Campylobacter jejuni in Poultry: Pathogenesis and Control Strategies

C. jejuni is the leading cause of human foodborne illness associated with poultry, beef, and pork consumption. C. jejuni is highly prevalent in commercial poultry farms, where horizontal transmission from the environment is considered to be the primary source of C. jejuni. As an enteric pathogen, C. jejuni expresses virulence factors regulated by a two-component system that mediates C. jejuni’s ability to survive in the host. C. jejuni survives and reproduces in the avian intestinal mucus. The avian intestinal mucus is highly sulfated and sialylated compared with the human mucus modulating C. jejuni pathogenicity into a near commensal bacteria in poultry. Birds are usually infected from two to four weeks of age and remain colonized until they reach market age. A small dose of C. jejuni (around 35 CFU/mL) is sufficient for successful bird colonization. In the U.S., where chickens are raised under antibiotic-free environments, additional strategies are required to reduce C. jejuni prevalence on broilers farms. Strict biosecurity measures can decrease C. jejuni prevalence by more than 50% in broilers at market age. Vaccination and probiotics, prebiotics, synbiotics, organic acids, bacteriophages, bacteriocins, and quorum sensing inhibitors supplementation can improve gut health and competitively exclude C. jejuni load in broilers. Most of the mentioned strategies showed promising results; however, they are not fully implemented in poultry production. Current knowledge on C. jejuni’s morphology, source of transmission, pathogenesis in poultry, and available preharvest strategies to decrease C. jejuni colonization in broilers are addressed in this review.


Introduction
C. jejuni was first recognized in 1886 by Escherich as he described the C. jejuni as a spiral bacteria isolated from the colon of dead children [1]. Escherich also identified the C. jejuni microscopically in stool specimens of children who suffered from diarrhea without being able to culture it on solid agar [1]. In 1909, a Vibrio-like bacterium was frequently isolated from aborted fetuses [2], later named Vibrio fetus [3]. Similar reports linked Vibrio-like organisms to sterility in cows [4], and dysentery in pigs, and later named Vibrio jejuni [5].
Similarly, several reports noted the presence of Vibrio fetus in the blood of pregnant women [6] and the blood of people associated with outbreaks related to consumption of milk contaminated with Vibrio fetus [7]. The absence of a proper isolation method for Vibrio fetus (C. jejuni) from feces resulted in fewer case reports despite the high prevalence of this pathogen. However, C. jejuni was successfully isolated from the stool of a patient suffering from acute enteritis [8]. The development of simpler isolation techniques for culturing C. jejuni led to the rapid isolation of this pathogen. In the mid-1980s, C. jejuni was recognized as one of the major causes of enterocolitis in humans [9].
C. jejuni is the leading cause of human foodborne illness associated with poultry, beef, and pork consumption [10]. C. jejuni is found in the gut of warm-blooded animals, with

Pathogenesis of C. jejuni in Broilers
C. jejuni pathogenesis consists of four main steps: (1) ingestion, (2) acid bile resistance, (3) reproduction in mucus, and (4) invasion of epithelial cells infection is transmitted between birds via the fecal-oral route [56]. A small do (around 35 CFU/mL) is sufficient for successful bird colonization [57]. As an ogen, C. jejuni expresses virulence factors regulated by a two-component sys diates C. jejuni's ability to survive the gut's harsh conditions [58]. Campylobac efflux pump (CmeABC) helps C. jejuni in eliminating toxic compounds such bials, bile salts, and heavy metals. CmeABC comprises three proteins, a per tein, an inner membrane protein, and an outer membrane protein [59]. C encodes the multidrug efflux pump in C. jejuni and it is regulated through C (CmeR) [60]. The presence of bile compounds stimulates the expression of creasing C. jejuni's resistance to bile salts [60]. Mutations in regulator genes r resistance block C. jejuni's colonization ability [61].
C. jejuni depends on the two-component system consisting of CheY (cy sponse regulator protein) and CheA (membrane-associated histidine auto k in responding to different chemoattractant/chemorepellents found in diffe ments [62]. In response to a stimulus, CheA is autophosphorylated, and group is transferred to activate CheY. CheY interacts with the flagellar moto teins leading to a clockwise rotation of the flagella [63]. The flagella play a c C. jejuni motility, adhesion, and invasion of the intestinal epithelial cells [64 lum consists of seven protofilaments of FlaA and FlaB subunits [64] and is a basal structure through FlgE, which serves as a hook [64]. FlaA is the major F

Pathogenesis of C. jejuni in Broilers
C. jejuni pathogenesis consists of four main steps: (1) ingestion, (2) acid tolerance and bile resistance, (3) reproduction in mucus, and (4) invasion of epithelial cells [12]. C. jejuni infection is transmitted between birds via the fecal-oral route [56]. A small dose of C. jejuni (around 35 CFU/mL) is sufficient for successful bird colonization [57]. As an enteric pathogen, C. jejuni expresses virulence factors regulated by a two-component system that mediates C. jejuni's ability to survive the gut's harsh conditions [58]. Campylobacter multidrug efflux pump (CmeABC) helps C. jejuni in eliminating toxic compounds such as antimicrobials, bile salts, and heavy metals. CmeABC comprises three proteins, a periplasmic protein, an inner membrane protein, and an outer membrane protein [59]. CmeABC gene encodes the multidrug efflux pump in C. jejuni and it is regulated through Cme repressor (CmeR) [60]. The presence of bile compounds stimulates the expression of CmeABC, increasing C. jejuni's resistance to bile salts [60]. Mutations in regulator genes related to bile resistance block C. jejuni's colonization ability [61].
C. jejuni depends on the two-component system consisting of CheY (cytoplasmic response regulator protein) and CheA (membrane-associated histidine auto kinase sensor) in responding to different chemoattractant/chemorepellents found in different environments [62]. In response to a stimulus, CheA is autophosphorylated, and a phosphate group is transferred to activate CheY. CheY interacts with the flagellar motor switch proteins leading to a clockwise rotation of the flagella [63]. The flagella play a central role in C. jejuni motility, adhesion, and invasion of the intestinal epithelial cells [64]. The flagellum consists of seven protofilaments of FlaA and FlaB subunits [64] and is attached to the basal structure through FlgE, which serves as a hook [64]. FlaA is the major Flagellin in C. jejuni and is regulated by σ28 promotor [20]. On the other hand, FlaB is the minor flagellin in C. jejuni and is regulated by the σ58 promoter [20]. Chemotaxis such as aspartate, glutamate, citrate, and L-fucose upregulates the σ58 gene [65]. FlaA plays a significant role in C. jejuni's initial colonization of the chicken GIT [66]. The FlaA mutant has a ability to decrease the C. jejuni colonization in chicken [67]. Furthermore, the flagella include the type III secretion system (T3SS), which is responsible for delivering effector proteins needed for cellular invasion [20]. Thus, mutations in the flagellum lead to a decreased ability in colonization and invasion of intestinal epithelial cells.
The absence of immortalized chicken intestinal cell line hinders the capacity to characterize the mechanism of C. jejuni invasion of epithelial cells. In vitro, C. jejuni was capable of invading primary avian cells [68,69]. The presence of avian mucus protected the human cell line against the C. jejuni invasion [70]. It is well-known that C. jejuni survives and reproduces in avian mucus [71]. However, several factors interfere with C. jejuni's capacity to invade the avian epithelial cells in vivo and might explain the near-commensal relationship of C. jejuni in avian species. Several differences are observed between humans and avian species in terms of body temperature (37 • C vs. 42 • C), mucus pH (the avian mucus is more acidic), and difference in mucus structure. C. jejuni upregulates genes related to metabolism and regulatory systems and downregulates genes related to periplasmic proteins at 37 • C in comparison with 42 • C [72]. This difference in gene expression may explain the C. jejuni adaptability and pathogenicity in humans' intestinal tract. C. jejuni upregulates the CadF gene, which is responsible for cell adhesion at 37 • C and 42 • C, indicating the ability of C. jejuni to adhere to intestinal cells in humans and avian species [72]. On the other hand, C. jejuni isolates showed different gene expressions at 37 • C vs. 42 • C [73]. The difference in gene expression might explain some of the differences in C. jejuni's pathogenicity. However, it might not be enough to justify the complete picture of C. jejuni pathogenicity in humans vs. the near-commensal relationship in avian species. It was hypothesized that the pH of the avian mucus confers protection for avian species against C. jejuni. However, through in vitro studies, the neutralization of the avian mucus did not diminish its anti-Campylobacter jejuni properties [68].
Purified chicken mucin inhibited the adherence and internalization of C. jejuni to a human intestinal cell line without affecting C. jejuni viability [70]. The oxidation of purified chicken mucin with sodium metaperiodate enabled C. jejuni to invade the intestinal cell line [70]. The results indicate the protecting role of o-glycosylated mucin structure in the intestinal cell line against C. jejuni.
The avian mucus is highly sulphated and sialylated compared with the human mucus [74]. The comparison between chicken and human mucin structures revealed thirtythree unique structures in chicken mucin [75]. The large intestine in chicken contains the highest sulphated structures, followed by the small intestine and cecum [75]. In chicken, C. jejuni colonizes mainly the ceca and, to a lesser extent, in the small and large intestines [76]. Evidence from in vitro studies shows that the purified chicken mucin from the large intestine had a higher inhibition ability against C. jejuni compared with the purified chicken mucin from the small intestine and cecum [75]. These results highlighted that presence of sulphated O-glycans is inversely correlated with the concentration of C. jejuni in the host. Furthermore, the increased sulfation and sialyation increase the anionic charge in the chicken mucin, creating a charge repulsion effect against C. jejuni [74]. These results indicate the role of chicken mucus in modulating C. jejuni virulence in avian species. C. jejuni pathogenesis is summarized in Figure 2.

Control of C. jejuni in Broilers: (Preharvest)
C. jejuni establishes colonization in the lower intestinal tract of the chicken, par larly in the ceca, within 24 h [65]. C. jejuni concentration can reach up to 1 × 10 9 CFU infected birds [20]. Birds are usually infected from two to four weeks of age and rem colonized until they reach market age [65]. Therefore, control methods are needed t duce C. jejuni prevalence across broiler farms.

Biosecurity
Strict biosecurity measures are the key role in preventing the transmission of C. j in broilers' houses [35]. Identifying the potential sources and methods to detect C. j at the farm level are the major steps needed for a successful biosecurity measures. Res ing access to poultry houses is key to maintaining a C. jejuni-free flock and following s biosecurity measures can decrease C. jejuni prevalence by more than 50% in broile market age [38]. Cleaning and disinfecting poultry houses between cycles can redu jejuni prevalence [38]. Furthermore, strict hygiene practices such as boot covers, h washing, and footbaths can decrease C. jejuni transmission [35]. Standard litter man ment practices are also critical to decrease the presence of C. jejuni in the litter, and tr mission is reduced with enough downtime between flocks [77].
Partial depopulation (thinning) of broiler flocks can increase the risk of C. jejuni tr mission on the farm [78]. A strict biosecurity measure during the thinning process i quired to ensure a low transmission rate of C. jejuni [78]. Furthermore, C. jejuni preval peaks during summer and early autumn time [79]. This seasonality of C. jejuni also c lates with the peak of insect populations [80]. Therefore, strict biosecurity measure needed during summer, early autumn, and thinning to ensure a low prevalence of C. j within the farm.

Probiotics, Prebiotics, and Synbiotic
Probiotics have the ability to improve gut health and prevent enteric diseases in p try. Several mechanisms of probiotics include (1) antagonism and competitive exclu

Control of C. jejuni in Broilers: (Preharvest)
C. jejuni establishes colonization in the lower intestinal tract of the chicken, particularly in the ceca, within 24 h [65]. C. jejuni concentration can reach up to 1 × 10 9 CFU/g in infected birds [20]. Birds are usually infected from two to four weeks of age and remain colonized until they reach market age [65]. Therefore, control methods are needed to reduce C. jejuni prevalence across broiler farms.

Biosecurity
Strict biosecurity measures are the key role in preventing the transmission of C. jejuni in broilers' houses [35]. Identifying the potential sources and methods to detect C. jejuni at the farm level are the major steps needed for a successful biosecurity measures. Restricting access to poultry houses is key to maintaining a C. jejuni-free flock and following strict biosecurity measures can decrease C. jejuni prevalence by more than 50% in broilers at market age [38]. Cleaning and disinfecting poultry houses between cycles can reduce C. jejuni prevalence [38]. Furthermore, strict hygiene practices such as boot covers, hand washing, and footbaths can decrease C. jejuni transmission [35]. Standard litter management practices are also critical to decrease the presence of C. jejuni in the litter, and transmission is reduced with enough downtime between flocks [77].
Partial depopulation (thinning) of broiler flocks can increase the risk of C. jejuni transmission on the farm [78]. A strict biosecurity measure during the thinning process is required to ensure a low transmission rate of C. jejuni [78]. Furthermore, C. jejuni prevalence peaks during summer and early autumn time [79]. This seasonality of C. jejuni also correlates with the peak of insect populations [80]. Therefore, strict biosecurity measures are needed during summer, early autumn, and thinning to ensure a low prevalence of C. jejuni within the farm.

Probiotics, Prebiotics, and Synbiotic
Probiotics have the ability to improve gut health and prevent enteric diseases in poultry. Several mechanisms of probiotics include (1) antagonism and competitive exclusion of enteric pathogenic bacteria, (2) pH reduction by producing organic acids, (3) bacteriocin production, (4) stimulation/modulation of host immune response, (5) and alteration of virulence factors of enteric pathogens [81]. On the other hand, prebiotics are non-digestible feed ingredients that confer a beneficial effect on the host by promoting the proliferation of beneficial bacteria in the gut [81]. The combination of probiotics and prebiotics is known as synbiotics [81].
The ability of probiotics, prebiotics, and synbiotics to combat C. jejuni has been demonstrated in vitro, in vivo, and in field studies. In vitro, the anti-Campylobacter activity of probiotics has been carried out in agar-plate diffusion assays [82], co-cultures assays [83], and adhesion and colonization assays using cell lines [82].
Probiotic species can disrupt the expression of virulence factors in enteric pathogens [87,88]. The cell-free supernatant media of L. acidophilus strain La-5 and Bifidobacterium longum strain NCC2705 downregulated ciaB (invasion) and FlaA (motility) [87]. Similarly, the cell-free supernatant of L. salivarius, L. johnsonii, L. crispatus, and L. gasseri downregulated flaA, flab, flhA (motility), ciaB (invasion), and AI-2 (quorum sensing molecule autoinducer-2) [89]. These studies demonstrate the ability of probiotics species to attenuate the C. jejuni virulence factors. The downregulation of C. jejuni motility and invasion genes results in a lower ability to colonize the GIT of broilers and invade human and chicken primary cell lines [89]. Lactobacillus spp. Supplementation can modulate the host immune system [89]. L. salivarius, L. johnsonii, L. crispatus, and L. gasseri supplementation increased nitric oxide production and phagocytic ability of chicken macrophages, leading to a decrease in the C. jejuni load [89]. Furthermore, a mixture of Lactobacillus spp. Increased the expression of costimulatory molecules, namely: CD40, CD80, and CD86, in macrophages [89]. The costimulatory molecules are essential to initiate an adaptive and humoral immune response; hence probiotic supplementation can initiate the innate and adaptive immune response against C. jejuni [90].
Probiotic bacteria adhere to and occupy gut mucosal surfaces and competitively exclude enteric pathogens. In vitro studies are fundamental to investigating the probiotics' mechanism of action. On the other hand, in vivo studies provide a comprehensive assessment of probiotics' ability to benefit the host. Not all promising results in vitro are replicated in vivo. For example, in vitro studies with E. faecalis strain decreased the C. jejuni load by two log CFU/g. However, the E. faecalis strain could not decrease C. jejuni's load in vivo [86]. In vitro studies with L. plantarum N8, N9, ZL5, and L. casei ZL4 adhered to the HT-29 cell line and competitively disrupted the adhesion and invasion of C. jejuni to the HT-29 cell line [91]. Similarly, L. paracasei JR, L. rhamnosus 15b, Y L. lactis, and L. lactis FOA decreased C. jejuni adhesion and invasion of the primary chicken cell line [92]. Probiotics enhance the integrity of the intestinal barrier by upregulating the expression of tight junction genes [93]. In a study, the supplementation of E.coli Nissle 1917 to the Ht-29 cell line upregulated tight junction genes expression resulting in lower C. jejuni intracellular invasion [93].
The supplementation of Poultry Star ® , (Overland Park, KS, USA) (E. faecium, P. acidilactic, L. salivarius, and L. reuteri) via drinking water from the day of hatch to slaughtering decreased C. jejuni cecal load by six log CFU/g at 35 days of age [84]. However, studies with Poultry Star ® supplementation only decreased C. jejuni cecal load by 2 log CFU/g at day 35, and no reduction in C. jejuni cecal load at 42 days of age [94]. The variability in probiotic efficacy between the two studies can be due to the difference in experimental design, challenge timing, and cecal microbiota of the broiler birds. Isolated B. subtilis exhibited an anti-Campylobacter activity in vitro [95]. The supplementation of B. subtilis isolates reduced C. jejuni cecal load by one log CFU/g [95]. Motile probiotic bacteria can migrate towards the ceca where C. jejuni resides, thus having more chances to eliminate C. jejuni [95]. Therefore, the same B. subtilis isolates were propagated ten times to increase their motility. The propagated B. subtilis supplementation reduced C. jejuni cecal load by 2.5 log CFU/g at 21 days of age [95]. Similarly, the supplementation of B. longum subsp. longum PCB133 + galactooligosaccharide decreased C. jejuni cecal load by 1 log CFU/g at 56 days of age [96]. The oral gavage of L. salivarius SMXD51, every 2-3 days from the hatch until day 35, resulted in a 2.5 log CFU/g reduction in C. jejuni cecal load [97]. Furthermore, the supplementation of L. paracasei J. R, L. rhamnosus 15b, L. lactis Y, and L. lactis FOA combination used seven days before slaughter decreased C. jejuni load by five log CFU/g [92]. These data may suggest that multispecies probiotics may be better than single species probiotics in decreasing C. jejuni load in the poultry.
Contradictory results were obtained when evaluating the efficacy of probiotics on C. jejuni load in broilers. The probiotic bacterial strain, the supplementation dose of probiotics, the route of administration, C. jejuni challenge strain, age, sex, and breed of birds used for the study should be considered when evaluating the efficacy of probiotics. Moreover, C. jejuni survival in the host depends on the host's microbiota [98]. Therefore, an interaction between C. jejuni and residing microbiota can influence the efficacy of the supplemented probiotic either positively or negatively.

Organic Acids
Organic acids are organic compounds that have acidic properties [99]. Organic acids are differentiated from other acids by having a carboxyl acid -COOH to which hydrogen or an organic compound might be attached [99]. Organic acids can be (1) short-chain fatty acids (SCFAs) (≤C6) such as acetic, lactic, butyric, fumaric, and propanoic acid, (2) medium-chain fatty acids (C7:C10) such as capric, caprylic acid, and (3) long-chain fatty acids (≥C11) such as lauric acid [99]. The gastrointestinal tract of avian species harbors millions of bacteria that produce different metabolites, including organic acids [99]. The antimicrobial mechanism in probiotics is mediated through the production of organic acids [81]. Therefore, supplementing organic acid is expected to impact the bird's health positively. Organic acids supplementation leads to a decrease in the gut pH, enhancing the proteolytic enzymes and nutrient digestibility [99]. Furthermore, organic acids can act as bacteriostatic and/or bactericidal against gram-negative pathogenic bacteria, making them a suitable antibiotic alternative [83].
The supplementation of 2% formic acid in combination with 0.1% sorbate prevented the colonization of C. jejuni [100]. However, supplementing 2% formic acid alone was insufficient to prevent C. jejuni colonization [100]. Formic acid lowers the pH of the gut affecting the acid-sensitive bacteria present in the environment. On the other hand, sorbate targets the bacteria by diffusing through the cell membrane and lowering the pH of C. jejuni.
In vitro, butyrate supplementation demonstrated a bactericidal effect against C. jejuni [101]. However, the supplementation of butyrate-coated micro-beads did not decrease C. jejuni colonization [101]. The ineffectiveness of in vivo butyrate supplementation can be attributed to the fast absorption of butyrate by enterocytes. Similarly, the feed acidification with 5.7% lactic acid and 0.7% acetic acid decreased the presence of C. jejuni in the feed [102]. However, the limited effect of organic acids on C. jejuni colonization in broilers might be attributed to the possibility of being absorbed by the gut microbes before it reaches the ceca [102].
Another study tested different combinations of organic acids, prebiotics, and probiotics against C. jejuni infection. Only Adimix ® Precision, (Dendermonde, Belgium) (sodium salt butyrate) decreased the cecal load of C. jejuni by 2 log CFU/g at 42 days of age [94]. Other compounds containing organic compounds such as lactic acid had a limited ability to decrease C. jejuni cecal load. The efficacy of organic acids in controlling enteric pathogens relies heavily on (1) type of SCFAs, (2) dose of SCFAs in feed, (3) buffering capacity of the feed and (4) complex microbiota in the host.
When choosing the type of organic acid for supplementation, the pathogen's metabolism also should be considered. C. jejuni cannot ferment carbohydrates and depends mainly on amino acids and some SCFAs to proliferate in the avian gut [103]. C. jejuni utilizes acetate, lactate, fumarate, succinate, and malate as part of its citric acid cycle to satisfy its energy needs [103]. The ability of C. jejuni to use SCFAs might explain the ineffectiveness of organic acid supplementation such as lactic acid, formic acid, and acetic acid in the presented studies. Butyrate supplementation might be the organic acid of choice to control C. jejuni. Yet, the fact that butyrate is the primary energy source for enterocytes [104] limits its presence in the gut and decreases its ability to fight pathogens.

Bacteriophages
Bacteriophages are viruses ubiquitously found in nature and infect bacterial and archaeal cells [105]. The world contains approximately 10 32 , which is almost 10 times more than the number of bacterial cells on earth [106]. Bacteriophages were discovered in 1915 by Frederick Twort and Félix d'Hérelle in 1917 [107]. Bacteriophages are considered non-pathogenic to humans as they are frequently isolated from human saliva [108] and feces [109]. Humans are frequently exposed to bacteriophages in food and drinking water without any adverse reaction to their consumption [110]. Moreover, bacteriophages dominate the human gut virome [110]. However, the chicken virome is yet to be characterized.
Bacteriophages are suggested as an antibiotic alternative in controlling foodborne pathogens, as they are easy to isolate, have narrow specific, and do not alter the microbiome of the treated host [111]. Though more than 170 C. jejuni phages have been documented, the majority of these phages have a narrow spectrum [105] to control foodborne pathogens. C. jejuni phages are divided into two categories: lytic and lysogenic. Lytic bacteriophages are preferred as they can lyse the targeted cell immediately [111]. In contrast, the lysogenic bacteriophages are not used as they incorporate into the bacterial genome and transfer virulence factors between bacteria [111].
Lytic Campylobacter phages are categorized based on their size into three categories [105]. The first category includes large phages, ranging between 320 and425 kbp, whereas the second category comprises phages that range between 175 and183 and show a high affinity towards C. jejuni and C. coli [105]. The third category includes Campylobacter phages with the smallest size and the greatest affinity and lytic ability toward C. jejuni [105]. Campylobacter phages are versatile tools that can be incorporated into the preharvest and postharvest control of foodborne pathogens.
The efficacy of two Campylobacter phages, CP8 and CP34, was tested for five days post-C. jejuni infection, resulting in a 0.5-5 log CFU/g reduction in C. jejuni based on the intestinal site and phage dose [112]. The best results in C. jejuni load reduction was obtained 24-48 h post bacteriophage supplementation [112]. Similarly, the efficacy was tested with two administration routes (drinking water vs. feed) of a phage cocktail (phiCcoIBB35, phiCcoIBB37, and phiCcoIBB12) [113]. The highest reduction (2 log CFU/g) of C. jejuni load was recorded when the cocktail phage was supplemented in the feed [113]. These results indicated the ability of bacteriophages to control C. jejuni and highlighted the need to determine the dose and route of administration to achieve the best results.
A comparison between a single phage and a cocktail phage to reduce C. jejuni colonization resulted in a maximum of 2.8 log CFU/g cecal C. jejuni load in both groups [114]. The single phage resulted in 43% phage resistance, whereas the cocktail phage led to 24% phage resistance [114]. The development of phage resistance limits the using Campylobacter phages in controlling C. jejuni prevalence. Though the use of phage cocktails might delay the C. jejuni development, the effectiveness of C. jejuni phages is yet to be determined at the farm scale [114].

Bacteriocins
Bacteriocins are ribosomal synthesized antimicrobial peptides secreted by bacteria. Bacteriocins can act as bacteriostatic and bactericidal against related bacterial species [115]. The secretion of bacteriocins confers the destruction of targeted bacteria without damaging the host. Bacteriocin mode of action is mediated through membrane permeabilization followed by cell lysis [116]. The supplementation of bacteriocins in C. jejuni infected broilers efficiently reduces C. jejuni's load and contamination in the food chain [117,118]. Seven-day-old broilers treated with purified encapsulated OR7 bacteriocins produced by L. acidophilus NRRL B-30514 significantly reduced C. jejuni load [118].
Recently, reuterin emerged as a promising bacteriocin in controlling C. jejuni colonization in broilers. Reuterin is an antimicrobial compound produced during the anaerobic formation of glycerol by L. reuteri [119]. Reuterin exhibits a wide antimicrobial spectrum against gram-negative and gram-positive bacteria, yeast, and mold [119]. The mechanism of action of reuterin is mediated through the reaction of acrolein with the thiol groups of glutathione, inhibiting the redox-base defenses and leading to oxidative stress in the targeted bacteria [120]. The genome analysis of C. jejuni revealed the absence of glutathione biosynthesis protein, suggesting that C. jejuni lacks the ability to detoxify acrolein [121]. The absence of glutathione biosynthesis protein might explain the susceptibility of C. jejuni during in vitro studies to reuterin [122].
Bacteriocins production has a high metabolic cost; hence probiotic species will not overproduce it. Supplementing encapsulated bacteriocins with probiotic species might play a prominent role in competitively excluding C. jejuni from the avian gut.

Vaccines
Vaccination remains a potentially effective strategy to mitigate the prevalence of foodborne pathogens (C. jejuni and Salmonella) in poultry production [123]. Vaccination aims to stimulate a mucosal anti-Campylobacter jejuni immune response and reduce the C. jejuni load at market age. Several vaccine strategies have been developed to control C. jejuni in broilers:

Whole Cell Vaccine and Live Attenuated Vaccine
A formalin-killed C. jejuni whole cell vaccine containing 2.7 × 10 8 CFU/mL C. jejuni combined with an oil adjuvant or aluminum hydroxide gel adjuvant was inoculated subcutaneously in Japanese Jordi chicken at 37 days of age [124]. The aluminum hydroxide gel adjuvant group received a booster at 58 days of age. The birds were challenged with C. jejuni on 72 days of age. Both vaccine groups induced high anti-C. jejuni IgG levels [124]. Similarly, a formalin-killed C. jejuni whole-cell vaccine was formulated with or without an E. coli heat-labile toxin as an adjuvant. The vaccine administration enhanced the anti-C. jejuni levels and reduced C. jejuni colonization from 16% to 93% in the vaccinated group compared with the non-vaccinated one [125]. However, the E. coli heat liable toxin did not increase the immunogenicity of the vaccine [125].
Oxidative stress response plays a significant role in C. jejuni's enteric lifestyle. C. jejuni oxidative stress defense mutant shows a low ability to persist in the avian gut. In this study, birds were orally gavaged with 0.5 mL of C. jejuni ∆ahpC mutant at 3 and 7 days of age, followed by a challenge (WT C. jejuni) at 14 days of age [126]. The pre-colonization of broilers with the C. jejuni ∆ahpC mutant decreased the C. jejuni by three log CFU/g reduction at 42 days of age [126]. These results suggest that the C. jejuni ∆ahpC mutant has the potential to be used at the farm level to control C. jejuni at the preharvest stage; however, more safety studies are required at a farm level.

Crude Cell Lysate
The efficacy of a nanoparticle vaccine composed of poly lactide-co-glycolide nanoparticle (NP) and encapsulated 25, 125, or 250 µg outer membrane of C. jejuni was evaluated against C. jejuni [127]. The subcutaneous route induced the highest immune response in vaccinated broilers and decreased the C. jejuni load by 5.7 logCFU/g in the ceca [127]. Similarly, the oral delivery of C. jejuni oral lysate reduced the C. jejuni load in layer and broiler chickens by 2.24 log CFU/g and 2.14 log CFU/g, respectively, at 22 days post-infection [128].

Subunit Vaccine
Type VI secretion system (T6SS) enables bacteria to infect neighboring cells and plays a vital role in inter-bacterial competition and bacterial communication with the host's cells [129]. In C. jejuni, the type VI secretion system (T6SS) plays a role in evading the immune system and bacterial survival [130]. A subunit vaccine was formulated from a purified 50 µg of recombinant hemolysin co-regulated protein (RHCP) entrapped in chitosan sodium tripolyphosphate nanoparticles [131]. The broilers were orally gavaged with subunit vaccine at 7 days of age and then boosted at 14 and 21 days of age [131]. The vaccinated broilers were then challenged with C. jejuni at 28 days of age. The vaccinated group had one log CFU/g reduction of C. jejuni load in the ceca [131].

Bacterial Vector-Based Vaccine
Live or genetically engineered bacterial strains emerge as potential vaccine candidates against enteric pathogens. Bacteria that are avirulent to chickens and elicit an immune response are considered suitable vectors. These vectors can present C. jejuni virulent antigens to the birds' immune system. C. jejuni mutants show a transient colonization pattern in the chicken gut and do not persist enough to activate an immune response [132]. Avirulent Salmonella and Lactobacillus strains are the bacterial vectors of choice for creating a bacterial vector-based vaccine against C. jejuni. An avirulent Salmonella Typhimurium χ3987 strain expressing CjaA was orally gavaged in broilers at 1 and 14 days of age (booster) [133]. At 28 days of age, the broiler was challenged by C. jejuni. The vaccine inoculation led to 6.0 log CFU/g reductions in the C. jejuni cecal load [133].
Finally, phase variation and strain differences in C. jejuni, C. coli, and C. lari complicate the development of a potential vaccine that can decrease campylobacteriosis around the globe.

Quorum Sensing Inhibitors
In broilers, C. jejuni cecal load can reach up to 1 × 10 9 CFU/g [134]. C. jejuni can detect and respond to rapid changes in bacterial densities using quorum sensing [135]. Quorum sensing is a cell-to-cell communication in which bacteria produce, detect, and respond to signaling molecules known as autoinducers [136]. The accumulation of autoinducers happens in a density-dependent manner [135]. When the autoinducer concentration reaches a certain threshold, it leads to the activation of a signal cascade [137]. The signal cascade alters gene expression, resulting in morphological changes in the bacterium that aids its survival in the environment [137].
At first, quorum sensing studies in C. jejuni identified a gene that encodes an orthologue of the LuxS system that mediates the production of autoinducer-2 (AI-2) [138]. In the same study, the C. jejuni luxS mutants showed a decreased motility in semisolid media, indicating a key role of luxS in regulating darting motility in C. jejuni [138]. Furthermore, the role of luxS in host colonization was evaluated in a study testing the colonization ability of the C. jejuni luxS mutant strain vs. the C. jejuni wild-type strain [139]. The luxS mutant showed decreased colonization capacity in chicken seven days post-inoculation; however, some birds inoculated with the luxS mutant strains maintained a similar level of colonization compared with groups inoculated with the wild-type strain [139]. Furthermore, a competitive fitness experiment between the wild-type and luxS mutant showed a decrease in the recovery of the mutant in comparison with the wild-type, indicating an important role of luxS in C. jejuni fitness [140].
The imminent role of luxS in C. jejuni's adaption to environmental conditions [141], expression of virulence factors [140], and biofilm formation [142] make it a potential target for controlling C. jejuni infection. In vitro, (-)-α-pinene showed an anti-quorum sensing activity against C. jejuni by decreasing the C. jejuni quorum signaling by more than 80% [143]. The supplementation of 250 mg/L of (-)-α-pinene in C. jejuni-challenged broilers resulted in 0.8 log CFU/g reduction in the cecal load [143]. (-)-α-pinene inhibitory activity against C. jejuni is attributed to (-)-α-pinene ability to inhibit efflux pump activity and quorum sensing, which play a crucial role in colonizing the host [143]. Thus, (-)-α-pinene can potentially contribute to the control of C. jejuni in broilers.
Citrus extracts decreased motility and biofilm formation in E. coli O157:H7, S. typhimurium, and P. aeruginosa. Similarly, citrus extracts inhibited C. jejuni autoinducer-2 quorum sensing, resulting in lower motility and lower biofilm formation [144]. Similarly, Sedum rosea (roseroot) extract decreased C. jejuni quorum signaling by more than 90% and decreased C. jejuni invasion of INT407 cells by 80% [145]. These results demonstrate the ability of natural phenolic compounds to alter the quorum sensing in C. jejuni, resulting in a lower fitness in C. jejuni. Quorum inhibitory compounds are a promising tool to control C. jejuni in poultry. However, additional studies are needed to determine the required dose and treatment period to decrease the load of C. jejuni in poultry. The control strategies of C. jejuni are summarized in Figure 3.
cating an important role of luxS in C. jejuni fitness [140].
The imminent role of luxS in C. jejuni's adaption to environmental conditions [141], expression of virulence factors [140], and biofilm formation [142] make it a potential target for controlling C. jejuni infection. In vitro, (-)-α-pinene showed an anti-quorum sensing activity against C. jejuni by decreasing the C. jejuni quorum signaling by more than 80% [143]. The supplementation of 250 mg/L of (-)-α-pinene in C. jejuni-challenged broilers resulted in 0.8 log CFU/g reduction in the cecal load [143]. (-)-α-pinene inhibitory activity against C. jejuni is attributed to (-)-α-pinene ability to inhibit efflux pump activity and quorum sensing, which play a crucial role in colonizing the host [143]. Thus, (-)-α-pinene can potentially contribute to the control of C. jejuni in broilers.
Citrus extracts decreased motility and biofilm formation in E. coli O157:H7, S. typhimurium, and P. aeruginosa. Similarly, citrus extracts inhibited C. jejuni autoinducer-2 quorum sensing, resulting in lower motility and lower biofilm formation [144]. Similarly, Sedum rosea (roseroot) extract decreased C. jejuni quorum signaling by more than 90% and decreased C. jejuni invasion of INT407 cells by 80% [145]. These results demonstrate the ability of natural phenolic compounds to alter the quorum sensing in C. jejuni, resulting in a lower fitness in C. jejuni. Quorum inhibitory compounds are a promising tool to control C. jejuni in poultry. However, additional studies are needed to determine the required dose and treatment period to decrease the load of C. jejuni in poultry. The control strategies of C. jejuni are summarized in Figure 3.

Conclusions
Chicken around the globe remains the main reservoir for campylobacteriosis in humans. With the increase in campylobacteriosis worldwide, antibiotic resistance in C. jejuni and increased post-C. jejuni infection complications (such as GBS and MRS), looking for a suitable control strategy becomes the need of the hour. A multi-hurdle approach is needed to ensure the control of foodborne pathogens from farm to fork. Strict biosecurity

Conclusions
Chicken around the globe remains the main reservoir for campylobacteriosis in humans. With the increase in campylobacteriosis worldwide, antibiotic resistance in C. jejuni and increased post-C. jejuni infection complications (such as GBS and MRS), looking for a suitable control strategy becomes the need of the hour. A multi-hurdle approach is needed to ensure the control of foodborne pathogens from farm to fork. Strict biosecurity combined with feed additives and a suitable vaccine (if developed) might be the method of choice to control C. jejuni in broiler production.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.