A review on development of novel strategies for controlling Salmonella Enteritidis colonization in laying hens: Fiber-based molt diets1

A review on development of novel strategies for controlling Salmonella Enteritidis colonization in laying hens: Fiber-based molt diets1 S. C. Ricke,*2 C. S. Dunkley,† and J. A. Durant‡ *Center for Food Safety and Department of Food Science, University of Arkansas, 2650 N. Young Ave., Fayetteville 72704; †Poultry Science Department, University of Georgia, 2360 Rainwater Rd., Tifton 31793; and ‡Biological Science Department, Oakwood College, 7000 Adventist Blvd., Huntsville, AL 35896 ABSTRACT Limiting Salmonella Enteritidis from table eggs can involve intervention approaches at several levels of the production cycle, beginning at the hatchery and ending at the processing or table egg production facilities. Likewise, interventions that limit Salmonella Enteritidis dissemination can be implemented at various stages during the life cycle of infection of Salmonella in the laying hen. However, achieving complete elimination of Salmonella infestation in egg products has remained elusive. There is a multitude of reasons for this, including adaptability of the organism, virulence properties, and persistence. Likewise, environmental factors in the layer house such as transmission routes, reservoirs, and feed sources can influence the exposure of susceptible laying hens to Salmonella Enteritidis. Consequently, successful applications of control measures depend not only on the timing of when

they are applied but also on effective surveillance to detect frequency and level of infection of Salmonella. Several studies demonstrated that molt induction by feed withdrawal altered the immune system and the gastrointestinal tract of hens, making them susceptible to Salmonella Enteritidis colonization of the gastrointestinal tract. To alleviate this, the development of alternative methods to induce a molt became necessary. The use of several fiber-containing diets was shown to effectively induce a molt with alfalfa-based diets being the most extensively studied. Further reduction of Salmonella Enteritidis levels in eggs will probably require application of multiple interventions at several steps during egg production and processing as well as a better understanding of the mechanisms used by Salmonella Enteritidis to persist in laying flocks.

Key words: fiber, egg, laying hen, molt, Salmonella Enteritidis 2013 Poultry Science 92:502–525 http://dx.doi.org/10.3382/ps.2012-02763

INTRODUCTION

each case of salmonellosis was costing between US $700 and $1,350 and depending on the economic model used, the total annual costs of salmonellosis in terms of medical costs and lost production ranged from 464 million to 2.3 billion (Todd, 1989, 1990). Over 15 yr ago, in 1996–1997, the CDC reported 35,621 culture-confirmed cases of salmonellosis (Frenzen et al., 1999). In the ensuing years of surveillance and reporting, the annual cost of salmonellosis in the United States has continued to climb. For the year 2003, the estimated cost was just above $3 billion (ERS, 2004). This cost is now even more; there are almost 42,000 culture confirmed cases of salmonellosis occurring per year in the United States (Scallan et al., 2011). More recently, an egg-associated outbreak led to almost 2,000 illnesses nationwide in the United States (CDC, 2010a). Salmonella enterica subspecies enterica serovar Typhrimurium (Salmonella Typhimurium) and Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis) continue to be the 2 of the most common serotypes isolated from humans suffer-

Salmonella species are important facultative intracellular enteric pathogens that continue to be a significant source of foodborne gastroenteritis in humans (Finlay and Falkow, 1989; D’Aoust and Maurer, 2007; Mead et al., 2010; Foley et al., 2011; Finstad et al., 2012; Howard et al., 2012). Almost 25 yr ago the Centers for Diseases Control (CDC) estimated that in the case of salmonellosis, 170,000 patients visited a physician, 16,400 cases required hospitalization, and 600 cases resulted in death (Cohen and Tauxe, 1986). Even then, estimates based on medical expenses, loss of human productivity, and cost of legal action indicated that ©2013 Poultry Science Association Inc. Received September 11, 2012. Accepted October 14, 2012. 1 Presented as part of the Tomorrow’s Poultry: Sustainability and Safety Keynote Symposium at the Poultry Science Association’s annual meeting in Athens, Georgia, July 9, 2012. 2 Corresponding author: [email protected]

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ing with infectious gastroenteritis, although other serotypes have emerged in more recent times (CDC, 1993; Gantois et al., 2009). Therefore, these Salmonella species continue to be recognized as primary public health concerns. This review will focus on general aspects of Salmonella in poultry, followed by Salmonella issues associated with preharvest egg production with the primary focus being on fiber-based molting diets and their impact on nutrition, egg production, and Salmonella infection.

SALMONELLA IN POULTRY Poultry Meat Products and Salmonellosis In general, poultry products are considered one of the major sources of Salmonella infections, but the Salmonella contamination of poultry meat products and a correlation with human disease are difficult to quantify with precision because nonpoultry sources also contribute (Gast, 2007; Hanning et al., 2009; Mead et al., 2010; Cox et al., 2011; Koo et al., 2012). However, Salmonella human infections are usually associated with poultry products because these organisms can colonize the intestinal tract of poultry (Barrow et al., 1988). The ceca of chickens are the major sites of Salmonella colonization (Fanelli et al., 1971), and chickens can become asymptomatic carriers of Salmonella. Therefore, cecal and intestinal contents have been considered to be the primary source of Salmonella contamination of broilers during the grow-out period and in the processing plant (Lillard, 1989; Izat et al., 1990; Cox et al., 2011). Jones et al. (1991) observed that 21% of whole broiler carcasses were contaminated with Salmonella, and Salmonella Typhimurium was the most common serotype. During processing, salmonellae can become entrapped in the poultry tissues and are not easily removed by normal processing methods (Lillard, 1988, 1989; Cox et al., 2011). The crop may also serve as an important source of Salmonella contamination of broiler carcasses (Hargis et al., 1995). The authors reported that at a commercial processing plant 52% of the crops cultured were positive for Salmonella compared with 15% of the ceca. Data from this plant indicated that the crops were far more likely to rupture than ceca (86-fold) during processing, thereby serving as a significant source of contamination. In an effort to reduce contamination of equipment and carcasses, it is a common practice to withdraw feed and water from broilers several hours before processing (Bilgili, 1988). However, Corrier et al. (1999) found that the incidence of Salmonella crop contamination increased as much as 5-fold during preslaughter feed withdrawal.

Egg Production and Salmonellosis In the early 20th century, avian-adapted serovars of Salmonella, such as Salmonella Pullorum and Salmo-

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nella Gallinarum, were endemic in the poultry flocks of the United States and Europe (Poppe, 2000; Rabsch et al., 2000; Mead et al., 2010). Because these serovars caused significant economic losses due to increased chick mortality, surveillance and eradication programs were initiated to control these diseases (Poppe, 2000; Shivaprasad, 2003; Hitchner, 2004). As these serovars were eliminated, Salmonella Enteritidis became more prevalent to the point that Salmonella Enteritidis became an increasing food safety problem in many parts of the world in the late 1970s (Rodrigue et al., 1990). Prior to 1980, Salmonella Enteritidis was a relatively infrequent cause of human illness in the United States (Altekruse and Swerdlow, 1996). Based on this timeline, it has been hypothesized that Salmonella Enteritidis occupied the ecological niche formerly occupied by the host-specific Salmonella Pullorum and Salmonella Gallinarum (Bäumler et al., 2000). Salmonella Enteritidis initially emerged in the New England region in 1979. In the years from 1985 to 1989, eggs were found to be the source of 82% of the human cases of Salmonella Enteritidis in the United States (Mishu et al., 1994; Dhillion et al., 2001). In the early 1990s, Salmonella Enteritidis was the most common serovar reported in the United States (Mishu et al., 1994). Ebel et al. (1992) found that the prevalence of Salmonella Enteritidis infections in layer flocks ranged from 3% for flocks in the southeastern United States to 44% in the northeastern regions. By 1995, Salmonella Enteritidis comprised 25% of all foodborne Salmonella isolates, compared with 5% in 1985 (Gomez et al., 1997). By the late 1990s, it was estimated that 500,000 cases of human illness were annually attributable to Salmonella Enteritidis (Kaiser and Lamont, 2001). More recent information indicates that Salmonella Enteritidis is the second most frequently isolated serotype of Salmonella from human illness in the United States, and by 2006 was responsible for almost 17% of reported cases of salmonellosis (CDC, 2010b; Howard et al., 2012). In the United States, Salmonella Enteritidis isolates most commonly associated with human outbreaks were phage types 8 and 13 (Mishu et al., 1994; Patrick et al., 2004). Epidemiological studies attributed outbreaks of Salmonella Enteritidis infections to the consumption of contaminated grade A table eggs, which were initially identified as the predominant source of Salmonella Enteritidis infections in humans, and eggs or products containing eggs are still considered the most common vehicle for transmission (St. Louis et al., 1988; Poppe, 1999; Guard-Petter, 2001; Braden, 2006). Although cases have arisen from the other sources of poultry-related exposure, processed chicken carcasses, this is less of a contributor to foodborne disease (Patrick et al., 2004; Altekruse et al., 2006). The primary mode of transmission is through eggs and humans contract the disease by consuming undercooked contaminated eggs (Ricke et al., 2001; Gantois et al., 2009). Salmonella Enteritidis is invasive in poultry, can colonize the gastrointestinal tract (GIT), ultimately infecting the reproductive

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tract, and in turn has the potential to contaminate eggs by transovarian transmission (Thiagarajan et al., 1994; Guard-Petter, 2001; De Buck et al., 2004; Gantois et al., 2009). Adult laying hens that shed Salmonella Enteritidis in their feces may contaminate the surface of the eggshell following oviposition. Salmonella Enteritidis can subsequently penetrate the eggshell during inadequate storage and contaminate the egg contents (Coyle et al., 1988). However, because the exterior of table eggs are washed and sterilized, incorporation of Salmonella Enteritidis into the egg is thought to also occur from contamination of the ovary or oviduct (Humphrey et al., 1989; Timoney et al., 1989; Gast and Beard, 1990a,b; Thiagarajan et al., 1994). Salmonella Enteritidis has been isolated from the contents of clean, intact eggs (Humphrey et al., 1989; Gast and Beard, 1990a,b). Thiagarajan et al. (1994) observed that after oral infections with Salmonella Enteritidis, the organism could be isolated more frequently from the membranes of the preovulatory follicles than from the yolks. The granulosa cells of the hen’s ovary have a diversity of surface structures necessary for attachment of Salmonella Enteritidis. Infected granulosa cells may slough off and contaminate the yolk during ovulation. Several strategies for controlling Salmonella Entertidis have been implemented over the years both at the preharvest level as well as during postharvest production. Clearly, at the postharvest level, proper refrigeration to prevent temperature abuse as well as avoiding undercooking are well-recognized recommendations (Chantarapanont et al., 2000; Ricke et al., 2001; Curtis, 2007; Howard et al., 2012). Intact egg treatments for Salmonella including disinfectants, egg washing, ionizing radiation, UV, and in-shell pasteurization, among others, have been extensively described elsewhere and will not be discussed here (Rodriguez-Romo and Yousef, 2005; Schroeder et al., 2006; Curtis, 2007; Howard et al., 2012). Likewise, poultry and laying hen preharvest approaches for Salmonella such as biosecurity, management, vaccinations, probiotics, prebiotics, and bacteriophage interventions have been extensively discussed elsewhere (Stavric, 1992; Stavric and D’Aoust, 1993; Stavric and Kornegay, 1995; Barrow and Wallis, 2000; Barrow et al., 2003; Holt, 2003; Joerger, 2003; Patterson and Burkholder, 2003; Curtis, 2007; Gast, 2007; Callaway and Ricke, 2012; Dewaele et al., 2012; Doyle and Erickson, 2012; Perumalla et al., 2012; Ricke et al., 2012; Siragusa and Ricke, 2012). This review will specifically focus on dietary modification preharvest intervention strategies that decrease the colonization potential of Salmonella Enteritidis as it enters the layer hen GIT of susceptible birds.

SALMONELLA COLONIZATION OF THE POULTRY GIT General Concepts Poultry can be exposed to a wide variety of sources and reservoirs of foodborne Salmonella including feed,

aerosols, insects, water, and animals, among others (Jones et al., 1991; Nakamura et al., 1997; Holt et al., 1998, 2007; Beaumont et al., 1999; Murray, 2000; Kwon et al., 2000b,c; Gast et al., 2004; Maciorowski et al., 2004; Li et al., 2007; Park et al., 2008). Other factors such as flock size, housing systems, and farms inhabited by hens of different ages represent potential risk factors for Salmonella Enteritidis infections in laying hens, but more research needs to be conducted to sort through the complexities of how these factors individually as well as collectively affect Salmonella Enteritidis dissemination (Mollenhorst et al., 2005; De Vylder et al., 2009; Van Hoorebeke et al., 2010; Holt et al., 2011; Jones et al., 2012). During initial infection in chickens, Salmonella encounters a variety of extracellular and intracellular environments in the different regions of the intestinal tract (Dunkley et al., 2009a). The ceca and the crop, which are the main sites of Salmonella colonization in birds, contain high concentrations of short-chain fatty acids (SCFA; Fanelli et al., 1971; Barrow et al., 1988). The crop is the first environment encountered by Salmonella after infection in poultry. The crop is colonized by lactobacilli and produces primarily lactic acid (Fuller, 1973). In the ceca, which are the primary site of Salmonella colonization in poultry, high concentrations of SCFA are present as a result of microbial fermentations, which produce acetate, propionate, and butyrate, as the major SCFA. Because bacterial virulence is regulated in response to a combination of environmental stimuli, discovering the interplay between environmental signals is important to understand the relevant features of the host that affect Salmonella invasiveness in vivo. Once in the GIT environment of a bird or human, Salmonella spp. encounter extremes of temperature, pH, oxygen tension, bile salts, and competing microorganisms (Foster and Spector, 1995; Park et al., 2008; Dunkley et al., 2009a). This hostile environment serves as a signal for Salmonella to initiate transcription of genes specifically adapted for host interactions (Galán, 1996). The simultaneous requirement for several environmental cues may activate Salmonella Pathogenicity Island 1 encoded genes only when expression of these genes is beneficial in promoting intestinal invasion (Hueck, 1998). The primary environmental conditions studied that have been studied (low oxygen, high osmolarity, and slightly alkaline pH), are thought to exist within the lumen of the human intestinal tract where Salmonella invasion occurs (Bajaj et al., 1996). In the avian GIT, SCFA produced by the indigenous fermentative microflora have been extensively examined as potential modulators for Salmonella virulence after it was shown that they could lead to Salmonella acid tolerance and cross protection to nonrelated stressors (Kwon and Ricke, 1998; Durant et al., 2000a; Kwon et al., 2000a; Ricke, 2003b). Durant et al. (2000a) using hilA (virulence regulatory gene and invF (invasion gene) promoter-β galactosidase structural fusion strains demonstrated that at pH 6, maximal hilA and invF

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gene expression was observed within the first hour of growth in the presence of acetate, propionate, or butyrate, and this expression was 1.4- to 3.7-fold higher than the Luria Bertani (LB) broth control. In conjunction with the Salmonella Typhimurium fusion strain studies, Durant et al. (1999b, 2000d,e) conducted a series of tissue culture studies with Salmonella Typhimurium and the interaction with SCFA. Salmonella Typhimurium invasion of cultured HEp-2 cells was decreased when Salmonella was grown for 4 h in LB broth supplemented with propionate, butyrate, or a mixture of the 3 organic acids. Acetate had little influence on cell association and the ability to invade cultured HEp-2 cells. The regulation of cell-association and invasion by SCFA was dependent on the concentration and the pH of the medium. Because of the complexity of the avian intestinal environment, it is still unknown how many in vivo environmental cues are actually required for full expression of the invasion phenotype (Dunkley et al., 2009a). Continuous culture studies have been conducted either with pure cultures of Salmonella Typhimurium or within a mixture of cecal microflora to simulate liquid phase digesta dilution rates and environmental conditions occurring in the avian cecum, but these do not represent the solid phase of the digesta that would also be present or the cecal lining (Nisbet et al., 1996a,b; Dunkley et al., 2008d, 2009b, 2012). More studies are required to determine the interaction between all of the different regulatory mechanisms and environmental signals that influence the invasion phenotype. The potential importance of the laying hen cecal and crop environments in determining Salmonella Enteritidis virulence has been investigated and is discussed in the following section.

Salmonella Enteritidis Virulence Expression in the Laying Hen GIT In a series of in vitro and in vivo studies by Durant et al. (1999a, 2000b,c; Ricke, 2003a), several environmental signals potentially relevant to the laying hen intestinal tract were measured for their ability to affect Salmonella Enteritidis invasion and regulation of invasion genes. Salmonella Enteritidis constructed with a hilA promoter gene-β galactosidase structural gene fusion was used to determine the effects on invasion gene expression. The expression of hilA was 2.9-fold higher in LB broth diluted 1:5 compared with undiluted LB broth. Addition of 0.2% glucose, fructose, or mannose to the media reduced hilA expression 1.5- to 2-fold. Addition of 0.2% casamino acids, arabinose, fucose, or lactose had little effect on hilA expression. In vivo studies by Durant et al. (1999a) involving force molt induced in 50-wk-old Leghorn hens by 9 d of feed withdrawal altered the crop environment, decreasing the concentrations of lactate, acetate, propionate, and butyrate and increasing the pH level. This was linked to a substantial increase in Salmonella Enteritidis crop and cecal colonization in addition to spleen and liver invasion. When the

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recovered crop contents were recovered and incubated in vitro with Salmonella Enteritidis, the expression of the hilA promoter gene-β galactosidase structural gene fusion was 1.6- to 2.1-fold higher in the crop contents from molted birds compared with control birds. This was supported later by in vivo studies demonstrating increases in quantitative PCR hilA expression levels of Salmonella Enteritidis recovered from infected laying hens after feed removal (Dunkley et al., 2007d). Taken together these series of studies suggested that fermentation capacity in the avian GIT in the form of SCFA in the ceca and the changes in crop pH and lactate concentrations that occur during feed removal could directly influence Salmonella Enteritidis colonization and invasion in the laying hen (Ricke, 2003b; Dunkley et al., 2009a). Clearly a better understanding of the systemic physiology of the laying hen as well as the GIT is needed to determine what changes are manifested as management practices such as induced molting that are conducive to increased levels of Salmonella Enteritidis.

INDUCED MOLTING PRACTICES IN THE LAYER INDUSTRY Molting in the Avian Species— General Concepts Avian molting involves the periodic shedding and replacement of feathers. It also involves the involution of the reproductive system of hens, resulting in a reproductive quiescence. During their natural lifespan, birds will undergo a series of different molts. From hatching up to their first annual cycle, birds will change at least 4 different plumages including natal down, juvenile, alternate, and basic plumages (Lucas and Stettenheim, 1972). Generally speaking, most avian species will have 2 molts per year, a prenuptial or prealternate molt and a postnuptial or postalternate molt. The postnuptial molt is associated with the reproductive quiescence. It is a more complete molt that follows a breeding or laying cycle and involves the loss of feathers from the wings, tail, and body of the bird. Molting is usually considered to be more than simply plumage replacement because it also involves physiological changes in the bird (Stettenheim, 1972). These physiological changes include vascularization of the feather follicles and papillae (Stettenheim, 1972) and cyclic osteoporosis (Murphy et al., 1992; Murphy, 1996). Fractional protein synthesis rate, not only for feather keratin synthesis rate, but primarily for skeletal muscle synthesis (Murphy and Taruscio, 1995; Murphy, 1996), also are changes that occur during molting. Others have reported changes in metabolic rate (Perek and Sulman, 1945), reduction in body fat (Kuenzel and Helms, 1974), and altered heterophil:lymphocyte (H:L) ratios (Gross and Siegel, 1983; Davis et al., 2000) during a molt. The postnuptial molt period is a critical period in the bird’s cycle because it involves major remodeling of

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the body and also the resetting of the neural system to respond to photostimulation (Kuenzel, 2003). A variety of hormones and neuropeptides are involved in the remodeling process. One function of vasoactive intestinal polypeptide is to shut down the reproductive system by initiating incubation behavior such as broodiness (desire of hens to sit on eggs to hatch them), which is usually followed by a postnuptial molt (Kuenzel, 2003). In wild birds, the primary initiating factor of molting is the establishment of broodiness. After the onset of this broodiness, the hen will undergo the involution of the reproductive system, followed by cessation of lay, and finally the shedding and replacement of feathers (Sherry et al., 1980). During this time, the bird will voluntarily reduce their intake of food, and this has been referred to as spontaneous anorexia by Mrosovsky and Sherry (1980). A hen can lose up to 20% of its BW during spontaneous anorexia; one-half of this lost weight is from the involution of the reproductive tract. After the chicks have hatched, the hens will once again begin to consume feed and the feathers will be replaced. This process is a natural phenomenon in species that live in environments where incubation and feeding are incompatible (Sherry et al., 1980).

Molting in the Commercial Egg Industry Over the years, researchers have developed methods of artificial molt induction to occur at times other than at the time of natural molting. These methods have resulted in the cessation of lay and the loss of feathers. Historically, induced molting involved the removal of feed, water, or both feed and water. It also involved reducing the photoperiod to 10 h or less. Hens would then be fasted for a preset period of time to cause the complete regression of the reproductive tract (Berry, 2003). In the 1950s, egg producers in the United States first adapted the practice of induced molting. Egg producers at that time used multiple-molting programs in which hens were molted more than once and the laying cycles were shortened (37 wk). The first molt was an average of 60 wk, and the second molt was induced at approximately 106 wk. Californian producers were the first to adopt the practice (Bell, 2003), which spread across the United States to the major egg-producing regions in the mid 1970s. In 2000, the USDA stated that 93% of the flocks in the Southeast region of the United States practice force molting intensively (USDA, 2000). In the early 2000s, Bell (2003) concluded that approximately 73% of the commercial laying facilities in the United States implement the practice of induced molting to rejuvenate their flocks. The practice was done to give the egg producer maximum production from hens on the commercial layer farms by enabling them to have a second or even a third laying cycle. Under optimal economic conditions, the useful reproductive life of laying hens can be extended from less than 80 wk to more than 110 wk or even 140 wk (Webster, 1995; Bell, 2003),

giving the producer second and third laying cycles. Lee (1982) stated that molting had beneficial effects on the individual hen’s performance and the practice was also beneficial for overall flock management because the hens were synchronized for the second laying cycle. If allowed to molt naturally, hens would begin molting at different times and this would prolong the process through the entire flock. Bell (2003) concluded that there was improved postmolt performance in molted hens compared with their nonmolted counterparts with the peak of egg production occurring during the second cycle of approximately 75 to 85% 13 wk into the second cycle. Roland and Bushong (1978) demonstrated that forced molting reduced the incidence of uncollectable eggs. Naturally during the periods of short day length, birds in the wild will experience weight loss along with feather loss and the regression of the reproductive system (Brake and Thaxton, 1979; Mrosovsky and Sherry, 1980; Etches, 1996), and during this time they will undergo a period of a natural lack of appetite. In the commercial layer industry, an induced molt will usually mimic these conditions that would have occurred naturally (Brake, 1993). Traditionally, there were 3 basic ways by which a molt was induced: feed removal or limitation, low-nutrient ration, and feed additives (Bell, 2003; Berry, 2003; Park et al., 2004c; Ricke et al., 2010). Each of these methods usually involves the alteration of the photoperiod from a long day to a short day. Whichever method is used should be easy to apply, low cost, result in low mortality, consider animal welfare issues, and ultimately result in enhanced performance during the second cycle (Swanson and Bell, 1975; Ruszler, 1998; McDaniel and Aske, 2000; Hester, 2005). The method should also avoid the potential feed refusal by the birds, be readily available, be economical with minimal feed processing, and provide a molt induction stimulus enough to cause sufficient reproductive tract regression during the molt (Ricke, 2003a; Koelkebeck et al., 2006).

Feed Withdrawal-Based Molts— Historical Perspectives In the past, commercial egg producers in the United States typically used feed removal as their method to induce a molt. It has been reported that in 1999 62.1% of egg producers that were surveyed molted their last flocks once, and 12.1% molted their last completed flocks twice (USDA, 2000). This was because feed withdrawal was considered easy to implement and would achieve BW loss of 25 to 35% to obtain the optimum postmolt performance in terms of egg production and shell quality (Baker et al., 1983; Webster, 2003). Feed withdrawal (deprivation) involved the removal of feed for a period of 5 to 14 d, and this may or may not have included a period of rest for up to 21 d after the fast (Bell and Kuney, 1992). Conventional feed withdrawal

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programs involve the removal of feed from the hens and also the reduction of the photoperiod in the houses to an 8-h lighted period or less. Removal of feed was allowed to continue long enough to cause the complete involution of the reproductive tract (Berry, 2003). Although feed withdrawal historically was the molting method of choice by the commercial egg industry, concerns arose regarding animal welfare (Webster, 2003). In attempt to address this, Webster (2003) summarized and compared existing studies on the physiology and behavior responses to feed withdrawal. A key point was the realization that the corresponding physiological responses could be separated into phases (Webster, 2003). Previously, Cherel et al. (1988) had concluded that fasting (deprivation) could be categorized into 3 phases: phase 1 is usually short (a few days) and involves a rapid decrease in body mass loss while at the same time there is a reduction in the rate at which the body mass is lost. Phase 2 is usually long term and may last several weeks or even months in some species. During this phase, most of the energy used is from the catabolism of fat (Webster, 2003). Phase 3 can last from days to weeks, and protein catabolism begins (Cherel et al., 1988). However, the degree of fasting that is imposed to induce a molt can be viewed as a physiological adaptation of the hens (Webster, 2003). It was concluded that a properly managed moderate period of feed withdrawal posed no threat to healthy laying hens but could impair less healthy birds (Webster, 2003). In addition, any alternative molting method being proposed would need to achieve similar physiological responses from the bird as well as not compromise animal welfare (Webster, 2003).

Feed Withdrawal and Salmonella Enteritidis There was evidence of systemic Salmonella Enteritidis infections occurring in laying hens when Gast and Beard (1990a) detected Salmonella Enteritidis in the albumin and yolks of eggs from hens after they were orally inoculated. This was an indication that internal contamination of eggs could occur before the laying of the egg, when Salmonella Enteritidis was deposited on the shells. Keller et al. (1995) observed that when eggs that were not yet formed were removed from the oviduct, they were contaminated with Salmonella Enteritidis much more often than freshly laid eggs. Early on, it was postulated and demonstrated that molting could significantly influence Salmonella Enteritidis infection at different times in the infection cycle (Holt and Porter, 1992a,b, 1993; Holt, 1993). In follow-up studies it was observed that molting exacerbated the infection as shown by the more rapid dissemination of the organism in the GIT as well as systemically, an indication that molting made these hens more susceptible (Holt et al., 1995). This was confirmed when infectious dose was determined and it was shown that feed withdrawal molt increased bird susceptibility to Salmonella Enter-

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itidis infection by 2 to 3 logs compared with unmolted control hens (Holt, 1993). Feed withdrawal was also shown to have detrimental effects on the hens’ immune system, which could be linked to increased susceptibility to Salmonella Enteritidis infection (Golden et al., 2008). In mammals and birds, starvation and nutrient-deficient diets were shown to reduce humoral immunity (Ben-Nathan et al., 1977; Gross and Newberne, 1980) and also cell-mediated immunity (DePasquale-Jardieu and Fraker, 1979; Gross and Newberne, 1980). Holt (1992a) observed that cell-mediated immunity was significantly depressed, whereas in another study (Holt, 1992b), the B-cells and CD8+ T cells were less infected. DePasquale-Jardieu and Fraker (1979) detected elevated levels of serum corticosterone, which suggested that the hormone played a role in the depression of cell-mediated immunity. Brake and Thaxton (1979) and Etches et al. (1984) also noted a similar response when molting hens by way of feed withdrawal. The intestinal shed rate of Salmonella Enteritidis is higher in birds that were exposed to exogenous sources of the pathogen concomitantly during molt induction. These hens had a more severe infection than unmolted hens (Holt and Porter, 1992b; Holt et al., 1995). These hens not only shed more organisms (Holt and Porter, 1992a; Holt, 1993; Holt et al., 1994, 1995) but they also showed significantly more inflammation, especially in the colon and cecum (Holt and Porter, 1992b; Porter and Holt, 1993; Holt et al., 1995; Macri et al., 1997). Feed-withdrawal-associated stress causes increased susceptibility to Salmonella Enteritidis infection (Holt, 1993; Corrier et al., 1997; Durant et al., 1999a; Holt, 2003; Ricke, 2003a), and this in turn can lead to more widespread problems. Salmonella Enteritidis infection is usually marked by increased intestinal shedding and colonization in internal organs such as the liver, spleen, and ovaries (Holt and Porter, 1992a; Holt, 1993; Thiagarajan et al., 1994; Holt et al., 1995). Once Salmonella Enteritidis becomes prevalent in the flock environment, exposure to larger numbers of susceptible hens in a flock is of major importance. Nakamura et al. (1993) observed that short-term feed removal can increase horizontal transmission to nearby hens, and Holt and Porter (1992a) observed a similar outcome when molting increased horizontal transmission to hens in neighboring cages. The impact of airborne transmission is also a major concern because transmission to birds in cages that were a distance away from infected birds during a molt by feed withdrawal was observed by Holt et al. (1998). Laying hens with Salmonella Enteritidis infected ovaries will not only lay contaminated eggs, but infected chicks will also be hatched from these eggs. These chicks will grow to become pullets that will subsequently lay contaminated eggs (Hopper and Mawer, 1988; Lister, 1988; O’Brien, 1988). O’Brien (1988) reported that postmortem of infected chicks showed the most con-

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stant lesion was a toxic indurated yolk sac membrane. O’Brien (1988) also detected early signs of pericarditis, and when cultured on selective media, 73% of the chicks were found to contain Salmonella Enteritidis, which indicates that the pathogen can persist in the chickens up to the time of slaughter. The process of molting can cause an increase in infectivity of these birds (Cason et al., 1994), and stress situations such as feed withdrawal to induce a molt can also cause a recurrence of a previous Salmonella Enteritidis infection (Holt, 1999).

REDUCING SALMONELLA ENTERITIDIS IN LAYING HENS BY MOLT DIET General Concepts Because of the increased pressure and criticism on commercial layer producers for their practice of feed withdrawal as a means of inducing or forcing a molt, researchers began investigating different methods to bring on a molt instead of depriving hens of feed. The goals of a successful molt are to achieve a complete cessation of lay long enough for the total regression of the reproductive tract and an acceptable and persistent second cycle performance (Berry, 2003). Generally speaking, egg production and quality deteriorate as the flock ages (Roland and Bushong, 1978; Bell, 2003). After the rejuvenation of the reproductive tract the hens will come into a second cycle and produce better quality eggs (Keshavarz and Quimby, 2002). Deterioration of the egg quality includes the internal and external traits of the egg, and improvement in egg quality parameters is evident after an induced molt (Swanson and Bell, 1975). The parameters that are of particular importance include albumin quality, candled grade, shell thickness, specific gravity, egg weight, and shell texture (Bell, 2003). As discussed previously, it became well established that feed withdrawal altered the microenvironment of the crop and ceca in the intestines of poultry, which in turn increased susceptibility to increased colonization and systemic infections (Corrier et al., 1997; Durant et al., 1999a; Ricke, 2003a; Dunkley et al., 2009a; Norberg et al., 2010; Ricke et al., 2010). Other researchers have shown that emptying of the GIT by removing feed is consistent with increased levels of Salmonella in a variety of animal species including chickens (Brownlie and Grau, 1967; Tannock and Smith, 1972; Moran and Bilgili, 1990; Humphrey et al., 1993; Corrier et al., 1999). Because induced molting by feed withdrawal was being practiced by commercial layers, research into different methods by which to ameliorate the increased incidence of Salmonella Enteritidis infection was deemed necessary. Consequently, it was proposed that one of the keys for developing non-feed withdrawal molt diets was to simultaneously initiate a fully complete molt process via a an alternative molt diet but retain normal GIT microbial gut fermentation activity to serve as a barrier

to Salmonella Enteritidis colonization (Ricke, 2003a). Whichever products were to be used must be part of an alternative method to induce molt and should be of economic advantage to the commercial farmers by successfully providing a second laying cycle of high production rates and high quality eggs while at the same time reducing the incidence of increased susceptibility to Salmonella Enteritidis (Ricke, 2003a).

Alternative Methods of Inducing a Molt Molting techniques have varied widely in an effort to initiate a molt and cease egg production to alleviate problems of increased Salmonella Enteritidis infectivity associated with feed withdrawal. Attempts have been made to restrict feed intake, thereby reducing BW and inducing a molt. This has been done by using nutritional imbalances, addition of dietary amendments that decrease appetite, incorporation of dietary fillers, administration of hormones to induce molts, or lowenergy diets (Bell, 2003; Holt, 2003; Park et al., 2004c). Specific examples of these methods include feeding diets that contain less than 0.3% calcium (Douglas et al., 1972; Martin et al., 1973; Gilbert and Blair, 1975; Wakeling, 1978), less than 0.04% sodium (Whitehead and Shannon, 1974; Dillworth and Day, 1976; Nesbeth et al., 1976; Campos and Baiao, 1979; Ross and Herrick, 1981), diets containing various levels and forms of zinc (Scott and Creger, 1976; Creger and Scott, 1977, 1980; Shippee et al., 1979; McCormick and Cunningham, 1984a,b; Stevenson and Jackson, 1984; Berry and Brake, 1985; Cunningham and McCormick, 1985; Goodman et al., 1986; Berry et al., 1987; Alodan and Mashaly, 1999; Bar et al., 2003; Moore et al., 2004; Park et al., 2004a,b,c; Ricke et al., 2004a,b), feeding diets containing iodine in the form of potassium iodine (Arrington et al., 1967), aluminum (Hussein et al., 1989), copper (Stevenson and Jackson, 1984), or magnesium (Shippee et al., 1979). However, the results of some of these diets do not induce a consistent molt in all hens in the flock; most were either costly or managerially unwieldy, generated adverse behaviors such as cannibalistic pecking, or inconsistently limited Salmonella Enteritidis infection (Ricke, 2003a; Webster, 2003; Biggs et al., 2004; Park et al., 2004c).

FIBER AND PLANT POLYSACCHARIDEBASED MOLT DIETS General Concepts and Criteria High-fiber diet components consist predominantly of plant cell walls, nonstarch polysaccharides, and noncarbohydrate compounds including lignin, protein, fatty acids, and wax (Bach Knudsen, 2001). By definition, dietary fiber cannot be digested by endogenous processes, but can be hydrolyzed, fermented, or both by the resident microorganisms (Wenk, 2001). Consequently a

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diet that is high in fiber is usually lower in ME than a diet that has low fiber content (Wenk, 2001). Langhans (1999) stated that high-fiber diets cause earlier satiety of an animal, whereas Rijnen et al. (1999) concluded that an animal that reaches satiety both physically and nutritionally is less stressed. However, these diets can alter the GIT by changing its microbial activities, the rate of passage, metabolites, and the overall effectiveness of the GIT (Bach Knudsen, 2001; Wenk, 2001). The general concept of incorporating fiber into poultry diets is not a new one. Several studies have examined the physiological responses of young chicks and laying hens to diets including various fractions of fiber and plant polysaccharides (Baker, 1977; Moran and Evans, 1977; Akiba and Matsumoto, 1978; McNaughton, 1978; van der Aar et al., 1983; Mateos et al., 2012). Such diets have been shown to not only support an active fermentation in vivo and in vitro by avian GIT microorganisms but also generate detectable shifts in GIT microflora (Ricke et al., 1982; Guo et al., 2003; Hume et al., 2003; Saengkerdsub et al., 2006; Dunkley et al., 2007c,e). Depending on level in the diet and particle size, the presence of fiber in poultry diets has also been shown to influence the avian GIT by exhibiting slower passage rates, expanding gut fill and gut capacity (Sibbald, 1979a,b, 1980; Jørgensen et al., 1996; Hetland and Svihus, 2001; Mateos et al., 2012). Feed particle size may also be a factor that directly influences GIT establishment of Salmonella. Mikkelsen et al. (2004) demonstrated that pigs fed coarse nonpelleted cereal grain diet particles yielded lower in vitro survival of Salmonella incubated in stomach contents derived from pigs fed these diets. This correlated with the higher concentration of undissociated lactic acid and greater populations of anaerobic bacteria. In an in vivo study, Huang et al. (2006) demonstrated that pelleting corn soy diets increased the incidence of Salmonella Typhimurium in gizzard and ceca contents of growing broilers. Although in vivo work with fiber and nonfiber molt diets combined with the passage rate marker hafnium was inconclusive (Dunkley et al., 2008c), it is intuitive that slower passage rates may support a more extensive GIT microbial fermentation. More fermentation activity should be inhibitory to Salmonella Enteritidis establishment. However, Nisbet et al. (2008) has speculated that the presence of fiber in the diets may also help to retain gut motility during feed restriction and hence restrict Salmonella Enteritidis colonization. How much this might influence the GIT cellular microenvironment remains to be determined; Rolls et al. (1978) did not observe any differences in rate of GIT epithelial cell renewal in germ-free or conventional birds fed wheat bran. Regardless of what mechanism(s) are involved, it would appear that fiber-containing components can certainly be fermented by the avian GIT microflora and because they are low energy would achieve gut fill without contributing extensively to the ME of the bird. With this in mind, the incorporation of high-fiber, low-energy di-

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ets to induce a molt evolved as a potential molting diet alternative to limit Salmonella Enteritidis infection in susceptible laying hens (Ricke, 2003a). However, such diets must not only provide a consistent GIT microbial fermentation concomitant with molt induction but also accomplish both goals and meet the criteria for optimal molt, namely avoiding feed refusal by the hen, causing sufficient reproductive tract regression, retaining egg quality, originating from readily available commercial sources, and being economical with minimal feed processing (Ricke, 2003a; Koelkebeck et al., 2006). A variety of fiber-based diets have been examined for potential incorporation into poultry feed for inducing molt, and these will be discussed in the following sections.

Cotton Seed Meal Cotton seed meal is lower in energy and protein than soybean meal and has been successfully incorporated into poultry diets (Davis et al., 2002). When cotton seed meal was fed to broiler chicks, Waldroup (1981) reported depressed weight gain due to the presence of gossypol, a potentially toxic agent found in the oil of the seed. Davis et al. (2002) examined the effects of cotton seed meal when incorporated in layer diets due to the low energy and protein requirements of layers when compared with broilers. Phelps et al. (1965) previously reported that laying hens fed a diet containing cotton seed meal with free gossypol produced eggs that exhibited a brown yolk color. Davis et al. (2002) observed laying hens fed cotton seed meal at different levels (20 to 30%) of the diet and reported reduced egg sizes and yolk discoloration. They reported no significant difference in egg production between the control hens and the hens fed feed containing cotton seed meal. There are, however, disadvantages of using cotton seed meal in a layer diet because it can result in the production of eggs with brown yolk discoloration.

Jojoba Meal Jojoba meal is a byproduct after the extraction of oil from jojoba seeds. It contains approximately 30% CP, which gives it a high possibility as an ingredient for animal feed. However, when Ngou Ngoupayou et al. (1982) fed jojoba meal to chickens, they observed impaired BW gain, reduced feed intake, and impaired feed efficiency. Arnouts et al. (1993) reported that broiler chickens fed a diet supplemented with 4% jojoba meal exhibited a reduced growth rate. Vermaut et al. (1998a) used jojoba meal to induce a molt in broiler breeder hens. These hens reached maximum BW loss 5 wk after the start of the molt and stopped laying 3 wk after molt induction. They observed that the oviduct regressed to the same degree as the pair-fed hens (3.7 g as opposed to 4.1 g). The oviduct regrew completely after the jojoba meal was removed from these hens. This result was not observed by Vermaut et al. (1998b), who fed

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jojoba meal to growing pullets and observed irreversible inhibition of oviduct development, resulting in no egg laying; they postulated that these effects may be as a result of factors present in jojoba that interfere with follicle maturation and deposition of yolk material.

Guar Meal Guar also known as cluster or guar bean (Cyamopsis tetragonolobus L.) is an annual leguminous crop and guar gum that includes mannan- and galactomannanbased polymers from the seeds of the bean (Ishihara et al., 2000). Guar meal is a dietary byproduct of guar with a protein content ranging from 33 to 45% (Gutierrez et al., 2008). Previous research indicated that feeding partially hydrolyzed guar gum to young hens reduced Salmonella Enteritidis incidence in organs as well as Salmonella Enteritidis agglutinating serum antibody titers and increased fecal Salmonella Enteritidis excretion and cecal Bifidobacterium and Lactobacillus populations (Ishihara et al., 2000). Feeding guar meal diets to laying hens to induce a molt proved to be effective in causing greater loss of body fat and decreased loss of body protein compared with a molt induced by feed withdrawal (McGinnis et al., 1983). Zimmermann et al. (1987) examined the effects of inducing a molt in hens that were given a layer ration that contained 10 and 15% guar meal. These diets suppressed the feed consumption of the hens until they obtained 30% BW loss. They observed that laying hens stopped laying after being fed for 8 d with a 15% guar meal to the layer diet. Zimmermann et al. (1987) also reported that these hens lost 31% of their BW within 21 d. The hens which were fed 10% guar meal had the best eggshell quality; however, these hens displayed the highest mortality rate. Egg weights were not different between treatments, and the hens that were fed guar meal and those on a thirdday feeding treatment displayed the best internal egg quality. A major disadvantage of feeding guar meal was that these hens were the slowest to return to lay. Gutierrez et al. (2008) demonstrated that molting diets containing 20% guar meal with or without a mannase enzyme limited Salmonella Enteritidis infection in molted hens, but the enzyme additive enhanced resistance to Salmonella Enteritidis and achieved a complete molt with only 16% BW loss compared with 19% loss for hens fed guar meal without enzyme addition and 27% loss by the feed withdrawal control group.

Grape Pomace Grape pomace is the solid remains of grapes after the juice or oil has been pressed out. It can be used as animal feed, but its use is limited to 30% of the diet due to its low nutritional value (Mole et al., 1993). Keshavarz and Quimby (2002) molted hens using grape pomace and observed that these hens stopped laying by d 3 to 4 after the initiation of the molt, which oc-

curred at a similar time as the feed withdrawal hens. They also reported that the hens fed grape pomace lost a similar amount of BW as the feed withdrawal hens (30.3 and 30.8%, respectively). No significant differences were observed between the feed withdrawal hens and the hens that were fed grape pomace in egg production during the first 4 wk after the initiation of molt (7.5 and 5.0% production, respectively). They also observed no differences in the specific gravity of eggs or any of the other internal egg qualities they investigated. When they examined the organ weights, they observed that the feed withdrawal hens had a mean ovary weight of 3.1 g and oviduct weight of 10.4 g compared with the hens fed grape pomace, which exhibited ovary and oviduct weights of 2.9 and 8.4 g, respectively, but the hens in these 2 treatments were not significantly different. Although grape pomace can effectively induce a molt and generate a postmolt performance that is comparable with feed withdrawal hens, accessibility and storage issues could be problematic due to its light and fluffy nature and thus considerable required space for storage. Also, based on the physiological response of hen on the grape pomace diet, it appears that this diet did not prove to be less stressful on the hens than the conventional method of feed withdrawal because even though the hemocrit counts for the hens fed grape pomace were lower than the feed-deprived hens, the corticosterone levels were not significantly different among treatments.

Cereal Grain Byproducts Cereal grain byproducts derive from components or processes involving cereal grains that are typically underutilized and may contain considerable structural fiber but still can have nutritional value as a feedstock for livestock. Wheat middlings are the byproducts of wheat flour manufacturing. Wheat bran is also a byproduct of wheat manufacturing and contains higher fiber than wheat middlings which are used as a source of energy in animal diets (Bai et al., 1992). The utilization of wheat middlings as a feed source in commercial layers had been explored previously by Patterson et al. (1988), who demonstrated that laying hens fed a diet containing wheat middlings at 25% of the diet achieved similar production, feed consumption per day, and feed per dozen eggs to other diets tested. Soy hulls as a source of soluble and insoluble fiber became a popular source of fiber due to the large quantities produced, their easy availability, and the fact that they elicited health benefits for humans and animals (Muzilla et al., 1989; Lo, 1989; Cole et al., 1999). Corn distillers byproducts have been available in animal diets for several years originally from the beverage-alcohol industry but more recently from the tremendous upsurge in fuel alcohol production from corn (Morrison, 1954; Bregendahl, 2008). After ethanol is distilled off following fermentation, corn dried distillers grain solubles (DDGS) are

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generated from the leftover stillage by centrifugation to separate the wet grains, subsequently recombined with the solubles produced from the thin stillage via evaporation and condensation, and finally dried into a DDGS product (Bregendahl, 2008). Biggs et al. (2004) evaluated the efficiency of wheat middlings, corn gluten feed, and DDGS as alternative methods of inducing a molt. Previously, Biggs et al. (2003) demonstrated that diets high in wheat middlings or corn were effective methods that could obtain postmolt performances comparable with a 10-d feed withdrawal molt. In a later study, Biggs et al. (2004) observed that all diets (wheat middling, corn gluten feed, and DDGS) yielded decreased egg production and BW during the molting period. However, they found that the DDGS diet had the highest oviduct and ovary weight (2.5 and 3.5%, respectively, of their BW), which suggested that there was limited ovarian regression using DDGS. Biggs et al. (2004) postulated that feeding hens with diets high in corn, wheat middling, corn gluten feed, or 71% wheat middling-23% corn combination could be used as effective alternative molt programs because they observed no significant differences in postmolt egg parameters among treatments. They observed that whereas the feed-deprived hens in their study ceased egg production within 6 d, the hens in the other treatments did not totally stop producing eggs. However, the hens in the wheat middlings treatments reduced production at a faster rate than the hens in the other treatments. Postmolt feed intake and feed efficiency of hens fed wheat middlings during a molt were not different from those of hens that were molted by conventional feed withdrawal. The initial acceptance of the feed by the hens was poor, and hens fed wheat middling ate 2-fold less feed than other treatments. Mejia et al. (2010, 2011) concluded that limit feeding of a corn and DDGS diet achieved a similar longterm postmolt performance as hens molted with an ad libitum 47% corn: 47% soybean hull mixture, but limit-feeding DDGS at 65 or 55 g per hen per day did not totally shut down egg production. Dickey et al. (2010, 2012) observed that soybean hull and wheat middling molt diets did not adversely influence behavior compared with hens undergoing feed withdrawal or increase the heterophil-to-lymphocyte ratio. However, feed withdrawal resulted in the greatest decrease in egg production during the molting period followed by soybean hulls and wheat middlings. Only limited studies have been done with cereal grain byproducts and prevention of Salmonella Enteritidis infection in laying hens. Seo et al. (2001) examined the efficiency of wheat middlings as an alternative diet, not only to induce a molt, but they also evaluated the efficiency of the diet to reduce Salmonella Enteritidis shedding. They observed that both the feed withdrawal hens and the hens fed wheat middlings stopped laying within 7 d after the beginning of the molt. The wheat middling-fed hens shed Salmonella Enteritidis but significantly less than the feed withdrawal hens,

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which shed 3 to 5 log more Salmonella Enteritidis than the other groups. Seo et al. (2001) also observed that the levels of Salmonella Enteritidis in the liver, spleens, ovaries, and liver in the hens molted by feed withdrawal were significantly higher than the hens fed wheat middlings and also higher than the nonmolted birds. They noted that 63% of the feed-deprived hens tested positive for Salmonella Enteritidis in the ovaries, whereas none of the wheat middling or the nonmolted hens tested positive and also resulted in reduced numbers of Salmonella Enteritidis in the feces. The hens fed wheat middlings consumed the diet readily without problems of feed refusal.

ALFALFA MOLTING DIETS Alfalfa as a Dietary Ingredient for Poultry Alfalfa is a forage crop that is easily accessible in most and poultry producing regions of the United States and is currently commercially available in mash, pelleted, and cube form (Matsushima, 1972; NRC, 1994). It contains 17.5% CP and 24.1% crude fiber and has low levels of ME, approximately 1,200 kcal/kg compared with layer ration, which has a ME of 2,965 kcal/kg (NRC, 1994). As early as the mid 1920s and early 1930s, it was demonstrated that alfalfa leaf, alfalfa meal, and alfalfa hay could serve as a source of vitamin A in poultry diets (Beach, 1924; Davis and Beach, 1925; Kennard and Lingle, 1928; Stuart, 1929; Heywang, 1933). Alfalfa leaf meal, when fed in sufficient amounts (5 to 10% of the diet) to provide 0.2 mg of carotene per bird daily, prevented the occurrence of deficiency lesions in the throats of birds (Williams et al., 1938). Sen et al. (1998) and Ponte et al. (2004) found that alfalfa was well balanced in amino acids, and rich in vitamins, carotinoids, and xanthophylls that could give the poultry carcass a desirable yellow color. Draper (1948) observed a significant decline in weight when chicks were fed alfalfa meal and the rate of weight reduction increased as the quantity of alfalfa in the diet increased from 5% to 15%. Cooney et al. (1948) reported that there was an unidentified factor in alfalfa that resulted in a reduced growth of New Hampshire chicks when fed at 10% of the diet. German and Couch (1950) and Heywang (1950) also observed a depressed growth in chicks, and the depression in growth was proportional to the levels of alfalfa in the diet. Alfalfa contains high levels of bioactive antinutritive factors such as saponins, which can cause nutritional digestive problems in several nonruminant animal species and interfere with rumen microbial fermentation in ruminants (Cheeke et al., 1977; Hegsted and Linkswiler, 1980; Lu and Jorgensen, 1987; Sen et al., 1998; Francis et al., 2002). Saponins are considered steroids or triterpenoid glycosides that possess hypocholesterolemic, anticarcinogenic, antiinflammatory, and antioxidant properties (Rao and Gurfinkel, 2000; Ponte et al., 2004). However, Ponte et al. (2004) did conclude that

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meat from broiler hens fed moderate levels of alfalfa was acceptable to consumers. Earlier work on laying hens focused on incorporating alfalfa as part of the layer ration for altering egg quality. The utilization of alfalfa in laying hen nonmolt nutrition was examined in an effort to improve yolk pigmentation and also as a protein concentrate (Miller et al., 1972; Burdick and Fletcher, 1984). Williams et al. (1938) concluded that when alfalfa was fed in sufficient quantities to laying hens to furnish 0.2 mg of carotene per bird daily, alfalfa also kept the birds in good health and resulted in fair egg production. Heiman and Wilhelm (1937) observed that yolk pigmentation, which was greater for hens that were fed alfalfa than hens that were fed corn, is considered a desirable trait for consumers in some regions. Heywang (1950) noted that White Leghorn laying hens fed diets containing 5 to 25% sun-cured alfalfa meal exhibited a reduction in total egg production as the level of the alfalfa in the diet increased. When high levels of alfalfa were incorporated in the diets of laying hens, Whitehead et al. (1981) observed that the saponins contained in the alfalfa depressed feed consumption, BW, egg production liver lipid concentration, and plasma triglyceride concentrations. However, Kuzmicky and Kohler (1977) concluded that an alfalfa leaf protein concentrate (Pro-XAN) containing a high level of xanthophylls could serve as an excellent pigment source for broilers and layers. Patterson et al. (1988) used a corn, soybean meal alfalfa meal mixture as a control diet to compare various levels of wheat middlings in layer diets on egg production parameters and observed that Haugh units were decreased and egg yolk color was increased in the eggs from alfalfa control birds versus eggs from the hens fed 43 and 89% wheat middlings. More recently Güçlü et al. (2004) reported that including 9% alfalfa meal into a laying quail diet could be used to enhance eggshell quality and reduce serum triglycerides and decrease cholesterol in the serum and egg yolk without adversely influencing performance.

Alfalfa Molt Diets—Reproductive Tract Response and Egg Production Work on molting with alfalfa diets began in the mid 2000s. Alfalfa was initially included at 100% of the molt ration, but given inconsistent and in some cases lower feed intake responses, eventually layer ration-alfalfa combinations were considered to be more practical (Donalson et al., 2005; Woodward et al., 2005). Donalson et al. (2005) examined a 100% alfalfa along with combined alfalfa layer rations at either 90 or 70% alfalfa with nonmolted and feed withdrawal treatments as controls over a 9-d molt period, and as the percentage of layer ration increased, feed intake increased. In general, hens molted with alfalfa diets achieved weight loss during the molt that was comparable with that of the feed withdrawal hens, which had been seen in other alfalfa molt studies as well (Donalson et al., 2005;

Woodward et al., 2005). Donalson et al. (2005) evaluated the changes in the internal organs of the hens fed alfalfa molt diets compared with those hens that were molted by feed withdrawal, and observed no significant differences in ovary or oviduct weight among the hens in the molting treatments (alfalfa or feed withdrawal), indicating that the reproductive tracts of the hens on the diet were regressed. Landers et al. (2005a,b, 2008b) reported similar results when they compared ovary regression weights between hens molted by feed withdrawal and hens molted using 100% pelleted or as an alfalfa meal product. In these studies, egg production generally ceased within a few days of molt induction. Likewise, Donalson et al. (2005) saw no significant differences between the feed-deprived hens and the alfalfa-fed hens for the length of time it took for the birds to return to 50 to 60% production. Donalson et al. (2005) observed that hens fed 100% alfalfa meal returned to lay an average of 14.8 d after molt induction, whereas Landers et al. (2005b) observed hens fed 100% alfalfa meal and 100% alfalfa pellets returned to lay 14.0 and 11.6 d, respectively. Donalson et al. (2005) reported that the feed-withdrawal hens were up to 74.29% egg production by 39 wk postmolt, which was not significantly different from the hens that were fed 90% alfalfa diets, and similar levels of egg production postmolt were also observed by Landers et al. (2005b). Both Landers et al. (2005b) and Donalson et al. (2005) reported that hens which were molted using alfalfa diets produced eggs with interior qualities that were comparable with the eggs produced by the feed-withdrawal hens. They also noted that hens which were fed 100% alfalfa diets consumed the least amount of feed and lost weight that was comparable with feed withdrawal hens. Donalson et al. (2005) also reported that hens fed 90 to 100% alfalfa had second egg laying cycles that were comparable with the feed-withdrawal hens, but hens that were fed 70% alfalfa lost the least amount of weight and took the longest time to cease egg production. Landers et al. (2005a) conducted a sensory study that evaluated the consumer assessment of eggs produced by the hens treated using alfalfa diets. The sensory investigation was based on the taste, texture, and color of the cooked egg. Taste/texture assessments revealed that egg from hens fed alfalfa diets exhibited taste/texture values that were similar to feed-deprived hens and the taste panelists could not differentiate between the samples in taste/texture and color evaluations. They concluded that the eggs produced by hens molted with alfalfa were not considered less desirable than those produced by hens molted by feed withdrawal.

Alfalfa Molt Diets—Laying Hen Behavior and Physiology A wide range of physiological and behavior studies have compared nonmolted with molted birds in an at-

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tempt to dissect out responses specific to alfalfa-fed birds undergoing molt. When examining the behavioral profile of domesticated hens, several specific parameters have been examined including feeding, drinking, comfort, social, reproductive, and antipredator behavior (Duncan, 1970). During molting, several behavior patterns are considered of particular interest and are potential welfare indicators of stress, including submission—putting up no resistance; aggression—aggressive behavior toward neighbor; escape; and avoidance—show of fear (Anderson et al., 2004). In addition, nonnutritive pecking (nonaggressive pecking at anything other than feed), preening (manipulation of the plumage with the beak), walking (locomotion involving one step or more), being still (complete immobility of an alert hen), feeder (behavior directed into the feed trough), and drinking water have been considered (Webster, 2000). In a series of studies, Dunkley et al. (2008a,b) video recorded and quantified a variety of hen behaviors (nonnutritive pecking, walking, drinking, feeder activity, preening, aggression, and head movement) in 10min intervals during the molt period comparing full-fed nonmolted birds with feed withdrawal and alfalfamolted birds. In an initial study Dunkley et al. (2008a) compared full-fed nonmolted birds with birds molted either by feed withdrawal or fed a 100% alfalfa crumble diet. Full fed birds and alfalfa-fed hens spent more time at the feeder, whereas hens undergoing feed withdrawal generally exhibited more nonnutritive pecking and head movements than alfalfa- or full-fed hens throughout the molt period. In a follow-up study, when hens molted with alfalfa-layer ration combination molt diets were compared with feed withdrawal hens (Dunkley et al., 2008b), 70% alfalfa:30% layer ration and 80% alfalfa:20% layer ration-fed hens spent significantly more time walking than hens in the 90% alfalfa:10% layer ration group. Full-fed control hens and 70% alfalfa: 30% layer ration hens spent half as much time preening, whereas feed withdrawal molted hens spent almost twice as much time involved in nonnutritive pecking compared with other treatments. Webster (2000) earlier had noted that hens undergoing feed withdrawal molting expressed increased aggressive behavior on d 1 of the molt followed by increased standing, head movement, and nonnutritive pecking, with nonnutritive pecking continuing to be higher throughout the feed-withdrawal period. Although some of this could be suggestive of frustration-type behavior, Webster (2000) cautioned that this could be more reflective of an adaptive response to increase behavior related to foraging for food. Consequently, it would be intuitive to assume that the presence of a molt diet in the feeder would reduce such behaviors. Attempts have been made to relate some of these behavioral changes to stress responses in the bird, and shifts in physiological parameters have been offered up as a way to more precisely quantitate levels of stress. Indeed, when feed removal occurs and a laying hen

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transitions into reproductive tract regression and egg production cessation, major metabolic and physiological shifts do occur (Dunkley et al., 2007a; Landers et al. 2008a). However, Thaxton and Puvadolpirod (2000) advised caution when attempting to diagnose stress by using physiological changes because stress consists of a series of nonspecific adaptive responses that can enable a return to homeostasis (Puvadolpirod and Thaxton, 2000a,b,c,d). Metabolic shifts that have been detected in feed-withdrawal-molted hens include changes in serum cholesterol, ketone bodies, uric acid, and triglyceride levels, which are believed to reflect physiological adjustments to loss in BW and reduction of the reproductive organs (Dunkley et al., 2007a; Landers et al., 2008a,b). Obviously, comparing these metabolic responses in a feed consumption-based molted hen versus a hen experiencing feed withdrawal could help to delineate some differences unique to feed withdrawal. However, when Dunkley et al. (2007a) compared serum metabolites collected from full-fed hens with hens molted either by feed withdrawal or fed a 100% alfalfa crumbles diet, calcium and total protein were generally always lower in the molted birds and uric acid was lower in all molted birds during the initial stages of molt. Although a general initial reduction in the uric acid concentration was observed in both the feed-withdrawal and alfalfa-fed hens, only uric acid concentrations of the feed withdrawal hens tended to remain lower throughout the trial. Dunkley et al. (2007a) concluded that the sustained low level of uric acid in feedwithdrawal hens could be due to the lack of a dietary protein source. Although alfalfa contains protein, hens initially did not accept the alfalfa diet as a feed source, which could account for the reduction in uric acid concentration observed at the beginning of the trial. The decrease in serum calcium in molted birds is a reflection of eliminating the calcium requirement for eggshell formation because egg production is stopped (Ricke et al., 2010). Calcium changes are critical to the health status of the hen; medullary bone in the laying hen serves as a labile source of calcium, and several studies have indicated that induced molt using feed removal significantly decreases bone quality in laying hens (Garlich et al., 1984; Park et al., 2003; Kim et al., 2005, 2006, 2007, 2008; Mazzuco and Hester, 2005a,b; Mazzuco et al., 2011). Consequently, enhancing bone mineral deposits initiation of egg production and after molting to not only improve eggshell quality but also avoid potential bone weakness issues should be a future goal, but more methodology development will be needed to understand fundamental bone metabolism (Park et al., 2004c; Kim et al., 2005, 2012). Immune responses including changes in H:L ratios and increases in acute phase proteins such as α1-acid glycoprotein (AGP) have been monitored as possible indicators of physiological stress in laying hens (Wolford and Ringer, 1962; Kogut et al., 1999; Holt and Gast, 2002; Mumma et al., 2006; Dunkley et al., 2007b;

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Mazzuco et al., 2011). Dunkley et al. (2007b) compared a series of immunological responses of hens either infected or not infected with Salmonella Enteritidis and either full fed or molted either by feed withdrawal or by feeding a 100% alfalfa crumbles diet. In general, bile IgA levels were greater in hens infected with Salmonella Enteritidis versus those not infected except for one group of feed withdrawal birds, whereas cecal IgA was greater only in the infected feed withdrawal birds except for one uninfected feed withdrawal group where higher levels were detected. The H:L ratios increased in the feed-withdrawal birds by d 9 of the molt, and feed withdrawal birds in general exhibited higher AGP than their counterparts by the later stages (d 9 and 12). Dunkley et al. (2007a) concluded that alfalfa crumbles fed as a molt diet appeared to reduce stress and inflammation based on the H:L ratios and AGP levels in the serum, when compared with feed withdrawal-­ molted hens. The immune response was delineated further when McReynolds et al. (2009) compared heterophil function by quantitating total and differential peripheral blood leukocyte as well as measuring the production of an oxidative burst and cellular degranulation. They observed that the alfalfa numerically increased heterophil functions over a 12-d period compared with nonfed control birds whereas heterophil functions declined in the control birds. Based on this, McReynolds et al. (2009) suggested that one of the roles for a molt diet may actually be to help the hen retain a more functional innate immune system that enables it to resist pathogen infection.

Alfalfa and In Vitro and In Vivo Microflora Responses Because alfalfa possesses a high fiber content, it could serve as a molt dietary source for GIT microbial fermentation that would potentially limit establishment of pathogens such as Salmonella Enteritidis (Vispo and Karasov, 1997; Ricke, 2003a). Retaining actively fermenting microflora during the molting period in turn should limit Salmonella Enteritidis colonization and persistence in the laying hen GIT (Ricke, 2003a). Dehydrated alfalfa is one of several feedstuffs that can reside longer than 24 h in the digestive tract of chickens (Sibbald, 1979a) and therefore would appear to have sufficient residence time for maximizing fermentation by avian GIT. Initial work by Saengkerdsub et al. (2006) demonstrated that using alfalfa as an in vitro substrate for laying hen cecal microflora supported substantial methane production, which is an indicator of a fairly complete anaerobic GIT ecosystem (Ricke et al., 2004c; Saengkerdsub et al., 2007a,b). In a survey of a multitude of potential dietary fiber sources, Dunkley et al. (2007c) compared in vitro fermentability properties of these high-fiber feed sources for molting including alfalfa using laying hen cecal microflora as the inocula. Al-

falfa and alfalfa-corn mixtures and soybean hulls were generally more fermentable followed by wheat middlings, with all feed substrates consistently producing considerably higher concentrations of acetic acid than propionic or butyric acid. Donalson et al. (2007, 2008a) conducted in vitro laying hen cecal microflora incubation studies and observed that alfalfa and alfalfa-layer rations combinations with prebiotic fructooligosaccahrides (FOS) were capable of limiting in vitro growth of Salmonella Typhimurium in the presence of fermenting microorganisms after 24 h of incubation before Salmonella Typhimurium was innoculated. When cecal fermentation was examined in in vivo trials, the relationship between alfalfa and GIT SCFA is less clear. Early work by Woodward et al. (2005) with 100% alfalfa meal did not for the most part increase cecal SCFA compared with feed withdrawal; SCFA concentrations from both treatments were consistently lower than the full-fed birds. This was attributed to low intake perhaps due to the saponin content, and when alfalfa was mixed with 10 or 30% layer ration, feed intake increased accordingly (Donalson et al., 2005; Woodward et al., 2005; McReynolds et al., 2006). Even though Donalson et al. (2005) did not collect cecal SCFA data, based on the generally much higher SCFA levels observed by Woodward et al. (2005) on hens fed 100% layer ration, it would be assumed that SCFA would have increased as more layer ration was apportioned into the alfalfa diet. When Donalson et al. (2008b) included the prebiotic FOS in a 90% alfalfa:10% layer ration, feed intakes were similar to those reported by Woodward et al. (2005), but there still was limited differentiation in cecal SCFA production between the feed withdrawal and alfalfa-molted birds. However, when Dunkley et al. (2007e) fed 100% alfalfa crumbles, daily feed intakes were similar to what had been reported earlier by Donalson et al. (2005, 2008b) and Woodward et al. (2005), but cecal total SCFA and acetate and propionate were consistently higher than the feed-withdrawal birds and were in some trials similar to the levels detected in full-fed nonmolted birds. As indicated earlier, physical form of a feed material can make a difference in terms of GIT microenvironment (Mikkelsen et al., 2004; Huang et al., 2006). Consequently, alfalfa in the form of a crumble may have been more evenly dispersed in the lumen, leading to more contact with fermentative microorganisms or this form may have been more consistently consumed on a daily basis leading to a more constant bulk fill in the hen ceca, or both. In vivo marker-based passage rate studies similar to the ones attempted by Dunkley et al. (2008c), but with compounds that would attach to feed particles more optimally, might be helpful to resolve this question. The composition of the cecal microorganisms is also shifted during molt. Initial work demonstrated that denatured gradient gel electrophoresis could be used to distinguish cecal microbial populations from hens fed

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different molting diets (Hume et al., 2003; Ricke et al., 2004a). Dunkley et al. (2007c) used this approach to demonstrate that cecal populations incubated in vitro with various feed and fiber sources could be grouped by banding patterns and differentiated from each other. When Dunkley et al. (2007e) applied denatured gradient gel electrophoresis analysis to in vivo studies, they were able to demonstrate that over the 12-d molt period microbial fecal populations among feed withdrawal, full-fed, and alfalfa-crumble-fed birds, as expected, were all similar in the beginning, but the feed-withdrawal and alfalfa-fed hen fecal populations began to shift and diverge more and more from each other as the molt period progressed. On the last day when respective cecal samples could be compared, full fed and alfalfa cecal populations were more than 90% similar. In a follow-up study, Callaway et al. (2009) was able to further delineate these population shifts among full-fed, alfalfa, and feed-withdrawal molted hens using bacterial tag-encoded FLX amplicon pyrosequencing. Sequencing analysis revealed that the number of microbial genera was the greatest in the alfalfa-crumble-fed birds and the feed-withdrawal molted birds displayed the most limited microbial diversity and were the only group inhabited by Salmonella but not Lactobacillus.

Alfalfa Molt Diets and Salmonella Enteritidis Infection As noted in the previous section, early attempts to molt with 100% alfalfa meal were effective at inducing molt (Landers et al., 2005b) but were less reliable for generating a consistent cecal fermentation response (Woodward et al., 2005). When Woodward et al. (2005) used 100% alfalfa meal to induce molting in hens more than 50 wk old, they observed that in 3 of the 4 trials, there was a significant decrease in cecal Salmonella Enteritidis colonization between the alfalfa diet and the feed withdrawal molted hens. Crop colonization was relatively limited for all birds, with no detectable Salmonella Enteritidis recovered from any of the full fed control birds and intermittent recovery from the molted birds. This may be reflective of the consistent crop pH level of around mid-4 for the control birds versus a crop pH over 5 for the most of the molted birds. Previously, Durant et al. (1999a) demonstrated that the rise in crop pH from 4 to 5 may be a contributing factor in Salmonella Enteritidis establishment in the crop of feed withdrawal-molted hens. In most of the 4 trials, there was a significant reduction in Salmonella Enteritidis infected organs (liver, ovary, and spleen) in birds fed the alfalfa diet compared with birds undergoing feed-deprived molt. In an effort to improve consistency several modifications of alfalfa and combinations with other compounds were examined. McReynolds et al. (2005) combined a 100% alfalfa molt diet with drinking water containing an experimental chlorate product that had been shown

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to reduce enteric pathogens in a variety of animals (Anderson et al., 2009). In the crop and organs, only the chlorate alone or in combination with alfalfa was effective in significantly reducing Salmonella Enteritidis compared with nonfed birds, whereas in the ceca alfalfa alone was not effective but the combination of chlorate and alfalfa were more effective than the chlorate alone. Although it was not measured, McReynolds et al. (2005) suggested that lowered feed intake of alfalfa may have accounted for it being less effective in limiting Salmonella Enteritidis. The fact that lactic acid concentrations in the crop and ceca were not different than the nonfed birds would tend to support this premise even though the alfalfa-chlorate combination was always the highest and almost doubled over the cecal lactic acid concentrations of birds receiving chlorate alone. As discussed earlier, Donalson et al. (2008b) had shown that prebiotic FOS when combined with a 90% alfalfa:10% layer ration only achieved limited differentiation in cecal SCFA production between the feed withdrawal and alfalfa molted birds in the 4 trials that these metabolites were measured. In this same study the Salmonella Enteritidis levels (positive birds and population enumerations) in the crop, ceca, and organs (liver, ovary, and spleen) were with few exceptions higher in the feed withdrawal birds than the full-fed (layer ration) birds, but the alfalfa and alfalfa-FOS molted birds tended to be intermediate depending on the individual trials with limited impact of FOS addition. However, Salmonella Enteritidis excretion patterns over 28 d with some exceptions declined fairly rapidly and remained low. Donalson et al. (2008b) speculated that the lack of impact by FOS may be a combination of avian GIT microflora not capable of directly utilizing FOS, preference for other substrates, or low intake of FOS. McReynolds et al. (2006) examined combinations of alfalfa and layer ration similar to the ratios that Donalson et al. (2005) had previously optimized as molt diets for egg production for the ability to limit Salmonella Entertidis colonization and infection. Combining alfalfa with layer ration in either 90% (alfalfa):10% (layer ration) or 70:30 ratios achieved ovary reduction almost identical to feed withdrawal molted birds but with less BW loss and 3-fold (90:10 ratio) and 6-fold (70:30) higher feed intake than birds fed 100% alfalfa. The feed intake of the birds fed 70:30 was almost one-third of the full-fed nonmolted birds. Although fermentation metabolites were not measured in the ceca and crops of these birds, the reductions in Salmonella Enteritidis in the crop, ceca, and organs were fairly consistent and for the majority of the time resulted in levels of Salmonella Enteritidis counts and positive birds as low as those seen in the full-fed nonmolted birds. Dunkley et al. (2007d) achieved a 2- to 4-fold reduction in Salmonella Enteritidis colonization in the ceca of birds fed 100% alfalfa crumbles as a molt diet and the levels were fairly similar to the low numbers of Sal-

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monella Enteritidis recovered from full fed nonmolted birds. Dunkley et al. (2007d) was also able to confirm using real-time PCR that the cecal feed withdrawal microenvironment greatly enhanced expression of hilA, the key virulence regulatory gene of Salmonella, whereas expression was suppressed in full-fed birds and for the most part in alfalfa-crumble-fed birds. The levels of hilA detected in the ceca by Dunkley et al. (2007d), as was seen earlier in the work by Durant et al. (1999a) with the crop, appear to correspond to the level of organ invasion. This suggests that virulence in Salmonella Enteritidis is modulated by the avian crop and ceca respective environments can influence not only the ability of this pathogen to colonize but also the extent of systemic invasion into the internal organs of the susceptible bird.

CONCLUSIONS From 1996 to 1999, Salmonella Enteritidis illness rates declined 48%, and from 1996 to 2000 the incidence per 100,000 population decreased from 2.5 to 1.8 (USDHHS-CDC 2000, 2003). It is believed that incorporation of a variety of control measures from farm to table including several specific management intervention strategies on the farm (purchasing replacement chicks from Salmonella Enteritidis-free breeder flocks, switching to more Salmonella Enteritidis-resistant laying hen breeds, use of Salmonella Enteritidis vaccines, or a combination of these) helped to bring about this decrease (Patrick et al., 2004). Unfortunately, based on more recent epidemiological reports, this previously reported dramatic decline in Salmonella Enteritidis incidence was eliminated by a renewed upsurge in Salmonella Enteritidis infections (USDHHS-CDC 2003). This made it much more difficult to achieve the President’s Council of Food Safety plan to reduce egg-associated Salmonella Enteritidis by half in 2005 and complete elimination by 2010 (President’s Council on Food Safety, 1999; Patrick et al., 2004). Instead, renewed efforts were needed to develop additional intervention technologies to limit Salmonella Enteritidis colonization in laying flocks. Given the connections made in the mid to late 1990s between feed withdrawal-based molting regimens and the ability of Salmonella Enteritidis to proliferate in these environments, alternative methods of inducing a molt that involved some attempt to continue to provide birds with a feed source during the molting process became the focus of molting research, and numerous strategies were developed and examined in the early 2000s. Although some of the early attempts at alternative diets that involved nutrient restriction or dietary additives were successful in inducing molts, they often were expensive, hard to adapt, or just simply impractical. By the mid-2000s approaches involving high-fiber, low-energy diets such as cereal grain processing byproducts, fruit and plant byproducts, and plant structural

polysaccharides were screened for their abilities to be effective in inducing a molt. In addition to successfully inducing a molt, criteria for evaluation included egg production responses and economics. Several proved to successfully meet these criteria, and several are now commercially used and others are being developed (Willis et al., 2008; Patwardhan et al., 2011). One of the most extensively studied fiber-based molt diets was alfalfa. Alfalfa was considered a potential candidate because it was high in crude fiber and CP and low in ME. It was shown early on to effectively induce a molt in laying hens and generate a postmolt performance that was comparable with the postmolt performance of feed-deprived hens and also reduce the incidence of Salmonella Enteritidis colonization in the GIT of hens during an induced molt. Further evaluation of physiological, immunological, and behavioral response of molting hens on high-fiber, low-energy diets was conducted to validate and optimize alfalfa combinations that were consistent in most facets of an optimal molt procedure. Although options for optimizing the alfalfa-based diets were identified, additional refinement is needed to improve consistency. However, despite the success of these research activities, Salmonella Enteritidis is still problematic as eggassociated outbreaks continue to occur (CDC, 2010a). To counter this, multiple interventions throughout the egg production cycle and at the retail level have been suggested to further reduce risk (Schroeder et al., 2006; Golden et al., 2008). However, there is more to be done with molt diets and the laying hen as well. The work with alfalfa demonstrated that preventing Salmonella Enteritidis colonization and invasion is much more complicated than simply retaining an actively fermenting GIT tract and its resident microflora to make the GIT more hostile to the pathogen. Numerous host factors are in play as well, including not only the immune system but also the microenvironment of the intestinal lining as well as general physiological state of the laying hen. Likewise, the Salmonella colonization process may be more complicated than was originally thought. For example, there is now evidence that acute inflammation may in fact impart competitive advantages to Salmonella to overcome indigenous GIT microflora by generating metabolites from the inflammation process that allow it to outgrow the anaerobic indigenous GIT microflora (Santos et al., 2009; Winter et al., 2010). How this may factor into the interaction between Salmonella Enteritidis and the laying hen GIT microflora is not known. Finally, dietary fiber sources can no longer be perceived as passive bulking agents but must be viewed as active entities that can directly interact with host functions such as the immune system. Fortunately, more analytical tools are in hand to begin to delineate some of these complicated and interactive host-GITpathogen factors including high-throughput sequencing to better define the GIT microbiome, metabolomics to sort out the metabolite reactions occurring in the GIT

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lumen and tissue surfaces, and gene expression profiling for elucidating animal host functional genes as well as microbial and pathogen functions (Cogburn et al., 2003; van der Werf et al., 2005; Sirsat et al., 2010; Kwon and Ricke, 2011). As these approaches come into play, specific targets can be identified for designing more improved molt diets as well as more targeted interventions at all stages of egg production.

ACKNOWLEDGMENTS This review was partially supported by Hatch grant H8311 administered by the Texas Agricultural Experiment Station (Texas A&M University, College Station), USDA-National Research Initiative grant # 2002-0614, US Poultry and Egg Association grant #485, a USDA Food Safety Consortium Grant, and USDA-National Integrated Food Safety Initiative Grant #2008-5111004339.

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