SALMONELLA IN CARCASS BONE MARROW AND NECK SKIN: POTENTIAL

SOURCES FOR GROUND CONTAMINATION

by

DIEZHANG WU

(Under the Direction of Walid Q. Alali)

ABSTRACT

Possible routes for contamination of ground chicken are through grinding chicken parts containing contaminated neck skin and bone marrow internalized with Salmonella. The objective of this study was to determine

Salmonella prevalence and serotype distribution of broiler bone marrow and neck skin samples. A total of 300 drumstick bone marrow samples, 299 neck skin samples, and bootsock samples from 26 broiler houses were tested according to the

USDA-FSIS standard protocols. Salmonella prevalence of bone marrow, neck skin, and broiler houses were 0.8%, 21.4% and 80.1%, respectively. Salmonella prevalence of rinsed skin samples (2.3%) and stomached skin samples (20.7%) were significantly different (p<0.05). Six Salmonella serotypes including S.

Kentucky, Typhimurium, Enteritidis, Agona, Ouakam, and Liverpool were identified.

Overall, Salmonella Kentucky was the most frequently isolated serotype. To

conclude, contaminated neck skin can contribute to ground chicken contamination; whereas the contribution of internalized drumstick bones is expected to be much lower.

INDEX WORDS: Salmonella, broiler carcass, bone marrow, neck skin, ground

chicken contamination

v

SALMONELLA IN BROILER CARCASS BONE MARROW AND NECK SKIN: POTENTIAL

SOURCES FOR GROUND CHICKEN CONTAMINATION

by

DIEZHANG WU

B.S., Purdue University, West Lafayette, 2011

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2013

i

© 2013

Diezhang Wu

All Rights Reserve

ii

SALMONELLA IN BROILER CARCASS BONE MARROW AND NECK SKIN: POTENTIAL

SOURCES FOR GROUND CHICKEN CONTAMINATION

by

DIEZHANG WU

Major Professor: Walid Q. Alali

Committee: Mark A. Harrison

Charles L. Hofacre

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia December 2013

iii

ACKNOWLEDGEMENTS

I would like to express my greatest gratitude to my major professor, Dr.

Walid Q. Alali, for his direction, comments, remarks and engagement through out my graduate study at the University of Georgia. I would also like to thank Dr. Mark A.

Harrison and Dr. Charles L. Hofacre for being on my committee, and offering suggestions and great support for finishing my study.

Thanks to Daniel Lefever, Da’Shundria Davis, Cagatay Celik, who accompanied my visits to the farm and plant, and helped me finish the project.

Special thanks to Hao Zhang, for being supportive and patient.

In the end, I would like to give the utmost appreciation to my parents,

Yuefeng Wu and Xiaoqing Wu for their unconditional love and support. Thank you for teaching me to work hard and never stop learning. I love you!

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iv

LIST OF TABLES ...... vii

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ...... 1

Salmonellosis and Products ……………...... 1

Salmonella Prevalence at Farm Level ...... 3

Salmonella Prevalence at Processing Plants ...... 6

Controlling Strategies at Farms ...... 10

Controlling Strategies at Processing Plants ...... 17

Conclusions ...... 23

References ...... 25

2 SALMONELLA IN BROILER CARCASS BONE MARROW AND NECK SKIN:

POTENTIAL SOURCES FOR GROUND CHICKEN CONTAMINATION ...... 43

Abstract ...... 44

Introduction ...... 45

v

Materials and Methods ...... 47

Results ...... 52

Discussion ...... 53

References ...... 58

3 CONCLUSIONS ...... 62

vi

LIST OF TABLES

Page

Table 1 Salmonella prevalence in broiler skin and bone samples ...... 63

Table 2 Salmonella serogroup isolated from broiler farm, skin and bone samples ...64

Table 3 Salmonella serotype isolated from broiler farm, skin and bone samples ..…65

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CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Salmonellosis and Poultry Products

Salmonella is a genus of gram-negative, rod shaped bacteria discovered by

American scientist Daniel Salmon. The genus is divided into two species, Salmonella enterica and Salmonella bongoria (CDC, 2011). The Salmonella genus is found to contain over 3,000 serotypes. Pathogenic Salmonella serotypes mostly belong to

Salmonella enterica subsp. enterica.

Salmonellosis is the infection caused by Salmonella, in which patients usually develop diarrhea, fever, and abdominal cramps 12 to 72 hours post infection (CDC,

2010). In severe cases, Salmonella cells may spread into the blood stream, invade other organs, and lead to chronic arthritis or even death. Young children, elderly, and people with a compromised immune system are more susceptible to severe

Salmonella infection (CDC, 2010). Salmonella is estimated to cause about 1.03 million foodborne illnesses including about 19,500 hospitalizations and 378 deaths in the U.S. every year (Scallan et al., 2011). According to the 2011 Foodborne

Disease Active Surveillance Network (FoodNet) surveillance report, a total of 7,813 lab-confirmed Salmonella cases were reported, among which S. Enteritidis (1,424

[18.2%]) was the most common serotype identified, followed by S. Typhimurium

(981 [12.6%]), S. Newport (959 [12.3%]), S. Javiana (753 [9.6%]), and S. I 4,[5],

12:i:- (314 [4.0%]) (CDC, 2012).

1

Poultry products are the primary sources of human Salmonella infections. In

2011, USDA-FSIS reported that the Salmonella prevalence in post-chill broiler carcasses and ground chicken were 6.5%, and 30.9%, respectively (USDA-FSIS,

2012a). The most common serotype isolated from broiler carcasses and ground chicken was S. Kentucky, and the prevalence of this serotype had an ascending trend from 36.8% in 2008 to 45.4% in 2010 on carcasses, and from 28.6% in

2008 to 35.0% in 2010 in ground chicken (USDA-FSIS, 2011a). Although S. Kentucky is frequently isolated from chicken carcass and ground chicken, it was reported that this serotype was over 100 times less likely to cause human illness compared to S.

Typhimurium, Enteritidis, and Newport (Sarwari et al., 2001).

The annual number of foodborne illness caused by major pathogens is estimated to be 9.4 million in the U.S. (Scallan et al., 2011). According to the food attribution model using foodborne outbreak data from 1988 to 2008, poultry meat was associated with 900,000 (10%) foodborne illnesses, following leafy vegetables

(2.2 million [22%]), dairy (1.3 million [14%]), and fruits-nuts (1.1 million [12%])

(Painter et al., 2013). With regard to Salmonella and poultry meat, Painter et al.

(2013) reported that 10-29% (approximately 103,000 to 298,000 illnesses) of all salmonellosis was associated with poultry meat. Furthermore, of the 278 deaths attributed to poultry between 1998-2008, Salmonella caused about 73 cases (26%)

(Painter et al., 2013).

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Salmonella Prevalence at Farm Level

The broiler industry in the U.S. is described as vertically integrated production system. The poultry company known as the “integrator”, provides chicks, feed, veterinary and field personnel service, makes decisions about frequency of flock rotation, and usually gives house and equipment specifications

(MacDonald, 2008; Vukina, 2001). The farmers who contract with a poultry company are responsible for land and housing facilities, utilities including water and electricity, operating expenses, and for growing the chicks to a required weight

(MacDonald, 2008; Vukina, 2001). Since the U.S. broiler industry is entirely vertically integrated from breeding and to feed mills, and then to transportation and processing (Vukina, 2001), both vertical and horizontal

Salmonella transmission are the routes responsible for the contamination of the final products. Vertical transmission of Salmonella occurs when pathogens disseminate from breeder flocks to broilers. Several studies found that: 1)

Salmonella was able to colonize the laying hen reproductive system (Gast et al.,

2004; Keller et al., 1995; Okamura et al., 2001), and 2) contaminated eggs could be produced by challenging laying hens with Salmonella orally, intravenously, intracloacally and intravaginally (Gast et al., 2004; Gast et al., 2002, 2003; Gast et al.,

2013; Miyamoto et al., 1997; Shivaprasad et al., 1990). Reiber et al. (1995) examined rooster semen and found that the most frequently isolated bacterial genera included

Escherichia, Staphylococcus, Micrococcus, Enterococcus, and Salmonella, which indicates that the aforementioned bacteria are endemic to poultry and may contaminate fertilized eggs. In the study conducted by Liljebjelke and colleagues, the

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authors revealed that the S. Typhimurium and S. Enteritidis strains isolated from one company’s breeder farms and the broiler houses had indistinguishable Pulsed

Field Gel Electrophoresis (PFGE) patterns (Liljebjelke et al., 2005). In addition,

Bhatia and McNabb (1980) reported that Salmonella serovars present in fluff and meconium samples from correlated with this those serovars present in litter samples from the broiler houses and carcass samples from the processing plant.

The spread of Salmonella within the same flock or between flocks is called horizontal transmission. Salmonella could be transmitted through direct or indirect contact. For example, Salmonella present in the breeder nest box, farm cold room, hatchery truck, or hatchery environment could contaminate broiler egg surfaces, which may enter eggs through cracks or pores (Cox et al., 2000). According to Cason et al. (1994), contaminated fertilized eggs were still able to hatch and Salmonella was able to colonize the digestive tract of the newly hatched chicks. Moreover, Cox et al. (1996) revealed that one-day-old chicks became Salmonella positive following inoculation of 50 S. Typhimurium cells in each eye indicating the possibility of

Salmonella contamination through dust particles in the air. Insects and rodents are regarded as important vectors facilitating Salmonella transmission to breeder and broiler birds. Salmonella spp. was isolated from flies at a relatively high frequency

(18.7%, n=150) at hatchery house (Bailey et al., 2001). Rodents were also found to shed S. Enteritidis and S. Infantis and were able to infect both breeders and broilers flocks (Henzler and Opitz, 1992; Umali et al., 2012).

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Several factors contribute to the prevalence of Salmonella at poultry farms, including geographic region, season, surrounding environmental condition, litter condition, flock disease history, rodent control, on-farm sanitary performance, feed and water management, biosecurity, facility maintenance, sampling method and types of samples collected (Berghaus et al., 2012). For instance, it was reported that litter with high water activity (Aw) was significantly associated with Salmonella positive flocks (Carr et al., 1995; Opara et al., 1992). Thakur et al. (2013) found that

S. Typhimurium isolated from broiler fecal samples had100% PFGE indistinguishable fingerprint patterns with isolates from outdoor environmental sources as well as isolates from other broiler houses.

The presence of Salmonella at the farm level was evaluated in a number of studies by determining its prevalence in breeder houses, hatcheries, and broiler farms (Bailey et al., 2001; Byrd et al., 1999; Liljebjelke et al., 2005). In a study that investigated Salmonella prevalence in , Alabama, Georgia, and North

Carolina, authors revealed that 88% of the 49 breeder farms sampled were

Salmonella positive, indicating the presence of Salmonella in breeder houses was ubiquitous (Berghaus et al., 2012). According to a study conducted by Byrd and colleagues from July 1995 and May 1996 in Texas, authors found that S. Heidelberg and S. Kentucky made up 50% of the Salmonella isolates (n=30) from 5 hatcheries and 59.6% of the Salmonella isolates (n=94) from 13 broiler houses (Byrd et al.,

1999).

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Salmonella Prevalence at Processing Plants

In 2012, USDA-FSIS implemented a new Salmonella performance standard for broiler carcass (USDA-FSIS, 2012b). The performance standard for young broilers is no more than 5 Salmonella positive carcasses out of 51 consecutive samples. Broiler carcasses are rinsed with buffered peptone water

(BPW) by USDA-FSIS employees and a 30 ml portion is then tested for the presence of Salmonella (USDA-FSIS, 2012c). For ground chicken, the new Salmonella performance standard is no more than 13 positives out of 26 (325 g) samples.

According to USDA-FSIS 2011 progress report on Salmonella and Campylobacter testing of raw meat and poultry products, Salmonella prevalence on broiler carcass and ground chicken in 2011 was 6.5% (n=4,744) and 30.9% (n=466) respectively

(USDA-FSIS, 2012a).

In 2010, the top five most common Salmonella serotypes on chicken meat

(n=458) during processing were S. Kentucky (45.4%), S. Enteritidis (27.1%), S.

Typhimurium (9.0%), S. Heidelberg (3.5%), and S. 4,5,12:i:- (2.2%). Among

Salmonella isolated (n=80) from ground chicken, S. Kentucky (35%) and S.

Enteritidis (30%) were the top two most prevalent serotypes, followed by S.

Heidelberg (10%), S. Typhimurium (7.5%), and S. 4, 12:i:- (3.8%) (USDA-FSIS,

2011a). According to the FDA 2011 National Antimicrobial Resistance Monitoring

System (NARMS) Retail Meat Report, 12% of 1,320 of retail chicken part samples

(breast, wing, or thigh) were Salmonella-positive (FDA, 2013). Among the

Salmonella isolated (n=158), S. Typhimurium (41.8%), S. Kentucky (28.5%) and S.

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Enteritidis (13.3%) were the three most frequently isolated serovars from retail chicken samples.

Skin and Neck Skin

During slaughter and processing, broiler carcasses (outside skin surface) may become contaminated with Salmonella from a number of sources such as fecal material from the incoming birds, cross contamination from processing equipment, and crop and intestinal rupture. During scalding of the birds, feather follicles are loosened to allow defeathering at the pickers. Salmonella may enter these follicles and become entrapped especially after carcass exposure to chilling water, which cause the follicles to tighten up. Neck skin is used in ground chicken production as a source of fat. As broilers are hung upside down during processing, neck skin is exposed to the wash fluids drained from the whole carcass. When neck skin harbors

Salmonella, it can be a source of ground chicken contamination. Kim et al. (1996) and coworkers successfully used confocal scanning laser microscopy (CSLM) to image Salmonella cells inside chicken feather follicles even though the skin surface was thoroughly rinsed. Carcass rinse and neck excision are two widely accepted standard methods for testing Salmonella on broiler carcass. As mentioned earlier, the USDA-FSIS requires that whole broiler carcasses are rinsed with 400 ml BPW, and to have 30 ml of the aliquot tested for Salmonella (USDA-FSIS, 2012c). In

Europe, 15 neck skins from 15 broilers (about 8.3 g per carcass) are pooled into 5 samples (3 neck composite skin samples of around 25 g each) for the analysis of

Salmonella (Commision, 2005). The two methods yielded non-significantly different

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Salmonella prevalence results when used on samples received from the same processing plant (Cox et al., 2010).

Internalization of Salmonella in Internal Organs Including Bone Marrow

There is limited research focused on the distribution of Salmonella spp. in the broiler bird’s internal organs. One reason for this could be that systemic Salmonella infection usually shows no clinical symptoms, and no effect on broiler body weight and breeder egg production.

Cox and other authors examined spleens, ceca, livers and gallbladders from two market-aged flocks, and found Salmonella in 48, 65, and 51% of the spleens, ceca, and livers and gallbladders, respectively (Cox et al., 2007a). Eight Salmonella serotypes were isolated, including S. Typhimurium, Muenster, Kentucky,

Montevideo, Thompson, London, Berta, and Schwarzengrund (Cox et al., 2007a). I another study, S. Enteritidis was recovered from experimentally inoculated (108 to

109 CFU/ml) layer birds’ spleen, liver, heart, gallbladder tissue, ovary and oviduct tissues (Gast et al., 2002; Keller et al., 1995). He et al. (2010) found that the spleen was the first organ showing positive Salmonella results at 12 h post oral-inoculation, followed by blood (14 h), liver and heart (16 h), pancreas (20 h), and kidney and gallbladder (22 h). Gast et al. (2011) and his coworkers exposed laying hens to different oral doses of S. Enteritidis to study internal organ colonization with this pathogen. The authors revealed that the recovery percentages for 108 CFU and 104

CFU/ml of oral S. Enteritidis inocula from the liver at 5 days post infection were 40% and 0%, respectively.

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Beside S. Enteritidis, other Salmonella serovars were also able to colonize internal chicken organs. Snoeyenbos et al. (1969) detected S. Heidelberg,

Typhimurium and Enteritidis from naturally infected chicken’s ovary and peritoneum. Keller et al. (1997) reported that S. Enteritidis and Typhimurium shared an equal ability to colonize the tissue of the reproductive tract and forming eggs in the oviduct when orally inoculated.

The presence and colonization of Salmonella into poultry bone marrow has been rarely studied. Velaudapillai (1964) tested lower leg bone marrow from 1000 fowls for Salmonella spp. Eight bones were Salmonella positive and the serotypes identified were S. Paratyphi B, Stanley, Newport, Gallinarum, and Waycross. Due to the lack of information provided involving the methodology of bone marrow testing in Velaudapillai’s study, it is not possible to determine if Salmonella positive samples were due to cross-contamination during bone extraction from the chicken lower legs. In a recent published experimental study by Kassem et al. (2012), broiler chicken bone marrow was found to harbor S. Enteritidis after oral inoculation with

105 CFU/ml. The authors also found that although the colonization level of S.

Enteritidis in bone marrow decreased over time, the presence of S. Enteritidis in bone marrow persisted until slaughter (Kassem et al., 2012).

In addition to poultry, Salmonella can also colonize the bone marrow of other animals such as dog, swine, horse and mice (Castro-Eguiluz et al., 2009;

Kondratenko and Tkach, 1975; Salehi et al., 2012; Zagaevskii, 1978). When

Salmonella Typhimurium was introduced into a dog’s gall bladder, this pathogen colonized the gall bladder and bone marrow (Kondratenko and Tkach, 1975).

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Zagaevskii (1978) reported that Salmonella were detected in both bone meal and feces of swine. After the withdrawal of the bone meal, the Salmonella excretion ceased. In addition, when the infected swine were slaughtered, Salmonella was present in the internal organs which included the kidneys, livers, gall bladders, spleens and bone marrow (Zagaevskii, 1978). Salmonella serogroup B was considered as the causative agent of typhoid disease and salmonellosis in horses.

Salmonella Typhimurium was isolated from the bone marrow, mensenteric lymph nodes, and intestinal contents from infected Caspian ponies (Salehi et al., 2012). In another study looking into the mechanism of the survival of Salmonella inside mice bone marrow, B cells were the targets for Salmonella infection and survival (Castro-

Eguiluz et al., 2009).

Controlling Strategies at Farms

Hatcheries

Contaminated egg surfaces could lead to Salmonella colonization in the broilers, thus disinfecting the egg surface is critical in controlling Salmonella vertical transmission. The most common approach to eliminate Salmonella from hatcheries is to disinfect the surfaces in the incubator, hatching cabinets and the hatching eggs

(Russell,2010). A number of interventions such as UV light, electrostatic space charge system, electrostatic spraying system, ozone and pulsed light were reported to induce 4 to 5 log reduction in Salmonella populations inoculated on egg surface

(Braun et al., 2011; Coufal et al., 2003; Lasagabaster et al., 2011; Mitchell et al.,

2002; Russell, 2003). Cox et al. (2007b) evaluated the bactericidal effect of seven

10

chemicals on hatching eggs inoculated with S. Typhimurium. The authors reported that quaternary ammonium compound, peroxyacetic acid and polyhexamethylenebiguanide hydrochloride (PHMB) kill 93.5 -100% of surface

Salmonella, whereas hydrogen peroxide, which has been used by the poultry industry, had a 70% reduction.

Water and Feed

Organic acids

Organic acids have been administrated into many matrices in poultry farms including feed, drinking water, and broiler litter. Although medium chain fatty acids showed greater antibacterial activity against Salmonella (Van Immerseel et al.,

2006), the most widely used organic acids in large scale studies are short chain fatty acids (SCFA) such as formic acid, acetic acid, butyric acid, propionic acid, citric acid and lactic acid (Byrd et al., 2001; Russell). In a study conducted by Byrd and coworkers, acetic, lactic, and formic acid were added to broiler drinking water during pre-slaughter feed withdrawal at low concentration (0.44-0.5%). The authors reported that the Salmonella prevalence in crop, number of Salmonella cells recovered from crop and prevalence on pre-chill carcass rinses were significantly reduced compared with the untreated control group (Byrd et al., 2001). Humphrey and Lanning (1988) conducted a large-scale study to evaluate the effect of formic acid added to breeder feed. The authors revealed that feed supplemented with 0.5% formic acid significantly reduced the Salmonella prevalence in breeder feed, breeder litter and newly hatched chicks.

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The mechanism of antimicrobial activity of organic acid is still not fully understood although several models have been established. One hypothesis is that organic acids pass through the cell membrane, dissociate and then acidify the cell cytoplasm (Kashket, 1987). Another hypothesis is that the fermentation product of the organic acids accumulate in the cell which causes not only the osmotic pressure experienced by the cell to increase, but has a secondary effect of interfering with normal cell metabolism (Flythe and Russell, 2006; Van Immerseel et al., 2006).

However, both of these two models lack enough data to completely explain the bactericidal effect of organic acids, because other factors including chain length, side chain composition, pKa values and hydrophobicity should be taken into consideration (Van Immerseel et al., 2006).

Dietary supplements

Antibiotics, probiotics and competitive exclusion (CE) products, prebiotics, and synbiotics are widely used dietary supplementations to control Salmonella in breeder and broiler flocks.

Antibiotics are used not only as a medication for bacterial infection, but also as a growth promoter mainly by reducing the number of normal microbiota to increase nutrient utilization by the animal (Gaskins et al., 2002; Vandeplas et al.,

2010). The sub-therapeutic use of antibiotics has been banned in Europe, and is carefully used in U.S. poultry production due to the concern of the rising of antibiotic-resistant bacteria that might facilitate the spread and prolonged carriage of resistant Salmonella (Cox and Pavic, 2010; Hazards, 2004; Smith and Tucker,

1978).

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Probiotics are defined as “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” (Fuller, 1989).

Probiotics usually contain a single or multiple strains belonging to the genera

Lactobacillus, Enterococcus, Pediococcus, Bacillus, and sometimes fungi such as

Saccharomyces (Vandeplas et al., 2010). It was proposed that the probiotics could exclude pathogens in the host intestines by competing for receptor sites and essential nutrients, modulating the host immune system, and producing antimicrobial compounds including bacteriocins, VFAs (i.e., organic acids), and hydrogen peroxide (Doyle and Erickson, 2006; Ouwehand et al., 1999; Vandeplas et al., 2010). Competitive exclusion products (CE) are mixed anaerobic cultures derived from poultry ceca contents or intestinal wall screened for the absence of avian and human pathogens (Doyle and Erickson, 2006). The CE bacteria can help establish a gastrointestinal system competitive or antagonistic to opportunistic pathogens when introduced to birds at early age (Doyle and Erickson, 2006). The CE strains could be administrated in drinking water and feed slurries, sprayed on hatching eggs or on agar plates for the chicks to eat (Doyle and Erickson, 2006), and

CE has been shown to effectively inhibit Salmonella cecal colonization (Corrier et al.,

1995; Higgins et al., 2007; Hofacre et al., 2000). The efficacy of CE treatment could be reduced if Salmonella colonization has already taken place (Bailey et al., 1998).

A prebiotic is “a nondigestible good ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health” (Gibson and

Roberfroid, 1995). Non-digestible fermentable carbohydrates, including polyols

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(sugar alcohols), disaccharides, oligosaccharides, and polysaccharides, could be used as prebiotics (Šušković et al., 2001). Spring et al. (2000) reported that mannanoligosaccharides could block the attachment of Salmonella to broiler gut mucosa by agglutinating with the pathogen. In addition, the fermentation of prebiotics by the normal gut microbes not only enhances its competitive exclusion activity against pathogens, but also adjusts the intestinal environment to be unfavorable for pathogens (i.e., lowering gut pH, producing SCFAs) (Šušković et al.,

2001).

Synbiotics refer to the combination of probiotic strains and prebiotic substrates, in which the prebiotics are selected for improving the growth of the probiotic strains (Šušković et al., 2001). Fructooligosaccharide (FOS) in conjunction with a bifidobacterial strain, or lactitol in conjunction with a Lactobacillus strain are examples of synbiotics (Collins and Gibson, 1999).

Vaccination

Both live-attenuated and killed vaccines have been developed for Salmonella in broiler production (Barrow, 2007). Live-attenuated vaccine consists of live

Salmonella strains with depleted metabolic function or virulence factor (Gamazo and Irache, 2007). When compared with killed vaccines, live-attenuated vaccines are more effective against Salmonella infection since they are easier to administer

(through drinking water or feed), and could induce humoral, mucosal and cellular immune response (Babu et al., 2004; Babu et al., 2003; Bailey et al., 2007; Gamazo and Irache, 2007). However, since live-attenuated strains could recover their virulence from the host or by acquiring genes from other microorganisms by

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horizontal gene transfer after they are released into the environment (Gamazo and

Irache, 2007), the safety of live-attenuated vaccines has been a concern (Barrow,

2007). Killed vaccines provide a broader range of protection by targeting multiple

Salmonella serotypes at the same time (Berghaus et al., 2011). Similar to live vaccines, the humoral and mucosal immune response triggered by killed vaccine could be passed on from breeders to progenies (Hassan and Curtiss, 1996; Inoue et al., 2008). Although the maternal antibodies are reduced over time (Hassan and

Curtiss, 1996; Inoue et al., 2008), it still offers good protection at the broilers’ early age and is important in Salmonella control at the epic of the production system. An experimental study conducted by Dórea and colleagues showed that the use of a live-attenuated Salmonella vaccine followed by a killed vaccine as a booster significantly lowered Salmonella prevalence in vaccinated hens, progeny broilers, broiler houses, and broilers entering the processing plant (Dórea et al., 2010).

Beside vaccine types, other factors such as the genetic origin of chickens and delivery method also influence the effectiveness of vaccines (Atterbury et al., 2010;

Kaiser et al., 1998) .

Litter

Poultry litter is the absorbent material used to line the floor of the poultry house, and may consist of wood shavings, peanut or rice hulls or other bedding material (Cox and Pavic, 2010). Studies have shown that litter could harbor

Salmonella and considered a significant risk factor in horizontal transmission of this pathogens to broilers (Kinde et al., 2005; Rodriguez et al., 2006; Thakur et al., 2013).

Fortifying litter with amendments (i.e., organic acids, sodium bisulfate) may help

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reduce Salmonella contamination (Ivanov, 2001; Pope and Cherry, 2000; Vicente et al., 2007). The reuse of litter is a standard practice in the U.S. (Macklin and Krehling,

2010), while it is banned in Europe (Union, 2007). The concern is that the moisture content and ammonia content of the litter will increase when used overtime, which could help facilitate the survival of Salmonella inside the litter (Carr et al., 1995;

Opara et al., 1992; Terzich et al., 2000). On the contrary, other researchers have found that Salmonella was not detected from reused litter by both culture method and molecular method (i.e., PCR) (Lu et al., 2003). Furthermore, it has been reported that newly hatched chicks raised on used litter had higher cecal volatile fatty acids (VFA) concentration and resistance toward Salmonella colonization compared with chicks raised on new litter (Corrier et al., 1992). Interestingly, in a 3- year longitudinal study evaluating Salmonella survival in litter reused for up to 14 consecutive flocks, the authors reported that Salmonella prevalence was significantly lower after the litter had been used for 6 consecutive flocks compared to…. (Roll et al., 2011). Hartel et al. (2000) suggested that stacking used litter for a reasonable time (> 8 days) was effective in eliminating fecal coliforms.

Pre-slaughter Feed Withdrawal

Pre-slaughter feed withdrawal is the process of terminating feeding the broilers several hours prior to shipping to the processing plant. Feed withdrawal is practiced to clear the bird’s intestinal tract from digesta and thus reduces fecal contamination on broiler carcasses (Doyle and Erickson, 2006). If the feed is removed too late (i.e., short feed withdrawal time), the broiler carcass will be contaminated by the digesta from a ruptured gut during processing (USDA-FSIS,

16

2010a). On the other hand, removing feed too early (i.e., long feed withdrawal time) will result in fragile internal organs, and the crop and ceca can easily tear during processing (USDA-FSIS, 2010b). Also, it has been demonstrated that compact (2- 8 h) and extended feed withdrawal (18 or 24 h) lead to higher Salmonella recovery from broiler crop (Barnhart et al., 1999; Corrier et al., 1999). An increased crop pH during feed withdrawal was associated with the incremental Salmonella recovery

(Hinton et al., 2000). However, Salmonella-positive percentage found in the ceca at the end of feed withdrawal period and at the post-chill had no significant difference from Salmonella-positive percentage in broiler ceca samples before feed withdrawal

(Corrier et al., 1999; Northcutt et al., 2003).

Controlling Strategies at Processing Plants

Processing Procedure from Live Chicken to Ground Chicken

The first step after the broilers arrive at the processing plant is weighing. The live weight is the basis for calculating the payment to the farmer. The weighed birds are then unloaded manually or automatically and hung upside down at the start of the processing line. The hanging room in some plants is dark, and a purple light can be used to reduce the broilers’ anxiety. The birds are then stunned electrically, mechanically, or under controlled atmosphere. Electrical stunning is the cheapest and most effective way (USDA-FSIS, 2010a). Controlled atmosphere stunning could be carried out by gas (i.e., carbon dioxide, argon, nitrogen). The most commonly used method for bleeding the stunned birds is called “Modified Kosher”. It results in cutting the jugular vein just below the jowls without contacting the windpipe and

17

esophagus (Barbut, 2002). The birds usually bleed out within 2 – 5 minutes and then enter the next stage of scald. Scalding is the process of loosening feather by hot water for better feather removal, and is done by single- or multiple- stage scald tank.

Broilers coming out of the scald tank then go through the picking machine. After the majority of feathers are removed by the rubber fingers inside the picker, some small feathers (i.e. neck feather) that are hard to pick out are singed. Then the birds are washed with hot water treated with chlorine or other chemicals. The birds then go through the hock cutter to remove the feet, and are then rehung in preparation of evisceration. The basic steps of evisceration includes removing the oil gland, opening the body cavity, extracting the viscera (USDA inspection occurs here), harvesting giblets, removing and discarding the intestinal tract and air sacs, removing and discarding the trachea, esophagus, crop, lungs and head (Barbut,

2002; USDA-FSIS, 2010a). The carcasses receive an inside-outside bird wash prior to entering the chill tank. The carcasses spend sometime in the chill tank, usually (1-

2 h) before coming out for packing or further processing. At the processing line, chilled carcasses are cut manually into different parts (i.e., drumstick, breast meat, tenders, and wings). These parts are then packed and shipped for commerce. At the end of the processing line, broiler frames with leftover meat, some skin-on parts such as breast meat (including neck skin pieces), and drumsticks are gathered and used for ground chicken production. The grinder separates the meat from the bones and cartilage to produce ground chicken with varying fat content.

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Transportation

Transportation of broilers to the processing plant for slaughter is a significant risk factor for in the dissemination of enteric pathogens through contaminated crates, trucks and catching/pickup crews (Cox and Pavic, 2010). The contamination of transport crates was identified as a risk factor to the end product

(i.e., carcass) contamination (Heyndrickx et al., 2002). According to the report by

Corry et al. (2002), inadequate fecal removal, insufficient disinfectant concentration and washing temperature, and the use of contaminated recycled water to soak the crates were major factors associated with ineffective crates decontamination.

However, an effective way to reduce the presence of Salmonella is simply washing the crates with water and let them completely dry for 48 hours (USDA-FSIS, 2010a).

Scalding

Scalding is used to break down the proteins that hold the feathers and to open up the feather follicles to facilitate feather removal (USDA-FSIS, 2010a).

Usually in-coming birds carry a large amount of organic materials (i.e., litter, feces), which are washed off in this process. The high level of organic material in the scalding tank is a concern of cross-contamination (Russell, 2007). In order to dilute the organic material and reduce the Salmonella load in scalding water, high-speed counter-current flow and multi-tank scalders are recommended (Cason et al., 2006;

Cason and Hinton, 2006). In addition, pre-scald brushing and chlorinated water washing process could be implemented to reduce organic load of in-coming birds

(Russell, 2007). The combination of scalding temperature and time is an important pathogen control point. Water with high temperature makes the carcass oily, which

19

facilitates the attachment of Salmonella to the skin. If the temperature is too low,

Salmonella could survive and propagate easily in the scald tank (USDA-FSIS, 2010a).

In the U.S., most broiler processor use hard scald (59-64°C, 30-73 secs). However,

Slavik et al. (1995) reported that hard scald has little effect on the reduction of

Salmonella on broiler carcass. The pH of scalding water was suggested to be either above or below the optimal pH for Salmonella growth (pH 6.5-7.5) (USDA-FSIS,

2010a). The proper water pH can be maintained by adding acidic disinfectant and frequent pH monitoring (Mead et al., 2010; USDA-FSIS, 2010a).

Picking

The removal of feathers is done by mechanical pickers with rubber fingers that rub feathers off the broiler carcass (Barbut, 2002). When picking, the broiler carcasses are hung upside-down and squeeze through the rotating drum of the picker, then the rubber fingers with grooved surfaces pick off the feathers from feather follicles (Barbut, 2002). This process could cause extrusion and scattering of fecal material onto the surface of the rubber fingers and serves as a possible source of cross-contamination (Mead et al., 2010). Campbell et al. (1984) found that

Salmonella were isolated from pickers before the start of the processing more often than from the other equipment. The authors explained that it was due to the complex construction of the pickers and the inadequate cleaning of the picker fingers (Campbell et al., 1984). To reduce the cross-contamination by the picker, it is suggested that worn fingers must be replaced and feather buildup in the picking area should be avoided (Mead et al., 2010; USDA-FSIS, 2010a).

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Eviscerating

Evisceration is the process of opening the broiler cavity and removing internal organs to prepare for chilling, with the head either saved or removed

(USDA-FSIS, 2010a). Crop and intestines could rupture during viscera extraction, which is considered as a major source of carcass contamination with enteric pathogens (Smith et al., 2007). Byrd et al. (2002) reported that 67% of broiler carcasses were positive for a fluorescent dye orally gavaged before processing. One method to reduce Salmonella contamination during evisceration is to undergo proper pre-slaughter feed withdrawal, which was discussed earlier in this review.

Another method is washing the broiler carcass after evisceration to remove excess organic debris (Mead et al., 2010). The proper use of an inside-outside bird washer

(IOBW) could effectively reduce Salmonella prevalence on broiler carcass by 75%

(Smith et al., 2005). Selected chemicals such as trisodium phosphate, lactic acid, sodium bisulfate, cetylphridinium chloride, and sodium chloride could be added to enhance the efficacy of IOBW (Northcutt et al., 2007; Yang et al., 1998). Other factors that can impact the bacterial removal are number and types of nozzles, nozzle pressure and arrangement, water pH and distribution, line speed, and flow rate (Cox and Pavic, 2010). Interestingly, Northcutt et al. (2005) stated that the use of chlorine compound and hot water did not significantly increase the Salmonella removal during IOBW. The authors suggested that the washing time (i.e., 5 s) was inadequate and probably the main reason for insufficient reduction in bacteria.

21

The USDA has a regulation of zero tolerance for visible feces on broiler carcass before entering the chiller (USDA-FSIS, 2011b). Carcasses with visible contamination must be reprocessed or condemned (USDA-FSIS, 2010a).

Chilling

The aim of chilling is to reduce the carcass temperature, usually to 4°C or below, within 30-75 mins (Barbut, 2002). Immersion chilling and air chilling are the two common methods used in broiler production. In the U.S., most companies prefer the former. Although immersion chilling is the most effective strategy to lower bacterial numbers on broiler carcass when compared with the other interventions

(i.e., IOBW, post-evisceration wash, chemical washes), Stopforth et al. (2007) reported that Salmonella prevalence on broiler carcass increased after immersion chilling. Thus, the chill tank was identified as a major site where cross- contamination between Salmonella-negative and Salmonella-positive birds occurred

(Sarlin et al., 1998). Chlorine is a common and relatively effective water additive used to control Salmonella cross-contamination in chill tank (USDA-FSIS, 2010a).

Mead and Thomas (1973) revealed that chilling water with 40-50 ppm chlorine led to a significant bacterial reduction on broiler carcass. In addition, chlorine dioxide, acidified sodium chlorite, trisodium phosphate (TSP), peroxyacids and lactic acid are potential interventions to use in chilling water for Salmonella reduction (Panel,

2005; Smulders, 1987). To obtain the maximum Salmonella inhibition effect in the chill tank, the pH of the water needs to be well maintained (i.e., 6.0-6.5), counter- current water flow should be used, and the flow speed should be high enough

(USDA-FSIS, 2010a). However, chemicals used in immersion chilling have little

22

effect on Salmonella attached firmly on the skin or trapped inside the feather follicles. It has been suggested that sonification in the chill tank may detach those cells and get better Salmonella reduction on the carcasses (Lillard, 1993).

Sanitation and Hygiene

Broilers are processed under a labor intense environment where workers are intensively involved in the production. Sanitation (i.e., reduce of bacterial load by chemicals) and hygiene (i.e., standard personal practice) are extremely important to prevent cross-contamination at the processing plant. In addition to establishing an effective Hazard Analysis and Critical Control Point (HACCP) program, Good

Manufacturing Practices (GMPs), appropriate training and Sanitation Standard

Operating Procedures (SSOPs) are all required (Mead et al., 2010). USDA established a set of guidelines regarding the sanitation and hygiene practices in poultry processing plants (USDA-FSIS, 2010a). For example, sanitation should always follow the cleaning of plant floor, walls and processing equipment. Mandatory hand washing and sanitizing should be enforced. People working inside the plants should wear coverings (i.e., hairnet, apron, gloves) to prevent cross-contamination.

Conclusions

The consumption of Salmonella contaminated poultry products pose a great public concern. Salmonella are transmitted both vertically, from breeders to broilers, and horizontally, between birds and from the contaminated environment to the birds. Multiple intervention strategies have been developed for use on the farm and at the processing plant to control and prevent Salmonella transmission

23

and cross-contamination. Interventions at the farm level includes adding antimicrobial compounds into drinking water and feed, proper use of litter, pre- slaughter feed withdrawal, and vaccination. At the plant, broilers are processed under a controlled environment with the application of hurdle technologies to reduce Salmonella cross-contamination.

Ground chicken is highly contaminated with Salmonella compared to post- chill carcasses. Understanding the source of Salmonella on the various chicken parts

(i.e., bone marrow and neck skin) that are used in ground chicken production is essential for control and prevention of this pathogen. The upcoming FSIS sample collection of 325 grams of ground chicken samples to replace the existing 25 grams sample, will increase the likelihood of detecting Salmonella. Data on Salmonella internalization of chicken internal organ are limited. Most of the studies have focused on observing immune response of artificially infected broilers. The presence of Salmonella in bones of naturally contaminated broilers was reported in only one published study. Salmonella present in bone marrow can be released during the grinding of the chicken bone-in parts which may lead to ground chicken contamination.

Salmonella is hard to remove on broiler carcasses when firmly attached to the skin or trapped in the feather follicle. Neck skin can be more contaminated with

Salmonella than other skin parts as chicken are processed while hung upside down, allowing wash fluid to drip over and through the neck skin. Thus, when used in ground chicken production, neck skin may contribute to Salmonella contamination of the ground product.

24

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