How to Control Campylobacter in Poultry Farms?: An Overview of the Main Strategies How to Control Campylobacter in Poultry Farms?: An Overview of the Main Strategies

It is now recognized that Campylobacter is one of the main bacterial hazard involved in foodborne diseases around the world leading to an increasing number of gastrointes‐ tinal campylobacteriosis in humans. Also, it is known that this disease has a very high‐ social cost. According to researchers of Emerging Pathogens Institute (EPI) (University of Florida, the United States), the combination poultry/ Campylobacter is the greatest cause of human campylobacteriosis. It is well known all around the world that intestinal carriage of Campylobacter is very large and frequent; it can be reached 100% of animal infected. Reducing this biological hazard can be exercised at different stage levels in the food chain. Intervention at the farm level by reducing colonization of the birds should be taken into account in the overall control strategy. This chapter gives an up‐to‐date overview of suggested on‐farm control measures to reduce the prevalence and colonization of Campylobacter in poultry.


Introduction
These days, the majority of human zoonotic microbial infections have a food origin ( Table 1). Contamination of the food matrix can occur at all stages of the food production chain. In the search for causes of contamination, any stage of the production chain must not be neglected. This fact requires a global approach of problems and a good knowledge of the characteristics of the microorganisms involved. For the latter, the precise knowledge of their privileged reservoir and their potential ability to colonize other reservoirs will identify or clarify some contamination scenarios. Thus, it is known that the psychrotrophic nature of Listeria monocytogenes and their affinity for inert surfaces and biofilms found in food industries are promoting the contamination of the food matrix during the industrial stages of product processing [1]. Contamination of food appears as a necessary step to trigger disease in humans. In some cases, and for certain microorganisms, this phase must necessarily be followed by another phase involving a multiplication of microorganisms in food, concomitantly, or not, with a toxin synthesis. This second phase will allow microorganisms to reach sufficient numbers (minimum infectious dose) to cause disease in consumers. Thus, some microbial hazards should multiply in food (such as Salmonella or L. monocytogenes) and others not (Campylobacter, VTEC, for example).  [2]. To illustrate the importance of Campylobacter infections in France, it is interesting to recall that the report of the "Institut de Veille Sanitaire" (InVS) in 2004 estimated the number of confirmed cases of campylobacteriosis in France to 21,652 cases, of which 17,322 cases were from food origin [3]. According to this report, 3516 cases had required hospitalization and 18 cases would have conducted to death.
Campylobacter infections not only indirectly cause a high processing cost but also a high number of days of work stopping. For example, the treatment of campylobacteriosis in the UK is 465 € while it is 77 € in the Netherlands [4]. In Europe, the annual cost of campylobacteriosis treatment is of the order of 2.4 billion euros [5].
In Africa, the situation is most worrying. It is known that the first Campylobacter infection occurs early in life, by food or nonfood way. Indeed, children under 5 years are the most exposed at the campylobacteriosis [6]. So, Goualie et al. [7] reported an estimated incidence of campylobacteriosis between 40,000 and 60,000 for 100,000 children in developing countries of this continent. These numbers are increasing in most African countries. So, the means of control and prevention are more than necessary even though it has been shown that repeated infections in some children gave them a protection in front of the next infection [8].
Transmission by direct contact with reservoirs like pets, human being, or contaminated bathing water, although rare, should not be neglected. It can cause disease, especially for high-risk professions, namely: farmers, veterinarians, and slaughterhouse workers [9]. Notwithstanding, in most cases, transmission to humans is done indirectly by ingestion of water or food contaminated by certain species of Campylobacter called thermophilic (named C. jejuni ssp. jejuni, C. coli, C. lari, C. upsaliensis), naturally present in many farm animals (poultry, cattle, swine, etc.) [10]. In fact, researchers from the Emerging Pathogens Institute (EPI), mentioned above, were then interested in food vehicles of microbial hazards. Of the 14 most frequent pathogens involved in infectious disease of food origin and the 12 most consumed foods in the United States (or the 168 food-pathogen combinations studied), Campylobacter-chicken combination is the one that is causing the greatest number of cases followed by Toxoplasmapork combination, Listeria-ready to eat meats combination, Salmonella-poultry, and Listeriadairy products combinations [11]. In the United States, the campylobacteriosis is mainly sporadic cases of which it turns out that the contaminated chicken consumption is the cause of most cases. It seems that in Europe we can also highlight the Campylobacter-chicken combination since a study conducted in Belgium during the dioxin crisis, has shown that the number of campylobacteriosis decreased by 40% during the withdrawal period of the sale of poultry [12]. Table 2 highlights the importance of chicken as Campylobacter vector compared with pork and beef meat ( Some gestures made during the preparation of foods in the kitchen is often the cause of contamination transfers, including the use, for cutting the roasted poultry, of the board on which was cut or eviscerated raw poultry. Furthermore, studies have shown that the transfer of Campylobacter from chicken skin to kitchen work surfaces was possible at significant rate (from 0.05 to 36%), as well as to the hands of users (2.9-3.8%) [13]. It has, moreover, been shown that Campylobacter was able to survive for several hours on surfaces and stainless steel utensils, and sponges used for cleaning surfaces could also be sources of contamination [14].
Campylobacter jejuni is responsible for over 85% of campylobacteriosis. When it occurs, campylobacteriosis occurs typically after incubation for 24-72 h, by intestinal manifestations during a week. The most frequently described manifestation is acute gastroenteritis characterized by inflammation, severe abdominal pains in the periumbilical region, mucous diarrhea that can be bloody, accompanied sometimes by fever. It should be noted that the clinical manifestation is often less severe in developing countries where campylobacteriosis is manifested only by a significant watery diarrhea, this could be related to immune protection settling in individuals with frequent contact with Campylobacter [8].
This disease can be serious for certain populations or during postinfection complications, like Guillain-Barré syndrome or Miller-Fisher syndrome [15]. It seems that some serogroups of C. jejuni, as the serogroup O19 Penner, are particularly involved in this type of complications. Having a minimum growth temperature of 30°C, being intolerant to ambient oxygen and also being sensitive to technological stress (such as cold, heat, acidification, and drying), Campylobacter jejuni was always considered as a delicate and fragile organism [16]. Despite these nutritional requirements and the sensitivity to environmental stresses which prevent to grow and multiply outside the host or in food, C. jejuni is still able to survive and persist throughout the food production chain to cause campylobacteriosis constituting, in fact, a real paradox [17].
Although Campylobacter is found in the intestinal tracts of most red meat animals (cattle, pigs, and small ruminants) and pets (cats and dogs), the avian reservoir remains predominant due to the high carrier rate in animals and to the bacterial load per gram of feces, up to 10 7 CFU/g [13,18]. Due to the large intestinal asymptomatic carriage in production animals, manure, slurry, soil, and water can also be reservoirs of Campylobacter. In fact, a study conducted in Italy showed that about 30% of water samples from rivers were contaminated with Campylobacter jejuni [19].
The colonization of the intestine of broilers by Campylobacter during rearing is responsible for contamination of carcasses after processing [20][21][22]. Worldwide, the average prevalence of Campylobacter on poultry carcasses is about 60-80% [2,23]. The carcass contamination occurs during the slaughtering process, even if certain operations are contaminating more than others. Thus, it is recognized that the contamination occurs more favorably during defeathering and evisceration, with feces leaking from the cloaca and the rupture of caeca, causing massive contamination by Campylobacter [24][25][26]. In addition, various transfers of contamination (or cross contamination) can intervene. Among these, there is a carcass to carcass contamination by contact and contamination transfer via vectors such as equipment and personnel, mostly [24].
All of these works clearly show that intestinal carriage of Campylobater by poultry is a key element of the transmission of this hazard to humans. Although beyond the breeding, control measures of this danger exist (good hygienic practices transformations, physical, and chemical treatments sanitizers), their effectiveness will be strengthened if the number of Campylobacter present at this stage is as low as possible. Therefore, the reduction or eradication of intestinal carriage in chickens is a strategic element of major importance for risk control Campylobacter in this sector especially as the contamination of poultry carcasses is proportional to the amount of Campylobacter present in caeca before slaughter [22]. In fact, a recent study of EFSA based on a quantitative microbiological assessment of risks (QMRA) evaluated the performance of implementation of interventions in primary production on reducing the risk of campylobacteriosis cases for the chicken consumers [27]. Following this study, the potential reduction of campylobacteriosis cases can be predicted after the application of breeding measures and is presented in Table 3.

Interventions in primary production Reduction of campylobacteriosis cases
Improved hygiene/biosecurity 16%  Table 3. Effect of interventions in primary production on the reduction of human campylobacteriosis cases.
The study found that the most effective measures are those aimed at reducing the number of Campylobacter in caeca of chickens. However, other simpler measures also have a real impact, we now do an overview of all these measures.

Control Campylobacter in chicken farms
The few quantitative risk assessment studies available on the Campylobacter-chicken combination, conducted in different countries, often focus on the effectiveness of physical and chemical means of eliminating this zoonotic agent during the slaughter and the transformation process [28]. These methods have often proven their efficacy but are often oversized and sometimes poorly accepted by the consumer [10]. Preventive approaches such good hygiene practices and biosecurity now find some interest and may be a strategy to prevent the colonization of animals by Campylobacter and participate in the control of this zoonotic agent in the production of poultry meat. This interest is reinforced by the development of indirect measures, complementary of best practices, to reduce the intestinal number of Campylobacter in poultry. For some authors, these interventions would significantly reduce campylobacteriosis [29]. Thus, a Belgian study showed that the incidence of human campylobacteriosis in the country could be reduced by 32, 53, and 77% if the prevalence of broiler batches colonized by Campylobacter was reduced by 25, 50, or 75%, respectively [30].

Good hygienic practices and biosecurity
Thus, in addition to reducing the risk Campylobacter obtained at later stages of the human food chain, this reduction can also be achieved by the establishment of biosecurity measures at the breeding stage. These interventions are designed to protect a population of animals from the introduction of infectious agents transmissible like Campylobacter. In poultry, the biosecurity program includes all measures that must or may be taken to prevent the entry of this agent and changing the health status of the chicken population. These measures, collectively known as biosecurity, cover the hygienic practices during the rearing period. These include washing hands before entering in a poultry house, the use of different boots to enter each house, the cleaning, and disinfection of shoes before entering the room, a high level of hygienic water quality beverage.
Other measures such as cleaning and effective disinfection of poultry house between two batches of animals, as reducing the number of visits, as strict control of entry into the breeding of rodents, wild birds, and flying insects. Thus, studies in Denmark have shown that the use of mosquito nets preventing the entry of flying insects in the broiler house, potential vectors of Campylobacter, significantly reduced the contamination of poultry by Campylobacter during the seasonal peak (Figure 1) [31].
The application of all these measures greatly reduces the risk of Campylobacter infections. So, Gibbens et al. estimated that this application would lower the prevalence of Campylobacter in batches of chickens from 80 to less than 40% [32]. The respect of a good personal and clothing hygiene for staff and good measures of biosecurity, including the control of rodents and insects in two Dutch farms, reduced prevalence of Campylobacter in batches of chickens from two different farms of 34% in the first farm, and of 20% in the second [33].

Treatment of drinking water
Another important factor is the quality of drinking water. Several studies have shown that poor quality water (untreated water from wells) may increase the transmission of Campylobacter in animals [34,35]. The microbiological quality of drinking water should be monitored by the analysis and can be improved on the farm by techniques such as filtration, chlorination, ozonation, UV rays. For some authors, the impact of these interventions on Campylobacter infection is uncertain, but they point out that the absence of interventions may be worse [32,36]. Studies by Byrd et al. showed that adding 0.44% (vol/vol) of lactic acid in drinking water prior to slaughter, has reduced the level of contamination of carcasses with Campylobacter [37]. Hilmarsson et al. showed that the addition of glycerol monocaprate (monocaprin) the last 3 days before slaughter, has reduced the number of C. jejuni in feces samples of chickens naturally or artificially infected [38].

Use of antimicrobial from vegetal origin
In addition to their application in drinking water, organic acids can also be used as additives in foods to reduce the prevalence of Campylobacter in poultry. Thus, 0.7% caprylic acid reduced colonization when used preventively on old chickens of 10 days and achieves a significant reduction of C. jejuni in broiler feces up to three to four decimal reductions [39]. In contrast, Van Deun et al. [40] observed that butyrate did not reduce colonization of caeca by Campylobacter in broiler chickens, but the addition of fatty acids short chain at a concentration of 1% reduces the risk of colonization of farms [41]. However, Hermans et al. [42] did not find any effect of these medium chain fatty acids (caproic, caprylic, and capric) on the number of Campylobacter in the caeca of broilers 28 days fed therewith 3 days before slaughter. Moreover, they observed that injection of a highly concentrated solution of sodium caprate directly into the caeca did not prevent colonization and did not reduce the caeca contents by Campylobacter. Thereafter, these authors showed that this ineffectiveness was explained by the presence of the intestinal mucus whom protecting C. jejuni in the caeca vis-a-vis of the bactericidal effect of organic acids observed in vitro. Conversely, another research group found a significant reduction (several logs) of Campylobacter in caeca of chickens when caprylic acid was given 3 days before slaughter [43]. In addition, another study showed that adding monocaprin to chicken feed the last 3 days before slaughter, resulted in a significant reduction of C. jejuni on feces samples of animals artificially or naturally infected, compared to controls [38]. These results, apparently contradictory, demonstrate the need to continue the investigations necessary to establish with greater certainty the effectiveness or lack of effectiveness of this strategy in the control of Campylobacter in chicken farms

Vaccination
The principle of vaccination of chickens against Campylobacter is to administer a product capable of inducing immunity directed specifically against this pathogen and confer immune More recent studies involving a larger number of animals were used to test the use of recombinant vaccines. Thus, 840 SPF chicks were used to evaluate the effectiveness of the vaccine derived from Salmonella enterica Typhimurium ΔaroA attenuated and expressing the immunogenic protein CJAA C. jejuni. Chicks who received the vaccine orally at the age of 1 day, then 2 weeks later, showed a reduction in fecal contamination by C. jejuni of 1.4log CFU/g, compared with unvaccinated chicks [46]. Layton et al. [47] used recombinant vaccines attenuated from Salmonella expressing three peptide epitopes of protein of Campylobacter (Omp18/CJAD protein, CJAA, and Cj0420 (ACE393)). These three vaccines were administered orally to chicks on the day 1, then 21 days later. The vaccinated chicks were inoculated with C. jejuni. Eleven days after the inoculation, an increase in IgG and IgA antibodies specifically against C. jejuni was observed and also a reduction in the number of C. jejuni in the ileum. Vaccination was most effective when the vectored vaccine expressing the epitope of the Omp18/CJAD protein was administered to chicks, with a considerable reduction of C. jejuni in the chicken intestine (4.8 decimal reductions of C. jejuni in the ileum) compared with unvaccinated controls and those vaccinated only with the vector (Salmonella 13A) or negative control (Figure 2).
These studies are promising and probably mean that a possible vaccination strategy for Campylobacter reduction is possible. They still face a lack of information on the immune system of the chick that such hinders the development of an attenuated vaccine expressing the linear peptide epitope Campylobacter (Omp18/CJA). In addition, advances in functional genomics Campylobacter suggest that other proteins of this agent could be excellent candidates for testing for future vaccines.

Use of phages
The lytic activity of bacteriophages can be used as a strategy to reduce the colonization of chickens with Campylobacter. Phages usually have a very narrow spectrum of activity, and they do not interact with other bacterial species in the intestinal flora. Phages bind and penetrate into bacterial cells by protein receptors and multiply within the cytoplasm until the death of the bacteria. At this time, the lysis of the bacteria permits the release of new bacteriophages.
Loc Carrillo et al. [48] and Wagenaar et al. [49] have shown three decimal reductions of Campylobacter in caeca of chickens that received the bacteriophage, compared with negative control. However, this reduction is not stable, they observed a reduction of only 1log/g 5 days later. Similarly, El-Shibiny et al. [50] observed an immediate reduction of 2log 10 (CFU/g) of Campylobacter in caeca 2 days after ingestion of phages. Then, the number of Campylobacter in caeca returns to the original level a few days later, reversing the improvement achieved. These results show that this strategy is more a short-term therapeutic strategy than a preventive long term one. It could be very interesting if, for example, the treatment is taken only 2-3 days before slaughter: resistance to bacteriophages then not have time to reverse the reduction of Campylobacter obtained by the treatment. In this matter, other studies have also shown that administration of phages in feed is more effective than oral gavage [37]. In these conditions of administration, use of phages few days before slaughter appears to be an excellent strategy for the reduction of Campylobacter in poultry, but the diversity of Campylobacter protein receptors requires a large diversity of phages, which also increases the complexity of this strategy.

Use of prebiotics and probiotics
Probiotics are defined as "live microorganisms which when administered in adequate amounts confer a benefit to human health." Prebiotics are generally oligosaccharides (fructo-oligosaccharide (FOS), galacto-oligosaccharides (GOS)) or polysaccharides such as inulin. These escape digestion in the small intestine and have a beneficial effect on the health of their host by stimulating the growth and/or activity of bacteria of the genera Lactobacillus and Bifidobacterium, naturally present in the colon or administered as probiotics.
The use of prebiotics and probiotics is a strategy that has been studied by several research teams in order to reduce the colonization of chickens by Campylobacter jejuni.
In 1997, Morishita et al. [51] used on 1-day-old chicks, a probiotic mixture containing Lactobacillus acidophilus and Enterococcus faecium. The chicks were randomly divided into two groups, one group was treated with the probiotic cocktail during the first 3 days of culture while the second batch received distilled water instead of the probiotic cocktail. Six hours after the first oral administration of probiotics, the number of C. jejuni in feces of the chicks was determined and was performed until the slaughter. The results showed a 70% reduction in the concentration of C. jejuni in chickens on day 3 and a 27% reduction for broilers at slaughter, compared to the control group. For Schoeni and Wong, administration of a mixture of different bacteria (Citrobacter diversus, Klebsiella pneumonia, and E. coli) appears to be effective in preventing or reducing the colonization of chickens with Campylobacter [52]. This protection has been strengthened by mannose which was given as a prebiotic. Again, this is very promising works, that requires further study in order to decide definitively on their use. They also have the merit of bringing forward an interesting and ongoing concept named "microbial solution for microbial problems."

Genetic selection of chicken
Selective breeding of resistant lines of chickens to Campylobacter colonization is a particularly modern intervention strategy to reduce Campylobacter problem in the poultry industry.
In 2005, Boyd et al. [55] have shown that the selection of chicken lines genetically resistant to Campylobacter germ, significantly reduces this risk in poultry. In their study, Boyd et al. [55] have inoculated 1-day-old chicks of different inbred lines with 10 7 -10 8 CFU of C. jejuni or C. jejuni 81-176 14 N and measured bacterial colonization levels of chickens over a period of 2-3 weeks. They have always been a difference of a factor 10-100 from four inbred lines in the number of C. jejuni present in chicken caeca between the four inbred chicken lines. The biggest difference was for the N line, which presented relatively high levels of Campylobacter, and the line 61, which had a relatively small number of bacteria. Among the four lines studied, the major histocompatibility complex does not appear to be a major factor in determining the resistance. The difference in the number of bacteria in fecal samples was observed after 24 h after inoculation and was still present at the end of the experiment. This work revealed that the difference in the number of bacteria was inherited in a consistent manner with the resistance (low number of bacteria), controlled by a single autosomal dominant locus. These data suggest that it may be possible to identify the responsible genes. Indeed, the recent knowledge of the whole sequence of the chicken genome has identified the genes involved in susceptibility to Campylobacter colonization [56]. These observations led to the suggestion that selective breeding could be used to select chickens resistant to Campylobacter colonization.

Use of bacteriocins
The use of antimicrobial peptides could be an interesting biological intervention strategy to reduce colonization of poultry by Campylobacter [57][58][59][60][61]. These studies highlight the ability of bacteriocins produced by lactic acid bacteria, such as Lactobacillus salivarius NRRL B-30514, Enterococcus faecium E50-52 E760, Lactobacillus salivarius, and L. salivarius SMXD51 1077 (NRRL B-50053)) to inhibit the growth of Campylobacter jejuni. In 2005, Stern et al. [62] studied the effect of the bacteriocin "SRCAM 602" produced by P. polymixa NRRL B-30509 on cecal colonization by Campylobacter of chicks artificially inoculated with 10 8 CFU of C. jejuni from day 1. These animals, colonized by Campylobacter, received from day +7 to day +10 a feed containing the bacteriocin purified (250 mg/kg). For the chicks that received during 3 days, this diet the number of Campylobacter in caeca was very low and undetectable (<2log 10 CFU/g), while the control animals showed high cecal colonization by Campylobacter (10 6 -10 8 log CFU/g) [62]. In 2008, Line et al. [57] have found similar results following administration of the bacteriocin "Enterococcine E-760" in broiler naturally infected with Campylobacter. Moreover, Svetoch et al. [63] administered 10.8 mg/chicken (oral gavage) of bacteriocin "E 50-52" produced by E. faecium NRRL B-30746 3 days before slaughter. The results showed a significant reduction of Campylobacter in the gut, greater than 10 5 CFU/g of feces.
Stern et al. [64] have studied the effect of the bacteriocin OR 7, produced by L. salivarius NRRL B-30514, in encapsulated form in a concentration of 250 mg/kg on eight groups of contaminated chicks by C. jejuni. In three of eight groups of chickens, there was no Campylobacter colonization and, in the other five groups, the level of contamination remained very low (10-100 CFU/g) compared with controls. By cons, L. salivarius NRRL B-30514 and Paenibacillus polymyxa NRRL-B-30509 showed no effect on colonization of artificially infected chicks by C. jejuni [64]. Finally, Svetoch et al. [63] showed that treatment with the bacteriocin L-1077, produced by L. salivarius strain NRRL B-50053, chickens inoculated with C. jejuni and Salmonella Enteritidis provides more than four decimal reductions in the number of bacteria per gram of cecal contents, compared with controls. Moreover, the presence of these bacteria in the liver and spleen of the animals is very greatly reduced.

Conclusion
Campylobacter is today a leading cause of foodborne diseases, all around the world. It is also a paradox for microbiologists, who see a contradiction between the apparent physiological fragility, its small genome and its obvious ability to survive outside its main habitat (digestive tract of birds) and to reach its main target (i.e., the consumer). Moreover, this impression is reinforced by the fact that the organism does not grow in foods and that his number would tend to decrease during processing operations, rather than increase. In fact, intestinal carriage of Campylobacter by animals becomes a key element of the contamination of the consumer and a series of strategies have been developed to reduce intestinal carriage in the past 15 years. Today, despite all the efforts and progress made, there is still no miracle solution but a set of interventions strategies, each with their advantages and disadvantages. The use of bacteriocins and bacteriophages is very promising because their implementation is simple: they can be easily administered with water or feed. However, their potential use requires further research work on their long-term effectiveness. In addition, the successful application of these methods as well as probiotics, prebiotics and even vaccination may be affected by genomic instability of C. jejuni [44] which may affect the effectiveness of these strategies in the long term. Finally, the reduction of Campylobacter contamination pressure of animals at the stage of livestock should be based on a strong base of Good Hygienic Practices and biosecurity, reinforced by targeted interventions selected on the criteria of efficiency, practicality, and cost in front of the type of poultry production.