time period. This highlights that the chance of relapse is 3.5 times greater when the calf is treated in the first four days post arrival

(Bateman et al., 1990).

When working with cattle used for meat purposes, how much meat is on the animal determines the animals end sale price. A Texas A&M review that looked at production costs from 1992-1995 found that calves that became ill with respiratory disease had a $0.19 to $0.35 less value per kilogram of purchase weight than healthy calves (Anonymous, 1993-1995). It is important to also take into consideration the addition cost of antibiotics, although this cost is hard to estimate because of different standard operating procedures.

Mycoplasma bovis Mastitis

Mastitis caused by M. bovis is one of the most prevalent dairy cattle diseases and typically affects all four quarters in lactating animals. The farm may also have problems with lameness, reproductive problems, calf pneumonia, and adult cow respiratory disease. Diagnosis is usually performed via milk culture or culture of udder parenchyma. In most cases of subclinical mastitis, M. bovis is spread mechanically through handling, milking machines, and udder wash solutions. Reports have also shown that the teat canal and genital tract are also possible routes of infection in which M. bovis can be passed to the fetus (Wrathall, 2007).

M.bovis has been isolated from the mammary gland of naturally infected cattle with mastitis. In 2002, out of 871 dairies in the US, 6.8% were M. bovis positive based on bulk tank samples (USDA, 2003). In other studies prevalence has been as great as 20% (Fox et al., 2003;

Wilson et al., 2009). How long mycoplasmas are shed in milk varies greatly depending on the study. Charmichael et al. found that shedding persists for up to six months whereas Cho et al. and Jasper et al. found similar results showing that shedding did not begin until eight months into infection with contamination for at least 13 months. In a small study by Biddle et al. with a

total of 10 cattle, who were all previously confirmed positive for mycoplasma species, did not shed any mycoplasma organisms 29% of the time. The cattle shed between 100 and 10,000

CFU/mL 10% of the time, between 10,000 and 100,000 CFU/mL 1% of the time, and >100,000

CFU/mL 60% of the time with average milk production at 19L/d. This study estimates that in

2003, the average cow in Washington State produced 33L/d with an average herd size of 330.

With these numbers, a single cow would have to shed between 10,000 and 100,000 CFU/mL for the bulk tank to contain at least 10 CFU/mL. This results in mycoplasma contaminated milk going undetected 39% of the time with only a single cow infected (Biddle et al., 2003). In 2002,

7.9% of dairy bulk tank milk samples tested positive for mycoplasma, with M. bovis accounting for 86% of positive mycoplasma samples (USDA-APHIS, 2002). Mycoplasma bovis positive samples were recovered from 76% of the states surveyed.

Microscopically acute infections of M. bovis which lead to abscess formation is caused by neutrophilic mastitis, which is characterized by neutrophilic infiltration of lobular interstitium, degeneration, and necrosis of alveolar epithelium and neutrophilic accumulation within alveoli. In subacute stages, macrophages predominate as inflammatory cells. Cows with acute infections are characterized by large drops in milk production heat production in all affected quarters, swollen and light brown in color of the udder (Brown et al., 1990). Upon palpitation, the parenchyma is often firm. Initially drawn milk appears normal but in a short amount of time will separate rapidly into a floccular deposit and clear supernatant. Since M. bovis mastitis is not easily detected, minor mastitis cases are often treated with antibiotics. It appears that the treatment is working because the associated bacteria are being killed.

However, the M. bovis is not affected by the antibiotic treatment which leads to chronic mastitis infections.

The inflammation at the acute stage is characterized by exudation of mostly eosinophils in the alveoli. Later on, an interstitial reaction with eosinophils and mononuclear cells, including plasma cells and lymphocytes, develops. The chronic stage is characterized by progressive fibroplasia around ductuli and alveoli, with hypertrophy of alveolar epithelium (Van der Molen and Grootenhuius, 1979). Some animals may return to secretion of normal-appearing milk within a few weeks; others may not do so until the next lactation, it at all. In either situation, the yield of milk is far below that prior to the disease (Boughton and Wilson, 1978).

Chronic mastitis infections are characterized by intermittent acute flare-ups or subclinical infections. Physiologically chronic mastitis cases have hyperplasia of alveolar and ductular epithelium, aggregation of lymphocytes within interstitium and around ducts, interstitial fibrosis, and lobular atrophy. Animals with subclinical infections can return to producing normal milk levels. However it should be noted that it is likely that M. bovis will be shed in the milk. Two closed herds in Finland were traced back to M. bovis contaminated semen that was used for artificial insemination, increased risk of mastitis is generally linked to larger herd size (Haapala, 2018 and Nicholas, 2016).

With mastitis, most of the economic loss is a result of decreased milk production and having to discard affected milk (either milk that is found to be contaminated with bacteria or antibiotics). Many times, the discarded milk is fed to calves as an economical alternative to milk substitutes. However, this can have detrimental effects on calf health because of their

underdeveloped immune system. In 1997, Waltz et al. were among the first to report that calves developed pneumonia and otitis media after consuming mycoplasma mastitic discarded milk. Butler et al. determined that pasteurization of discarded milk kills any mycoplasma species. However, in 1984, Pfutzner found M. bovis to be the only bacteria present in sterile milk. This information suggests that even pasteurized milk still has the possibility to be infected with mycoplasma, likely due to the varying strains.

Mycoplasma bovis was infused at a level of 3x104 cfu into 1 quarter on each of 10 cows.

Within 84 hours of infusion, M. bovis was recovered from all 10 experimentally challenged quarters, all of which remained infected throughout the study. Maximal number of M. bovis were recovered 193 hours (8 days) postinfusion. Milk samples from all 3 non-infused quarters on each animal were periodically collected and screened for the presence of M. bovis. Milk samples collected from these quarters 120 hours (5 days) post infusion were all free of M. bovis. By day 10 post infusion, M. bovis was recovered from 3 nonchallenged quarters, each from a different animal (Kauf et al., 2007).

Following infection, mean body temperature did not fluctuate throughout the study >_

1˚C from control measurements. The highest means body temperature recorded was 39.42 +-

0.20 ˚C and was observed at 156 hours postinfusion. M.bovis failed to elicit a febrile response, which is generally defined as an increase of >1 to 1.5 ˚C in body temperature (Ryan and Levy,

2003) and in cattle has been characteristically defined as temperatures >39,5 ˚C (Drillich et al.,

2006). Transient neutropenia and sustained lymphopenia and thrombocytopenia were observed within 84 hours of intramammary infusion of M. bovis. Decreased concentrations of circulating neutrophils were observed from 84 to 168 hours postinfusion. Significant and

sustained decreases in circulating concentrations of lymphocytes and platelets were observed from 84 hours postinfection until the end of the study (Kauf et al., 2007).

The systemic response of M. bovis is also characterized by the induction of acute phase protein synthesis of LBP and SAA. Blood LBP concentrations increased within 96 hours of M. bovis intramammary infusion and remained elevated throughout the study. Maximal blood concentrations of LBP were observed at 168 hours postinfection. Blood SAA concentrations were increased when compared to control. Blood SAA concentrations remained elevated throughout the study. Local sign of inflammation, milk was screened for increases in SCC.

Increased milk SCC were evident within 66 hours of M. bovis infection and remained elevated throughout the study. Maximal elevations in milk SCC were observed 90 hours postinfusion

(Kauf et al., 2007).

To determine if M. bovis could alter the integrity of blood- mammary gland barrier, milk concentrations of 2 blood derived proteins, BSA and LBP, were assayed via ELISA. Increases in milk BSA concentrations were initially detected 78 hours postinfection and were sustained from

90 hours until the end of the study. Maximal concentrations of milk BSA were detected on the final day of the study. Increases in milk LBP were initially detected at 90hr postinfection and were sustained from 102 hours until the end of the study. Increases in milk LBP were highly correlated with increases in milk BSA and circulating concentrations of LBP (Kauf et al., 2007).

To determine if M.bovis could evoke localized complement activation and IL-8 production, milk samples were assayed via ELISA for C5a and IL-8. Milk C5a concentrations were transiently elevated at 102 and 126 hours post infection and were consistently elevated from

144 hours until the end of the study. Maximal concentrations of C5a were detected at 216 hours postinfection. Milk IL-8 concentrations initially increased 120 hours after M. bovis infections and remained elevated for >36 hours (Kauf et al., 2007).

To assess the ability of M. bovis to evoke a proinflammatory cytokine response, TNF-α,

IL-1β, and TGF-α concentrations were determined. Initial elevations of TNF-α and IL-1β were observed between 96 and 102 hours and 90 and 102 hours postinfection, respectively, and sustained increases in both cytokines were detected from 120 hours until the end of the study.

Peak concentrations of TNF-α and IL-1β were both detected at 216 hours postinfection.

Increases in milk TGF-α concentrations were initially observed 90 hours after infection and were sustained from 102 hours until the end of the study. Peak concentrations of IFN-γ were detected 168 hours after infusion of M. bovis. Milk IL-12 concentrations increased within 78 hours of infection and remained elevated throughout the study. To assess the ability of M. bovis to evoke a counter regulatory anti-inflammatory cytokine response, TGF-β1, TGF-β2, and IL-10 concentrations were determined in milk samples. Significant increases in TGF-β1 were observed at 216 hours and 240 hours postinfection. Significant increase in TGF-β2 was detected only in milk from the final sampling of day 10. A transient increase in IL-10 was observed at 78 hours post infection, and sustained increases were detected from 90 hours until the end of the study

(Kauf et al., 2007).

There is a profound difference in the lag time between initial intramammary infusion and the earliest time at which bacteria growth reaches concentrations that are detectable in milk. It was not until 84 hours after infusion that all quarters had detectable amounts of M. bovis. M. bovis strain-dependent differences in the ability to spread through the blood may

account for the differential rates of infectivity of nonchallenged quarters reported in the current and previous studies. M. bovis was not detected in milk from those quarters until 10 to

15 days after infection of the single experimental quarter (Bennet and Jasper, 1978; Boothby et al., 1986; Byrne et al., 2005). Maximal increases in milk SCC occurred within 6 hours of initial decreases in circulating neutrophils. The prolonged decrease in circulating concentrations of these cells and sustained increases in milk SCC may reflect the continual attempt of the host cell to recruit effector cells to control and eradicate M. bovis, which persisted in the gland at high concentrations throughout the study (Kauf et al., 2007).

Changes in milk BSA concentrations have been shown to parallel those of IgG; the increase in milk BSA concentrations is consistent with previous studies demonstrating increases in milk IgG concentrations following M. bovis infection. M. bovis intramammary infection was also characterized by sustained activation of complement and production of cytokines TNF-α,

IL-1β, TGF-α, IFN-γ, IL-12, IL-10, TGF-β1, and TGF-β2. Increases in milk SCC, cytokine production and complement activation were all detected >66 hours after M. bovis infusion. Once M. bovis reaches a critical amount the innate immune system seemingly responds by mounting an inflammatory response. Prolonged inflammatory response was observed and may represent persistent, but futile, attempts of the innate immune system to control M. bovis (Kauf et al.,

2007).

Milk acidification lowers the milk pH so that it is unsuitable for bacterial growth and survival. Milk acidification, which involved lowering the pH of milk to a level that is unsuitable for bacterial growth and survival but still of nutritional benefit to calves (Anderson, 2008). Milk maintained at 23˚C and incubated at 37 ˚C saw a rapid decline in M. bovis growth with no viable

organisms detectable at 24 hours. Although M. bovis was not shown to proliferate in milk, its ability to remain viable in milk for up to 8 hours at ambient temperature explains how contaminated milk is able to infect calves, as milk is often fed within a couple of hours of collection. The milk has the potential to become reinoculated once placed into nonsterile collection and feeding equipment. Therefore, acidification of the milk has benefits in providing a continous preservative effect when combating the challenging issue of bacterial contamination, commonly experienced when feeding calves. Milk acidification against M. bovis strain (ATCC 25523), using Salstop with a pH 3.5 and pH 4, led to the elimination of viable M. bovis after just 1 hour of exposure time (Parker et al., 2016). Ideal pH for the growth of M. bovis in broth is 7.6 (Nicholas et al., 2008).

Chapter II. Essential Oils

Background

In spite of modern improvements in slaughter hygiene and food production techniques, food safety is an increasingly important public health issue (WHO, 2018). It has been estimated that as many as 30% of people in industrialized countries suffer from food borne diseases each year and in 2000 at least two million people died from diarrheal disease worldwide (WHO,

2002). There is still a need for new methods of reducing or eliminating food borne pathogens, possibly in combination with existing methods (Leistner, 1978). At the same time, Western society appears to be experiencing a trend of ‘green’ consumerism (Tuley de Silva, 1996; Smid and Gorris, 1999), desiring fewer synthetic food additives and products with a smaller impact on the environment. Furthermore, the World Health Organization has recently called for a

world-wide reduction in the consumption of salt in order to reduce the incidence of cardiovascular disease (WHO, 2002). If the level of salt in processed foods is reduced, it is possible that other additives will be needed to maintain the safety of foods. There is, therefore, score for new methods of making food safe which has natural or ‘green’ image. One such possibility is the use of essential oils as antibacterial additives.

Essential oils are steam-volatile, or organic solvent extracts, of plants. They are commonly derived from herbs and spices, and also present, to some degree, in many plants for their protective role against bacterial, fungal, or insect attack. They are mainly cyclic hydrocarbons and are alcohol, aldehyde or ester derivatives. The main effects of essential oils in the rumen involve reduction of protein and starch degradation and inhibition of amino acid degradation due to selective action on certain rumen micro-organisms (Hart et al., 2008). The mode of action suggested for essential oils is an effect on the pattern of bacterial colonization of substrates, in particular, starch rich substrates as they enter into the rumen. A second possible mode of action is their inhibition of hyper ammonia producing bacteria (HAB) involved in amino acid deamination (Wallace et al., 2002). The HAB are present in low numbers in the rumen, but they possess a very high deamination activity, producing ammonia (Wallace et al.,

2002). The reduction of protein, and starch degradation and inhibition of HAB bacteria by essential oils could be beneficial nutritionally in high protein diets (Patra and Saxena, 2009).

Terpenoids are terpenes with added oxygen molecules, or that have had their methyl groups moved or removed by specific enzymes (Caballero and Trugo, 2003). Thymol, carvacrol, linalool, methol, acetate, and citronellal are the most common and well-known terpenoids. The antimicrobial activity of most terpenoids is related to their functional groups, and the hydroxyl

group of the phenolic terpenoids and the presence of delocalized electrons are important elements for their antimicrobial action.

Phenylpropenes are named as such because they contain a six-carbon aromatic phenol group and a three-carbon propene tail from cinnamic acid, which is produced during the first step of phenylpropanoid biosynthesis. These compounds represent a relatively small portion of essential oils including eugenol and cinnamaldehyde. Most antimicrobial activity of eugenol can be ascribed to the presence of a double bond in the α, β positions of the side chain and to a methyl group located in the γ position (Hung and Fahey, 1983). The antimicrobial activity of the phenylpropenes also depends on the type and number of substitutions on the aromatic ring and on the microbial strain and conditions in which the essential oil is tested (Pauli and

Kubeczka, 2010).

Historical use of essential oils

The term ‘essential oil’ is thought to derive from the name coined in the 16th century by

Paracelsus von Hohenheim; he named the effective components of a drug Quinta essential

(Guenther, 1948). An estimated 3,000 EOs are known, of which about 300 are commercially important- destined chiefly for the flavors and fragrances market (Van de Braak and Leijten,

1999). It has long been recognized that some EOs have antimicrobial properties (Guenther,

1948; Boyle, 1955). The antibacterial properties of essential oils and their components are exploited in such diverse commercial products as dental root canal sealers (Manabe et al.,

1987), antiseptics (Bauer and Garbe, 1985; Cox et al., 2000) and feed supplements for lactating sows and weaned piglets (Van Krimpen and Binnendijk, 2001; Ilsley et al., 2002).

Although spices have been used for their perfume, flavor and preservative properties since antiquity (Bauer et al., 2001), of known EOs, only oil for turpentine was mentioned by

Greek and Roman historians (Guenther, 1948). Distillation as a method of producing EOs was first used in the East (Egypt, India, and Persia) (Guenther, 1948) more than 2000 years ago and was improved in the 9th century by the Arabs (Bauer et al., 2011). The first authentic written account of distillation of essential oil is accredited to Villanova (ca. 1235-1311), a Catalan physician (Guenther, 1948). By the 13th century, EOs were being made by pharmacies, and their pharmacological effects were described in pharmacopoeias (Bauer et al., 2001). However, their use does not appear to have been widespread in Europe until the 16th century from which time they were traded in London (Crosthwaite, 1998). Two Strassburg physicians, Brunschwig and

Reiff, mention only a relatively small number of oils between them; turpentine, juniper wood, rosemary, spike (lavender), clove, mace, nutmeg, anise and cinnamon (Guenther, 1948).

According to the French physician, Du Chesne, in the 17th century, the preparation of EOs was well known, and pharmacies generally stocked 15-20 different oils (Guenther, 1948). The use of tea tree oil for medicinal purposes has been documented since the colonization of Australia at the end of the 18th century, although it is likely to have been used by the native Australians before that (Carson and Riley, 1993). The first experimental measurement of the bactericidal properties of the vapors of EO is said to have been carried out by De la Croix in 1881 (Boyle,

1955). However, in the course of the 19th and 20th centuries, the use of EOs in medicine gradually became secondary to their use for flavor and aroma (Guenther, 1948).

Composition of Essential oils

Steam distillation is the most commonly used method for producing EOs on a commercial basis. Extraction by means of liquid carbon dioxide under low temperature and high pressure produces a more natural organoleptic profile but is much more expensive

(Moyler, 1998). The difference in the organoleptic profile indicates a difference in the composition of oils obtained by solvent extraction as opposed to distillation and this may also influence antimicrobial properties. This would appear to be confirmed by the fact that herbs

EOs extracted by hexane have been shown to exhibit greater antimicrobial activity than the corresponding steam distilled EOs (Packiyasothy and Kyle, 2002).

EOs can compose more than sixty individual components, also termed plant derived antimicrobials (Senatore, 1996; Russo et al., 1998). Major components can constitute up to 85% of the EOs, whereas other components are present only as a trace (Senatore, 1996; Bauer et al.,

2001). The phenolic components are chiefly responsible for the antibacterial properties of EOs

(Cosentino et al., 1999). There is some evidence that minor components have a critical part to play in antibacterial activity, possibly by producing a synergistic effect between other components. This has been found to be the case for sage (Marino et al., 2001), certain species of Thymus (Lattaui and Tantaouo-Elaraki, 1994; Paster et al., 1995; Marino et al., 1999) and oregano (Paster et al., 1995).

The composition of EOs form a particular species of plant can differ between harvesting seasons and between geographical sources (Arras and Grella, 1992; Marotti et al., 1994;

McGimpsey et al., 1994; Cosentino et al., 1999; Marino et al., 1999; Juliano et al., 2000; Faleiro et al., 2002). This can be explained, at least in part, by the formation of antibacterial substances from their precursors. P-Cymene (1-methyl-4-(1-methylethyl)-benzene) and γ-terpinene (1-

methyl-4-(1-methylethyl)-1, 4(cyclohexadine) are the precursors of carvacrol (2-methyl-5-(1- methylethyl)-phenol) and thymol (5-methyl-2-(1-methylethyl)phenol) in species of Origanum and Thymus (Cosentino et al., 1999; Jerkovic et al., 2001; Ultee et al., 2002). The sum of the amounts of these four compounds present in Greek oregano plants has been found to be almost equal in specimens derived from different geographical regions (Kokkini et al., 1997) and to remain stable in plants harvested during different seasons (Jerkovic et al., 2001). This indicates that the four compounds are biologically and functionally closely associated and supports the theory that thymol is formed via p-cymene from γ-terpinene in T. vulgaris (Kokkini et al., 1997). Generally, EOs produced from herbs harvested during or immediately after flowering possess the strongest antimicrobial activity (McGimpsey et al., 1994; Marino et al.,

1999). Enantiomers of EO components have been shown to exhibit antimicrobial activity to different extents (Lis-Balchin et al., 1999). For example, EO obtained from the seeds of coriander (Coriandrum sativum L.) has a quite different composition to EO of cilantro, which is obtained from the immature leaves of the same plant (Delaquis et al., 2002).

Testing Essential Oils

Testing and evaluations of the antimicrobial activity of essential oils is difficult because of their volatility, in-water solubility, and complexity. Four factors are especially important when testing essential oils: the assay technique; the growth medium; the micro-organism; and the essential oil.

The commonly used assay techniques can by classified by the fact whether or not they require a homogenous dispersion in water. A technique which does not require a homogenous

dispersion in water is the agar overlay technique using: discs, holes or cylinders as the reservoir; discs as the source of vapor. The reservoir containing the essential oil to be tested is brought into contact with an inoculated medium and, after incubation, the diameter of the clear zone around the reservoir (inhibition diameter) is measured. This method was originally designed to monitor the amount of antibiotic substances in crude extracts. Because all conditions cannot always be fulfilled, predictions are unreliable when using the proposed model (Janssen et al.,

1986).

Different types of reservoirs have been used: filter paper discs or cylinders placed on the surface of the medium, and holes punched in the medium. In all cases, the amount of oil and the diameter of the reservoir are important parameters. In some studies, the amount of oil adhering to a filter paper disc was estimated. Irrespective of whether a precisely measured volume of oil was applied to a disc or the disc was soaked in the oil, the amounts adhering to the disc did not differ. No difference was found between the amounts of a polar or a non-polar compound (Janssen et al., 1986).

In the case of cylinders, a certain minimum volume of oil is necessary to observe any inhibition, and there is an optimum volume beyond which no further increase in inhibition diameter takes place. However, the significance of these volumes in the description of the antimicrobial activity of an essential oil is not known. When cylinders and discs of similar diameter were used for the same oil, equal inhibition diameter was found (Banerjee et al.,

1976; Banerjee et al., 1978; Banjerjee et al., 1978).

In a homogenous system, various assay techniques can be applied. Most often MIC and minimal lethal concentration (MLC) values are determined. The principles of the techniques have been described by Kavanagh (Kavanagh, 1963). The MIC values can be determined either in a liquid or in a solid medium, the MLC values only in a liquid medium. The term MID

(maximum inhibitory dilution) has also been employed (Villar et al., 1986) because usually volume over volume percentages are used.

Use of a homogenous system also allows growth curves of a micro-organism to be recorded. In general, the turbidity is taken as an indication of bacterial density (Onawunmi et al., 1984; Simeon de Buochbery et al., 1976; Andrews et al., 1980). In every case, the growth curves are compared with those obtained in a medium without essential oil. Differences with respect to the lag time, the growth rate, and the maximum density attained are seen. For all oil types, the lag times for E. coli and Staph. Aureus increase with increased with increasing concentrations of oil. However, no decrease in the growth rate in the exponential phase was seen, and all curves eventually attained the same maximum density. In another study

(Onawunmi et al., 1984), the growth curves were not sufficiently neat to allow the calculation of lag times and/or growth rates.

The growth medium

The medium, the direct environment of the test organism, is an important factor in determining the antimicrobial activity of a substance. Medium constituents may react with essential oil components and activate of inactivating them. Thus, it is known that some

aldehydes, such as perillaldehyde, can react with the sulfhydryl groups in proteins (Kurita et al.,

1979).

In early studies on the agar overlay technique, the influence of the agar content on the solubility of some oil constituents was established. The higher the agar content, the lower the solubility and thus the smaller the inhibition zones (Schuermann and Spitzner, 1960).

The pH of the medium seems to play an important role as well. For phenols (Polster et al., 1986) and carboxylic acids, it is known that only the uncharged form is able to penetrate the microbial cell and to exhibit activity. It was found (Jay and Rivers, 1984) that the pH can also have an influence when testing neutral molecules. For Gram-positive, non-lactic bacteria the activities of eugenol, l-carvone, d-carvone, and methanol, respectively, were larger at pH 6 than at pH 8.

Tests of antibacterial activity of EOs in food systems

However well EOs perform in antibacterial assays in vitro, it has generally been found that a greater concentration of EO is needed to achieve the same effect in foods (Shelef, 1983;

Smid and Gorris, 1999). The ratio has been recorded to be approximately twofold in semi- skimmed milk (Karatzas et al., 2001), 10-fold in pork liver sausage (Pandit and Shelef, 1994), 50- fold in soup (Ultee and Smid, 2001) and 25- to 100- fold in soft cheese (Mendoza-Yepes et al.,

1997). Several studies have recorded the effect of foodstuffs on microbial resistance to EOs, but none appears to have quantified it or to have explained the mechanism, although suggestions have been made as to the possible causes. The greater availability of nutrients in foods compared to laboratory media may enable bacteria to repair damaged cells faster (Gill et al.,

2002). Not only are the intrinsic properties of the food (fat/protein/water content, antioxidants, preservatives, pH, salt and other additives) relevant in this respect- the extrinsic determinants (temperature, packaging in vacuum/gas/air, characteristics of microorganism) can also influence bacterial sensitivity (Shelef, 1983; Tassou et al., 1995). Generally, the susceptibility of bacteria to the antimicrobial effect of EOs also appears to increase with a decrease in the pH of the food, the storage temperature and the amount of oxygen within the packaging (Tassou et al., 1995, 1996; Skandamis and Nychas, 2000; Tsigarida et al., 2000). At low pH the hydrophobicity of an EO increases, enabling it to more easily dissolve in the lipids of the cell membrane of target bacteria (Juven et al., 1994).

It is generally supposed that the high levels of fat and/or proteins in foodstuffs protect the bacteria from the action of the EO in some way (Aureli et al., 1992; Pandit and Shelef, 1994;

Tassou et al., 1995). For example, if the EO dissolves in the lipid phase of the food, there will be relatively less available to act on bacteria present in the aqueous phase (Mejlholm and

Dalgaard, 2002). Another suggestion is that the lower water content of food compared to laboratory media may hamper the progress of antibacterial agents to the target site in the bacterial cell (Smith-Palmer et al., 2001).

A reaction between carvacrol, a phenolic component of various EOs and proteins has been put forward as a limiting factor in the antibacterial activity against Bacillus cereus in milk

(Pol et al., 2001). Carbohydrates in foods do not appear to protect bacteria from the action of

EOs as much as fat and protein does (Shelef et al., 1984). High water and/or salt level facilitates the action of EOs (Shelef et al., 1984; Wendakoon and Sakaguchi, 1993; Tassou et al., 1995;

Skanndamis and Nychas, 2000).

The physical structure of food may limit the antibacterial activity of EO. A study of the relative performance of oregano oil against S. typhimurium in broth and in gelatin gel revelated that the gel matrix dramatically reduced the inhibitory effect of the oil. This was presumed to be due to the limitation of diffusion by the structure of the gel matrix (Skandamis et al., 2000).

It is known that colonial growth restricts diffusion of oxygen (Wimpenny and Lewis, 1977) and cells situated within a colony may be shielded to a certain extent by the outer cells from substrates in the emulsion. If the oil droplets in a food emulsion are of the appropriate size, it could be possible of bacteria growth within colonies to be protected from the action of EOs in this way.

Eugenol and coriander, clove, oregano, and thyme oils were found to be effective at levels of 5-10ul g-1 in inhibiting L.monocytogenes, A.hydrophila and autochthonous spoilage flora in meat products, sometimes causing a marked initial reduction in the number of recoverable cells (Aureli et al., 1992; Stecchini et al., 1993; Hao et al., 1998; Tsigarida et al.,

2000; Skandamis and Nychas, 2001) whilst mustard, cilantro, mint and sage oils were less effective or ineffective (Shelef et al., 1984; Tassou et al., 1995; Gill et al., 2002; Lemay et al.,

2002). A high fat content appears to markedly reduce the action of EOs in meat products.

In view of the published data on EOs in foods, the following approximate general ranking (in order of decreasing antibacterial activity) can be made: oregano/clove/coriander/cinnamon>thyme>mint>rosemary>mustard>cilantro/sage. An approximate general ranking of the EO components is as follows (in order of decreasing antibacterial activity): eugenol>carvacrol/cinnamic acid>basil methyl chavicol>cinnamaldehyde>citral/geraniol.

Antioxidant and Anit-inflammatory activities of Essential oils

Probiotics, as living microorganisms, pose a significant challenge to feed manufacturers in terms of their viability/survival during feed processing. EOs, on the other hand, can be handled and included in the feed similarly to other feed ingredients. The tested Gram positive potential pathogenic strain were found to be more sensitive to the rested EOs than to the tested Gram-negative strains. The naturly identical EOs which appear to have the highest growth reducing capacity were carvacrol, cinnamaldehyde, and thymol. Citral, eugenol, and limonene were also active but less so compared to the above three. All three tested natural extracts exhibited a strong antimicrobial activity. This may, in part, be explained by the fact that thymol is one of the main components of oregano and thyme oil (Chorianopoulos et al., 2004).

Although the reductions in growth were in general statistically significant, they were small, and the biological relevance of this reduction remains, therefore, to be established. There is also a relative resistance of the tested beneficial members of the intestinal microbiota, lactobacilli and bifidobacterial (Ouwehand et al., 2010). L.plantarum has been found to be resistant to thymol, eugenol, and carvacrol at 300mg/l under aerobic conditions. L.acidophils, however, was highly sensitive to thymol and carvacrol under aerobic conditions at 300 mg/l (Si et al., 2006).

The antioxidant activity of EOs is another biological property of great interest because they may preserve food from toxic effects of oxidants (Maestrin et al., 2006). Moreover, essential oils being also able of scavenging free radicals may play an important role in some disease prevention. Increasing evidence has suggested that these diseases may result from cellular damage caused by free radicals (Aruoma, 1998; Kamatou and Viljoen, 2010).

If essential oils are able to scavenge some free radicals, they can also act as anti- inflammatory agents, because one of the inflammatory responses is the oxidative burst that occurs in diverse cells (monocytes, neutrophils, eosinophils, and macrophages). Phagocytosis of bacteria, which occurs during inflammation, is accompanied by a dramatic increase in oxygen consumption resulting in the formation of superoxide anion radical which is quickly converted to hydrogen peroxide spontaneously, or by the enzyme superoxide dismutase. Hydrogen peroxide can then also be reduced by transitions metal ions generating the hydroxyl radical, one of the strongest oxidizing agents that can rapidly react with polyunsaturated fatty acids, resulting in the production of peroxyl radicals. Peroxide hydrogen can also oxidise halide ions to hypochlorous acid, which is a strong oxidant that can react with amines producing chloramines, some of them being very toxic (Quinn and Gauss, 2004; Fridovich, 1997; Chen and Schopfer,

1999; Gomes et al., 1995). These ROS (reactive oxygen species). During an inflammatory process, reactive nitrogen species are generated. Nitric oxide is produced in large quantities by the inducible nitric oxide synthases in activated macrophages and neutrophils during defense and immunological reactions. However, this reactive species can also exert its toxicity by generating the peroxynitrite anions after reacting with the superoxide anion radical (Gomes et al., 2008; Kostka, 1995; Nagano, 1999; Miyasaka and Hirata, 1997).

ROS and RNS are generated in phagocytes in order to neutralize the invading organism, having, therefore, an important role in the host defense mechanism. Nevertheless, their over production may be responsible for damage at inflammatory sites. In addition, these reactive oxygen species play important roles in inflammation by being trigger elements or by being signaling messenger molecules. ROS and RNS act as modulators of proteins and lipid kinases

and phosphatases, membrane receptors, ion channels, and transcription factors, including nuclear factor-κβ (NK- κβ), which regulate the expression of key cytokines (Gomes et al., 2008).

Primary antioxidants can donate a hydrogen atom rapidly to a lipid radical, forming a new radical, which is more stable. Secondary antioxidants react with the initiating radicals (or inhibit the initiating enzymes) or reduce the oxygen level (without generating reactive radical species). Therefore, these secondary antioxidants can slow the rate of radical initiation reaction by eliminating of initiators. This can be performed by deactivating high energy species (singlet oxygen); absorbing UV light; scavenging of oxygen; chelating metal that catalyzes free radical reaction, or inhibiting enzymes, such as peroxidases, NADPH oxidase, xanthine oxidase, among other oxidative enzymes (Singh and Singh, 2008).

The substrate of oxidation may be individual lipids, lipid mixtures (oils), proteins, DNA, blood plasma, LDL, and biological membranes. Lipid oxidation is a complex reaction that can be generated via three different pathways: 1) Non enzymatic free radical-mediated chain reaction;

2) Non-enzymatic, non-radical photo-oxidations; 3) Enzymatic reactions (Miguel, 2010).

The first pathway leads to initiation of rapidly progressing, destructive chain reactions, generating hydroperoxides and volatile compounds, generally through a three-phase process: initiating, propagation and termination. The initiation phase involves the homolytic breakdown of hydrogen in the α-position relative to the fatty acid chain double bond, leading to the formation of an allyl radical. These species are highly unstable, short-lived intermediated that stabilize themselves by abstracting hydrogen from another chemical species or rapidly react with oxygen to form a peroxyl radical (propagation phase). In the propagation phase, peroxyl

radicals formed can further oxidize the lipid, producing hydroperoxides. These are stable via double-bond rearrangement (electron delocalization), originating conjugated dienes and trienes. These intermediated decomposed, originating alcohols, aldehydes, alkyl formats, ketones, hydrocarbons, alkoxyl radicals, and formic acid. All of these compounds are considered secondary products of lipid oxidation (Miguel, 2010).

In the inflammatory response, there is an increase of permeability of endothelial lining cells and influxes of blood leukocytes into the interstitium, oxidative burst, and release of cytokines (TNF-α). At the same time, there is also induction of the activity of several enzymes

(oxygenases, nitric oxide synthases, peroxidases) as well as the arachidonic acid metabolism. In the inflammatory process, there is also the expression of cellular adhesion molecules, such as intercellular adhesion molecules (ICAM) and vascular cell adhesion molecule (VCAM) (Gomes et al., 2008). The anti-inflammatory activity of essential oils may be attributed not only to their antioxidant activities but also to their interactions with signaling cascade involving cytokines and regulatory transcription factors, and on the expression of pro-inflammatory genes

(Murakami and Ohigashi, 2007).

Cinnamaldehyde is reported to inhibit the secretion of IL-1β and TNF- α within LPS or lipoteichoic acid (LTA) stimulated murine J774A.1 macrophages. Cinnamaldehyde also suppressed the production of these cytokines from LPS simulated human blood monocytes derived primary macrophages and human THP-1 monocytes (Chao et al., 2008). The anti- inflammatory effects of diverse combination of thyme (p-cymene and thymol as main components) and oregano (carvacrol) oils on mice with TNBS- induced colitis showed that some combinations lowered the amounts of IL-1β and IL-6 cytokines (Burkovska et al., 2007).

The inhibitory effect of some of the essential oils on the production of pro-inflammatory cytokines seems to be mediated by suppressing gene expression of these cytokines. Such was described by some authors because they found that the essential oils studied significantly suppressed the protein and mRNA expression of the cytokines in stimulated cells, assuming, therefore, that the inhibitory effect of these essential oils on the expression of the pro- inflammatory cytokines occurs mainly at transcriptional level (Dung et al., 2009; Yoon et al.,

2010; Dutra et al., 2009; Burkocska et al., 2007). Thyme essential oil, mainly constituted by p- cymene and thymol, only at high concentrations (5,000 mg/L) significantly inhibited total mRNA

IL-1β expression in the mouse colon in which colitis was induced by TNBS, not inhibiting IL-6 expression (Juhas et al., 2008). Cinnamaldehyde-mediated inhibition of cytokines production found by Chao et al. (2008), may be due to the reduction of ROS release as well as of those of

JNK and ERK within LPS stimulated J774A macrophages.

Peroxisome Proliferator- Activated Receptor (PPARs) are ligand- activated transcription factors that belong to the superfamily of nuclear hormone receptors, which also includes receptors for vitamin D, vitamin A, thyroid hormone, bile acids, and steroid hormones. The

PPAR subfamily comprises three isotypes, PPARα, PPARβ/δ, and PPARγ, which play various roles in lipid and carbohydrate metabolism, cell proliferation and differentiation, and inflammation

(Hotta et al., 2010; Mangelsdorf et al., 1995).

Phenols are able to donate H-atoms of phenol hydroxyl groups in reaction with peroxyl radicals that can produce stabilized phenoxyl radicals, thus terminating lipid peroxidation chain reactions. The antioxidant activity of phenols depends on the electronic and steric effects of the

ring, substituents, and the strength of hydrogen boding interactions between the phenol and the solvent (Pedulli et al., 1997; Lucarini et al., 2002; Snelgrove et al., 2001; Avila et al., 1995).

Many essential oils exhibit antioxidant and antimicrobial activities (Baratta et al., 1998;

Baratta et al., 1998; Burt, 2004). Phenols, such as thymol, carvacrol, and eugenol, and monocyclic hydrocarbons, such as terpinolene, belong to the most active natural antioxidants found in the essential oils (Yanishlieva et al., 1999; Juven et al., 1994; Ruberto and Baratta,

2000). However, due to their poor water solubility and the requirement of high concentration to reach a therapeutic effect, the efficiency of these compounds in treatment is limited.

The free radical scavenging activity was determined by the DPPH assay (Brandwilliams et al., 1995). Thymol, carvacrol, and eugenol derivatives scavenged the DPPH radical in a dose- dependent manner, and the DPPH radical scavenging activity was decreased in the following order: 4-allyl-2-(hydroxymethyl)- 6-methoxyphenol > eugenol> BHT> 4-(hydroxymethyl)-2- isopropyl-5-methylphenol> 4,4′-methylenebis(5-isopropyl-2-methylphenol)> 4-

(hydroxymethyl)-5-isopropyl-2-methylphenol> carvacrol> thymol. Eugenol and BHT reduce two or more DPPH radicals, despite the availability of only one hydrogen on a hydroxyl group. All derivatives are better antioxidants than the corresponding parent compounds according to

DPPH. Derivatives have lower antiproliferative activity and toxicity than starting compounds

(Mastelic et al., 2008).

Essential oils on ruminal function

Several EOs selected for their effectiveness against gut pathogens need not have toxic effects on commensals in the mixed colonic community at a concentration that would be

expected to suppress the growth of pathogens. Possibly beneficial commensals also colonize the small intestine at much lower abundance (van den Bogert et al., 2013). With the exception of the higher concentrations of thymol and geraniol, the EOs had minor or no effects on the fermentation, in terms of either species abundance of VFA formation. Specifically, thymol has major inhibitory effects on pathogens at 100p.p.m. but which did not affect the overall fermentation at this concentration in the present study (Thapa et al., 2012).

Oxygenated monoterpenes, particularly monoterpene alcohols and aldehydes, strongly inhibited growth and metabolism of rumen microbes, whereas monoterpene hydrocarbons slightly inhibited and, sometimes, stimulated the activity of rumen microbes. These findings were some of the first to demonstrate that the chemical composition of EO greatly influences their effects on activity of ruminal microorganisms (Oh et al., 1967, 1986; Nagy and Tengerdy,

1968).

Early work by Borchers (1965) showed that the addition of thymol to ruminal fluid (1g/l) containing casein resulting in an accumulation of amino acids and a decrease in ammonia N concentration, suggesting inhibition of amino acids deamination by ruminal bacteria. Borderick and Balthrop (1979) also observed that thymol inhibited deamination of amino acids to NH3-N.

More recently, McIntosh et al. (2003) observed a 9% reduction in the rate of amino acids deamination when casein acid hydrolysate was incubated in vitro for 48 hr in batch cultures of ruminal fluid collected from cows fed a silage-based diet supplemented with 1g/day of a commercial mixture of EO compounds. Newbold et al. (2004) also reported a reduction, 24%, in the in vitro rate of AA deamination when casein acid hydrolysate was incubated for 24 hr with ruminal fluid collected from sheep fed diets containing 100mg of EO. In addition, there is no

additional reduction in the rate of deamination when the ionophore monensin was added to the ruminal fluid, suggesting that the bacterial species affected by EOs were the same as those inhibited by monensin. EOs inhibited the growth of some (Clostridium sticklandii and

Peptostreptococcus anaerobius) hyper-ammonia producing (HAP) bacteria, but other HAP bacteria (Clostidium aminophilum) were less sensitive. Hyper-ammonia producing bacteria are present in low numbers in the rumen, but they posses a very high deamination activity (Russell et al., 1988). Wallace (2004) reported that the number of HAP bacteria was reduced by 77% in sheep receiving a low protein diet supplementation with EO at 100mg/day, but that EO had no effect on HAP bacteria when sheep were fed a high-proteins diet. Collectively, results of the studies by McIntosh et al. (2003), Newbold et al. (2004), and Wallace (2004) suggested that effects of EO on ruminal protein metabolism are on AA degradation and these effects are likely due to inhibition of HAP bacteria.

Other studies used the rumen in situ bag technique to investigate the effects of EO on ruminal N metabolism. Molero et al. (2004) used growing heifers to evaluate the influence of

EO (700mg/day) on in situ ruminal degradability of proteins in soybean meal, corn gluten feed, fish meal, green peas, sunflower meal, and lupin seeds. Of the five protein supplements examined, EOs only tended to reduce the effective ruminal protein supplements examined, EO only tended to reduce the effective ruminal protein degradability of lupin seeds, green peas, and soybean meal, and the reductions were too small to have any likely nutrition impact on the ruminal protein metabolism in the animal. Newbold et al. (2004) and Benchaar et al. (2006) reported no change in the kinetics of protein degradation from soybean meal incubated in the rumen of sheep or dairy cows supplemented daily with 110mg or 2 g of E0 respectively.

EOs and their components have been shown to affect ruminal N metabolism in a dose- dependent manner. Busquet et al. (2006) demonstrated that some EOs (anise oil, cade oil, capsicum oil, cinnamon oil, clove, bud oil, dill oil, garlic oil, ginger oil, oregano oil, and tea tree oil) and their main components (anethol, benzyl salicylate, carvacrol, carvone, cinnamaldehyde, and eugenol) markedly inhibited NH3-N concentration was, however, associated with a reduction in total volatile fatty acid (VFA) concentration, suggesting a reduction in overall fermentation of the diet. Since VFA are the principal source of energy for ruminants, decreasing ruminal VFA production could have adverse nutritional consequences if this effect was expressed in vivo. The flow of bacterial N was not changed by the addition of garlic EO or cinnamaldehyde (Busquet et al., 2005). Newbold et al. (2004) and Benchaar et al. (2006) observed no change in duodenal bacterial N flow of sheep and dairy cows fed daily dosages of

100mg and 2 g of EO respectively.

Protozoa engulf and digest large amounts of ruminal bacteria thereby decreasing net microbial protein flow from the rumen to the duodenum (Ivan et al., 2000). Protozoa also possess proteolytic and deaminating activities (Williams and Coleman, 1992). Thus, removal of protozoa from the rumen (defaunation) prevents recycling of N between bacteria and protozoa, which results in increased flow of microbial N from the rumen. Ando et al. (2003) reported that feeding 200g/day (30g/kg of total dietary DM) of peppermint to Holstein steers decreased the total number of protozoa. Mohammed et al. (2004) observed no change in the number of protozoa in ruminal fluid of steers supplemented with cyclodextrin encapsulated horseradish oil at 20 g/kg of dietary DM. McIntosh et al. (2003) observed that the bacteriolytic activity of rumen ciliate protozoa was unaffected in dairy cows supplemented with 1 g/day of EO.

Newbold et al. (2004) and Benchaar et al. (2007) reported that ruminal protozoa counts were not affected when sheep and dairy cows were fed at 100 and 750 mg/day of EO, respectively.

Supplementation of dairy cow diets with 1g/day of cinnamaldehyde had no effect on the number, or genetic composition, of ciliate protozoa (Benchaar et al., 2005). Cardozo et al.

(2006) observed that addition of a mixture of cinnamaldehyde (100mg/day) and eugenol

(90mg/day) to the diets of beef heifers increased numbers of holotrichs and had no effect on entodiniomorphs, but that there was no effect on numbers of these protozoal species when the mixture contained higher concentrations of cinnamaldehyde (600mg/day) and eugenol

(300mg/day).

Supplementation with EO or EO compounds has increased ruminal total VFA concentration, which may indicate improved feed digestion. Addition of 1.5mg/l of EO increased total VFA concentration in continuous cultures maintained at constant pH, although there was no concomitant increase in organic matter digestibility (Castillejos et al., 2005). Two in vivo studies with EO reported no effect when fed to sheep (110mg/day) or cattle (1g/day) on total VFA concentration or proportions (Newbold et al., 2004; Beauchemin and McGinn, 2006)>

It is possible that the effects of EO on total VFA concentration may depend on the composition of the diet. Benchaar et al. (2007) reported that RO (750mg/day) tended to increase total VFA concentration in the rumen of lactating cows when the diet contained alfalfa silage but tended to decrease total VFA concentrations when the diet contained corn silage. Mohammed et al.

(2004) reported that increasing levels (0.17 to 1.7 g/l) of cyclodextrin encapsulated horseradish linearly increased total VFA concentrations in batch cultures. When the same product was fed to cattle, there was a very small increase in total VFA, but no change in feed digestibility.

Overall, supplementation with EO or their components has caused either a decrease or no change in total VFA concentration in most studies. Whether VFA concentration decreases as a result of the antimicrobial effects of EO may be dose dependent. Busquet et al. (2006) studied effects of various plant extracts (anise oil, cade oil, capsicum oil, cinnamon oil, clove, bud oil, dill oil, garlic oil, ginger oil, oregano oil, and tea tree oil) and their main components (anethol, benzyl salicylate, carvacrol, carvone, cinnamaldehyde, and eugenol) on ruminal fermentation in a 24 hour batch culture. Each treatment was supplied at varying doses up to 3g/l of culture fluid. None of the EO or EO compounds increased total VFA concentration but, at the highest concentration, most treatments decreased total VFA concentration, a possible reflection of decreased feed digestion. Similar effects were reported by Castillejos et al. (2006) for eugenol, guaiacol, limonene, thymol, and vanillin using doses up to 5g/l. These EO compounds generally had no effect on total VFA concentration, with the exception of the highest dose, which decreased total VFA concentration in cultures for all compounds. Eugenol (500mg/l) reduced the proportion of propionate, without affecting total VFA concentration (Castillejos et al.,

2006). In vitro that effects of EO and their components of VFA profile are pH-dependent. At pH7 cinnamon EO and its main components cinnamaldehyde resulted in higher acetate to propionate ratio whereas, at pH 5.5, the acetate to propionate ratio was lower with cinnamon

EO and cinnamaldehyde (Cardozo et al., 2005).

When dairy cows were fed 750mg or 2g of EO daily no changes in DM intake, milk production, and milk components were seen (Benchaar et al., 2006, 2007). Similarly, supplementation of dairy cows with peppermint at 20g/kg DM had no effect on milk yield and milk composition (Hosoda et al., 2005). Yang et al. (2006) observed that addition of garlic

(Allium sativa, 5g/day) and juniper berry (Juniperus communis, 2g/day) oils to dairy cow diets had no effect on DM intake, milk production or milk composition. In these studies, the lack of effect of EO and their active components on milk performance was consistent with the absence of effects of these plant extracts on feed intake and ruminal fermentation. When cows were supplemented daily with 750mg of EO, there is no reported changed in milk FA profile

(Benchaar et al., 2007). Supplementing the same mixture at a higher concentration (2g/day) increased the concentration of conjugated linoleic acid (CLA), a health-promoting FA, in milk fat.

In beef cattle fed a silage-based diet supplementation with 2 or 4g/day of a commercial mixture of EO compounds (Vertan, IDENA, Sautron, France) consisting of thymol, eugenol, vanillin, and limonene there is no change in DM intake and ADG. However, the gain to DM intake ratio was affected quadratically with a dose of 2g/day maximizing feed efficiency.

Those EO and EO compounds that exhibit the highest antimicrobial activity against ruminal bacteria (carvacrol, oregano, thyme oils) are also generally the most potent against pathogens (Oussalah et al., 2007). In some instances, EO may also increase the sensitivity of bacteria to other antimicrobials (Rafii and Shahverdi, 2007; Santiesteban-Lopez et al., 2007) and it is possible that they may also enhance the sensitivity of pathogens within the digestive tract to other microbialcides. EO from peppermint was shown to exhibit activity against Giardia

(Vidal et al., 2007) a protozoa parasite that is highly prevalent in cattle (Olson et al., 2004). It is possible that EO, or their active components, may also have activity against other parasites that reside in the intestine such as Cryptosporidium, coccidia or nematodes. Effects of EO on parasites in the lower digestive tract would be dependent on the ability of the antimicrobial

components that they contain to remain active after passage through the rumen (Benchaar et al., 2007).

Mode of antibacterial action

Considering the large number of different groups of chemical compounds present in

EOs, it is most likely that their antibacterial activity is not attributable to one specific mechanism but that there are several targets in the cell (Skandamis et al., 2001; Carson et al.,

2002). An important characteristic of EOs and their components is their hydrophobicity, which enables them to partition in the lipids of the bacterial cell membrane and mitochondria, disrobing the structures and rendering them more permeable (Knoblch et al., 1986; Sikkema et al., 1994). Leakage of ions and other cell contents can then occur (Oosterhaven et al., 1995;

Gustafson et al., 1998; Helander et al.,1998; Cox et al., 2000; Lambert et al., 2001; Skandamis et al., 2001; Carson et al., 2002; Ultee et al., 2002). Although a certain amount of leakage from bacterial cells may be tolerated without loss of viability, extensive loss of cell contents or the exit of critical molecules and ions will lead to death (Denyer and Hugo, 1991). There is some evidence from studies with tea tree oil on E. coli that cell death may occur before lysis

(Gustafson et al., 1998).

Molecules rich in phenolics can pass through the phospholipid bilayer of bacterial cell walls and bind to proteins to prevent them from performing their normal functions (Juven et al., 1994). The alteration of membrane permeability and the loss of functional proteins transporting molecules and ions perturbs the microbial cell. This subsequently leads to

cytoplasmic coagulation, denaturation of several enzymes and proteins, and the loss of metabolites and ions (Burt and Reinders, 2003).

Lipid biosynthesis pathway is an important target for the development of novel antimicrobials (Heath and Rock, 2004; Heath et al., 2001). Due to their hydrophobic nature, EOs and/or their components can affect the percentage of unsaturated fatty acids (UFAs) and alter their structure (Burt and Reinders, 2003). The adaption of the cells to the presence of these compounds at a concentration lower than the MIC could result in an increase in the percentage of UFAs responsible for the fluidity of the membrane (Di Pasqua et al., 2007; Heath and Rock,

2004). The lipid profile of resting cells shows reorganization after treatment with EO compounds, and many authors have described the bacterial FA synthesis pathway as an optimal target of many antibacterial agents (Campbell and Cronan, 2001; Bayer et al., 2000; Heath et al., 2002). Treatment with thymol, carvacrol, and eugenol, which are all phenolic compounds, may increase the amount of saturated C16 (and shorter length) FA, increase the amount of saturated C18 and decrease the amount of unsaturated C18 FA in the bacterial membrane.

Generally, the EOs possessing the strongest antibacterial properties against food borne pathogens contain a high percentage of phenolic compounds such as carvacrol, eugenol, and thymol (Farag et al., 1989; Thoroski et al., 1989; Cosentino et al., 1999; Dorman and Deans,

2000; Juliano et al., 2000; Lambert et al., 2001). It seems reasonable that their mechanism of action would, therefore, be similar to other phenolics; this is generally considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force (PMF), electron flow, active transport and coagulation of cell contents (Denyer and Hugo, 1991; Sikkema et al.,

1995; Davidson, 1997).

The chemical structure of the individual EO components effects their precise mode of action and antibacterial activity (Dorman and Deans, 2000). The importance of the presence of the hydroxyl group in phenolic compounds such as carvacrol and thymol has been confirmed

(Knobloch et al., 1986; Dorman and Deans, 2000; Ultee et al., 2002). The relative position of the hydroxyl group on the phenolic ring does not appear strongly to influence the degree of antibacterial activity. However, in one study carvacrol and thymol were found to act differently against Gram-positive and Gram-negative species (Dorman and Deans, 2000). The significance of the phenolic ring itself (destabilized electron) is demonstrated by the lack of activity of methanol compared to carvacrol (Ultee et al., 2002). In one study the addition of an acetate moiety to the molecule appeared to increase the antibacterial activity (Dorman and Deans,

2000). As far as non-phenolic components of EOs are concerned, the type of alkyl group has been found to influence activity (Dorman and Deans, 2000).

Components of EO also appear to act on cell proteins embedded in the cytoplasmic membrane (Knobloch et al., 1989). Enzymes such as ATPases are known to be located in the cytoplasmic membrane an to be bordered by lipid molecules. Lipophilic hydrocarbon molecules could accumulate in the lipid bilayer and distort the lipid-protein interaction; alternatively, direct interaction of the lipophilic compounds which hydrophobic parts of the protein may occur (Juven et al., 1994; Sikkema et al., 1995). Some EOs have been found to stimulate the growth of pseudomycelia (a series of cells adhering end-to-end as a result of incomplete separation of newly formed cells) in certain yeasts. This could be an indication that EOs act on the enzymes involved in the energy regulation or synthesis of structural components (Conner and Beuchat, 1984). Cinnamon oil and its components have been shown to inhibit amino acid

decarboxylases in Enterobacter aerogenes. The mechanism of action was thought to be the binding of proteins (Wendakoon and Sakaguchi, 1995). Indications that EO components may act on proteins where also obtained from studies using milk containing different protein levels (Pol et al., 2001).

Carvacrol mode of action

The mode of action of carvacrol, one of the major components of oregano and thyme oils, appears to have received the most attention from researchers. Thymol is structurally very similar to carvacrol, having the hydroxyl group at a different location on the phenolic ring. Both substances appear to make the cell membrane permeable (Lambert et al., 2001). Carvacrol and thymol are able to disintegrate the outer membrane of gram-negative bacteria, releasing LPS and increasing the permeability of the cytoplasmic membrane to ATP. The presence of magnesium chloride has been shown to have no influence on this action, suggesting a mechanism other than chelation of cations in the outer membrane (Helander et al., 1998).

Studies with B. cereus have shown that carvacrol interacts with the cell membrane, where is dissolves in the phospholipid bilayer and is assumed to align between the fatty acid chains (Ultee et al., 2000). This distortion of the physical structure would cause expansion and destabilization of the membrane, increasing membrane fluidity, increasing passive permeability

(Ultee et al., 2002). Measurement of the average phase transition temperature of the bacterial lipids confirmed that membranes instantaneously become more fluid in the presence of carvacrol (Ultee et al., 2000). The passage of. B. cereus cells metabolites across the cell membrane of exposure to carvacrol has also been investigated. Intracellular and extracellular

ATP measurements revealed that they level of ATP within the cell decreased while there was no proportional increase outside the cell. It is therefore presumed that the rate of ATP synthesis was reduced or that the rate of ATP hydrolysis was increased.

Measurements of the membrane potential of exponentially growing cells revealed a sharp decrease in the addition of carvacrol and indicated a weakening of the proton motive force. The pH gradient across the cell membrane was weakened by the presence of carvacrol and was completely dissipated in the presence of 1mM or more. Intracellular levels of potassium ions dropped while extracellular amounts increased proportionally, the total amount remaining constant (Ultee et al., 1999). It was concluded that carvacrol forms channels through the membrane by pushing apart the fatty acid chains of the phospholipids, allowing ions to leave the cytoplasm (Ultee, 2000). Oregano EO, containing carvacrol as a major component, causes leakage of phosphate ions from Staph. aureus and P. aeruginosa (Lambert et al., 2001).

When tested in a liposome model system at concentrations above the MIC, carvone dissipated the pH gradient and membrane potential of cells. The specific growth rate of E. coli,

Streptococcus thermophilus, and L.lactis decrease with increasing concentrations of carvone, which suggests that it acts by disturbing the metabolic energy status of cells (Oosterhaven et al., 1995). In contrast, another study found that carvone was ineffective on the outer membrane of E. coli and S.typhimurium and did not affect the intracellular ATP pool (Helander et al., 1998).

Eugenol mode of action

Eugenol is a major component (approximately 85%) of clove oil (Farag et a., 1989). The sub-lethal concentration of eugenol has been found to inhibit the production of amylase and proteases by B. cereus. Cell wall deterioration and a high degree of cell lysis were also noted

(Thoroski et al., 1989). The hydroxyl group on eugenol is thought to bind proteins, preventing enzyme action in E. aerogenes (Wendakoon and Sakaguchi, 1995). Juven et al., (1994) examined the working of thymol against S. typhimurium and Staph. Aureus and hypothesized that thymol binds to membrane proteins hydrophobically and by means of hydrogen bonding, thereby changing the permeability characteristics of the membrane. Thymol was found to be more inhibitive at pH 5.5 than 6.5. At low pH, the thymol molecule would be undissociated and therefore more hydrophobic, and so may bind better to the hydrophobic areas of proteins and dissolve better in the lipid phase (Juven et al., 1994). Eugenol alters the membrane, affects the transport of ions and ATP and changes the fatty acid profile of different bacteria. It also acts against different bacterial enzymes, including ATPase, histidine carboxylase, amylase and protease (Thoroski, 1989; Wendakoon and Sakaguchi, 1995).

Trans-cinnamaldehyde mode of action

Cinnamaldehyde has at least three mechanisms of action against bacteria. At low concentrations, it inhibits enzymes involved in cytokine interaction or other less important cell functions, and at higher concentrations, it acts as an ATPase inhibitor. At a lethal concentration, cinnamaldehyde perturbs the membrane. Cinnamaldehyde is indeed capable of altering the lipid profile of the microbial cell membrane (Wendakoon and Sakaguchi, 1995). Trans-

cinnamaldehyde (TC), an unsaturated aldehyde, was shown to be responsible for the antibacterial activity, with the acrolein group in these molecules being essential for the activity

(Bae et al., 1992). Some studies have demonstrated that instability of TC when exposed to air, as the reactive unsaturated aldehyde is readily oxidized to cinnamic acid, which causes volatile loss and the instability of TC (Smith et al., 2000). In addition, in vivo, decomposition might also occur before it is able to perform a bactericidal activity, as absorbed TC can rapidly and irreversibly become cinnamic acid through enzyme catalysis; thus, it is unstable in the blood

(Yuan et al., 1992).

An increase in bacterial membrane rigidity after the addition of TC to growth medial was attributed to a large increase in the proportion of saturated fatty acids in the membrane phospholipids. This modification of fatty acids was probable a compensatory effect to maintain the membrane structure because of the fluidifying effect of TC (Di Pasqua et al., 2006; Di

Pasqua et al., 2007). The accumulation of numerous molecules inside the cell can perturb its selective permeability, potentially reaching a critical level to induce cell death (Trinh et al.,

2015).

In cellular respiration, the electron transport chain generates a transmembrane proton gradient necessary for ATP synthesis, performed by multiple enzymes with ATPase activity, including ATP-dependent transport proteins and the F1F0-AT-ase complex (Sikkema et al., 1995;

Andres and Fierro, 2010). The F1F0-AT-ase complex is a reversible proton translocating pump that may extrude protons from the cytoplasm by use of energy form ATP hydrolysis, and such proton efflux enhances the proton gradient and assists in the regulation of cytoplasmic pH

(Plesiat and Nikaido, 1992). The inhibition of membrane-bound or membrane-embedded

enzymes by TC may result in changes in the proton conformation, which causes the inhibition of

ATPase activity, the inhibition of other enzymes, and altered bacterial growth (Helander et al.,

1998).

TC downregulated F1F0-AT-ase, thereby inhibiting ATP synthesis in Cronobacter sakazakii

(Amalaradjou and Venkitanarayanan, 2011). The treatment of E.coli and Listeria monocytogenes with TC caused a disruption of the proton motive force with rapid depletion of

ATP and prevented an increase in cellular ATP concentration, which suggested that ATPase inhibition may play a significant role in reducing the growth rate at the sub-lethal concentrations (Gill and Holley, 2004; Gill and Holley, 2006). At high concentrations, TC also acts as an ATPase inhibitor (Nazzaro et al., 2013). The effects of both cinnamon water extract and TC on mitochondrial F1F0-AT-ase in rat liver are similar to those observed in bacterial cells: they dissipate the proton gradient, uncouple the mitochondrial, stimulate ATP hydrolysis, lower the level of ATP, and consequently damage mitochondrial functions, which may be attributable to either the release of protons into the matrix or the interaction of cinnamon and TC with sulfhydryl groups of membrane proteins (Usta et al., 2003). The dissipation of membrane potential across the mitochondrial membrane, such as that caused by TC, would increase ATP hydrolysis by uncoupling oxidation from phosphorylation (Hatefi, 1985). These uncouplers are reversed by thiols (Usta et al., 2003). TC was found to target thiols of cysteine residues on protein molecules (Weibel and Hansen, 1989), leading to possible destabilization of ATP activity.

Although cinnamaldehyde is known to be inhibitive to the growth of E. coli and S. typhimurium at similar concentrations to carvacrol and thymol, it did not disintegrate the outer

membrane or deplete the intracellular ATP pool (Helander et al., 1998). The carbonyl group is thought to bind proteins, preventing the action of amino acid decarboxylases in E. aerogenes

(Wendakoon and Sakaguchi, 1995).

Susceptibility of gram-negative and gram-positive organisms

The hydrophobicity that is typical of EOs is responsible for the disruption of bacterial structures that leads to increased permeability due to an inability to separate the EOs from the bacterial cell membrane. The permeability barrier provided by cell membranes is indispensable to many cellular functions, including maintaining the energy status of the cell, membrane- coupled energy-transducing processes, solute transport, and metabolic regulation. The cell membrane is also essential for controlling the turgor pressure (Poolman et al., 1987;

Trumpower and Gennis, 1994). The external capsule of some gram-negative bacteria limits or prevents the penetration of EOs into the microbial cell. The compounds present in the EOs are also capable of interfering with proteins in the wall that are often involved in the transport of essential molecules into the cell.

The integrity of the cell membrane is essential for the survival of bacteria because it is a key element of the fundamental biological activities taking place within the cells. The membrane represents an effective barrier between the cytoplasm and the external environment; the import and export of the metabolites and ions essential for all activities taking place within the cells. The membrane represents and effective barrier between the cytoplasm and the external environment; the import and export of the metabolites and ions essential for all activities occurring in the microbial cell occur through the cell membrane. When

antimicrobial compounds are present in the environment surrounding microorganisms, the bacteria may react by altering the synthesis of fatty acids and membrane proteins to modify the fluidity of the membrane (Mrozik et al., 2004). The hydrophobicity of the EOs and their components allow them to diffuse through the double lipid layer of the membrane. The EOs can alter both the permeability and function of membrane proteins. Some EOs, particularly oils that are rich in phenolics, are able to insert into the phospholipid bilayer of bacterial cell walls, where they bind to proteins and prevent them from performing their normal functions (Juven et al., 1994).

Approximately 90-95% of the cell wall of Gram-positive bacteria consists of peptidoglycan, which involves the linkage of molecules such as teicoic acid and proteins. The structure of the Gram-positive cell wall allows hydrophobic molecules to easily penetrate the cells and act on both the cell wall and within the cytoplasm. Phenolic compounds, generally present in essential oils, generally show antimicrobial activity against Gram-positive bacteria.

Their effect depends on the amount of the compound present; at low concentrations, they can interfere with enzymes involved in the production of energy, and at high concentrations, they can denature proteins (Tiwari et al., 2009).

The cell wall of Gram-negative bacteria is more complex. An outer membrane (OM) lies outside of the thin peptidoglycan layer. Small hydrophilic solutes are able to pass through the

OM via abundant porin proteins that serve as hydrophilic transmembrane channels, and this is one reason that Gram-negative bacteria are relatively resistant to hydrophobic antibiotics and toxic drugs (Nikaido, 1994; Vaara, 1992). The OM, however, almost but not totally impermeable

to hydrophobic molecules, some of which can slowly traverse through porins (Plesiat and

Nikaido, 1992; Nikaido, 1996).

Thymol and carvacrol have similar antimicrobial effects but have different mechanisms of action against Gram-positive and Gram-negative bacteria. The location of one or more functional groups on these molecules can affect their antimicrobial activity. Thymol is structurally analogous to carvacrol, but the location of the hydroxyl groups differs between the two molecules. However, these differences do not affect the activity of either antimicrobial agent (Dorman and Deans, 2000). Trans-cinnamaldehyde can inhibit the growth of E. coli and

S.typhimirium without disintegrating the OM or depleting intracellular ATP pools (Helander et al., 1998; Helander et al., 1997).

The mechanism of the EO is not isolated but instead involves a series of events both on the cell surface and within the cytoplasm. The alteration of membrane permeability and the defects in the transport of molecules and ions result in a ‘disbalance’ within the microbial cell.

This leads to cytoplasm coagulation, the denaturation of several enzymes and of cellular proteins and the loss of metabolites and ions (Burt and Reinders, 2003). In many conditions, such as in the presence of sub-lethal concentrations of EOs or other antimicrobial compounds, microorganisms react by increasing their expression of the stress-response proteins (Burt et al.,

2007) to repair the damaged proteins (Lambert et al., 2001). When the concentrations of EOs or other natural antimicrobials is higher, this response is unable to prevent cell death. This effect is more evident from Gram-positive bacteria. The cell wall of Gram-negative bacteria. The cell wall of Gram-negative bacteria is more resistant to the activity of EOs and their components.

The Gram-negative cell wall does not allow for the entrance of hydrophobic molecules as

readily as Gram-positive bacteria; thus, EOs are less able to affect the cell growth of Gram- negative bacteria (Burt and Reinders, 2003).

Most studies investigating the action of whole EOs against food spoilage organisms and food borne pathogens agree that, generally, EOs are slightly more active against gram-positive than gram-negative bacteria (Shelef, 1983; Shelef et al., 1984; Farag et al., 1989; Mendoza-

Yepes et al., 1997; Ouattara et al., 1997; Smith-Palmer et al., 1998; Marino et al., 1999, 2001;

Negi et al., 1999; Juliano et al., 2000; Ruberto et al., 2000; Senatore et al., 2000; Canillac and

Mourey, 2001; Demetzos and Perdtzoglou, 2001; Lambert et al., 2001; Mariano et al., 2001;

Cimanga et al., 2002; Delaquis et al., 2002; Pintore et al., 2002; Harpaz et al., 2003). That gram- negative organism are less susceptible to the action of antibacterials is perhaps to be expected, since they possess an outer membrane surrounding the cell wall (Ratledge and Wilkinson,

1988), which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering (Vaara, 1992). However, not all studies on EOs have concluded that gram-positives are more susceptible (Wilkinson et al., 2003). In another study, no obvious difference between gram-positives and gram-negatives was measured in the susceptibility after 24hr, but the inhibitory effect was more often extended to 48hr with gram-negative than with gram-positive organisms (Ouattara et al., 1997). A study testing 50 commercially available EOs against 25 genera found no evidence for a difference in sensitivity between gram-positive and gram- negative organisms (Deans and Ritchie, 1987). However, a later study using the same test method and the same bacterial isolates but apparently using freshly distilled EOs, revealed that gram-positive bacteria were indeed more susceptible to two or the EOs tested and equally sensitive to four other EOs than were gram-negative species (Dorman and Deans, 2000). It is,

therefore, possible that variation in composition between batches of EOs is sufficient to cause variability in the degree of susceptibility of gram-negative and gram-positive bacteria.

Synergism and antagonism between components of EOs

An additive effect is observed when the combined effect is equal to the sum of the individual effects. Antagonism is observed when the effect of one or both compounds is less when they are applied together than when individually applied. Synergism is observed when the effect of the combined substances is greater than the sum of the individual effects

(Davidson and Parish, 1989). Some studies have concluded that whole EOs have greater antibacterial activity than the major components mixed (Gill et al., 2002); Mourey and Cannilac,

2002), which suggests that the minor components are critical to the activity and may have a synergistic effect or potentiating influence.

The two structurally similar major components of oregano EO, carvacrol and thymol, were found to give and additive effect when tested against staph. aureus and P. aeruginosa

(Lambert et al., 2001). It appears that p-cymene, a very weak antibacterial, swells bacterial cell membranes to a greater extent than carvacrol does. By this mechanism p-cymene probably enables carvacrol to be more easily transported into the cell so that a synergistic effect is achieved when the two are used together (Ultee et al., 2000).

A mixture of cinnamaldehyde and eugenol at 250 and 500ul ml-1 respectively inhibited the growth of Staphylococcus sp., Micrococcus sp. Bacillus sp., and Enterobacter sp. For more than 30 days completely, whereas the substrates applied individually did not inhibit growth

(Moleyar and Narasimham, 1992).

A number of potential synergists have been suggested the use of EOs: low pH, low water activity, chelators, low oxygen tension, milk heat, and raised pressure, although not all of these have been researched in foodstuffs (Gould, 1996). Thymol and carvacrol have been shown to have a synergistic effect with high hydrostatic pressure (HHP). The viable numbers of mid- exponential phase L. monocytogenes cells were reduced more by combined treatment with 300

MPa HHP and 3mmol 1-1 thymol or carvacrol than by the separate treatments. Since HHP is believed to cause damage to the cell membrane, is it suggested that this common target is the root of the observed synergism (Karatzas et al., 2001).

The antibacterial activity of EOs is influenced by the degree to which oxygen is available.

This could be due to the fact that when little oxygen is present, fewer oxidative changes can take places in the EOs and/or that cells obtaining energy via anaerobic metabolism are more sensitive to the toxic action of EOs (Paster et al., 1990). The antibacterial activity of oregano and thyme EOs was greatly enhanced against S. typhimurium and Staph. Aureus at low oxygen levels (Paster el al., 1990). The use of vacuum packing in combination with oregano EO may have a synergistic effect on the inhibition of L. monocytogenes and spoilage flora on beef fillets;

0.8% v/w oregano EO achieved a 2-3 log10 initial reduction in the microbial flora but was found to be even more effective in samples packed under vacuum in low-permeability film when compared to aerobically stored samples and samples packaged under vacuum in highly permeable film (Tsigarida et al., 2000).

The MIC values presented here for carvacrol, thymol, and TC agree with previously reported values for E. coli and S.typhimurium (Karapinar and Aktug, 1987; Didry et al., 1993).

MIC value of carvacrol on E. coli is 3Mm. MIC value of carvacrol on S.typhimurium is 1mM. MIC

value of TC on E. coli is 3mM. MIC value of TC on S. typhimurium is 3mM. MIC value of thymol on E. coli is 3Mm. MIC value of thymol on S.typhimurium is 1mM. (Helander, 1998).

Cinnamaldehyde decreased the MIC of carvacrol (from 0.16-0.31 mg/mL to 0.078-

0.010mg/mL), but also the MIC of cinnamaldehyde itself was reduced (from 0.16-0.63 mg/mL to

0.078-0.0009mg/mL) by carvacrol for the most tested bacteria. The results showed that cinnamaldehyde and carvacrol had a synergistic inhibitory effect of 7 kinds of tested bacteria: E. coli, S.aureus, Y.regenburgei, S.intermedius, K.keistinae, S.haemolyticus, and S.enteritidis. All the FIC values were smaller than 4.0, which indicate that there is no antagonism between cinnamaldehyde and carvacrol. The combination sample showed better bactericidal effect compared with the cinnamaldehyde and carvacrol alone (Ye et al., 2013). The exact synergistic mechanism is likely due to some structural changes in the bacteria. For examples, the natural antimicrobials may have facilitated penetration of the drug through the outer layers of the bacterial cell wall or acted by blocking the inhibitory effects of protective enzymes or interfered with single or multiple metabolic targets of the antibiotic (Palaniappan and Holley, 2010).

Toxicity

Carvacrol is a hydroxylayed p-cymenes, and little is known about its metabolic activity.

In 1932 Schroder and Vollmer indicated that it is rapidly excreted in the urine of rats and rabbits. Earlier studies of thymol metabolism, phenol that has been studied more, were summarized by Williams (1959) who noted that it was excreted in the urine of rabbits, dogs, and man as glucuronide and sulphate conjugates and that small amounts of ring oxidation, giving thymoquinol also occurred. Robbins (1934) reported that about a third of the dose (1g)

was excreted unchanged in the urine by dogs whereas none could be detected in the feces.

Scheline (1978) reported some preliminary findings which showed that rats formed two monohydroxylated derivatives of thymol. Walde et al., (1983) showed that p-cymene undergoes extensive aliphatic hydroxylation at all available sites. This leads to the excretion of a complex pattern of alcohols, acids and hydroxy acids. These additional metabolic routes may be operative in the metabolism of the hydroxylated derivatives of p-cymene as well. More specifically with carvacrol, a newer study demonstrated that both of the aliphatic groups present undergo extensive metabolism. The oxidation of the isopropyl moiety is susceptible to oxidation. With carvacrol metabolism the detection of far fewer metabolites from the phenols than from hydrocarbon. Aromatic hydroxylation is a pathway of little of no importance. The present results indicate only the relative amounts of the urinary metabolites excreted whereas the p-cymene study reported quantitative values, the two reports do not allow for exact comparisons on this point. Carvacrol and thymol and their metabolites undergo rapid excretion

(Austgulen et al., 1987).

Eugenol is a constituent of many plants (Gildemeister and Hoffmann 1960). It is the principal constituent (70-90%) of clove oil. Other allylbenzenes, ex safrole, and estragole, are hepatoxins for animals an man (Homburger et al., 1961; Long et al. 1963; Taylor et al., 1964;

Borchert et al., 1973; Drinkwater et al., 1976; Miller et al. 1983), and metabolic activation of these compounds seems to be necessary for toxicity and carcinogenicity (Homburger et al.,

1961; Long et al., 1963; Borchert et al., 1973; Drinkwater et al., 1976; Miller et al., 1983;

Stillwell et al., 1974). The distribution of eugenol in rats and rabbits was studied as early as

1932 by Schroder and Vollmer. The LD50 of eugenol in rats and other animals have also been

determined (Sorber et al., 1950; Caujolle and Meynier, 1960; Taylor et al., 1964; Jenner et al.,

1964; Fugii et al., 1970). LaVoie et al., (1986) determined the LD50 of eugenol administered by intratracheal instillation, which in rats was: 11mg/kg body wt, and in hamsters was: 17mg/kg body wt. These results have shown that eugenol was nearly 250 times more toxic for rats when administered by inhalation than when administered orally.

Studies in vitro and in vivo with eugenol administered to rats or rat liver cells (Sutton et al., 1985; Delaforge et al., 1980; Weinberg et al., 1972) showed metabolic transformation of eugenol. As the metabolic pattern evolved, three groups of different polarity become apparent.

Isoeugenol, the epoxide, the benzylic alcohol, the HCl adduct to the epoxide, and dihydro- eugenol behaved very much like eugenol. Eugenol contains a free phenolic hydroxyl group, therefore can be readily conjugated and excreted as are other phenols (Sutton et al., 1985;

Williams, 1959). Studies in rats and guinea pigs showed that eugenol was effectively glucuronidated in these species (Sutton et al., 1985; Hariala et al., 1966) and the conjugation of eugenol with glucuronic acid and sulphate is also the major metabolic pathway of eugenol in man. Half of the dose was excreted in this way within 24hrs so that only half of the dose had any possibility of metabolic activation.

The oral LD50 for cinnamaldehyde in rats and mice has been reported to range from 2.22 to 2.4 g/kg body weight (Jenner et al., 1964; Sporn et al., 1965; Zaitsev and Rakhamanina,

1974). A prenatal toxicity study in rats showed a significant increase in the incidence of poor cranial ossification after oral treatment of pregnant Sprague-Dawley rats with cinnamaldehyde at doses of 5m25m and 250mg/kg body weight/day (Mantovani et al., 1989). After I.P. administration of 40 mg cinnamaldehyde to rats in four successive injections at 30 min

intervals, cinnamaldehyde was initially oxidized to cinnamic acid and then further metabolized mainly to hippuric acid, which could finally be recovered from urine. Approximately 29% of the ip dose was excreted in urine as hippuric acid within 24 hr (Teuchy et al., 1971). In male mice given ip injection of 383mg/kg of trans-cinnamic acid, of the total, injected dose 89% to 101% was recovered in the urine with 78%-93% recovered within 24 hours. A further 1.7%-3.5% of the dose was excreted within 72 hours in feces (Nutley et al., 1994).

Cinnamaldehyde is a potential alkylating agent that could react with cellular macromolecules. Protein-binding experiments showed that the binding sites on protein appeared to be mostly thiol groups of cysteine residues (Weibel and Hansen, 1989).

Cinnamaldehyde also causes the depletion of glutathione levels (Boyland and Chasseaud, 1968,

1970). In vitro, when cinnamaldehyde was added to fresh blood collected from untreated male

F344 rats, only a portion of added cinnamaldehyde was recovered as cinnamic acid; the other portion of cinnamaldehyde was believed to be conjugated to blood protein through the terminal thiol or amino group. The disappearance of cinnamaldehyde in fresh rat blood followed first order kinetics with a half-life of 4 min (Yun et al., 1992).

The bioactivity of cinnamaldehyde administered by the oral route was low. However, a high percentage of cinnamaldehyde dose was recovered as hippuric acid in rat urine, indicating a high systemic absorption of cinnamaldehyde and/or its gastro-intestinal metabolites.

Cinnamaldehyde is a reactive aldehyde and can be easily oxidized to cinnamic acid (Morrison and Boyd, 1973). As a typical reactive aldehyde, cinnamaldehyde can also readily conjugate within various amino acids or proteins trough amino or thiol groups to form Schiff bases and thiol conjugates (Weibel and Hansen, 1989). It has been reported that the conjugation sites for

bovine serum albumen protein appear mostly to be the thiol groups of cystine residues. The conjugates formed are eventually decomposed, and cinnamyl alcohol and cinnamic acid are released (Weibel and Hansen, 1989). In vivo, it can be expected that cinnamyl alcohol would be oxidized back to cinnamaldehyde in the liver under the catalysis of alcohol dehydrogenase

(Goldstein et al., 1974). The overall effect is the regeneration of cinnamaldehyde from protein conjugates in vivo. Furthermore, Schiff bases can by hydrolyzed to release aldehydes under acid conditions; such hydrolysis could also occur in vivo under the action of certain enzymes.

Therefore, the protein-conjugation reaction may be reversible in vivo. Since no regeneration of cinnamaldehyde was observed in studied in vitro of fresh rat blood spiked with cinnamaldehyde

(Yuan et al., 1992), it could be concluded that the corresponding enzymes are probably not present in the blood.

There are two proposed pathways for cinnamaldehyde administered both orally and

I.V.: one leads to cinnamic acid, and the other to conjugated protein products followed by regeneration of cinnamaldehyde by certain enzyme activities. The existence of the second pathway is supported by the fact that the terminal elimination half-life of cinnamaldehyde in rats (approximately 99minutes) after I.V. injection is much longer than the half-life of cinnamaldehyde (approximately 4 minutes) obtained in vitro after spiking fresh rat blood with cinnamaldehyde (Yuan et al., 1992). The large difference between the two half-lives could only be explained by the regeneration of cinnamaldehyde from protein conjugates in vivo. Since cinnamaldehyde is a lipophilic liquid, its gastrointestinal absorption should be relatively rapid.

This is also confirmed by the rate of excretion of hippuric acid in urine. It is likely that cinnamaldehyde can also conjugate with gastro-intestinal proteins since thiol and amino groups

are abundant in all proteins. The conjugation of cinnamaldehyde with these proteins can explain the fact that cinnamaldehyde was still detectable in rat blood even at 24 hr after dosing.

The conjugation of cinnamaldehyde to proteins is more likely to be a saturable process, and its rate is probably lower than that of oxidation of cinnamaldehyde since the estimated percentage of cinnamaldehyde oxidized, based on the first 30-min AUC value of cinnamic acid, was relatively high and also increased with I.V. dose (37 and 60% for 5 and 15 mg cinnamaldehyde/kg body weight, respectively). Concentrations of cinnamaldehyde in blood are lower after administration by gauge because all of the cinnamaldehyde absorbed must pass through, the liver because being distributed to the whole system. During the first passage, a large percentage of absorbed cinnamaldehyde could be oxidized. A portion of the absorbed cinnamaldehyde could conjugate with blood proteins before entering the liver, and most of these proteins’ conjugates would escape hepatic first-pass metabolism and slowly regenerate cinnamaldehyde. Since the release of cinnamaldehyde from gastrointestinal protein, conjugates would also be expected to be slow, the net effect of this whole process would be to increase significantly the absorption time and maintain cinnamaldehyde concentrations in blood at very low levels for a long period. Cinnamic acid could also be expected to be present in the blood for a long period since its concentration in blood should be mainly dependent on its rate of formation because its biological half-life is very short (7 min.) This proposed scheme would also easily account for the long duration of hippuric acid excretion (Yuan et al., 1992).

Cinnamic acid is converted to its acyl CoA, and it is this high-energy intermediate that is the key to the fate of cinnamic acid. Cinnamoyl CoA undergoes either glycine conjugation by a glycine N-acyl transferase, a reaction only seen in the mouse, or the addition of water across

the double bond to yield 3-hydroxy-3-phenylpropionyl CoA in the first step of β-oxidation. This

CoA may be cleaved to yield the free acid as a very minor metabolite, but most of it loses two protons to FAD to give 3-keto-3-phenylpropionyl CoA. If this CoA is cleaved, the free β-keto acid is, chemically unstable and decarboxylates to acetophenone, a minor urinary metabolite. 3- keto-3-phenylpropionyl CoA then loses a two carbon acetyl moiety by reaction with acetyl CoA, yielding benzoyl CoA and malonyl CoA. Benzoyl CoA is then in turn conjugated with glycine, giving hippuric acid, or hydrolyzed to free benzoic acid, excreted as such or after glucuronic acid conjugation (Nutley et al., 1994).

In both rat and mouse the elimination of cinnamaldehyde is essentially quantitative, and after 72 hours less than 2% of the dose remains in the carcass. Cinnamaldehyde is excreted rapidly, with 80% of the dose in the rat and 87% in the mouse being excreted in the first 24 hours after dosing, predominantly in the urine. The elimination of radioactivity is slightly faster in male animals at 2mg/kg after oral administration, compared with I.P. administration of

250mg/kg, possibly indicating that at the high I.P. dose, saturation of metabolism has slowed excretion (Peters and Caldwell, 1994).

Hippuric acid is the major metabolite (79% and 72% in rat and mouse, respectively) and is suggested to arise from oxidation of the aldehyde to cinnamic acid and further metabolism by way of β-oxidation of the side chain flowed by glycine conjugation to yield hippuric acid. This is analogous with the sequence in the catabolism of fatty acids (Dakin and Knoop, 1900) with cinnamic and other ω-phenyl FA (Williams, 1959). Xenobiotic carboxylic acids that enter the cell are converted to their acyl-CoA thioesters and undergo the various steps of the β-oxidation pathway of these high energy intermediates (Cladwell, 1984). Cinnamoyl-CoA undergoes either

conjugation with glycine by an N-acyl transferase, a reaction seen only in the mouse, or the addition of water across the double bond to yield 3-hydroxyl-3-phenylpropionyl-CoA in the first step of β-oxidation. This CoA may be cleaved to yield the free acid as a very minor metabolite, but most oxidized, lose two protons to NAD to give 3-keto-3-phenylpropionyl-CoA.

Acetophenone, the decarboxylation product of this intermediate was not detected in rat or mouse urine (although it has been reported as a metabolite of cinnamic acid; Nutley et al.,

1994). 3-keto-3-phenylpropionyl-CoA then loses an acetyl group, presumably by reaction with acetyl -CoA, yielding is then in turn conjugated with glycine giving hippuric acid, or hydrolyzed to free benzoic acid, excreted as such or after glucuronic acid conjugation. The main features of the pattern of oxidative metabolites in the rat and mouse very similar, with the exception of cinnamoylglycine which was important only in the mouse (Peters and Cladwell, 1994).

Comparison of oral and I.P. administration suggests that oxidative metabolism may be saturated for a short time after I.P. administration when a bolus dose reaches the liver rapidly, leading to small changes in the metabolic pattern. In general, the metabolic profile of cinnamaldehyde was simpler in males and females. The most marked difference was in the formation of cinamoylglycine, which was higher in the male than female mouse (Peters and

Caldwell, 1994).

Liver toxicology

Since the liver has large oxidative capacity, most cinnamaldehyde would be oxidized on the first pass (hepatic first pass effect), and the systemic exposure from a low concentration of absorbed cinnamaldehyde should be negligible. However, with a sufficiently high concentration

of cinnamaldehyde in the portal vein, some cinnamaldehyde could escape the hepatic first pass effect, and low concentrations of cinnamaldehyde in circulating blood could occur. It is also possible that cinnamaldehyde will conjugate with blood proteins prior to entering the liver and be protected from oxidative metabolism. The slow hydrolysis of cinnamaldehyde protein conjugates may provide a prolonged presence of cinnamaldehyde in the blood (Yuan et al.,

1993).

After doing hepatocyte suspension derived form male Fischer 344 rats, it was concluded that TC is the active species responsible for glutathione depletion and cytotoxicity. TC spontaneously reacts with glutathione in a concentration- dependent manner the extent of the reaction being dependent on the relative molar concentrations of the two reactants. TC depletes glutathione at a greater rate than cinnamyl alcohol. The rate of glutathione depletion by TC decreased as the cellular glutathione content fell. The lower rate at which cinnamyl alcohol may be attributed to its conversion to TC before it can conjugate with glutathione. The lower cytotoxicity of cinnamyl alcohol can also be attributed to its oxidation of TC since TC is produced at a slow rate (Peters, 1993). The cytoprotective mechanisms of the hepatocyte may be more effective against the slow production of TC from cinnamyl alcohol than TC introduction. Thus, a relatively higher cinnamyl alcohol concentration is required before this compound can cause overt cytotoxicity. Delbressine et al., 1981 observed a marked decrease in the excretion of thiol metabolites when female Wistar rats were given cinnamyl alcohol after pretreatment with the alcohol dehydrogenase inhibitor pyrazole, indicating that cinnamyl alcohol does indeed require oxidation to TC before conjugation with glutathione.

The co-incubation of ethanol and TC sets up a redox couple (Goon et al., 1992) in which ethanol is oxidized to acetaldehyde by alcohol dehydrogenase, and NADH so produced acts as a cofactor for the reduction of TC to cinnamyl alcohol. The effect of ethanol diverts TC away from its major pathway of oxidation and sets up a pool of cinnamyl alcohol from which Tc is relatively slowly metabolized (Peters, 1993). This diversion of TC to cinnamyl alcohol and aldehyde dehydrogenase inhibition by acetaldehyde produced from ethanol (thus inhibiting TC oxidation to cinnamyl alcohol) results in a prolonged glutathione depletion from 0 to 6 hours, as opposed to a transient depletion.

Preferential induction of biotransformation enzymes (GST, glucuronyl transferase over cytochrome P-450 isoenzymes) by eugenol in rat liver in vivo was reported (Rompelberg et al.,

1993). Induction of GST activity 1.3fold in rats treated daily with 1000mg/kg for 10 days was found. Total cytochrome P-450 contents of liver microsomes form rats treated with eugenol were not significantly different from those of the controls. Eugenol is not an effective inducer of cytochrome P-450 enzyme activities. An induction of phase-II biotransformation enzymes was found. In general, the fold induction of phase-II biotransformation enzymes is low. For GST a small induction may still be significant in view of the relatively large amounts of GST present in most cells. The preferential induction of phase-II biotransformation enzymes may be an indication that eugenol possesses antimutagenic/ anticarcinogenic properties (Prochaska and

Talalay, 1988).

An in vitro experiment with different treatment schedules was performed with Hep G2 cells to study further the effect of eugenol on B[a]P-induced genotoxicity. The Hep G2 cell lin is an immortalized liver cell line of human origin (Aden et al., 1979). It has high B[a]P-metabolizing

activity that converts B[a]P to intermediates that bind to DNA and are mutagenic (Diamond et al., 1980). Pretreatment with eugenol in the SCGE assay with Hep G2 cells has no effect on the genotoxicity induced by B[a]P.

Cinnamaldehyde- induced peroxidant state (Devaraj et al., 1992), glutathione depletion, and lipid peroxidation in rat liver (Raveendran et al., 1993). The bioactivity of cinnamaldehyde administered by the oral route to the rats was low in blood since all of the cinnamaldehyde absorbed must pass through the liver before being distributed to the whole system. The low levels of cinnamaldehyde that remained in the blood was retained for a long time. Since cinnamaldehyde has to pass through the hepatic first phase metabolism, prolonged exposure and also the concentration of cinnamaldehyde have an impact on its effect on kidney (Yuan et al., 1992).

Kidney toxicity

Reactive oxygen species are mediators of tissue damage in toxic renal failure (Jo et al.,

2004). Niknahad et al., 2003, studies the formation of reactive oxygen species by cinnamaldehyde-treated rats. The non-enzymatic antioxidants ascorbic acid, reduced glutathione, and α-tocopherol was reduced significantly in the kidney. Cinnamaldehyde- induced glutathione depletion in mammalian cells were observed by Janzowski et al., 2003.

During free radical scavenging action, ascorbic acid is suggested to be transformed into semidehydro ascorbate; reduced glutathione is required for the conversion of semidehydroascrobate back to ascorbate (Halliwell 2001). The reduction in the level of reduced glutathione affects the conversion of semidehydro ascorbate, and thus the level of ascorbic acid

is lowered in the cinnamaldehyde-treated rats. Depletion of α-tocopherol in the kidney of test rats revels that onset of lipid peroxidation (Galleano & Puntarulo, 1994).

During free radical scavenging action, enzymes such as superoxide dismutase, glutathione peroxide, and catalase are involved in the direct elimination of active oxygen species. Glutathione S-transferase detoxifies reactive oxygen species by decreasing peroxide levels (Sun, 1990). In this study, superoxide dismutase is evaluated in the kidney of test rats, and hence there may be an increase in the production of reactive oxygen species and hydrogen peroxide. The increase in the activity of glutathione peroxidase suppresses the level of hydrogen peroxide which is cytotoxic. Since catalase also detoxifies hydrogen peroxide

(Macedo et al., 2005), depletion of catalase is compensated by the increased glutathione peroxide activity in the detoxification process of hydrogen peroxide. Glutathione S-transferase in considered to be a marker for proximal tubule cell damage (Bruning et al., 1999) and these are the cells responsible for the rapid glutathione turn over in kidney. The increase in the level of glutathione S-transferase results in the detoxification of hydrogen peroxide with glutathione peroxidase. The nephrotoxic effect of structurally related compounds of cinnamaldehyde such as styrene and coumarin is also studied (Trucon et al., 1990; NTP; 1993). Gentamicin (a nephrotoxicant)- disturbed enzymatic defense system in the kidney of guinea pigs (Kavutcu et al., 1996).

Decreased food intake, decreased body and kidney weight, and many pathological changes of kidneys were seen in rats treated with cinnamaldehyde at the dose of 73.5mg/kg body weight/day for 90 days. The pathological changes include congestion of glomerular

capillaries, mild degenerative changes with the appearance of fibrin casts in tubules and occurrence of high mitotic activity.

In weaning male albino rats that were given either 2.14, 6.96, 22.62, and 73.5 mg/kg body weight for 10, 30, and 90 days kidney analysis showed a decrease in non-enzymatic antioxidants ascorbic acid, α-tocopherol, and glutathione. However, there is an increase in antioxidant enzymes superoxide dismutase, glutathione peroxide, and glutathione S- transferase. In rats treated with 73.5mg/kg body weight for 90 days, catalase was decreased, and TBARS was increased.

Safety Data

Even though a considerable number of EO components are GRAS and/or approved food flavorings, some research data indicates irritation and toxicity. Cinnamaldehyde, carvacrol, carvone, and thymol appear to have no significant or marginal effects in vivo while in vitro they exhibit milk to moderate toxic effects at the cellular level.

The flavor of beef fillets treated with 0.8% v/w oregano oil was found to be acceptable after storage at 5˚C and cooking (Tsigarida et al., 2000). The flavor, odor, and color of minced beef containing 1% v/w oregano oil improved during storage under modified atmosphere packaging and vacuum at 5˚C and was almost undetectable after cooking (Skandamis and

Nychas, 2001).

Mycoplasma hominis (M. hominis), the first human mycoplasma species, has been isolated in the case of a variety of diseases, including bacterial vaginosis, pelvic inflammatory disease, and pyelonephritis. It has been reported with increasing frequency to cause

complicated pregnancies, chorioamnioitis, postabortion and postpartum fevers. Implicated in extragenital infections such as postoperative wound infections, arthritis, prosthetic valve endocarditis, respiratory and other infections in immunosuppressed patients. In neonates, M. hominis has been associated with neonatal meningitis, pneumonia, and skin abscess. Much like

M. bovis., M.hominis is also antibiotic resistant. Cinnamon bark oil, carvacrol, eugenol were all variously able to inhibit the growth of M. hominis isolates. Cinnamon bark oil was found to be the most effective natural substance tested against M. hominis (MIC90=MBC90=500ug/ml).

Carvacrol also possesses strong antimicrobial activity against M. hominis strains in the study with Mic90 and MBC90 equal to 600 ug/ml. A somewhat lower value was determined for eugenol (MIC90=MBC90=1000ug/ml).

Neurological effects of EOs

Ion channels are integral membrane proteins designed to catalyze ion flux and, as a consequence produce changes in the membrane potential. These molecular nanostructures are present in all types of cells, but they have a very important role in excitable cells where they are the main components responsible for the generation of action potentials. Action potentials are the result of the activity of many different types of ion channels working in concert to carry information from one cell to another in a very controlled way. There are two major super- families of ion channels, including voltage-dependent and ligand dependent.

Ion channels are involved in many distinct physiological processes such as neurotransmitter release, excitation-contraction coupling, excitation-transcription coupling, control of gene expression, and cell development. Another important point is the realization

that ion channel dysfunctions could lead to serious pathological disorders compromising the whole organism.

In excitable cells such as neurons, voltage-gated K channels serve to repolarize or hyperpolarize the membrane. Therefore, pharmacological activation of K channels in excitable cells reduces excitability. On the other hand, K channels inhibition would cause an increase in excitability. Overall, K channels constitute potential drug targets for the treatments of diverse diseases from cancer to cardiovascular disorders (Wulff et al., 2009). Essential oils freely cross cellular membranes and may serve various signaling roles inside the cell. In addition, there are some reports indicating that essential oil plant components are active towards ion channels and receptors (Goncalves et al., 2008; de Almeida et al., 2008; Alves et al., 2010).

Dosal root ganglion (DRG) neurons transduce peripheral stimuli and transmit the sensory information to the central nervous system. Voltage-gated Na channels play a key role in the initiation and propagation of action potentials in the BRG. It has been reported that eugenol inhibits action potentials and Na currents in rat dental primary afferent neurons (Park et al.,

2006).

Buchauer found that the constituents of the EO (carvacrol and eugenol), accumulate in the nerve cell membrane, thus causing a sterical blocking of embedded function proteins, which are the ion channels. This blocking causes a change in the physical properties of the membrane, especially a modification of its ion permeability. Inhibition of the inflow of calcium ions leads to a spasmolysis. Suppressing the generation of an action potential causes local

anaesthesia, and the suppression of the irritability of mast cells, which are responsible for inflammation, brings about an anti-inflammatory effect.

Lipophilic substances such as EOs, interact with lipids of the cell membranes in the cortex (Panizzi et al., 1993; Teuschner et al., 1990). For this reason, esters which are more lipophilic than their corresponding alcohols, can, therefore, pass membrane barriers more easily, in particular, the blood-brain barrier, and consequently show and increased membrane activity that correlates well with their better sedative properties (Teuschnet et al., 1990).

Consequently, the lipophilicy of such substances could be of importance in their efficiency in decreasing the motility of the test animals.

To investigate if carvone is involved in the nervous excitability provoked by other monoterpenes mice that were treated with carvone showed a significant reduction of writhers in the acetic acid-induced writhing test when doses of 100 and 200mg/kg were applied

(Gonvalves et al., 2008). It was observed that carvone prevented the licking response of the injected paw after doses of 100 and 200 mg/kg were applied I.P. to mice in the formalin test. It follows from this observation that the opioid system did not participate in the analgesic effect of carvone, whereas linalool is involved in a central antinociceptive effect associated with glutamate NMDA receptors (Gonvalves et al., 2008).

The rapid actions of the neurotransmitter GABA are mediated by ionotropic GABA receptors; these are pentameric transmembrane proteins with an integral, GABA-gated, anion channel (Moss and Smart, 2001). In vertebrates, all ionotropic GABA receptors are designated type A, and metabotripic receptors type B. Vertebrate GABAA receptors are confined to the

nervous system (Sieghart, 1995), whereas insect inotropic GABA receptors are present in the nervous system and on muscle cells (Sattelle, 1990). To date, 20 different vertebrate GABAA receptor subunit isoforms have been cloned: α(1-6), β(1-4), γ(1-3), δ(1), ε(1), π(1), θ(1), ρ(1-3).

Further diversity can arise due to alternative splicing of some subunit genes (Korpi et al., 2002).

In humans and other mammals, behavioral effects which are typical of positive allosteric modulators of GABAA receptors include anxiolysis, cessation of convulsions, sedation and general anaesthesia (Sieghart, 1995). Some positive GABAA receptor modulators can also act as an antagonist on the same receptors when tested at higher concentrations (Robertson, 1989;

Frank and Leib, 1994) ant this activity may influence the spectrum of clinical effects observed

(Sanna et al., 1999).

Thymol elicited either zero or minimal agonist activity, indicating that the enhanced response to GABA is likely to be the result of a positive allosteric action of thymol. The significant leftward shift of the GABA dose-response curve obtained from human a1b3g2s- expressing oocytes demonstrates that thymol can decrease the apparent EC50 of GABA but does not greatly affect the maximum response. This suggests that thymol is specifically facilitating the manner in which GABA binds to or activates the receptor but cannot increase the current flow above the maximum possible achieved by GABA alone. The evidence reported here suggests that thymol does not share sites of action with many of the most widely investigated allosteric modulators of GABAA activity. From this study, thymol has tentatively been termed a positive allosteric modulator of GABA receptors even though no putative allosteric site has been defined simply by the virtue that it stimulated GABA induced currents at GABA receptors

in a concentration- dependent fashion and induced negligible currents by itself (Priestley et al.,

2003).

Eugenol blocks neuronal action potential generating by acting directly in voltage-gated sodium channels without any changes in resting potential or input resistance (Leal-Cardoso et al., 2010; Moreira-Lobo et al., 2010; Cho et al., 2008). Eugenol has a blocking effect on the transmission of evoked impulses in nerve tissue in concentration ranging from 100% to 0.01% in

Ringer's solution. Concentrations of 100%, 50%, and 25% showed the same effectiveness in completely blocking impulse transmission, while lower concentrations had a decreasing effect but in a direct dosage-response relationship. It appears that the three highest concentrations of these does not appreciably shorten the time necessary to extinguish the compound action potential as compared with the next two lower concentrations. Thus, it may be that beyond the maximal level of toxicity the rate of penetration of eugenol acts as the determining factor in its effect on blocking transmission. Concentrations from 25% to 0.05% all extinguished the action potential at progressively longer periods within the 3-hour test period. Concentration of 0.01%, however, extinguished the action potential in only one of four experiments. In the remaining three tests the amplitude of the action potential was reduced by 20%,25%, and 80% and then stabilized at a plateau to the end of the observation period. These tests were regarded as partially effective and labeled PE to indicate that evoked neural impulses were, to some degree, still being transmitted through the test segment by the third hour. Tests conducted with

0.005% produced neither reduction in amplitude nor changes in the configuration of the compounds action potential are regarded as noneffective (NE) (Kozam and Newark, 1977).

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At 100% concentration eugenol is cytotoxic and causes denaturation of cytoplasmic proteins, loss of cell boundaries, swelling and cell necrosis (Kozam and Mantell, 1978). Eugenol inhibits high-voltage-activated Ca currents in dental primary afferent neurons, which is not prevented by capsazepine, and N-type Ca currents in the cell line C2D7 (Lee et al., 2005). N-type

Ca channels mediate nociceptive signaling in sensory neurons (Altier and Zamponi, 2004). Thus, the inhibition of Ca channels may contribute to the eugenol analgesia. In addition, eugenol inhibits NMDA receptors but potentiates GABA receptors, which are both involved in pain sensitivity (Aoshima and Hamamoto, 1999; Wie et al., 1997).

Their findings clearly indicate that eugenol inhibits voltage-gated Na currents through its interaction with both resting and inactivated Na channels. The authors also evaluated the recovery from inactivation for both Na currents. Eugenol slowed the recovery from inactivation.

One possible implication that one would expect is a decrease in neuronal excitability leading to nerve conduction blockade. To complete their investigation Cho and colleagues demonstrated that the eugenol inhibition of Na currents did not show any stimulation frequency dependence which is a behavior distinct from other typical anesthetics (Hille, 2001).

Eugenol moved the steady-state inactivation curves of both Na currents to a hyperpolarizing direction. In addition, eugenol reduced the maximal Na current at negative holding potentials at which the channels were relieved from inactivation. Thus eugenol appears to inhibit Na currents through its interaction with both resting and inactivated Na channels. The recovery from inactivation of both Na currents was slowed by eugenol, which would decrease the neuronal excitability. The eugenol inhibition of Na currents was not dependent on the

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stimulus frequency. The inhibition of Na currents is considered as one of the mechanisms by which eugenol exerts analgesia (Cho et al., 2008).

Dallmeier and Carlini along with Guenette et al. have reported a eugenol-induced general anesthetic effect on rats. Other eugenol- induced effects related to depressant activity on the central nervous system such as sedation, reduction of convulsion threshold and hypothermia has also been described. Concerning the local- anesthetic effect, pharmacologically characterized as a direct blockade of voltage-dependent Na channels, the first suggestion that eugenol induced this type of activity came from earlier studies, which demonstrated that eugenol ability to block compound action potential of the rat vagus nerve.

Park et al., suggested a local-anesthetic effect of eugenol presumably related to the action on both tetradotoxin- sensitive and -restraint Na currents in the trigeminal ganglion neurons. More recently. Cho et al., demonstrated in the rat dorsal root ganglion that eugenol directly blocks the inward Na current. In that study Cho et al., have reported that it takes approximately 10min for the effect of a given eugenol concentration to reach a steady state.

Ohkubo and Shibata showed that eugenol is able to reduce acetic acid induced abdominal contortions and such effect was antagonized by capzazepine. Eugenol-induced inward current in the neurons of the dorsal root ganglia which was partially sensitive to capsazepine. Eugenol-induced current was strongly inhibited by extracellular calcium depletion but only slightly inhibited by EC sodium depletion. Yang et al. showed an eugenol- activated inward calcium current sensitive to capsazepine on both TRPV1 channels heterologously expressed in HEK293 cells and in neurons isolated from the trigeminal ganglion.

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Eugenol showed concentration-dependent suppression of neuronal excitability. At

0.1mM eugenol induced no significant alteration in the action potential waveform in all 14 cells tested. However, at 0.6 and 2.0mM eugenol induced reversible AP blockade in 6/17 and 10/12 cells tested, respectively. Important to note that in the concentration range of eugenol used in this study there was no significant alterations of the resting membrane potential. Conversely, eugenol (0.6mM) induced a reversible increase (approximately 2-fold) in threshold current for action potential generation. Surprisingly, only at 0.6mM eugenol provoked a significant decrease of the input membrane resistance. After exposure to eugenol (0.6mM and 2mM), those cells showing no obvious blockade, presented an average AP amplitude equal to 90.6% and 93.5% of control, respectively (Moreira-Lobo et al., 2010).

Overall eugenol blocks the compound action potential of the rat sciatic nerve very slowly; and it blocks the action potential of the rat SCG neurons without significant alterations of the membrane resting potential but altering the input membrane resistance with a time- course that suggests two phases of eugenol activity on the globe electrophysiological parameters related to excitability of peripheral neurons (Moreira-Lobo et al., 2010).

Eugenol blocks rat sciatic nerve compound action potentials probably by acting on the voltage dependent Na channels. At high concentrations (2mM) and during brief application eugenol blocked the action potential without interfering in the resting membrane potential or membrane input resistance. However, at low concentration (0.6mM) and longer applications, the authors observed a significant reduction in the input membrane resistance which raises the possibility of a secondary effect involved in the reduction of neuronal excitability when eugenol was present (Moreira-Lobo et al., 2010). Elucidative studies also showed that eugenol inhibited

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thermal nociception and capsaicin-induced thermal hyperalgesia in the orofacial area indicating that this phenylpropene derivative could also be used for other pathological pain conditions

(Park et al., 2009).

Eugenol has both excitatory and inhibitory effects (Lee et al., 2005; Park et al., 200; Li et al., 2007). The excitatory effect attributed to eugenol comes to its activation of TRPV1 channels that evokes an inward current capable of provoking a membrane depolarization. However, it was reported that eugenol inhibits voltage-gates K currents causing a prolongation of the action potential (Li et al., 2007). Interestingly, eugenol only increased the action potential duration in a small subpopulation of trigeminal ganglion neurons. The authors raised an important issue when comparing the concentration ranges where eugenol blocked Na and Ca currents indicating that eugenol is less potent towards Ca channels than Na or K channels.

With Carvacrol the full block of CAP demonstrates that carvacrol exerts its inhibitory effect on all types of myelinated fibers that contribute to the first and second CAP components of the sciatic nerve. The first component has an average conduction velocity of 90.3 m/s, which is compatible with Aa fibers (Nokes et al., 1991; Aidley, 1998). The second components showed a mean conduction velocity of 29.6 m/s, in agreement with values reported for the myelinated sensorial fibers AB and Aγ (Nokes et al., 1991). When exposed to the highest concentration of

1mM carvacrol, the positive amplitude of both CAP components was fully inhibited, but the time to develop this effect was different. While the second components were completely inhibited within 60min, the inhibition of the first component amplitude was remarkably slower, and only after 180 min was fully blocked. Carvacrol, in a concentration-dependent manner, significantly decreased conduction velocity for both components. The conduction velocity

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cannot be measured when using 1mM carvacrol due to the complete blockade of the CAP waveform (Joca et al., 2012).

A previous study has reported that 10mM carvacrol blocked excitability in the sciatic

(Goncalves et al., 2010). However, Joca et al., 2012 demonstrate that carvacrol induced inhibition in peripheral nerve fibers can be achieved with a 10-fold lower concentration (1mM), and compared with other essential oil components such as eugenol, estragole, and 1,8- cineole, have similar effects on the CAP amplitude, time course, and pharmacological potency, at a submillimolar range (Leal-Cardoso et al., 2004; Lima-Accioly et al., 2006; Moreira-Lobo et al.,

2010). When the present results are taken together, one can conclude that carvacrol blocks nervous conduction, and this may explain its reported antinociceptive activity in models related to neurogenic pain (Guimaraes et al., 2010). Carvacrol showed and inhibitory effect on all myelinated fibers that contribute to the first and second CAP components of the sciatic nerve

(Joca et al., 2012).

In addition, changes in threshold values in the presence of carvacrol indicate that this monoterpenoid effectively blocks excitability in the peripheral nervous system. Alteration in resting membrane potential and/or in input membrane resistance would be implicated in a reduction of neuronal excitability. These mechanisms have been described already to explain how 1,8-cineole inhibits action potentials in peripheral neurons (Lima-Accioly et al., 2006;

Ferreira-da-Silva et al., 2009).

High carvacrol concentrations (3mM), all tested neurons showed an action potential blockade within 2 to 5 min of exposure, while the membrane passive response was maintained

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(Joca et al., 2012). After washing carvacrol, the action potential slowly recovered its usual shape, and all measured parameters reached similar values to the control. The percentage of neurons that showed a blockade in action potential generation was dependent on carvacrol concentration used in each experiment. At the lowest concentration used (0.03mM), there was no action potential blockade and no significant change in any of the measured parameters.

When the carvacrol concentration was raised to 3mM, DRG neurons failed to generate action potentials. Carvacrol induced a progressive elevation in current threshold level as a function of concentration, reaching more than 60% of control threshold value at the highest concentration tested (Joca et al., 2012).

Overall Carvacrol reversibly blocks excitability and nerve conduction in peripheral neurons in a concentration dependent manner. Carvacrol- induced blockade of neuronal excitability occurs without altering the resting potential and/or the input resistance.

Furthermore, this effect is the result of direct inhibition of the voltage-gated sodium current, which suggests that carvacrol acts as a local anesthetic (Joca et al., 2012).

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III. Effects of intramuscularly injected plant-derived antimicrobials in the mouse model

Johnson, J., Elizabeth1, Duan, Jingyue1, Srirattana, Kanokwan1, Venkitanarayanan, Kumar1,

Tulman, Edan.2, Geary, J., Steven 2, & Tian, Xiuchun (Cindy) 1*

1. Department of Animal Science, 2. Department of Pathobiology and Veterinary Science,

University of Connecticut, Storrs, CT, 06269, USA

Author information: Elizabeth J Johnson ([email protected]) Jingyue (Ellie) Duan ([email protected]) Kanokwan Srirattana ([email protected]) Kumar Venkitanarayanan ([email protected]) Edan Tulman ([email protected]) Steven J Geary ([email protected]) Xiuchun (Cindy) Tian ([email protected])

Corresponding author: Xiuchun (Cindy) Tian, PhD, Department of Animal Science, University of Connecticut, 1390 Storrs Rd, Storrs, CT 06269-4163, USA. Phone: (860) 486-9087, Email: [email protected].

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Abstract

Plant derived antimicrobials (PDAs) such as trans-cinnamaldehyde, eugenol and carvacrol, possess strong antibacterial properties yet are generally regarded as safe. In the current crisis of antibiotic resistance, the use of PDAs as alternatives to prevent and control bacterial infections in livestock has gained momentum. The inhibitory effects of PDAs on common bacteria such as C.difficile, Listeria, Salmonella have been widely studied. PDAs have also been found effective in wall-less bacteria such as Mycoplasma, for which common antibiotics are not effective and vaccines are not always effective. In animal husbandry, PDAs can be potentially added in feed to metaphalactively prevent bacteria infection, but they could also be potentially used to treat infection in the form of an injectable. Previous reports of PDA injection were focus on toxicity or effectiveness after intraperitoneal administration in the mouse model. Here we investigated the toxicity trans-cinnamaldehyde, eugenol and carvacrol after intramuscular in mice. Two levels of control (DMSO) and each PDAs were injected into the hind limbs of CD-1 mice of 7-8 weeks of age. Mice were monitored for weight, eating, behavior, appearance, and foot/leg changes for 2 hours post-injection and twice daily for the next 4 days. The liver, kidney and hind limb were observed for discoloration, swelling, size (mm), weight (g), and relative weight (percentage of organ weight/body weight). A total of 13 parameters were studied for each treatment group. Significant differences between controls and treated were only observed for the relative liver weight at 3.04 mM eugenol (p<0.01). Taken together, little toxicity was seen at the treatment regimen examined. This initial study paved the foundation for future and more extensive work with more animals, varied treatment duration and additional treatment levels.

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Introduction

Plants have been used as medicine for centuries (Sofowora et al., 2013). Essential oils have been extracted from many different plants and possess anti-inflammatory, anti-microbial and anti-tumor effects (Langeveld et al., 2014). The active ingredients in essential oils, referred here as plant-derived antimicrobials (PDAs), are phenolic compounds such as carvacrol

(Lambert et al., 2001), eugenol (Thoroski et al., 1989), and trans-cinnamaldehyde (Wendakoon and Sakaguchi, 1995). Each PDA may have a different mechanism of action (Denyer and Hugo,

1991). The overall mechanism of antimicrobial actions of these PDAs includes the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport and coagulation of cell contents (Denyer and Hugo, 1991).

With the increase of resistance to antibiotics, alternatives are needed to control pathogenic infection in both humans and animals. Although essential oils and plant-derived antimicrobials have been studied for their growth-promoting (Burt, 2004), methane inhibiting

(Patra and Yu, 2012) and disease-preventing effects as feed additives in livestock (Patra and

Saxena, 2009), no studies have been conducted to test the possibility of PDAs as injectable to treat infections.

Previous PDA toxicity studies were mostly conducted by oral administration and intravenous (iv) injections (Janssen et al., 1986). A very limited number of reports document intraperitoneal (ip) injection in the mouse model, mainly exploring the beneficial effects of

PDAs (Sell and Carlini, 1976; Nutley et al., 1994; Stoner et al., 1973). For example, to use PDAs as an injectable in livestock, intramuscular (im) injection is the most appropriate. The aim of our study was to investigate if any local or systemic side effects will be elicited by im PDA injection at levels found to inhibit 100% bacterial growth in vitro. We found a slight toxicity

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effect in the 0.05% eugenol group with percentage of liver weight over body weight was found to be higher than control group. No other statistically significant changes were seen in any other treatment group in any of the parameters measured indicating that the selected PDAs are a potential source of im injection in cattle.

It is expected that the PDAs will be absorbed at the injection site, enter the blood stream and travel systemically. This will allow the PDAs to reach common sites of infection such as the lung, joints and the mammary gland. It has been shown that trans-cinnamaldehyde fed to mice exerted systemic effects and were effective in the treatment of urinary tract infection

(Mooyottum et al., 2017). Additionally, eugenol oil ip injection exerted antinociceptive effects on acetic acid and formalin injection in mice (Kurian et al., 2006). Furthermore, carvacrol ip injection to Parkinson rats improved memory loss (Haddadi et al,, 2018). If PDAs incorporated in feed or given ip can travel systemically, the im injected PDAs are expected to do so as well.

The injection study proposed here aims to treat already infected/sick individual animals. In separate cattle studies to be conducted we aim to incorporate PDAs in feed to metaphylactically prevent infection.

Materials and Methods

Chemicals

Carvacrol (CAR), eugenol (EU), trans-cinnamaldehyde (TC), dimethyl sulfoxide (DMSO), and phosphate buffer saline (PBS) were purchased from Sigma-Aldrich (Sigma-Aldrich, Inc., St.

Louis, MO, USA).

Animals

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A total of 72 CD-1 mice of 7-8 weeks of age, weighting from 25g-40g, were obtained from

Charles River Laboratory (Boston, MA, USA). Mice were housed in a biohazard level II AALAC- accredited facility, under 12:12-h light-dark cycle in a temperature and humidity-controlled room.

Mice had food and water ad libitum. Animal weighing and injections were conducted in a Class II biosafety cabinet. Decontamination and sterilization of the cabinet surface was done using 10% bleach to prevent cross-contamination between consecutively handled mice. Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the

University of Connecticut (protocol number A18-055).

Experimental design

Experimental design is illustrated in Figure 1. Briefly, mice were sexed upon arrival (Day

-2), separated into male and female cages and given 48 hours (h) of acclimation time. The 72 mice were divided into nine groups of 8 each (4 females and 4 males) including three control groups and six treatment groups. The levels of PDA injected were based on in vitro studies and are shown in Table 1. PDA or control (DMSO) injections were given on the morning of Day 0. Mice were observed every hour for 2 h post-injection and twice daily for 4 days thereafter.

Figure 1. Animal treatment and observation schemes. Mice arrived on Day -2 and were sexed.

The mice then had a 48h acclimation time. On the morning of Day 0 the mice were weighed and injected. Mice were observed hourly for 2 h, and twice daily post-injection until Day 4 when

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mice were weighed, euthanized for dissection. Red vertical bars indicate the times of observation.

A total of 12 parameters were observed as follows: 1) inflammation of injection site

(bump/raise), 2) eating/drinking behavior, 3) levels of activities, 4) overall physical appearance

(grooming/fur), 5-8) body weight (Days -2, 0, 2 and 4), and 9) foot/leg function/appearance.

Mice were euthanized on the morning of Day 4 with CO2 and necropsied subsequently. Upon dissection, additional parameters were observed: 10) inflammation/necrosis of the muscles of the injected legs, 11-12) weights of the liver and kidney.

For treatments, a 40% stock solution of each PDA was made in DMSO. The working dilution was made with PBS shortly before injection. The injectable was an emulsion and was fully mixed before administration into muscles of one of the hind limbs. The control mice received the same percentage of DMSO as in the corresponding treated mice.

Table 1. Levels of PDA treatments. DMSO was used as the vehicle control.

Treatment Level n Control DMSO 3X8 (M=12, F=12)* TC 2.27 mM 8 (M=4, F=4) 3.78 mM 8 (M=4, F=4) CAR 2.0 mM 8 (M=4, F=4) 3.33 mM 8 (M=4, F=4) EU 1.83 mM 8 (M=4, F=4)

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3.04 mM 8 (M=4, F=4) *: Three different control groups were needed in order to control for the same concentrations of DMSO used in different PDA treatments. The control groups were: 10.56 mM (or 0.075%) DMSO to control for 3.78 mM TC and 3.04 mM EU, 6.34 mM (or 0.045%) DMSO for 2.27 mM TC, 1.83 mM EU, and 2.0 mM CAR, and 12.68 mM (or 0.09%) DMSO for 3.33 mM CAR, respectively.

Animal Scoring System

The scoring system of eating, activity and appearance was adopted from Morton (1998) and Hankenson et al (2013; Table 2). We also observed appearance/functionality abnormalities of the foot/leg after injection and developed a scoring system (Table 2). During each observation, mice were scored individually on a scale of 0-3 for all parameters in Table 2. A score of 0 indicates normalcy whereas a score of 3 indicates not eating/drinking, severe dehydration and foot/leg problem. At any observation point, a mouse would be humanely euthanized if the following score was received: a single score of 3 in any of the categories of eating/drinking, activities, and appearance; a combined score of ≥10; a body condition score of 1 or 5.

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Table 2. Scoring systems for eating/drinking, physical activities, physical appearance, and

foot/leg function/appearance

Clinical Score Eating/drinking Physical activities Physical appearance Foot/leg function 0 Drinking and eating Moves quickly around cage, frequently Normal Unaffected well stands at sides, close to cage-mates 1 Change in eating or Reduced movements, little/no Ruffled fur Closed digits, walking drinking habit investigation of surroundings, seeks normally shelter, prefers cage corner, moves around cage with gentle handling 2 Inappetence No movements around cage, move Weeping eyes Closed digits, no grip slightly by gentle handling, isolated from cage-mates 3 No eating or drinking, No movement by gentle handling, unable Weeping/closed eyes, Closed digits, no grip, severely dehydrated to right self within 30 seconds after being urine staining, difficulty dragging leg, foot placed on back defecating flipped (palm side up)

All scoring systems were adapted from Morton (1998) and Hankenson et al (2013) with the exception for foot/leg scores which were developed in this study.

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On the morning of Day 4, mice were weighed and euthanized. Animals were dissected for the weight of liver and kidney. The skin of both hind legs was removed and the color, swelling/necrosis of muscles were compared.

Statistical Analysis

Inflammation of injection site (bump/raise), eating/drinking behavior, body weight (Days

-2, 0, 2 and 4), and weights of the liver and kidney between treatment and their respective controls were analyzed using paired t-test in Minitab18 (Minitab, LLC., 2010).

Inflammation/necrosis of the muscles of the injected legs were analyzed using a z-test in Minitab

18 (Minitab, LLC., 2010). Differences were considered significant when P<0.01. For the foot/leg scores and the behavior scores, over time, an ordered logistic regression was used in R (R Core

Team, 2013).

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Results

Effects of PDAs on mortality, body weight, eating/drinking, physical activity, appearance, injection site, and foot/leg

No mortalities were observed in any of the control, TC, and EU groups. However, CAR exhibited more acute toxicity in males than females (Table 3). In the 3.33 mM CAR group one male mouse was euthanized on the morning of Day 1 for receiving clinical score that exceeded study parameters of an overall total behavior score equal to or exceeding 10 or any score of 3. A second and third male mice were found dead in the 2.0 and 3.33 mM CAR groups on the evening of Day 0 and morning of Day 2, respectively.

Table 3. Mortality from CAR treatments (dead/total). No mortality was found in the corresponding DMSO controls.

2.0 mM 3.33 mM CAR

Female 0/4 0/4

Male 1/4 2/4

The weights of control mice ranged from 30.22g ± 4.33g and 32.75g ± 4.61g, respectively, before and after the experiment. No significant differences were seen between any treated groups and their respective controls at any time of data collection on Days 0, 2 and 4

(data not shown).

No significant differences were observed between any treated groups and their corresponding controls in eating/drinking, activity, appearance and foot/leg scores. Minor behavioral changes such as reduced movements and ruffled fur (lack of grooming), however,

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were observed in two males and one female treated with TC (Table 4). Most of these changes

occurred within an h of treatment and all animals returned to normal by 12 h of injection.

Similarly in the 3.33 mM CAR group, reduced activities/ruffled fur were also observed in two

male mice and reduction in eating/drinking in a third male. These effects seemed latent

compared to those by TC and lasted longer but all animals returned to normal by 24 h post

injection (Table 4).

Table 4: The averaged behavior scores and the number of animals receiving a score of >0 during

the first 12 h of TC and CAR treatments.

1 h post-injection 12 h post-injection

Treatment Eating Activity Appearance Eating Activity Appearance

Control 0 0 0 0 0 0

TC 2.27mM 0 0.125 (1/8) 0.125 (1/8) 0 0 0

3.78mM 0 0.125 (1/8) 0.125 (1/8) 0 0 0

Control 0 0 0 0 0 0

CAR 2.0 mM 0 0 0 0 0 0

3.33mM 0 0.125 (1/8) 0.125 (1/8) 0.44 (2/8) 0.31 (2/8) 0.38 (2/8)

No bumps or swelling (local inflammation) were observed at the injection sites during the

entire experimental period. The observation of digit-closing and foot-flipping was not expected

but carefully recorded and a scoring system developed (Table 2). Overall, the higher PDA doses

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always elicited a higher foot score and in more mice. Although various degrees of foot abnormalities were seen after injections of TC (Figure 2), CAR (Figure 3), or EU (Data not shown), none of the foot scores in any of the treated groups were significantly different from controls. Most changes in the foot occurred within the first h of injection and in the majority of the cases, the foot abnormalities returned to normal by the end of the observation period (Day 4)

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Control 4 2.38mM TC 3 3.97mM TC 2

Average Average foot score 0 D0 D0H1 D0H2 D0PM D1AM D1PM D2AM D2PM D3AM D3PM D4AM Time of observation

Day 0 Day 1 Day 2 Day 3 Day 4

H0 H1 H2 PM AM PM AM PM AM PM AM P-value

Control 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8

2.4 mM TC 0/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8 1/8 0.8904

4.0 mM TC 0/8 2/8 2/8 2/8 7/8 7/8 7/8 7/8 3/8 3/8 0/8 0.8904

Figure 2: The averaged foot/leg scores (means+SD; upper panel) and number of mice with scores of >0 (lower panel) in controls and TC treated mice. H and D: hour and day post-injection. Scoring system: 0=normal; 1=closed digits while walking well; 2=closed digits, no grip; 3=closed digits, no grip, dragging leg, foot flipped.

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4 Control 3.24mM CAR 3 1.94mM CAR

1 Average Average foot Score

0 D0 D0H1 D0H2 D0PM D1AM D1PM D2AM D2PM D3AM D3PM D4AM

Time of observations

Day 0 Day 1 Day 2 Day 3 Day 4

H0 H1 H2 PM AM PM AM PM AM PM AM P-value

Control 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8

2.0mM CAR 0/8 5/8 5/8 5/8 4/8 4/8 4/8 4/8 4/8 4/8 4/8 0.9585

3.33mM CAR 0/8 2/8 2/8 3/8 6/8 6/8 6/8 5/8 2/8 3/8 1/8 0.9586

Figure 3: The averaged foot/leg scores (means+SD; upper panel) and number of mice with scores of >0 (lower panel) in controls and CAR treated mice. H and D: hour and day post-injection. Scoring system: 0=normal; 1=closed digits while walking well; 2=closed digits, no grip; 3

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Effects of PDAs on the liver, kidney and leg muscles

The muscles on the entire injected legs were compared to those of the non-injected counterparts. No inflammation, necrosis or discoloration was observed in any animal, PDA treated or controls.

The weights of the liver and kidney, and the relative weight of the kidney were not significantly different between treated and their corresponding controls. The relative liver weight of mice treated with 3.04 mM EU, however, was significantly higher than that of the corresponding controls (p<0.01).

Table 5. Weights (g) and relative weights (%) of the liver and kidney in control and PDA treated mice. Treatments Liver (g) Kidney (g) Relative Liver (%) Relative kidney (%)

Con:6.34 mM 2.52±0.77 0.62±0.19 7.29±1.08 1.80±0.30

EU 1.83 mM 2.43±0.39 0.48±0.09 7.76±0.62 1.68±0.29

Con:10.56 mM 2.2±0.65 0.53±0.18 6.84±1.03 1.67±0.36

EU 3.04 mM 2.55±0.67 0.57±0.19 8.22±0.87 1.75±0.35

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Discussion

Carvacrol (Austgulen et al. 1987), eugenol (Schroder and Volmer, 1932), and trans- cinnamaldehyde (Teuchy et al. 1971) are all metabolized in the liver and excreted in the kidney within 24 h. With 13 parameters each observed for trans-cinnamaldehyde, eugenol, and carvacrol at two different levels, a total 117 parameters were monitored and the only statistically significant difference was found in the relative liver weight at 3.04 mM EU group. The most severe side effects were the death of 3 males out of 16 CAR treated mice. Other changes in behavior, activities, and foot/legs were minor, in few mice and all recovered by completion of the study, or 4 days after injection.

Carvacrol is generally regarded as safe for consumption and has been used in low doses as a flavoring constituent and natural preservative for foods. For potential effective therapeutic applications, higher amount need to be used. Toxicity studies of Carvacrol are limited (Suntres et al., 2015). In the mouse, it was estimated that the median lethal doses for intravenous and intraperitoneal administration were 80.0 and 73.3 mg/kg of body weight, respectively (Andersen,

2006). White it was not the purpose here to establish an LD50 for im administration of carvacrol, 3 of the 8 male mice died with 300-500 mg/kg (2.0-3.33 mM) while all females did not exhibit toxicity, suggesting a high tolerance from im administration. It is unclear why male responded more strongly than females. The exact mechanism of the toxicity could be a combination of carvacrol’s effects in cytotoxicity, energy production effects by mitochondria,

(Suntres et al., 2015).

No local inflammation or necrosis was observed throughout the experimental period, suggesting that PDAs were quickly absorbed and became systemic. The only local effect was the partial loss of function of the feet and theeir abnormal appearance, which occurred in all

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PDA treated groups. PDAs have been reported to accumulate in the nerve cell membrane, thus cause a sterical blocking of embedded function proteins, which are the ion channels (Buchauer et al., 1993). This blocking causes a change in the physical properties of the membrane, especially a modification of its ion permeability. Inhibition of the inflow of calcium ions leads to a spasmolysis. Suppressing the generation of an action potential thus causing local anaesthesia, and the suppression of the irritability of mast cells, which are responsible for inflammation, brings about an anti-inflammatory effect (Buchauer et al., 1993).

With specific regard to eugenol one study found that eugenol blocks rat sciatic nerve compound action potentials probably by acting on the voltage dependent Na+ channels. At high concentrations (2 mM) and during brief application eugenol blocked the action potential without interfering in the resting membrane potential or membrane input resistance. However, at low concentration (0.6 mM) and longer applications, it was observed that a significant reduction in the input membrane resistance which raises the possibility of a secondary effect involved in the reduction of neuronal excitability when eugenol was present (Moreira-Lobo et al., 2010).

Similar occurrences have been found in carvacrol. A concentration of 10 mM blocked excitability in the sciatic (Goncalves et al., 2010). However, Joca and colleagues (2012) demonstrate that carvacrol induced inhibition in peripheral nerve fibers can be achieved with a

10-fold lower concentration (1 mM). Carvacrol showed and inhibitory effect on all myelinated fibers that contribute to the first and second action potential components of the sciatic nerve

(Joca et al., 2012). In addition, changes in threshold values in the presence of carvacrol indicate that this monoterpenoid effectively blocks excitability in the peripheral nervous system.

Alteration in resting membrane potential and/or in input membrane resistance would be implicated in a reduction of neuronal excitability (Lima-Accioly et al., 2006).

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The highest PDA concentration that can be dissolved in the DMSO was 40% (or 2.66M

CAR; 2.44M EU and 3.03M TC). When the working concentration of PDAs were prepared by adding PBS, the PDA, DMSO and PBS mix became an emulsion. This, however, did not seem to have affected the absorption of PDAs because no local accumulation was found and systemic effects were quickly observed.

Conclusion

With 12 parameters observed at each level of control/treatment, a total of 117 parameters were studied. Overall, little toxicity was observed when PDAs were injected intramuscularly at the levels used. This study provides the first known reports of toxicity effects in mice intramuscularly injected with carvacrol, eugenol, and trans-cinnamaldehyde. This shows that intramuscular injection of trans-cinnamaldehyde, and eugenol is relatively safe overall with minor side effects improving over the span of a couple of days. CAR, however, has more acute toxicity in males.

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Acknowledgements

This work was supported by the USDA-ARS grant: 1265-31000-091-02S, summer fellowship from the Connecticut Center for Entrepreneur and Innovation, and the EIA award from CTNext. Conflict of interest: The corresponding author, Dr. Xiuchun (Cindy) Tian, is the founder of a start-up company, VigorSential, LLC. All other authors declare no conflict of interest. Authors' contributions X.C. Tian, E. J. Johnson, J.Duan designed the study; E.J. Johnson, J.Duan, Srirattana. K performed the experiment; E.J. Johnson, J. Duan analyzed the data; and E.Johnson drafted the manuscript. All authors read, revised, and approved the final manuscript.

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Chapter IV. VigorSential

University Support

Accelerate UConn is UConn’s National Science Foundation Innovation Corps site. Its mission is to catalyze the transition of new scientific discoveries and capabilities from the lab to the market place. Over the course of 7 weeks, teams will learn how to determine the market opportunity for their product or technology. Each workshop provides hands-on training in the fundamental components of new venture creations and it delivered by experienced entrepreneurs and faculty members. Participants are provided with personalized coaching and feedback which will help them construct an evidence-based business model and market entry strategy. Participating teams also receive $3,000 in seed funding for their ventures.

The Connecticut Center for Entrepreneurship and Innovation focuses its resources on high-impact ventures tackling fundamental problems. The goal of the Summer Fellowship

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E. Johnson program is to help entrepreneurs move out of the conceptual stage of venture development, and into the marketplace. Sessions include goal setting, defining their business model, mapping out key milestones, sale and growth strategy, customer acquisition, data- driven decision making, and forward planning. The top ten startups from across the university come together to spend 3 days a week over 8weeks developing skills needed to bring new products and technologies to market and receiving one-on-one coaching from industry experts. The Wolff

New Venture Competition features 10-minute presentations by the top five entrepreneurial teams from the CCEI Summer Fellowship program, chosen on a basis on venture viability and value added to the market.

CTNext is a support network for Connecticut entrepreneurs that gives the states most promising startups grants to fund projects that help advance businesses. Selected applicants are then invited to one of the Entrepreneur Innovation Awards events for the final phase of review, where the entrepreneur's ideas are presented to a live panel comprised of entrepreneurial experts.

In order to turn a passion into a successful business, it needs to be set up in a way where it makes a profit and is sustainable long term. In the world we live in today it can be extremely difficult to promote a new product, especially if your potential customers have outside views/ knowledge regarding the specific product you’re trying to sell. By checking out the competition you will know: 1) what is already on the market, 2) what your innovation will have to offer, 3) if your idea will truly meet the desires or needs of your potential customers, 4) the general price customers are willing to spend on similar products, 5) how customers will hear/ gain knowledge about your product, 6) how customers will obtain the product, 7) how

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A business model is the profit formula. It’s the method used to acquire customers, serve them, and make money doing so. A business can be broken down into three primary areas: offering, monetization, and sustainability. The business model is the core concept upon which you develop your business plan, and therefore is a significant portion of your business plan. The business models answer the same basic questions of 1) what problem are you trying to solve, 2) who needs this problem solved, 3) what segment is the model pursing, 4) how will you solve this problem better, cheaper, faster or differently than others, 5) what is the value proposition,

6) where does your offer place you on the value chain, 7) what is your revenue model, 8) what is your competitive strategy, 9) how will you maintain your competitive edge, and 10) what partners or other complementary products should be used. To make a truly successful business model the product/business itself needs to be differentiated from competitor products and the real interworkings of the model need to be difficult to replicate.

Competitive advantage allows a company to perform at a higher-level than others in the same industry or market. This can allow you to oversell, out profit, and out perform others in the same industry of the market typically by three main areas: 1) cost leadership, 2) differentiation, and 3) focus. Cost leadership means that the business has the capability to deliver similar products or services as your competitors for a lower cost, successfully done by

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E. Johnson lowering the cost of goods sold, instead of lowering the sales price. Differentiation means that the customer feels your product has superior and different attributes than the competing products. By focusing on the company’s specific purpose, you can gain the life-long trust of your customers because they will know your brand over time. To enhance the company’s competitive advantage ability to innovate is a must. Innovation can be done at several levels within a company from product design, to product re-framing, to team management. A company should embrace innovation because it is viewed by customers as a sign that the company knows/ and is willing to design products that are useful and necessary to customers.

Below is the business plan for the product MycoZAP from VigorSential.

Executive Summary

VigorSential, LLC is based in Storrs-Mansfield, Connecticut. The company is in the process of offering natural preventions for Mycoplasma bovis associated diseases in cattle. Its goal is to use plant derived antimicrobials (trans-cinnamaldehyde, eugenol, and carvacrol) in an easy, cost effective, and sustainable way to reduce overall antibiotic use along with increasing animal welfare.

MycoZAP is the first product presented by VigorSential that incorporates the unique biological activities of PDAs into a loose mineral power to be used on both beef and dairy farms.

These PDA’s can be mixed either into loose mineral that the cattle already receive or into water troughs. This unique technology is easily adaptable to both dairy and beef farms all over the globe because it can be easily scaled to any level and is economical for both the farmers and

VigorSential.

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The initial target customers are large beef farmers that have at least 2,500 head with a strong emphasis on calf operations, where the calves are shipped to auction follow by feedlots.

This customer segment is most affected by the negative consequences of M.bovis because the operations are not closed systems, unlike dairy farms where cows are less frequently transported in and out. These farmers need an inexpensive preventative teatment that is quick and easy to use. Since MycoZAP has unique biologically active properties, it will change from season to season with the current active form of M.bovis, therefore, making it a long-term investment and product for the farmers. Unlike traditional antibiotics that only target specific forms of M.bovis and become quickly outdated. VigorSential has developed a great team of scientific researchers, business expertise, and veterinarians that allow every aspect of the product and business to optimally work.

Company description

VigorSential was created based on years of knowledge on the benefits of natural compounds such as PDAs. M.bovis has been an issue for several decades; however recent outbreaks and the general publics shift away from antibiotic use, prompted the merge of PDAs to prevent M.bovis. The specific PDAs being used in MycoZAP have been scientifically proven to inhibit the growth of other bacteria species. However, until recently their effectiveness in

M.bovis had not been shown. By using PDAs on M.bovis, it allows the farmers an easy, effective, and reliable way to prevent the diseases caused by M.bovis infection.

The general business model is that VigorSential will gain the selected PDAs from is numerous suppliers. From there VigorSential will combine the PDAs into their specific

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E. Johnson combination to create MycoZAP. Initially, this loose powder will be supplied to farmers to either use in their current mineral bales or to be mixed with water.

Products and Services

The original form of MycoZAP is intended to be a loose mineral that farmers can place into existing mineral mixes or even into the water. The product can also be turned into a mineral block for greater ease. After future research, the product has the potential to be used as an injectable prevention/ treatment, which could be extremely beneficial for dairy farmers with frequent cases of mastitis.

Typically a beef farm with an average herd size of 2,500 head will spend $16,500 annually on vaccines, with veterinarian costs at more than $250 and a withhold time of the meat of 60 days costing $13,700 in just meat sales. This means that on average with all factors considered is cost farmers $30,450 annually to vaccinate their entire herd. Farmers will spend even more annually on antibiotic costs. On average the same farmer will spend $30,000 on just antibiotics, with more than $250 in veterinarian costs and a withhold time of 3-5days costing

$26,010 in meat sales. All of this combined brings the farmers annual antibiotic costs to

$56,230 annually. With MycoZAP the farmer will spend $2,800 annually on MycoZAP alone.

However, there is no additional veterinarian cost and no additional withhold time on meat sales, meaning the meat is safe for sale/consumption throughout the entire process. By using

MycoZAP, the farmers will still have to pay for the yearly vaccine costs; however with consistent use, antibiotics can be reduced saving the farmer $53,430 yearly.

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There is no other product like this currently on the market. Researchers/ companies have made previous attempts in other species; however the attempts were unsuccessful because of implementation issues and biological changes that were made to the natural components during processing. MycoZAP can be used long term, season after season, on the farms because it will change as the M.bovis changes, therefore, reducing the chance of bacterial resistance.

Direct sales of MycoZAP will be made with company representatives who can supply the product directly to the farmers. Farmers will also have the option to buy the product online at www.vigorsential.com. Sales of the product are expected to be stable throughout the year because the large cattle operations being targeted operate year-round. Based on M.bovis seasonal projections (stated above) there do not appear to be any seasonal variations on

M.bovis prevalence. Initial customers include large beef/calf farmers in the US, with expansion to include large dairy industries, followed by global marketing. The technology around

MycoZAP can also be adapted to target chickens, pigs, sheep, goats, and horses worldwide.

Since the PDAs are natural compounds, they themselves are not patentable. However, their specific combination and ratios (formula) used to create MycoZAP is. This will be initiated, once all animal trials are completed.

Market analysis

There are 692.3 million beef cattle and 265 million dairy cattle in the world. In the US alone, there are 24 million dairy cattle and 22.7 million beef cattle. Of the beef cattle, there are another 22.7 million beef calves. MycoZAP will be sold in 10 pound buckets for $399/bucket,

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E. Johnson which is only $1.12 per calf. In the first year of sales (2020) the target is to acquire 10% of the calf market giving projected sales at $3.5 million. If all of the calf sales were acquired total projected sales is $35 million. If sales are expanded to all beef in the US and then to all cattle in the world projected sales are $104 million and $1.08 billion respectively. After the total cattle population is acquired the technology can be modified to target the 19 billion chickens, 2 billion pigs, 1 billion sheep, 450 million goats, and 58 million horses across the globe.

Strategy of implementation

MycoZAP will be implemented into the market over the course of 2 years. The project began in 2018, and first sales are projected to be in 2020. In 2018 and 2019, the cell work will be completed, providing preliminary information around the PDAs interaction with M.bovis. In

2019, the small animal (mice) toxicology study was completed to show no significant toxicology reactions (see study above). In the remainder of 2019, the large animal trials will be planned, patent planning will continue, along with the first phase of marketing/advertising. Production of

MycoZAP will be in the first part of 2020, with the first sales projected to occur in the later half.

Marketing and advertising will occur by utilizing four main channels: 1) newsletters and brochures, 2) attending seminars and trade shows, 3) vigorsential.com, and 4) visiting farms and large animal veterinarians to continue gaining customer knowledge. Magazines such as

Country Folk and Agweb will also be utilized, closer to production and sales. In 2021, the first phase of expansion will occur, where MycoZAP will be incorporated into a mineral block.

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Financial Plan and projections/ Organization and Management team

Research and design costs are estimated at $363,455 with selling, general and administrative expenses estimated at $779,600. This brings total cost projections at $1,143,055.

Specific break down of costs can be found in the summary table below. MycoZap will be initially sold in 10 pound buckets for $399 with the cost of production at $55, which provides a gross profit of $344 per unit. In 2020, with the sale of 10,000 units expenses are projected at

$1,143,055 with sales at $3.9 million. Year two of sales is estimated at 19,700 units, with expenses at $1,200,207, and sales at $7.88 million. Year three of sales is estimated at 34,400, units with expenses at $1,314,511, and sales at $15.73 million. After year two of sales the sales associates, regional manager, and lab workers employment numbers will be doubled.

Possible sources of funding include USDA: NIFA, American Jersey Cattle Association,

Organic Farming research Foundation, New York Farm Viability Institute, Spark Program

(UCONN), UConn Innovation Fund. Thus far, funding has come from Accelerate Uconn, CCEI

Summer Fellowship, CTNext EIA, and USDA (grant for lab worker).

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