Campylobacter is a leading cause of human gastroenteritis in the United States and worldwide (Corcionivoschi et al., 2012; Sibanda et al., 2018). In the United States alone, the Centers for Disease Controls and Prevention (CDC) estimates an annual 1.3 million cases occurring per year (Centers for Disease Control and Prevention [CDC], 2018). From 1999 to 2008, Campylobacter was estimated to cause an annual 8,463 hospitalizations and 76 deaths in the United States, along with an annual $1.7 billion financial cost to the United States (Batz et al., 2012). In 2015, of the 4,598 hospitalizations caused by foodborne disease 1,087 were the result of Campylobacter (Centers for Disease Control and Prevention [CDC], 2017a). This burden on public health was second only to Salmonella when considering bacterial infections (Batz et al., 2012; Scallan et al., 2015). When aggregating the loss of life and health due to illness, Scallan et al. (2015) determined that annually, Campylobacter caused 22,500 disability-adjusted life years (DALY). Campylobacter was ranked 3^rd as the leading impact of public health due to foodborne illness behind non-typhoidal Salmonella (32,900 DALY) and Toxoplasma (32,700 DALY). A systematic analysis from 1990 to 2013 identified Campylobacter as the fourth leading cause of diarrheal disease behind rotavirus, typhoid fever, and cryptosporidiosis (Murray et al., 2015). According to the World Health Organization (WHO), diarrheal diseases, viewed as a whole, cause an estimated 550 million illness each year resulting in the annual death of approximately half a million infants under the age of two (World Health Organization [WHO], 2013).

In a population-based study on patients with diarrheal illness induced by Campylobacter, over 95% of the 1,316 cases were caused by Campylobacter jejuni (Friedman et al., 2004). This species, along with pathogen Campylobacter coli, have been found in the gastrointestinal tract (GIT) of poultry (Wang et al., 2002; European Food Safety Authority [EFSA], 2010; Centers for Disease Control and Prevention [CDC], 2017b). Broilers and layer flocks have consistently been shown to contain Campylobacter prevalences greater than 70% (Corry and Atabay, 2001; Stern et al., 2001; Ansari-Lari et al., 2011; Sahin et al., 2015). Schets et al. (2017) found that 97% of layer and 93% of broiler flocks tested positive for Campylobacter. Across eight flocks in the Netherlands, 55 cecal samples were taken and found that C. jejuni was the Campylobacter isolate for 100% of cecal broiler samples where in layer hens 52% were C. coli, 40% were C. jejuni (40%) and 7% were C. lari. Identical sequence types were found in the soil, sediment, and surface water which indicates potential contamination of the bird through environmental means. Through contamination of the poultry carcass, Campylobacter can cause foodborne illness and has been the etiological agent in outbreaks caused by poultry products (Nauta et al., 2009; Geissler and Powers, 2011). Therefore there is a continued urgency to implement pre- and post-harvest technologies to prevent Campylobacter contamination at all stages of poultry production.

With the rise of multidrug-resistant bacteria, consumer preference for antibiotic-free chicken, and government regulations such as the European Union’s 2006 ban of antibiotics, alternative antimicrobials have become necessary (Cervantes, 2015; Johnson, 2015; Shin et al., 2015). One alternative to conventionally implemented pre- and post-harvest antimicrobial strategies is the use of essential oils (EOs) (Tiihonen et al., 2010; Amerah et al., 2012; Calo et al., 2015; O’Bryan et al., 2015; Thibodeau et al., 2015; Micciche et al., 2018b). The EOs industry had a United States market share of $6.6 billion in 2016, a 286% increase from the 2004 market share of $2.3 billion (Zviely, 2004; Grand View Research, 2018). They are often used as flavoring agents in food products and perfumes, embalmment, anti-inflammatory and anesthesia remedies (Burt, 2004; Bakkali et al., 2008). Some EOs, such as eugenol, have been reported to have a preventative effect against cancer (Burt, 2004; Tsuneki et al., 2005; Bakkali et al., 2008). Plant-based EOs such as eugenol, thymol, carvacrol, and cinnamaldehyde have been examined and screened for their antimicrobial properties against a number of pathogens including Campylobacter (Friedman et al., 2002; Chouliara et al., 2007; Thibodeau et al., 2015; Kelly et al., 2017; Upadhyay et al., 2017). They are also commercially attractive as alternative antimicrobials, because they are considered acceptable for organic and non-conventional applications (Micciche et al., 2018b; National Organic Program, 2018). They may be useful not only in preventing human health-related diseases but in improving the performance of the bird (Alcicek et al., 2004; Hernandez et al., 2004; Diaz-Sanchez et al., 2015; National Organic Program, 2018). As such, the objective of this review was to discuss EOs as an intervention approach for limiting Campylobacter contamination in the poultry industry pre- and post-harvest. By investigating the metabolic activity of Campylobacter, a greater understanding of the mechanistic effects of EOs may be elucidated for more optimal application approaches in the future.

Campylobacter belongs to family Campylobacteraceae, which also includes Arcobacter and Helicobacter (Fitzgerald and Nachamkin, 2011). They are non-sporulating Gram-negative microorganisms, with an s-shape and cell dimensions between 0.5 and 5 μm and ranging in width from 0.2 to 0.9 μm (Vandamme et al., 2006). A single polar flagellum is found in 20 of the 22 species with the exception being the non-motile C. gracilis and C. showae that possess multiple flagella (Debruyne et al., 2008; Facciolà et al., 2017). Campylobacter spp. are microaerophilic and grow optimally in a gas composition of 5% O₂, 10% CO₂, and 85% N₂, pH of 6.5 to 7.57 and from 37 to 42°C (Garénaux et al., 2008; Davis and DiRita, 2017; Facciolà et al., 2017). Campylobacter will not grow at temperatures below 30°C, due to an absence of a cold shock protein gene, or with a water activity below 0.987, which is necessary to maintain turgor pressure (Hazeleger et al., 1998; De Cesare et al., 2003; Wallace, 2003; Levin, 2007; Facciolà et al., 2017). Cold shock proteins range from 65 to 75 amino acids serve as nucleic acid chaperones that prevent mRNA secondary structures from forming which enable efficient transcription and translation (Phadtare and Severinov, 2010; Keto-Timonen et al., 2016). Interestingly, despite the lack of a cold shock protein, biofilms of C. jejuni have been found to form and persist in 13°C conditions and formed biofilms with the largest surface area compared to 20, 37, and 42°C (Sanders et al., 2008). Attachment of planktonic cells to the biofilms was not significantly impacted by temperature (Sanders et al., 2008).

Compared to other foodborne pathogens occupying the poultry GIT, Campylobacter possesses a metabolism that is somewhat different and thus may be a challenge for some intervention strategies. Campylobacter spp. do not utilize the traditional glycolysis pathway, instead favoring amino acids such as aspartate, glutamate, serine, and proline for their cellular respiration pathways (Stahl et al., 2012). The traditional glycolytic pathway is incomplete as genes encoding glucokinase and 6-phosphofructokinase are absent (Parkhill et al., 2000). Furthermore, despite a complete tricarboxylic acid cycle (TCA), gluconeogenesis from glucose-6-phosphate to glucose is not observed, and the genes necessary for encoding the proteins associated with the pathway are absent (Parkhill et al., 2000; Velayudhan and Kelly, 2002). Some of the gluconeogenesis and glycolysis genes and respective proteins are present which has led to the hypothesis that C. jejuni can catabolize intermediary molecules (Stahl et al., 2012). For instance, Hofreuter et al. (2006) suggested that the presence of a glycerol-3-phosphate transporter (GlpT) in C. jejuni 81176 could be indicative of the breakdown of glycerol-3-phosphate to generate pyruvate and potential further breakdown through the TCA cycle. Additionally, the non-oxidative portion of the pentose pathway shunt (transaldolase, transketolase, ribulose-3-phosphate epimerase, and ribose-5-phosphate isomerase) is suggested to have the ability to metabolize pentose sugars (Line et al., 2010).

Two studies independently identified novel l-fucose pathways with a l-fucose permease that is homologous to the one characterized in E. coli (Dang et al., 2010; Muraoka and Zhang, 2010; Stahl et al., 2011). Fucose is a component of eukaryotic glycoproteins and is found in mucin (Allen and Griffiths, 2001; Robbe et al., 2004). Campylobacter jejuni utilizes fucose as a chemoattractant for mucin attachment (Hugdahl et al., 1988; Tu et al., 2008; Gangaiah et al., 2010; Kassem et al., 2013). While fucose uptake was phenotypically observed in C. jejuni strain NCTC 11168 (isolated from human feces), it was not detected in strains 81116 and 81176 (Muraoka and Zhang, 2010). This is due to the presence of a genomic island cj0480c–cj0490, which has been linked to an increase in virulence (Parker et al., 2006). Stahl et al. (2011) noted that this metabolic pathway improved colonization of the piglet model for human disease indicating its importance in human-associated pathogenesis. Moreover, when the l-fucose pathway genes were mutated, C. jejuni was still able to colonize the ceca of chickens but was outcompeted in a co-colonization experiment by the wild-type strain (Muraoka and Zhang, 2010). Despite the presence of this metabolic pathway, Campylobacter is largely asaccharolytic and relies primarily on amino acids and organic acids for its energy and carbon needs (Lin et al., 2009; Stahl et al., 2012; Kassem et al., 2013; Kelly et al., 2017).

Amino acids utilized by Campylobacter as carbon and energy sources include asparagine, glutamine, serine, aspartate, and proline (Wright et al., 2009). Campylobacter jejuni will preferentially utilize serine, aspartate, asparagine, and glutamate (Guccione et al., 2008; Wright et al., 2009; Stahl et al., 2012). Parsons (1984) determined the most common amino acids within the ceca of leghorn hens through ion-exchange chromatography. Serine ranked 4^th (92.2 mmol/mol ceca content), aspartate ranked 2^nd (109.9 mmol/mol ceca content), and glutamate ranked 1^st (137.5 mmol/mol ceca content) as the most concentrated amino acids within the ceca. Asparagine, glutamine, glycine, cysteine, and tryptophan concentrations were not reported. While these concentrations will vary depending on diet, GIT microbial population, and bird type, the data provides insight into why amino acid metabolism of C. jejuni could play an essential role in its ecological niche within the poultry GIT (Ravindran and Bryden, 1999; Stahl et al., 2012). Whereas other species must rely on carbohydrate fermentation, the plentiful concentration of amino acids in the poultry ceca, due to protein-rich diets, allows Campylobacter to thrive (Józefiak et al., 2004; Vegge et al., 2009; Hermans et al., 2012).

Not all poultry dietary components contribute directly to the nutrition of the bird but may still interact with the GIT microbial population including resident foodborne pathogens. For example, there are primary plant compounds and secondary plant metabolites present in the poultry GIT that may not necessarily serve directly as nutrients but can still nutritionally influence bird performance (Smithard, 2002). While some fiber components, particularly lignin, are generally considered indigestible in the avian GIT, they can still impact GIT microbial composition, alter fermentation patterns, and influence metabolite absorption (Ricke et al., 1982, 2013; Jung and Fahey, 1983; van der Aar et al., 1983; Dunkley et al., 2007; Baurhoo et al., 2008; Sima et al., 2018). These high fiber sources have also been shown to limit the establishment of foodborne pathogens such as Salmonella Enteritidis in laying hens when serving as the primary dietary source in laying hens (Ricke, 2003; Woodward et al., 2005; Ricke et al., 2013). Plants also contain phenolic monomers (Jung and Fahey, 1983). Phenolic monomers can not only cross-link carbohydrates with lignin in the plant, decreasing fiber degradation in the GIT, but in their free form can be antimicrobial to aerobic and anaerobic bacteria (Zemek et al., 1979; Jung and Fahey, 1983).

Aromatic compounds can also be modified anaerobically (Tschech and Schink, 1985; Netzer et al., 2016). Often phenolic compounds such as chlorophenol and chlorobenzoate can be utilized as electron sinks, resulting in their reduction and modification within the lower GIT (Häggblom et al., 1993; Frazer, 1994). While Campylobacter has been shown to utilize some aromatic compounds such as resorcinol and β or γ-resorcylate, this was only shown in the presence of Clostridium spp. (Tschech and Schink, 1985; Evans and Fuchs, 1988). However, other aromatic compounds can be utilized as energy sources by some bacteria (Overhage et al., 2006). MetaCyc identifies five complete aromatic degradation pathways present in E. coli including cinnamate, phenylethylamine, nitroaromatic, and phenols (version 22.5; BioCyc; Menlo Park, CA, United States) (Caspi et al., 2015). Chlorobenzene, paraoxon, parathion, and shikimate degradation pathways have been identified in L. monocytogenes and degradation of p-cymene, along with other aromatic compounds, have been characterized in Pseudomonas spp. In Campylobacter, only the catechol degradation pathway has been detected. Numerous biodegradation enzymes have been elucidated in E. coli; for instance, eugenol has been shown to be degraded to ferulic acid by the vaoA gene-encoded enzymes (Díaz et al., 2001; Overhage et al., 2003, 2006). However, only resorcinol, protocatechuate, and phloroglucionol degradation enzymes are known to occur in Campylobacter (Evans and Fuchs, 1988; Villemur, 1995). Given the limited metabolic capacity of Campylobacter for modification, it would be intuitive that aromatic compounds that exhibit antimicrobial properties could effectively reduce Campylobacter populations in poultry production. This is important since aromatic compounds are one of the constituents present in phytobiotics.

Phytobiotics are plant-based compounds or extracts that have been suggested for use in commercial, and possibly organic, poultry production (Windisch and Kroismayr, 2007; Bakkali et al., 2008; Diaz-Sanchez et al., 2015; Micciche et al., 2018b). Most phytobiotics are Generally Recognized As Safe (GRAS) by the U.S Food and Drug Administration [FDA], 2018 and are less toxic and typically more residual-free compared to synthetic antibiotics (Diaz-Sanchez et al., 2015; U.S Food and Drug Administration [FDA], 2018). Botanicals, a subset of phytobiotics, are leaves, roots, bark, or other parts of a plant, and the terminology is often used interchangeably with phytobiotics (Windisch and Kroismayr, 2006; Mohammadhosseini et al., 2017). Other types of phytobiotics include herbs, which are derived from flowering non-persistent plants, oleoresins which are non-aqueous extracts such as balsam, and EOs (Prior and Cao, 2000). In this review, the focus will primarily be on the activity of EOs, but other phytobiotic compounds such as herbs will also be mentioned.

Essential oils, also known as volatile or ethereal oils, are oily plant-based liquids that possess aromatic properties (Burt, 2004; Hardin et al., 2010). The term “essential oils” was first coined by Paracelsus von Hohenheim in the 16^th century (Guenther, 1948). The term ‘essential’ relates to the effective element in a medical preparation of the drug and is therefore loosely defined (Oyen and Dung, 1999). Currently, there are over 3,000 known EOs with approximately 300 being commercially relevant (Bakkali et al., 2008; Diaz-Sanchez et al., 2015). They include oils such as turpentine, eugenol, and cinnamaldehyde, and can be derived from other botanical compounds and herbs such as thyme, oregano, rosemary, and lemon (Burt, 2004; Fisher and Phillips, 2008; Diaz-Sanchez et al., 2015). Essential oils are extracted from their corresponding plants by steam distillation, hydrodistillation, or solvent extraction which all can create a concentrate of aromatic and volatile compounds including terpenoids and phenylpropanoids (Nakatsu et al., 2000; Hardin et al., 2010; Raut and Karuppayil, 2014). The concentrations of the ‘essential’ compounds in EOs vary wildly and are not typically defined (Lee et al., 2004a; Benavides et al., 2012).

Essential oils have a wide range of applications including turpentine for paint mixing or linalool and linalyl acetate that are used as alternative sleep aides (Buchbauer et al., 1991; Burt, 2004). Many are incorporated as ingredients for their palatable tastes and smells in foods and aromatic sprays (Franz et al., 2010). Oregano, thyme, and cinnamon are well-known flavor enhancers (Khan and Abourashed, 2011; Wang et al., 2013). Bergamon is used for its aromatic properties of Earl Gray Tea, while synthetic based citrus oils are an ingredient in soft drinks (Fabricant, 2008; Gonzalez-Molina et al., 2009; Callaway et al., 2011). While 32.9% of EOs are applied in the food and beverage industry, the second largest application of EOs in 2015 was for spa or relaxation purposes (30.84%) (Grand View Research, 2018).

In poultry, EOs have been utilized in preharvest management settings for non-pathogen related benefits (Diaz-Sanchez et al., 2015). Digestibility of poultry feed has been shown to be improved by the addition of EOs (Williams and Losa, 2001). CRINA^® (Akzo Nobel, Crina S.A, Switzerland) is a commercial blend of EOs containing thymol, eugenol, and piperine. Broilers fed 50 mg/kg of CRINA^®, exhibited improved activity of total amylase, trypsin, and maltase of 40kU/pancreas, 63U/pancreas, and 12.6 μM/g mucosa respectively, compared to the control group activity of 29kU/pancreas, 42U/pancreas, and 10.6 μM/g mucosa, respectively (Jang et al., 2004, 2007). This effect was not observed; however, when 0.1% of lactate was also introduced into the diet (Jang et al., 2004). The use of 200 ppm of a blend of oregano, cinnamon, and pepper improved fecal digestibility of dry matter (Hernandez et al., 2004). Ileal absorption levels of amino acids such as threonine, serine, asparagine, phenylalanine, histidine, and lysine were positively improved by 7.8 and 8.8% by the addition of 150 and 300 ppm of a plant extract containing capsaicin, carvacrol, and cinnamaldehyde (Jamroz et al., 2003).

Amino acid absorption studies have also been performed using rat intestines. A catheter was attached to both ends of a sector of the jejunum of anesthetized rats (Kreydiyyeh et al., 2000). In the treatment groups, the jejuna were preincubated with 1000 ppm cinnamaldehyde or 850 ppm of eugenol or a saline control. Alanine was then fed through the jejuna, and it was observed that after 40 min the jejuna preincubated with cinnamaldehyde and eugenol absorbed 22 to 25 nmol of alanine compared to 60 nmol in the control. This suggests reduced nutrient absorption (Kreydiyyeh et al., 2000; Lee et al., 2004a). However, conclusions drawn from this study may be difficult to apply to production animals due to the high concentrations necessary in the feed to result in similar concentrations in the GIT as utilized in Kreydiyyeh et al. (2000). Lee et al. (2003) observed that 100 ppm of thymol or cinnamaldehyde exhibited no significant effect on pancreatic digestive enzyme activity at day 21 or day 40 within female broilers (Lee et al., 2004a). This study shows that thymol alone is unable to impact pancreatic digestive enzymes within poultry and a combination of multiple EOs, such as in Jang et al. (2004, 2007), may be necessary to impact digestibility responses in broilers. However, it may be specific combinations of EOs that impact digestibility and nutrient absorption.

Improvements in nutrient absorption and digestibility can result in improved growth rates within broilers (Gous, 2010). Feed conversion ratio (FCR), the ratio between feed intake and average weight gain, is one of the more commonly used metrics to determine if a feed additive is beneficial to commercial livestock production (Leenstra, 1986). Several studies have reported no improvement in growth rate or FCR when diets have been supplemented with EOs (Lee et al., 2003; Jang et al., 2004, 2007). However, FCR and body weight gain (BWG) improvements have been observed in other studies that utilized EOs (Denli et al., 2004; Cabuk et al., 2006; Basmacioğlu Malayoğlu et al., 2010). Cabuk et al. (2006) demonstrated that 24 or 48 mg/kg of Heryumix^TM significantly improved FCR on days 21 to 1.53 and 1.56 compared to the control at 1.62. On day 42, compared to the control at 1.87, the FCR was also improved to 1.80 and 1.77 with 24 or 48 mg/kg of Heryumix^TM (Cabuk et al., 2006). Heryumix^TM is composed of oregano, laurel leaf, sage, myrtle, fennel, and citrus peel extracts (Herba Gida Maddeleri; Seferihisar, Turkey). Denli et al. (2004) observed similar improvements in quail when their diet was supplemented with 60 mg/kg of thyme or black seed oil.

This discrepancy may be due to certain variables including bird type, feed composition, and EO type. The intestinal mucosa and GIT microbiome composition of birds vary depending on the species and age of the bird (Zoetendal et al., 2004; Stanley et al., 2014). While the phyla Bacteroidetes and Firmicutes are dominant within quail and broiler chicken cecal microbiomes, the represented genera are different (Oakley et al., 2014; Liu et al., 2015). Over 117 different genera were identified by Wei et al. (2013) in broiler chicken ceca. The quail microbiome, however, is not as taxonomically rich at the genus level with only 32 genera being detected by Wilkinson et al. (2016). Furthermore, while the top five most dominant genera based on taxa analyses were Lactobacillus, Rumiunoccocus, Clostridium, Bacteroides, Faecalibacterium in the chicken cecal microbiome, the quail ceca microbiome was dominated by Bacteroides, Ruminococcus, Faecalibacterium, Enterococcus, and Clostridium (Wei et al., 2013; Wilkinson et al., 2016). These microbiome differences along with unknown host-species interactions should be considered when evaluating and comparing the effectiveness of any particular feed amendment across species (Koutsos and Arias, 2006).

Additionally, the feed may play a major role in how EOs improve FCR. As elucidated in Jamroz et al. (2003), EOs can impact the absorption of amino acids and potentially other nutrients in the ileum. As a consequence, the FCR should be improved in birds on typical commercial diets amended with EOs. Furthermore, improved absorption of amino acids could conceivably also allow for the use of non-conventional diets that contain less protein. In-depth studies employing not only commercial bird performance measurements but intestinal pathway and tissue profiling to screen and compare feed composition and EO blend combinations would be necessary to address this hypothesis.

Finally, while terpenoids are the main constituents of EOs, their chemistry can vary widely (Jager, 2010). For instance, carvacrol is a monoterpene alcohol and, in rats, its aliphatic group readily undergo aromatic hydroxylation while its alcohol group undergoes carboxylation (Jahrmann, 2007; Jager, 2010). Thymol on the other hand forms derivatives of benzyl alcohol and 2-phenylpropanol when reduced (Austgulen et al., 1987). When carvacrol was fed to rats, their excreted urine contained more of 2-(3-Hydroxy-4-methylphenyl)propan-2-ol then carvacrol but when thyme was fed to rats it was in the highest concentration out of its five derivates (Austgulen et al., 1987). This suggests, in rats, that carvacrol undergoes chemical interactions and is metabolized more than thymol. This functional difference between EO metabolism, along with the importance of bird type and feed composition, may in part explain the variation in EOs benefit to broiler nutrition.

Although there is variation in EOs benefit to poultry growth, the antioxidant activity of many EOs is well-known (Baratta et al., 1998; Ruberto et al., 2000; Martucci et al., 2015). Rosemary oil, thymol, carvacrol, oregano, ginger, and coriander all possess antioxidant activity (Wei and Shibamoto, 2007). An oxidation deterioration test involving the application of EOs to lard indicated that 0.20% oregano possessed the most antioxidant capacity followed by thyme, dittany, marjoram, spearmint, then lavender (Economou et al., 1991). Economou et al. (1991) also found that combinations of thyme and marjoram and thyme and spearmint EOs also had potential synergistic properties in protecting lard from oxidation. These properties benefit bird health and can be marketable if EOs are used on the finished product (Lee et al., 2004b; Diaz-Sanchez et al., 2015).

Additionally, the sensory characteristics of EOs mean that they can enhance the sensory characteristics of the final products if used in the appropriate concentrations. The addition of 300 mg/kg of oregano, garlic, or an equal combination in the diets was shown to significantly improve the flavor of frozen chicken breasts up to 60 days (Kirkpinar et al., 2014). Birds fed the EOs were processed, and the breasts were stored at -25°C for sensory analysis. Overall flavor evaluated on days 1, 15, and 30 indicated that all EOs treatments scored significantly higher than the control. Overall acceptability scores of breast meat indicated that only garlic resulted in a more palatable final product on days 1, 15, and 30. Overall acceptability scores were based on flavor, appearance, and tenderness. Days 45 and 60 scores were not analyzed statistically due to spoilage. On organic seabass filets, the addition of 0.2% thyme oil improved the sensory characteristics (Kostaki et al., 2009). A panel of seven judges evaluated the filets on nine days within a 21-day storage trial where the filets were held at -30°C. While the control group reached the acceptability limit on taste in 6 days the addition of 0.2% thyme improved the sensory characteristics scores extending shelf life by 2 days. When the filets were placed in modified atmospheric packaging (MAP) (60% CO₂; 30% N₂; 10% O₂), sensory characteristics were improved to allow for a shelf life of 14 days. When thyme oil was added to the MAP, shelf life was extended by 3 days. Despite these potential benefits, their antimicrobial activities might be some of their more important attributes to commercial poultry production.

Campylobacter contamination on poultry products is one of the more common causes of campylobacteriosis in humans (Keener et al., 2004). Incidence rates in a study of 425 broiler carcasses over 12 months revealed 87.5% of the post-chill carcasses were contaminated with Campylobacter in a French slaughterhouse (Hue et al., 2010). In the United States, 52% of post-chill carcasses (n = 325) were contaminated with Campylobacter with 100% of the carcasses being contaminated pre-chill (Son et al., 2007). The most frequent method for this contamination event is for the GIT to rupture during processing and contaminants to spill onto the carcass (Berrang et al., 2001). If not properly treated during carcass rinses, cross-contamination can occur, especially within chiller tanks (Bashor et al., 2004). As such, to prevent cross-contamination, it is vital that carcass rinses and sprays be applied as appropriate sanitation techniques. Essential oils may serve as an alternative sanitizer for processing washes.

In vitro laboratory experiments may have more relevance to post-harvest interventions than pre-harvest mediations due to the complexity of the poultry GIT that cannot easily be modeled in a laboratory environment (White et al., 1997). For instance, in a series of benchtop studies orange oil was found to be inhibitory to C. jejuni, C. coli, L. monocytogenes, Salmonella, and Pseudomonas (O’Bryan et al., 2008; Nannapaneni et al., 2009; Chalova et al., 2010). Nannapaneni et al. (2009) tested seven orange oil fractions on 3 Arcobacter strains and 21 Campylobacter strains, including 14 C. jejuni strains, four of which were isolated from poultry. When viewed on disk diffusion agar, cold pressed (CP) terpeneless Valencia orange oil produced the largest zones of inhibition (in mm) of C. jejuni including those isolated in poultry, whereas other orange oil fractions produced more limited zones of inhibition. Valencia orange oil was also reported to be inhibitory toward C. coli and Arcobacter. Kurekci et al. (2013) found tea tree oil at a concentration of 0.001% to be inhibitory against two strains of C. jejuni on nutrient agar. Wild carrot oil, when used in agar plates as an antimicrobial against Campylobacter spp. including a multidrug-resistant strain, C. jejuni 99T403, exhibited a minimum inhibitory concentration (MIC) of 125 to 500 μg/mL depending on the species or strain (Rossi et al., 2007). Isoeugenol and E-methyl isoeugenol are extracts of carrot oil and when tested against C. jejuni resulted in an MIC of 125 μg/mL (Rossi et al., 2007). Thymol, in a concentration of 0.25 μmol/mL, reduced C. jejuni and C. coli by 5 logs CFU/mL in Bolton broth from an initial concentration of 7 log CFU/mL (Anderson et al., 2009; Carocho et al., 2014). An EO extract from Origanum minutiflorum, composed primarily of carvacrol and p-cymene, has been shown to be effective in inhibiting C. jejuni in concentrations as low as 12.5 μg/mL on Mueller-Hinton agar (Aslim and Yucel, 2008). However, these MICs were strain-specific, as of the 12 C. jejuni strains tested, only C. jejuni 118d was inhibited at the concentration of 12.5 μg/mL, while C. jejuni 113k, 7d, and 9a required 700 μg/mL to be inhibited (Aslim and Yucel, 2008). Cinnamon, clove, thyme, and bay leaf oils were found to be bacteriostatic at a concentration of 0.075% against C. jejuni, S. Enteritidis, E. coli, Staphylococcus aureus, and L. monocytogenes when tested in tryptic soy broth (TSB) (Smith-Palmer et al., 1998).

Other studies reported similar inhibitory and bacteriostatic effects of oregano, eucalyptus, marigold, ginger, jasmine, cedarwood, carrot, mugwort, bergamot, and other EOs, against Campylobacter and other foodborne pathogens (Friedman et al., 2002; Moreira et al., 2005; Fisher and Phillips, 2006; Thanissery et al., 2014). The MIC of EOs on other foodborne pathogens were collected and reviewed in Hyldgaard et al. (2012). Friedman et al. (2002) tested 119 EOs against C. jejuni, E. coli O157:H7, L. monocytogenes, and S. enterica in PBS for 60 min at 37°C (42°C for C. jejuni) followed by plating on appropriate media for each species, including an iron-supplemented Brucella agar for C. jejuni. The EO concentration that resulted in the bactericidal activity of a 50% CFU decrease relative to the control was determined for each EO bacterial species combination. Marigold taegetes (0.003%), ginger root (0.005%), jasmine (0.006%), patchouli (0.007%), and gardenia (0.007%) were the most effective oils against C. jejuni RM1221 followed by cedarwood (0.0075%), carrot seed (0.0078%), celery seed (0.0085%), mugwort (0.009%), spikenard (0.009%), and orange bitter oils (0.009%). In this study, 39 EOs were tested against all four foodborne pathogens. When their bactericidal activities were averaged, the five most effective EOs were cinnamaldehyde (0.03%), thymol (0.05%), Spanish oregano (0.05%), carvacrol (0.06%), and Oregano origanum (0.06%). Of these five EOs, C. jejuni was impacted the most by the lowest percent concentration compared to the other tested foodborne pathogens.

This suggests that not only may C. jejuni be a prime target for EO remediation, but EO that have not been extensively tested may be more optimal candidates to utilize, such as marigold and jasmine. Disk diffusion methods indicated that lemon, sweet orange and bergamot were effective against L. monocytogenes, S. aureus, B. cereus, E. coli O157:H7, and C. jejuni (Fisher and Phillips, 2006). Each oil was added to a 2 cm disk and then placed on agar plates in the presence of the bacteria of interest, with C. jejuni SR 117 being plated on Campylobacter agar base with 5% horse blood and incubated at 42°C. No zones of inhibition were detected for C. jejuni when orange or citral was added. Bergamot produced a zone of inhibition of 23 mm, and lemon produced a zone of inhibition of 18 mm, which were the smallest zones compared to the other tested bacteria. The zone of inhibition for linalool was greater than 90 mm. The MIC was also determined for bergamot (greater than 4%), lemon (greater than 4%), and linalool (0.06%). While no zone of inhibition was visually detected for orange oil by Fisher and Phillips (2006), when orange oil was supplemented with thyme, the combination EOs demonstrated antimicrobial activity against Campylobacter (Thanissery et al., 2014). Similar to Fisher and Phillips (2006), a disk diffusion test was performed using thyme, orange, rosemary, clove, and a 1:1 ratio of thyme and orange oil. This was tested on C. jejuni 11601 MD, C. jejuni RM1221, and C. coli RM2228 along with a cocktail of the three strains. No visible growth on disk diffusion assay across all strains and in the cocktail was visualized when exposed to thyme or clove. For the cocktail, orange oil, rosemary, and the combination of thyme and orange oil produced zones of inhibition of 17, 11, and 20 mm, respectively. A macro-broth dilution assay was also performed using Mueller Hinton broth to determine the MIC and minimum bactericidal concentration (MBC) of these oils. However, the exact MIC and MBC was not determined as 0.0008% concentrations of the oils were sufficient for inhibiting bacterial growth.

Synergistic effects have also been detected (Nguefack et al., 2012). For instance, a combination of oregano and thyme or oregano and cinnamaldehyde required 80% less EOs to produce the same inhibitory effects in nutrient broth with a Campylobacter growth supplement (Navarro et al., 2015). Thymol and geraniol reduced Clostridium difficile in feces at 500 ppm, which was five times the concentration utilized for a significant reduction in pure cultures. When 0.16 mg/mL of rosemary oil, consisting of carnosic acid, carnosol, and rosmarinic acid was applied in the laboratory, a 2 log CFU/mL reduction in C. jejuni was observed in Mueller-Hinton broth when the initial concentration was at 7 log CFU/mL (Klancnik et al., 2009; Piskernik et al., 2011). However, four times the concentration was needed to achieve the same reduction in chicken meat juice, isolated from thawed previously frozen carcasses, unless supplemented with nisin, a bacteriocin (Piskernik et al., 2011). While the complete composition was not provided in Piskernik et al. (2011), it was suggested that lipids and proteins within the juice matrix might have partly inhibited the EO therapy. Burt (2004) suggested that the decreased EO effectiveness in the food matrix compared to the broth experiment may be because in an oil-in-water emulsion that allows the bacteria to grow as films and in colonies, which can shield interior cells from therapeutics. As a consequence, while in vitro laboratory models are important, to elucidate the potential impact of EOs, poultry product matrices as well as processing environments must be considered.

The use of EOs in the post-harvest environment has focused on their bactericidal activity within carcasses washes and finished products (Calo et al., 2015; Dima and Dima, 2015). A 0.5% 50:50 mixture of thyme oil and orange oil was used in a marinade for chicken wings dip-inoculated with 10⁷ CFU/mL of a nalidixic acid resistant strain of C. coli (Thanissery and Smith, 2014). While cross-contamination events were observed via the marinade, C. coli concentrations on treated wings were reduced by 3.0 log CFU/mL, as determined through plating of rinsates. Skinless chicken breasts experimentally infected with 5 × 10⁵ CFU/g of C. jejuni were subjected to stinkwort (0.2%; Inula graveolens), bay leaf (0.6%; Laurus nobilis), mastic tree (0.6%; Pistacia lentiscus), and winter savory (0.6%; Atureja gontana) (Djenane et al., 2012). After 8 days of refrigerated storage under microaerophilic conditions, greater than 5 log CFU/g reductions of C. jejuni were observed compared to the control inoculated with 5 × 10⁵ CFU/g. Sensory analysis indicated these EOs improved or did not impact the odor of the refrigerated samples according to a six member trained panel. Within 60 s, 0.06% linalool oil reduced C. jejuni on 2 cm × 2 cm pieces of cabbage leaf and chicken skin by greater than 5 log CFU and 2 log CFU, respectively (Fisher and Phillips, 2006). Cold pressed Valencia orange oil has also been shown to reduce C. jejuni UAF 244 on retail chicken thighs and legs (Nannapaneni et al., 2009). Chicken thighs and legs were dipped in a 0.8% saline solution containing 10⁶ CFU/mL C. jejuni UAF 244 for 5 min and then submerged for 2 min in 20% (v/v) Valencia orange oil or 20% (v/v) limonene. The samples were then rinsed and plated. Across both types of chicken pieces, 1.5 to 2 log CFU/mL reductions were observed, compared to the control. Treatments with limonene resulted in reductions without detectable recovery of viable bacterial cells, although the limit of detection was not provided by the authors. Moreover, while taste panels have found concentrations of orange oil up to 0.1% to be acceptable in milk, chicken patties and marinades, a 20% part dip has not been investigated for impacts on sensory characteristics (Jo et al., 2004; Fisher and Phillips, 2008; Rimini et al., 2014).

Other factors may confound EO efficacy toward Campylobacter in poultry products. For example, one major concern for poultry industries is the ability for pathogens to form biofilms (Srey et al., 2013). Biofilms are bacterial communities within a polysaccharide matrix that can readily form and attach on processing surfaces (Costerton, 1995; Donlan and Costerton, 2002). Biofilms can be difficult to remove by antimicrobials and processing sanitizers such as chlorine and peracetic acid, which are commonly used in the industry (Frank et al., 2003; Ryu and Beuchat, 2005; Scher et al., 2005; Deborde and Von Gunten, 2008). Campylobacter is known to form biofilms on stainless steel, polystyrene, and glass (Gunther and Chen, 2009). Coriander oil and its antimicrobial component linalool were found to affect biofilm formation of Campylobacter planktonic cells and pre-established biofilms (Duarte et al., 2016). When coriander and linalool were used at 2 μg/mL (approximately four times the MIC) C. jejuni and C. coli biofilms were reduced in size by 70 to 80% after 48-h incubation in a crystal violet assay (Duarte et al., 2015, 2016). Even when using half the MIC, coriander, and linalool reduced the biofilm by 20 to 25% (Duarte et al., 2016). Results of biofilm inhibition varied more wildly from planktonic cells, but all concentrations (0.025 to 2 μg/mL) successfully inhibited biofilm growth compared to the control, with linalool reducing some biofilm formation between 10 and 20% of the control (Duarte et al., 2016). Thyme, oregano, and cinnamon EOs, when used at concentrations below the MIC, were also found effective against biofilms of Acinetobacter, Sphingomonas, and Stenotrophomonas spp. that were isolated from biofilms within the food industry (Szczepanski and Lipski, 2014). Sphingomonas biofilms were reduced by 50% by thyme oil at a concentration of 0.001% where the MIC was 0.008% (Szczepanski and Lipski, 2014). Similar results were found using thyme and balsam on Pseudomonas and S. aureus biofilms (Kavanaugh and Ribbeck, 2012; Kerekes et al., 2015).

The effectiveness of EOs in poultry has not been clearly defined yet. Product advantages have been noted in several studies, but studies also exist that display no impact on FCR or BWG. Other advantages of EOs include the potentially improved flavor of the carcass, antioxidant capacity, and improved feed digestibility. There is also evidence that EOs can be utilized in vitro to impact pathogen concentrations, including Campylobacter. However, this depends largely on the EO utilized as the mechanism(s) of action are not well-defined. With over 300 commercially available EOs, precisely elucidating the underlying mechanisms may prove difficult (Bajpai et al., 2012). Less information is available regarding the mechanistic role of EOs used in vivo. Their potential ability to improve amino acid absorption in the ileum may allow for generating a GIT environment unfavorable to Campylobacter in the ceca due to diminished substrate availability, which is further downstream. To fully elucidate the impact EOs have on Campylobacter concentrations in vivo, further research on the mechanism(s) and effects of EOs must be performed.

More targeted delivery of EOs to certain sites in the avian GIT may be warranted as well. Microencapsulation may help to stabilize the chemical activity of the EO until it reaches its target site in the GIT, thus ensuring less variability. Microencapsulation also holds promise in addressing the intestinal absorption of an antimicrobial. However, investigations into the duration the encapsulated EOs remains in the GIT need to be performed to see when the remediation should be administered. In post-harvest settings, further studies should be performed involving the addition of EOs to sprays, washes, or within the chiller tank.

To determine the specific antimicrobial effects of EOs, the mechanism(s) must be elucidated. This is essential because, with a wide variety of EOs, there may be multiple mechanisms at work and thus synergistic potential which cannot be determined without proper identification of the mode of inhibitory action for each individual EO. Toward this effort, molecular approaches such as transcriptomics and proteomics may be employed to determine which pathways EOs inhibit will lead to further understanding of their impact on Campylobacter. While there have been suggestions to utilize EOs as a hurdle technology in poultry production pre- and post-harvest, mechanisms of action against Campylobacter and the optimal GIT locations and processing steps must first be established before any practical recommendations can be given.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

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