Foodborne illness and its implications on food industry and to the general public is a concern and this impact could be reduced by establishing effective, novel intervention hurdles to enhance food safety in a "farm to fork" approach (Oliver et al., 2008). Conventionally, the majority of the efforts in controlling the pathogens have primarily focused on post-harvest; however, limiting the spread of foodborne pathogens prior to harvest (as they are reservoirs/asymptomatic carriers of several foodborne pathogens) has increasingly gained interest and focus among food safety researchers, policy makers, government officials (FSIS/USDA), and consumers (Tuohy et al., 2005; Choct, 2009; FSIS/USDA, 2010). Pre-harvest food safety is now being considered more essential to protect food supply as foodborne pathogens can enter the food supply during slaughter from the hide, workers or machinery in the processing environment or by direct contact with feces or digesta from the intestinal tract (Elder et al., 2000). In addition to these factors, animal husbandry practices, handling and processing can also contribute to the incidence of foodborne illness (Beier and Pillai, 2005 and Park et al., 2008).
2. FOODBORNE PATHOGENS ASSOCIATED WITH POULTRY
The Centers for Disease Control and Prevention (CDC) estimated that there are 76 million cases of food borne diseases with 325,000 hospitalizations and 5,000 deaths occurring every year in the United States (CDC, 2009). A majority of these foodborne illness/outbreaks have been linked to contaminated poultry products or contact with food animals, waste, and enteric pathogens in poultry (Doyle and Erickson, 2006). Primary foodborne pathogens mainly transmitted through raw and processed poultry products are Salmonella and Campylobacter (Mead, 1999; Bryan, 2001; Park et al., 2008). In 2008, the incidences of foodborne diseases associated with Salmonella and Campylobacter were 16.2 and 12.68 % respectively (Vugia et al., 2009). In addition to the common bacterial pathogens, zoonotic parasitic infestations such as Trichinella spiralis and Toxoplasma gondii can pose health risks to the consumers (Gebreyes, 2008).
Salmonellosis is the second commonly reported foodborne disease associated with consumption of meat, poultry, eggs, milk and sea foods in the United States (Vugia et al., 2007). According to CDC estimates there are approximately 1.4 million cases of salmonellosis resulting in 17,000 hospitalizations and 585 deaths each year in the United States (Mead et al., 1999; Voetsch et al., 2004). Farm animals such as chicken and turkey represent a major reservoir of Salmonella (Table 1) and can also act as asymptomatic carriers in the absence of clinical disease (Oliver et al., 2008).
Salmonella in chickens can contaminate meat and eggs and have become a persistent problem associated with the poultry industry in the United States on an annual basis (USDA, 2007a; USDA, 2007b). Factors affecting the susceptibility of poultry to Salmonella colonization include age, serotype and intial challenge dose level, stress, presence of feed additives, such as antimicrobials and anticoccidials, survival through low pH of the stomach, competition with gut microflora, and presence of compatible colonization sites (Bailey, 1988).
Campylobacter jejuni is the major foodborne agents associated with diarrhea and gastroenteritis and represents a major concern to the poultry industry (Altekruse, 1999; Newell and Fearnly, 2003). The prevalence of Campylobacter in the U.S. is 32 to 53 % in poultry (Miller and Mandrell, 2005). Over the years, Campylobacter have evolved and adapted to colonize in the intestine in poultry which in turn can pose serious public health hazards (Heuer et al., 2001; Newell and Davison, 2003).
According to the preliminary surveillance data by FoodNet in 2008, the estimated incidence associated with Salmonella, Campylobacter, and other foodborne pathogens did not change significantly when compared to the previous 3 years (Vugia et al., 2009). This reinstates the importance and demand for effective control strategies and interventions to produce wholesome food products.
3. PRE-HARVEST CONTROL STRATEGIES
Developing interventions that have potential in reducing pathogens substantially in the live animal can improve food security and safety (Lonergan et al., 2005; Ricke and Jones, 2010). A wide range of intervention strategies have been developed to reduce the burden of foodborne pathogens in poultry, including genetic selection of animals that are resistant to colonization, breeding treatments to prevent vertical transimission of enteric pathogens, sanitation practices, additives (feed or water), and biological treatments that directly or indirectly inactivate the pathogen within the host (Doyle and Erickson, 2006). However, use of antibiotics and chemotherapeutics in prophylactic doses for prolonged periods has led to concerns across the world regarding cross resistance and multiple antibiotic resistances among foodborne pathogens (Mathur and Singh, 2005). Furthermore, use of antibiotics as growth promoters in feed to reduce pathogens impacts the export of meat and poultry products to Eurpoean countries (EC 2001, 2003a). This in turn has generated interest in development of novel, innovative and safe alternatives that would boost natural defense mechanisms which includes, acidification of feed by organic acids, feeding probiotic organisms and feeding prebiotic compounds (Williams et al., 2001; Oliver et al., 2008; Ricke and Jones, 2010). In addition to food safety issues, high protein prices and environmental concerns have caused the poultry industry to consider the adoption of feed supplements such as probiotics that would positively influence the animal performance including modulating gut microflora (Tuohy et al., 2005).
4. CHICKEN GUT MICROFLORA
Chicken gut (also referred as digestive tract or gastrointestinal tract) begins with the mouth and ends at the cloaca with several important organs [such as esophagus, crop, proventriculus (true -stomach), gizzard/ventriculus, small intestine, caeaca, large intestine] in between. Based on the microflora dynamics and their colonization perspective, the poultry intestine can be divided into three parts: a) the duodenum and the small intestine, where bacteria numbers are relatively low (less than 108/g), b) the ceca, major site of bacterial colonization and microbial fermentation (approximately 108/g; wet weight), c) and the large intestine (Barnes, 1972).
The GI tract and its associated tissues in poultry during hatching time are relatively sterile and underdeveloped (Cressman, 2009). However, as the chick or poult grows, the gastrointestinal tract provides the required conditions for bacterial colonization, including attachment sites, optimal pH, substrate/nutrients, and waste removal. At this stage, healthy broilers and poults exhibit significant changes such as more rapid proportional weight increases of gastrointestinal tissues when compared to total body mass and increases villus volume (3 to 5 fold) between 2nd and 14th day and crypt depth (2 to 3 fold by day) (Uni et al., 1998). Similar increases have also been observed in poults although not to the same extent (Uni et al., 1998).
Development of normal GI microflora of poultry has been extensively studied in specific pathogen free (SPF) chickens as the results would be unbiased due to the absence of competitive microflora. The use of SPF birds is advantageous compared to using conventionally raised chickens since there is no risk of additional infectious agents such as viruses and parasites that may be present in the latter (Coloe et al., 1984). In a study conducted by Coloe et al. (1984) regarding the development of normal gut microflora in SPF chickens, no bacteria were detected at hatching (day 1) and development of significant levels (108 CFU/g) of facultative anaerobes such as fecal streptococci and coliforms by day 3 and Proteus sp (greater than 107 CFU/g) were accompanied by day 7 in the cecum.
In poultry, major sites of colonization by gut microflora in GI tract are the crop, proventriculus, gizzard, small intestine, colon and ceca (Chichlowski et al., 2007a; Gaskins et al., 2002; Heczko et al., 2000; and Rastall, 2004). Normal GI microflora and predominant species in various parts of healthy chicken intestine are presented in Table 2. In the proximal part of the intestine (crop, gizzard, proventriculus) there are usually low numbers of anaerobic bacteria due to the presence of oxygen, low luminal pH, and hydrochloric acid originating from the proventriculus (Rastall, 2004). In spite of these unfavorable conditions, Lactobacilli can still survive in the chicken crop due to surface receptors on Lactobacilli that have the ability to adhere to the squamous epithelial cells of the crop to be retained in high numbers (107 to 08) (Fuller, 2001) and exhibit stable, persistent and host-specific adhesion effects (Fuller, 1973). Consequently, predominance of Lactobacilli in the crop results in the production of lactic acid that can reduce Escherichia coli and Salmonella significantly during contamination (Fuller, 1977; Durant et al., 1999; 2000).
Microbial colonization of the poultry GI tract starts with microbial contact from eggshell, feed, and other environmental sources immediately after hatching (Cressman, 2009). Normal microflora colonize the GI tract beginning with the early post-hatch period, form a symbiotic relationship with the host and have a significant impact on the uptake and utilization of energy and nutrients (Choct et al., 1996; Smits et al., 1997; Apajalahati and Bedford, 2000; Torok et al., 2007). Development of the small intestinal microflora is observed in the first two weeks of post-hatching until several weeks (Ochi et al., 1964; Smith, 1965; Smirnov et al., 2006). Immediately after hatching there is evidence that bacteria, particularly Streptococci and Enterobacteria, multiply initially in the ceca and spread throughout the alimentary tract within 24 hr (Smith et al., 1965). Lactobacilli can become established by the 3rd day, while the streptococci and enterobacteria slowly decline in the GI tract except in the ceca (Barnes et al., 1972). By 2 weeks of age, Lactobacilli became the predominant microflora with occasional Streptococci and Enterobacteria in the duodenum, and lower portions of the small intestine (Barnes et al., 1972). In the cecum, Bifidobacterium establish as predominant microflora by 30 days (Ochi et al., 1964). Recent evidence based on real-time PCR analyses of feces from 3 to 12-day-old broilers also indicates the presence of methanogens (Saengkerdsub et al., 2007b). In adult birds most of the methanogens have been identified as Methanobrevibacter woesei (Saengkerdsub et al., 2007a). Overall, the composition of the microflora undergoes major changes during the time of hatch and the anerobic microflora becomes established which requires significant amounts of substrates such as carbohydrates (Apajalahti et al., 2002).
The diverse microbial community profile thus developed over time can be identified through molecular techniques such as Denatured Gradient Gel Electrophoresis (DGGE) % G (Guanine) + C (Cytosine) profiling and 16S rDNA sequencing (Apajalahti et al., 2002; Gong et al., 2002; Hanning and Ricke, 2011; Holben et al., 2002; Zhu et al., 2002). Studies conducted based on these methods revealed that: (1) only 10 % of the gastrointestinal bacteria represent previously known bacterial species (Maidak et al., 1999), (2). Thirty-five % represent previously unknown species within a known bacterial genus; (3) and the remaining 55 % represent bacteria for which even the genus is completely unknown. Furthermore, a total of 640 different species and 140 different bacterial genera were found in the chicken gastrointestinal tract.
The microbial community profile in the chicken gastrointestinal tract is chiefly influenced by the diet (grain base) and the age of the bird (Barnes et al., 1972). Apajalahati and Bedford (2000) studied the effect of grains (wheat, corn or rye) on the microbial community profile and concluded that incorporation of rye in the diet increased the abundance of bacteria with a 35 to 40 % G + C content significantly when compared to wheat and corn. In the same study, wheat increased the proportion of bacteria with G+C between 55 and 59 % and at 69 %. However, this study did not reveal the bacterial identity, but concluded that incorporation of diet with corn favored low % G+C microorganisms (Clostridia and Campylobacter), rye stimulated the growth of Lactobacilli and Enterococci, whereas the wheat-based diet favored higher % G+C microorganisms (Propionibacteria and Bifidobacteria). In addition to diets, processing of grains have also demonstrated significant effects as different processed diets favored different bacteria in the gastrointestinal tract of chicken regardless of whether they originated from the same raw material (Apajalahti et al., 2001). Furthermore, anaerobes and Lactobacilli were found to be significantly lower in gizzards of broilers fed with sorghum and wheat based diets when compared to broilers raised on barley and maize diets (Shakouri et al., 2008). Similar differences were observed in cecum while in the ileum there was no effect of grains on anaerobic and Lactobacilli populations (Shakouri et al., 2008). Supplementation of the diets with fats and their source has been observed to influence microbiota structure (Knarreborg et al., 2002; Dänicke et al., 1999). Knarreborg et al. (2002) studied the effect of animal and plant derived fats on the microbiota within the ileum of broilers (14 to 21 d) and reported that the source of dietary fat significantly altered the viable populations of Clostridium perfringens while Lactobacilli species were not affected. Dänicke et al. (1999) demonstrated that broilers fed diets with beef tallow when compared to soybean oil had significantly more Gram positive cocci in the crop, jejunum, and ileum (1.18, 1.05, 1.36, and 2.10 CFU/log10 higher) at day 16. Enterobacter was substantially higher in the crop and duodenum (1.05 and 1.30 CFU/log10 higher respectively) in birds fed with soybean oil and the total number of anaerobes did not vary substantially across intestinal segments due to the source of fat.
