ABSTRACT
THE ESTABLISHMENT OF SALMONELLA AND LISTERIA PREVALENCE IN GOAT AND LAMB MEAT IN THE U.S.
Small-ruminant meat, or more specifically, goat and lamb, has always been a staple food source in various underdeveloped countries. Currently, consumption of small-ruminant meat is on the rise in the U. S. Many studies have researched the presence of various pathogens in other red meats; however, limited research has been made available for small-ruminants. Understanding the prevalence of Salmonella and Listeria in small-ruminants at a retail level helps contribute to baseline data and can allow for the food industry to find areas of improvement. A total of 100 U. S. retail samples were obtained from local California markets and online retail markets outside of California. Salmonella and Listeria analysis was performed using polymerase chain reaction though the DuPont BAX System Q7 and confirmation testing through traditional plate methods. Presence of Salmonella and Listeria were 0% and 1%, respectively. Generic E. coli, total coliforms, and mesophilic aerobic bacteria were enumerated using 3M petri film.
The total generic E. coli plate count was 0.10 log10 CFU/g, coliform count was
0.49 log10 CFU/g, and APC count was 4.60 log10 CFU/g. The results of this study indicate that there is a presence of microorganisms once it has reached the retail level. Controlling the environment and food handling from processing methods to retail is key to a safer food supply. Overall, further research should focus on a larger retail sample size. Understanding where contamination occurs is vital to creating a safer food supply.
Brittney Cemo December 2018
THE ESTABLISHMENT OF SALMONELLA AND LISTERIA PREVALENCE IN GOAT AND LAMB MEAT IN THE U.S.
by Brittney Cemo
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Food and Nutritional Sciences in the Jordan College of Agriculture Sciences and Technology California State University, Fresno December 2018 APPROVED For the Department of Food and Nutritional Sciences:
We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree.
Brittney Cemo Thesis Author
Erin Dormedy (Chair) Food Science and Nutrition
Steven Pao Food Science and Nutrition
Sara Shinn Food Science and Nutrition
For the University Graduate Committee:
Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS
X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship.
Permission to reproduce this thesis in part or in its entirety must be obtained from me.
Signature of thesis author: ACKNOWLEDGMENTS Completing this project and level of education has been one of the most difficult and exciting journeys I have embarked on. While still studying as an undergraduate I knew this is where I wanted my journey to go but did not understand the patience, time commitment, or strength it would take. Thankfully with the support from a few people who have pushed me along the way this journey has been bearable, and I am happy to finally close this chapter. Thank you to all my committee members Dr. Erin Dormedy, Dr. Sara Shinn, and Dr. Steven Pao for the guidance and support in the duration of my time as a graduate student. Dr. Dormedy, the encouragement and guidance you have provided me these past 9 years has made me more optimistic, stronger and responsible. I would like to thank my God mother, Susan Mooney, who convinced me that achieving this part of my education was the right path for myself. I remember sitting in a coffee shop with you discussing what life after college would look like and the reassurance it took to convince me I was on the right path for myself. I would also like to thank my mama for her support throughout my entire education. You are my number one cheerleader, my rock, and my shoulder to cry on. You are an incredible woman and I am incredibly proud to be your daughter. Lastly, my Aunt Linda who took the time to help me apply for colleges as I was exiting high school. I was so lost in such a big world and your guidance helped to keep me in line with exactly what I knew I always wanted. I would not be here if it was not for you. TABLE OF CONTENTS Page
LIST OF TABLES ...... vi
LIST OF FIGURES ...... vii
INTRODUCTION ...... 1
Foodborne Illness ...... 1
Number of Outbreaks, Illnesses, and Deaths ...... 1
Foods Associated with Foodborne Illness ...... 2
LITERATURE REVIEW ...... 6
History, Origin, and Diet ...... 6
Population ...... 7
Consumption on the Rise ...... 8
Foodborne Pathogens ...... 10 Studies Finding the Prevalence of Salmonella, Listeria, and E. coli in Meat ...... 17
Processing Methods ...... 27
Controlling the Processing Methods ...... 29
Analysis Methods Available ...... 34
METHODOLOGY ...... 38
Sample Collection ...... 38
Sample Processing ...... 38
Statistical Analysis ...... 43
RESULTS AND DISCUSSION ...... 44
CONCLUSION ...... 47
REFERENCES ...... 48
LIST OF TABLES
Page
Table 1. Presence of Salmonella and Listeria, in all samples tested from the U.S...... 44
LIST OF FIGURES
Page
Figure 1. Mesophilic aerobic plate counts for 86 retail and online small- ruminant samples. Reported numbers are the mean log and error bars on the sample mean represent standard error...... 45 Figure 2. Generic E. coli and coliform counts for 29 retail and online small- ruminant samples. Reported numbers are the mean log and error bars on the sample mean represent standard error...... 46
INTRODUCTION
Foodborne Illness Foodborne sickness is a serious issue that can lead to severe illnesses, hospitalization, or death (Centers for Disease Control and Prevention, 2015b). Although foodborne illness is common we do not have to let the illness statistics increase, it can be prevented (Centers for Disease Control and Prevention, 2015c). Bacteria is one of three reasons why food becomes contaminated and in turn becomes toxic to humans (Centers for Disease Control and Prevention, 2015c). As with many other types of illness, some people are more likely than others to be susceptible to foodborne illnesses due to undeveloped or weakened immune systems such as infants, the elderly, those who are immunocompromised, and pregnant women. When a harmful pathogen enters the gastrointestinal tract we begin to see evidence of “nausea, vomiting, abdominal cramps and diarrhea,” (Centers for Disease Control and Prevention, 2015c).
Number of Outbreaks, Illnesses, and Deaths The Center for Disease Control and Prevention (CDC), “estimates that each year 1 in 6 Americans get sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases,” (Centers for Disease Control and Prevention, 2015b). When these numbers decline and decrease in illnesses begin to show we believe this is partly due to food suppliers and researchers working diligently to make the food supply safer for consumption. The CDC has recognized eight major pathogens in three different categories that are of great importance to be aware of (Centers for Disease Control and Prevention, 2014). The different categorize includes: illnesses, hospitalizations, and deaths (Centers for Disease Control and Prevention, 2014). Norovirus is the most common attained foodborne illness at 58%, with 2 2
Salmonella in second at 11% (Centers for Disease Control and Prevention, 2014). Salmonella contributes to 35% of foodborne illness requiring hospitalizations, with Norovirus following with 26% (Centers for Disease Control and Prevention, 2014). Salmonella causes 28% of foodborne illnesses that result in death with Toxoplasma gondii following closely behind with 24% and Listeria monocytogenes with 19% (Centers for Disease Control and Prevention, 2014). Salmonella is either first or second in each of these three categories, this is important to understand and a great reminder that Salmonella is a killer and an enemy of our wellbeing. As for Listeria during 2016 the CDC estimated that, “1,600 people get listeriosis each year and about 260 die,” (Centers for Disease Control and Prevention, 2016a). Looking at Listeria recalls many of them are refrigerated or frozen foods. Since 2014 Listeria has had an increase of outbreaks in food and more data should be collected so we can be cautious and aware of Listeria (Centers for Disease Control and Prevention, 2017).
Foods Associated with Foodborne Illness Any food can be associated with foodborne illness if the right conditions are present for microorganism growth. All meat processing plants should have a developed hazard analysis critical control point (HACCP) plan in place to check safe food parameters properly. Not only is it up to the processors to keep the food safe but consumers should be aware and cautious of how to keep their food safe as well. The foods typically associated with the virus causing the most foodborne illness, Norovirus, are leafy greens, fresh fruits, and shellfish. Foods that are most commonly associated with Salmonella outbreaks include but are not limited to ground beef, poultry, bean sprouts, and eggs (Centers for Disease Control and Prevention, 2016d). Lastly, foods associated with Listeria include but are not 3 3 limited to frozen vegetables, soft cheese, raw milk, prepackaged salads, sprouts, and cantaloupe (Centers for Disease Control and Prevention, 2016c, 2017). Food can be contaminated at any point in the farm to consumer chain including growing, shipping, handling, processing, or preparation. It is important for each food contactor to be aware of cleanly and proper handling parameters. Small-ruminants, or more specifically, lambs and goats, are a very important food resource eaten throughout the world. The purpose of this research is to establish if there is a presence of Salmonella or Listeria in small-ruminant meat once it has reached the consumer. Currently there is little research done on Salmonella in small-ruminants meat at the retail level and no research discussing the possible presence of Listeria in small-ruminants at the retail level. With Listeria being a pathogenic nuisance to the frozen food industry, it is fundamental to understand its inhabitance in raw frozen meat. In addition to determining the presence of these two pathogens, data were collected on mesophilic aerobic bacteria, coliforms and generic E. coli. Instead of primarily studying lamb or goats separately, both types of meat were analyzed for this project. Lamb and goat are often considered the same animal in developing countries (Hanlon, 2015). Those cultures have migrated to the U.S. and have brought their traditional foods along with them. When in the field collecting samples from local markets, merchants were asked if they had goat and they often identified it as lamb. In some cases, the mutton purchased was deemed a mutton mix by the label. When asking what the mutton mix consisted the answer received was lamb and goat. Also, lamb and goat are often studied together in developing countries under one branch called small-ruminants (Hanlon, 2015). For the purpose of this research project, they are combined and considered small-ruminants. 4 4
The main objective of this research project is to help the Food Safety and Inspection Services (FSIS) of the United States Department of Agriculture (USDA) establish an estimated baseline of the national prevalence of Salmonella and Listeria in small-ruminant meat products. Salmonella has been one of the chosen microorganism for this project for several reasons. The first reason is because FSIS determined that a strategic plan would be put in place to determine at what point contamination occurs and how to prevent it from occurring (United States Department of Agriculture, 2010). Salmonella is the pathogen that was chosen because, “FSIS published the (PR/HACCP) Systems, Final Rule based on the baseline studies which established the reduction performance standards for Salmonella,” (United States Department of Agriculture, 2016b). The second reasoning is because goat and lamb is considered a red meat by the USDA (United States Department of Agriculture, 2011a). Salmonella has been found in other red meats, so a reasonable hypothesis is that there is Salmonella in goat and lamb. The third reason for choosing Salmonella is because this organism is the first leading cause of hospitalizations and deaths and the second leading cause of foodborne illnesses (Centers for Disease Control and Prevention, 2014). Listeria has been chosen as the second microorganism for several reasons as well. First, there is a growing awareness of this organisms presence in the refrigerated and frozen food industry in recent years (Centers for Disease Control and Prevention, 2017). Knowing if this organism is in red meats will be important for processors and consumers to know when handling the product. The second reason for studying Listeria is in the United States it has become one of the top three leading causes of death due to foodborne illness (Centers for Disease Control and Prevention, 2016b). Out of the 3,000 deaths each year 260 are due to Listeria (Centers for Disease Control and Prevention, 2016b). Lastly, both Salmonella and Listeria in 5 5 goat and lamb will be analyzed because consumption of this particular meat type is on the rise (United States Department of Agriculture, 2011a). With goat meat being the primary source of animal protein in North African and Middle Eastern nations a high importance should be placed upon this meat type to ensure it is safe for consumption (United States Department of Agriculture, 2011a). “There has been an increase in the influx of ethnic groups from areas of the world where goat meat comprises a significant portion of the diet,” (United States Department of Agriculture, 2011a). The new generation of American consumers tends to explore new foods more often and wants to continually broaden their horizons seeking the latest dish, usually of ethnic origins, to spark their taste buds needs (United States Department of Agriculture, 2011a).
LITERATURE REVIEW
History, Origin, and Diet Goats were one of the first farm animals to be domesticated (Nomura et al., 2013; United States Department of Agriculture, 2011a; Yang et al., 2015). According to the USDA, there is cave art depicting goats as an important food source that dates back 10,000 to 20,000 years ago (United States Department of Agriculture, 2011a). After 10,000 years of breeding, there are approximately 1,400 genetic diversities in goats from 45 breeds (Ajmone-Marsan et al., 2014; Yang et al., 2015). These 45 breeds are believed to have originally come from 15 countries in Europe and Asia (Ajmone-Marsan et al., 2014). More specifically the Fertile Crescent which includes the Persian Gulf, southern Iraq, Syria, Lebanon, Jordan, Israel and northern Egypt (Gordon Luikart, 2001; Nomura et al., 2013; Yang et al., 2015). Today goats live in diverse regions around the world such as tropical, dry desert, cold temperatures, high altitudes, and more (Morand-Fehr & Boyazoglu, 1999; Nomura et al., 2013). Farming of small ruminants is simple compared to most livestock animals. If there is enough area for them to roam, they usually have access to what they need, including diet. Small-ruminants prefer to graze on growing vegetation like weeds, shrubs, and other plants that most other domesticated farm animals refuse to eat (Pollott & Wilson, 2009). Since goats prefer to live off land vegetation they are a far more sustainable livestock commodity to raise for people living in underdeveloped and poverty-stricken countries. If a farmer has the financial means and no vegetation available they usually feed their goats food similar to what cattle eat (Goetsch, Merkel, & Gipson, 2011). 7 7 Population
World Population Goats have had a major rise in population over the past 20 years. The rise was not particularly in the U.S., but over the population of the entire world. The Food and Agriculture Organization of the United Nations (FAO) collected data that showed estimates of world livestock numbers. In 1990, small-ruminants, sheep and goats, were at 1795 million head (Food and Agriculture Organization of the United Nations, 2014). In 2000 there were 1811 million head count, and in 2012 there were 2165 million head (Food and Agriculture Organization of the United Nations, 2014). This recorded data over a 22 year span showed an overall increase of 20.6% (Food and Agriculture Organization of the United Nations, 2014). Although the increase of small-ruminant livestock was up 20.6% it still came in second to poultry at 104.2% (Food and Agriculture Organization of the United Nations, 2014). According to Morand-Fehr and Boyazoglu (1999), in 1996 sheep and goats were the world’s second and fourth largest livestock, respectively. Sheep have a heavier growth factor and are primarily bred in developed countries while goats show a 95% presence in underdeveloped or developing countries (Morand-Fehr & Boyazoglu, 1999). The numbers may be rising slowly but have showed steady growth and are likely to show more growth in the future.
Domestic Population: In the United States In 2007 the United States shows goats were farmed primarily in the eastern states and Texas with just over one-third of the goat population (United States Department of Agriculture, 2011c). Texas likely has the majority of goats because of the climate and terrain available (United States Department of Agriculture, 2011c). Angora goats which are breed for their furs were primarily found in 8 8
Texas, Arizona, and New Mexico (United States Department of Agriculture, 2011c). Meat markets heavily resemble the total markets with being primarily in the eastern U.S. and Texas (United States Department of Agriculture, 2011c). Lastly, the dairy market in the U.S. has a little less than one-fourth in California and Wisconsin combined (United States Department of Agriculture, 2011c).
Domestic Population: In California In the field for this research, it was determined that goat meat was primarily found in small family owned Hispanic markets and a few large Hispanic grocery chains. Dairy products such as milk, cheese and ice cream made from goat milk were found in high-end supermarkets and retail stores. When looking for goat milk there was usually only one size and brand available. Goat cheese was more available in both retail and high-end grocery stores, with different brands and flavors to choose from. Lamb meat was often found in family owned Armenian and Caucasian markets. Lamb meat was also found at high-end grocery chains. No other specialty products such as milk or cheese were found while purchasing samples.
Consumption on the Rise Increase in goat consumption is characteristically found in international populations, not domestic in the U.S. This is understandable since domestication and origination started in Europe and Asia. In 2011, the USDA stated At the present time, goats provide the principle source of animal protein in many North African and Middle Eastern nations. Goat is also important in the Caribbean, in Southeast Asia, and developing tropical countries. Three- fourths of all the goats in the world are located in the developing regions of the world. (United States Department of Agriculture, 2011a) 9 9
This leads us to question what cultures eat small-ruminants, and what the reasons and benefits are. There are three major reasons why goat meat is on the rise including ethnicity, religion, and ease of farming. Many of those consuming goat meat in the United State are not born in this country but are immigrants from places such as Middle East, Southeast Asia, Africa, Mexico, Western Europe and the Caribbean (United States Department of Agriculture, 2011c). There are three ethnicities driving the market for goat products including: Hispanic, Caribbean, and Chinese (United States Department of Agriculture). Hispanic ethnicities prefer the younger milk-fed goats, “cabritos” (United States Department of Agriculture). They are only about 25 pounds and are preferred due to the lighter color of fat (United States Department of Agriculture). The Caribbean ethnicities prefer their small- ruminants to be older males, bucks, and are cooked for an extended time period with lots of spices (United States Department of Agriculture). Religion plays a major role in consumption of meat, specifically in the Islamic Religion. The Islamic religion has the second largest amount of followers at 1 billion (ReligionFacts.com, 2015). The religion follows laws put forth by the Qur’an. The law forbids Muslims from eating any products that have come from a pig (Nakyinsige, Man, & Sazili, 2012). Ensuring that their food is processed by a manufacture who is halal certified is key to keeping away from eating pig (Nakyinsige et al., 2012). Often times manufactures in other countries will use pork collagen and fat to mix along with their other meats because it is cheaper (Nakyinsige et al., 2012). Muslims are roughly 1/7 of the world population and are growing in numbers everyday (ReligionFacts.com, 2015). Since Muslims are forbidden to eat pork, we can assume they turn to other types of meats for consumption like beef, lamb, and goat. Our third reason why goat meat is on the 10 10 rise is that goats are easy to farm. Small-ruminants can provide a vast amount of wealth for the right farmer. Ultimately, they require little to no maintenance. They are self-grooming, can feed on the land, and get enough exercise on their own when they can roam freely upon the land. Most of the care they will need is when it comes time to being milked. Almost 20% of the world population lives on less than one dollar a day and small ruminants are primarily farmed by poverty stricken rural homes (Pollott & Wilson, 2009). Farmers living on less than a dollar a day means they most likely do not have the funds to be feeding an animal. Contribution of small-ruminants to the home consists of a number of apparatus’ including: direct products, by-products, indirect benefits and intangible benefits (Pollott & Wilson, 2009). Products simply include: meat, milk, skins, fiber, horns, and offal (Pollott & Wilson, 2009). By-product like manure is utilized as fertilizer and for biogas production (Pollott & Wilson, 2009). Indirect benefits entail weed control and lastly intangible benefits consist of: generation and accumulation of capital, income, social, cultural and religious obligations and needs, providing status in the community, and lastly used for sport or recreation (Pollott & Wilson, 2009).
Foodborne Pathogens
What is Salmonella?
Organism overview. Salmonella belongs to the family Enterobacteriaceae and most strains are motile via flagella (Haiping Li, 2013). Several strains are nonmotile due to dysfunctional flagella such as Salmonella gallinarum and Salmonella pullorum. Salmonella is a gram-negative, rod shaped bacterium that grows within the temperature range of 7-48°C (Lawley, 2013). Salmonella is able 11 11 to withstand temperatures 4°C and below, but will be dormant (Lawley, 2013). The ideal pH for this microorganism to grow is at 6.5 – 7.5 (Lawley, 2013). Salmonella species catabolize D-glucose and other carbohydrates to produce both carbon and gas (Haiping Li, 2013). In order to identify Salmonella isolates we need to know a few basic traits of this bacteria such as: it is “oxidase negative and catalase positive, grow on citrate as a sole carbon source generally produce hydrogen sulfide (H2S), decarboxylate lysine, and ornithine, and do not hydrolyze urea,” (Haiping Li, 2013). Salmonella contains two species, enterica and bongori. S. enterica is divided into 6 subspecies that include enterica, salamae, arizonae, diarizonae, houtenae, and indica (Haiping Li, 2013). There are more than 2,500 serovars causing different diseases in humans collectively known as Salmonellosis (Wang 2013). There are around one million occurrences of Salmonellosis each year, where about 400 cases result in death (United State Department of Agriculture, 2013). Salmonellosis is the result of being infected by Salmonella where most people will have diarrhea, abdominal cramps, and fever within 8 – 72 hours after eating contaminated food lasting between four to seven days (United State Department of Agriculture, 2013). Life threatening risk may come to those who are: pregnant women, infants, young children, elderly, and immunocompromised patients (United State Department of Agriculture, 2013).
Species. Salmonella typhi and Salmonella paratyphi cause typhoid fever and only infect humans. The transmission is primarily from fecal to oral. Non- typhoidal Salmonella is primarily found in food and exotic pets. There are more than 2,300 serotypes of Salmonella the two most common being Salmonella enteritidis and Salmonella typhimurium (or typhi), these two serotypes cause almost half of all human infections (United State Department of Agriculture, 12 12
2013). Several other species include heidelberg, montevideo, newport, among many others (Centers for Disease Control and Prevention, 2015e). Salmonella has built multiple barriers in order to survive in its preferred environments. For example, “S. typhimurium at pH 5.8 engendered an increased thermal resistance at 50°C, an enhanced tolerance to high osmotic stress (2.5 M NaCl), a greater surface hydrophobicity, and an increased resistance to antibacterial lactoperoxidase system and surface-active agents such as crystal violet and polymyxin B (143). Salmonella cells surviving from long-term starvation and desiccation stresses demonstrate significant resistance to thermal, chemical, and other intervention processes,” (Haiping Li, 2013). Having to overcome multiple barriers presents a major challenge for food industry professionals who need their food products to be free of microorganisms and safe to eat.
Foods associated. The intestinal tract of humans and animals provides the perfect environment for Salmonella to live (United State Department of Agriculture, 2013). Salmonella infections are usually passed to humans via food vehicles (United State Department of Agriculture, 2013). Any food that provides a proper environment could contain Salmonella including but not limited to meat, dairy, fruits, vegetables, and nuts. Food outbreaks have included poultry, bean sprouts, ground beef, cantaloupe, pine nuts, and peanut butter, among others (Centers for Disease Control and Prevention, 2016d).
What is Listeria?
Organism overview. Listeria was given its first full description in 1923 by Murray, Webb, and Swann (Jay, Loessner, & Golden, 2005). Listeria is a gram- 13 13 positive, non-spore-forming, facultative anaerobic, rod shaped microorganism (Crum, 2002; Jay et al., 2005). It is catalase-positive and oxidase-negative (Crum, 2002). Listeria monocytogenes has a tumbling motility and can endure and grow in many different temperatures (Crum, 2002). The temperature range includes - 1.54°C to 50°C and at a pH range of 4.3 to 9.6 (Donnelly, 2001). Although Listeria can tolerate low temperatures it will not survive once temperatures reach 60°C for 30 minutes (Neusely de Silva, 2013). For Listeria to flourish in an environment it has to meet a few nutritional requirements: at least four B vitamins (biotin, riboflavin, thiamine, and thioctic acid), and 5 amino acids (cysteine, glutamine, isoleucine, leucine, and valine (Jay et al., 2005). Common media used to grow Listeria includes: brain heart infusion, trypticase soy, and tryptose broths (Jay et al., 2005). All species can grow in the 10% NaCl conditions and some can grow in 20% NaCl (Neusely de Silva, 2013). Listeria can also grow with 10% or 40% bile (Neusely de Silva, 2013). An infection from the Listeria microorganism is known as Listeriosis (Centers for Disease Control and Prevention, 2018). Listeriosis is typically most severe for pregnant women, newborns, older adults, and people with weakened immune systems (Centers for Disease Control and Prevention, 2018). If Listeriosis is contracted during pregnancy it can cause miscarriage, stillbirth, or newborn death(Centers for Disease Control and Prevention, 2018). Fetal loss and newborn death occurs in 20% and 3%, respectively (Centers for Disease Control and Prevention, 2018). People infected will experience fever, fatigue, muscle aches, diarrhea or gastrointestinal symptoms (Centers for Disease Control and Prevention, 2015d). Onset is typically within a few days of eating contaminated food but the CDC reports that it can extend as far out as 2 months (Centers for Disease Control and Prevention, 2015d). Environmental Listeria can be found in several areas including: difficult to clean 14 14 processing equipment, soil, water, and some animals (Meyer, Fredriksson- Ahomaa, Sperner, & Märtlbauer, 2011) (U. S. Department of Health & Human Services, 2018).
Species. Listeria is a microorganism that has six different species: L. monocytogenes, L. seeligeri, L. welshimeri, L. innocua, L. grayi, and L. ivanovii (Jay et al., 2005). Of the six species, the only species that is known to be harmful to humans is L. monocytogenes (Crum, 2002). There are 13 different serovars of L. monocytogenes (Crum, 2002). The three types of antigens that are the primary contributor to human sickness are1/2a, 1/2b, 4b (Crum, 2002). Many people consider the CAMP (Christie-Atkins-Munch-Petersen) test to be the ultimate indicator of L. monocytogenes. If this test is preformed and comes back positive with S. aureus or R. equi it must be considered presumptive positive for L. monocytogenes and further testing is required (Jay et al., 2005).
Foods associated. Listeria can be found in many environments including: soil, water, vegetation, animal feed, fresh or frozen food, among others (Neusely de Silva, 2013). However, the primary transport vehicles for infection in humans is food (Neusely de Silva, 2013). Many foods are associated with Listeria monocytogenes. Major outbreaks that have occurred in North America consist of soft raw milk cheeses, frozen vegetables, raw milk, packaged salads, soft cheeses, ice cream, prepackaged caramel apples, bean sprouts, cantaloupe, and others (Centers for Disease Control and Prevention, 2017).
What is Escherichia coli?
Organism overview. First to discover Escherichia coli was Theodor Escherich (Hacker 2007). Escherichia coli (E. coli) is a microorganism that was 15 15 first recognized as a foodborne pathogen in 1971 when 400 people in America fell sick while consuming imported cheese (Jay et al., 2005). E. coli is a gram negative, rod shaped bacteria typically found in the gastrointestinal tracts of hot- blooded animals, including humans (Neusely de Silva, 2013) (Tortora, Funke, & Case, 2013). Most strains of E. coli are not harmful and actually are needed in the gut flora, however there are some pathogenic strains that use food or water as a vehicle and cause illness in humans (Centers for Disease Control and Prevention, 2015a). Generally once the pathogenic strain has been ingested it takes three to four days for the incubation period to be completed. Symptoms of infection begin with acute stomach pain and non-bloody diarrhea that will worsen over several days. If infected with a Shiga toxin-producing E. coli (STEC) symptoms could consist of: severe stomach cramps, bloody diarrhea, vomiting, and a mild fever of less than 101°F (Centers for Disease Control and Prevention, 2015a). Also, if a person becomes infected with STEC a much more severe complication could occur called hemolytic uremic syndrome (HUS) (Centers for Disease Control and Prevention, 2015a). Symptoms of HUS are decreased frequency of urination, feeling tired, losing pink color in the cheeks and lower eyelids, and indications of possible kidney failure (Centers for Disease Control and Prevention, 2015a). If HUS is suspected hospitalization and treatment should be sought out immediately to prevent permanent damage to the kidneys or death (Centers for Disease Control and Prevention, 2015a). Reports of bloody diarrheal infections and post diarrheal hemorrhagic colitis usually correlate with O-antigenic serotypes, particularly O157:H7 (Hunt, 2010).
Species. Most strains of E. coli are harmless. However, few members of this species are deadly. Certain strains of E. coli can cause diarrheal infections 16 16
(Hunt, 2010). There are eight pathogenic varieties of E. coli that have been heavily studied and grouped together based on disease syndromes and characteristics, five of them are enterovalent pathovar groups (Jay et al., 2005; Tortora et al., 2013). These five virulence groups are: enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), and Shiga-toxin-producing E. coli (STEC) (Tortora et al., 2013). E. coli is known to have around 200 O serotypes. The O refers to the antigen type found in the cell wall of the bacteria, and the H is associated with antigens found on the flagella of the bacteria (Hunt, 2010; Jay et al., 2005). Serotype O is usually associated with foodborne illness (Hunt, 2010). The STEC group forms a toxin called shiga-toxin. The most infamous in this group is E. coli O157:H7, notably linked to many foodborne outbreaks (Hunt, 2010). The STEC group often causes food-borne gastroenteritis, bloody diarrhea, hemolytic uremic syndrome (HUS) and renal failure in children (Centers for Disease Control and Prevention, 2015a; Hunt, 2010). Anyone can contract the illnesses from E. coli and symptoms can appear as soon as one day or as late as 10 days (Centers for Disease Control and Prevention, 2015a).
Foods associated. There are many foods associated with pathogenic E. coli that include beef (Youssef 2013), sprouts (Buchholz 2011), spinach (Kinsey 2011), raw milk (Baylis 2009), drinking water (EPA 2013) and others. When foods are contaminated with harmful E. coli, it generally is due to fecal cross- contamination (McClure 2000). During slaughter, beef can become contaminated from the hands of processors (Youssef 2013). This is often due to the lack of hygiene. The processing course can also be a source of contamination though equipment such as gloves, knives, and conveyor belts (Youssef 2013). Raw meat 17 17 and other foods can be contaminated in the home as well (CDC). Prevention of E. coli is simple and accomplished through preventing cross contamination, washing hands and foods well, and cooking thoroughly (CDC).
Studies Finding the Prevalence of Salmonella, Listeria, and E. coli in Meat
Overview of Other Studies There has been limited research completed on the presence of Salmonella, Listeria, and generic E. coli in small-ruminants, including processing facility and retail studies. However, numerous studies have been published on a variety of meats, including national baseline studies. National baseline data reports have published for several types of meat: beef, beef trimmings, veal, young turkey, young chicken, chicken parts, market hogs, goose, and swine (United States Department of Agriculture, 2018). With small-ruminants growing in consumption (United States Department of Agriculture, 2011c), it is critical to understand the prevalence of Salmonella and Listeria in goats and lamb. Small-ruminants are considered red meat; previous studies of Salmonella, Listeria, and generic E. coli in processing facilities and retail samples of other red meats will help predict what this study may find. Certain pathogenic microorganisms such as Salmonella, Shiga-toxin producing Escherichia coli (STECs), Campylobacter jejuni, Yersinia enterocolitica, Listeria monocytogenes, and Staphylococcus aureus are attracted to red meats like beef and pork and are likely to be present during this study of small- ruminants (United States Department of Agriculture, 2013, 2016a). 18 18 Salmonella in Beef
Processing facility studies. In 2015, USDA reported baseline data for beef carcasses a total of 2,736 samples were collected (United States Department of Agriculture, 2015). Samples were analyzed for Salmonella, E. coli O157:H7 and non-O157 STEC (United States Department of Agriculture, 2015). Post-hide- removal and pre-chilling had 1,368 samples each from 139 abattoirs (United States Department of Agriculture, 2015). For Salmonella, post-hide removal samples showed 27.12% positive, while pre-chilling samples recorded 3.36% positive (United States Department of Agriculture, 2015). A study performed by Texas Tech University personnel found that out of 47 pre-evisceration and 47 post- evisceration samples Salmonella was found at a rate of 6.7% and 0%, respectively (Maradiaga et al., 2015). Another study collected 750 samples in a commercial Irish abattoir found that the beef carcasses swabbed showed Salmonella in 2% of the fecal samples, 2% in the rumen samples, and 7.6% on carcass samples (McEvoy, 2003). In 2011, Valerie Bohaychuk conducted a study in Canada which showed that 1 out of 1,036 (0.1%) beef carcass swab samples were positive for Salmonella post-evisceration (Valerie M. Bohaychuk, 2011).
Retail studies. A study performed by Texas Tech University personnel found that out of 555 retail beef samples 10.1% were contaminated with Salmonella, at a 95% confidence interval (Maradiaga et al., 2015). A second study sampled beef, pork, turkey and chicken in 1999 to 2000 from local Maryland and Washington, D. C. retail grocery supermarkets (Zhao et al., 2001). Beef collection consisted of 210 samples where only 4 (1.9%) were positive for Salmonella (Zhao et al., 2001). In 2010, a study sampled 2,885 retail ground and whole muscle beef samples, 2,199 and 686 respectively (Vipham et al., 2012). Salmonella was 19 19 detected using polymerase chain reaction (PCR) based method “with a sensitivity and specificity rates >98%” (Vipham et al., 2012). Salmonella was detected at 0.55% in retail ground beef and 1.02% in retail whole muscle samples (Vipham et al., 2012). All of these studies show that Salmonella is present in beef, which means the organism is probably occurring in small-ruminants as well.
Salmonella in Pork
Processing facility studies. In 2011, the USDA baseline data reported that pre-evisceration has a Salmonella prevalence of 69.64% and at post chill numbers had been reduced to 2.70%, this was with a 95% confidence interval (United States Department of Agriculture, 2011b). Another study done in 2009 by the University of Wisconsin-Madison found that pork carcasses in their study were higher than the FSIS baseline for Salmonella in pork, which is at 8.7% (Algino, Badtram, Ingham, & Ingham, 2009). For prewash, the carcasses contained 9.4% and chilled carcasses were at 18.8% (Algino et al., 2009). Not only does this show that contamination was high but that recontamination was happening within the process (Algino et al., 2009). Lastly for discussion, a study done in Canada showed that with a 95% confidence interval there were 17 out of 1,076 pork carcasses (1.6%) which tested positive for Salmonella (Valerie M. Bohaychuk, 2011). These carcass swab samples were taken post-evisceration while in the cooler (Valerie M. Bohaychuk, 2011). Looking at this data it is understandable that there is a need to control Salmonella in pork. In 1993 Copenhagen, Denmark took a major outbreak of Salmonella in pork so seriously that they put a response program into place to reduce possible incidences (Hurd et al., 2008). Denmark is the only country with a program that focuses from pre-harvest to post-harvest 20 20
(Hurd et al., 2008). With studies published for national baseline data we should be informed what retail sample prevalence is.
Retail studies. A study mentioned earlier performed in Maryland and Washington D.C. area collected 209 retail raw pork samples (Zhao et al., 2001). Of the 209 samples, 7 were positive for Salmonella, 3.3% (Zhao et al., 2001). Research from New Zealand collected 231 retail pork samples where results showed zero positive for Salmonella (Wong, Nicol, Cook, & MacDiarmid, 2007). Lastly, a study from Canada collected 133 retail pork samples (Aslam et al., 2012). Using a combination of PCR and traditional plating methods 3 of the 133, 2.2%, were found to be positive for Salmonella (Aslam et al., 2012).
Salmonella in Small-Ruminants (Goats and Sheep)
Processing facility studies. Prevalence of Salmonella in beef, pork, and lamb have been vastly studied. However, few research papers have been published on Salmonella in goats. Very few publications have shown the prevalence of Salmonella in red meat at the retail level. Publications primarily show Salmonella prevalence at pre- and post- intervention strategies to show the control in slaughter methods. A study done in two Australian abattoirs showed a prevalence of Salmonella with: 59.5% in rumen, 56.7% in fecal, and 32.2% in carcasses (Duffy, Barlow, Fegan, & Vanderlinde, 2009). The data found in this particular study showed a significant difference (P < 0.05) in the rumen and fecal than in the carcass (Duffy et al., 2009). Since Salmonella in rumen and fecal is more prevalent it is possible that these products of waste are contaminating the carcasses. Between the two abattoirs there were five dominant serotypes of Salmonella found including: saintpaul, chester, anatum, typhimurium, and agona 21 21
(Duffy et al., 2009). Another study performed in 2012 by Bonke established that 43 out of 100 sheep were positive for Salmonella in their tonsils. This particular study does show that there is a prevalence of Salmonella in sheep. In the same study performed by Bonke in 2012, Salmonella in goats was studied as well. Out of 100 goats that were studied two had Salmonella in their tonsils and one had Salmonella in their feces (Bonke et al., 2012). Salmonella enterica subspecies diarizonae was isolated from 20% and 1% of sheep and goat tonsils respectively(Bonke et al., 2012). Our partner school for this research, Texas Tech University, tested for Salmonella prevalence in goat and lamb carcasses, testing was done on fecal matter, hide, lymph nodes, and retail carcass swabs (Hanlon, 2015). In fecal samples Salmonella was detected at 10.33% in goats and 11.41% in lamb (Hanlon, 2015). Hide samples showed 3.31% in goats and 25.47% in lamb (Hanlon, 2015). Salmonella in goat carcasses showed 2.17% during pre- evisceration swabbing, 4.10% at post-evisceration, and no positive samples during post-intervention (Hanlon, 2015). Prevalence of Salmonella in lamb carcasses had 3.55% at pre-evisceration, 5.20% at post-evisceration, and 3.45% at post- intervention, with a 95% confidence interval (Hanlon, 2015).
Retail studies. Hanlon (2015) not only collected pre-evisceration, post- evisceration, and post intervention samples but also collected small-ruminant retail samples from the Bahamas, California, and Texas. Retail small-ruminant samples had an overall 16.82% presence of Salmonella out of 107 samples collected (Hanlon, 2015). Hanlon noted that retail sample results were comparatively high to other retail market studies and seemed concerning compared to the post- intervention results obtained at 2.04% (Hanlon, 2015). Not many retail studies have been performed on small-ruminants in the United States, but several have 22 22 been performed throughout the world. In New Zealand, 230 retail lamb or mutton samples were collected with 3 or 1.3% detected positive for Salmonella (Wong et al., 2007). Salmonella spp. in lamb samples were identified as Brandenberg isolates (Wong et al., 2007). In Katmandu, 1 of 31 retail goat samples were positive for Salmonella, 3.2% with a 95% confidence interval (Maharjan, Joshi, Joshi, & Manandhar, 2006). Similar to three chicken samples and 2 buffalo samples, Salmonella spp. for goat was not able to be differentiated between subgenus I or II (Maharjan et al., 2006). Understanding that there is Salmonella in all red meats previously discussed after post-evisceration is important to note. It does show that there is a great possibility that there will be similar findings in small-ruminant retail meat. These numbers show that there needs to be a baseline set to have a common goal between processors to keep the small-ruminant food supply safe.
Listeria in Beef
Processing facility studies. In 2011, a study performed in Germany collected 501 beef samples from within slaughterhouses and processing plants (Meyer et al., 2011). Samples taken “consisted of: meat cuts, kidneys, livers, hearts, and tongues” (Meyer et al., 2011). These samples showed that 15% of 501 beef samples were positive for Listeria species (spp.). Those samples were further tested which showed 3% positive for Listeria monocytogenes (Meyer et al., 2011).
Retail studies. In 2009, Karakolev spent 5 years collecting various meat samples included 253 raw and minced beef (Karakolev, 2009). Of the 253 samples, 33 were positive for Listeria. When typing the genes 21 were found to be L. monocytogenes (Karakolev, 2009). A second study published in 2013 collected 23 23
417 samples of raw beef, pork, mutton, and chicken meat from several cities in China (Wang et al., 2013). Raw beef samples showed 10.3% (11/107) positive for L. monocytogenes which ranked third highest out of the four meat types (Wang et al., 2013). Lastly, an older study from 1990 analyzed for Listeria spp. in three types of retail red meat including beef, pork, and lamb (Johnson, Doyle, & Cassens, 1990). Of the 50 retail beef samples 3 were found positive for Listeria (Johnson et al., 1990). The samples types consisted of two beef English roasts and one beef sirloin tip roast (Johnson et al., 1990). Lastly, a study from 2012 sampled 243 retail raw meat samples from the metro Detroit area (Da Rocha, Gunathilaka, & Zhang, 2012). One hundred thirty-three beef samples were collected where 45 (33.8%) were found positive for genes Listeria, of the 45, 18 were typed for L. monocytogenes (Da Rocha et al., 2012).
Listeria in Pork
Processing facility studies. In 2011, Meyer and others not only collect beef samples from slaughterhouses and processing plants but also pork samples. Types of samples taken entailed the same as beef: meat cuts, kidneys, livers, hearts, and tongues (Meyer et al., 2011). Data showed that 13% of 484 pork samples were positive for Listeria spp. Those samples were further tested which showed 4% positive for Listeria monocytogenes (Meyer et al., 2011).
Retail studies. In 2009, Karakolev not only collected beef samples but also 252 raw and minced pork samples (Karakolev, 2009). Of the 252 samples, 36 were positive for Listeria. When typing the genes 18 were found to be L. monocytogenes (Karakolev, 2009). A second study, previously discussed in the beef section published by Wang, showed raw pork samples positive for L. 24 24 monocytogenes at 20% (20/100) (Wang et al., 2013). Pork had the highest prevalence out of the four meat types (Wang et al., 2013). Lastly, older research from 1990 studied 50 pork samples at the retail level (Johnson et al., 1990). Roast samples were collected and showed that 3 out of 50 were positive for Listeria (Johnson et al., 1990). Positive samples consisted of two boneless pork loin roasts and one pork bone-in sirloin roast (Johnson et al., 1990).
Listeria in Small-Ruminants (Goats and Sheep)
Retail studies. In 2015, a study was completed at Virginia State University comparing goat and lamb retail samples purchased on the internet and local markets (Kim, Stein, & Pao, 2015). Out of 134 samples, 45 were positive for Listeria spp., 35% (Kim et al., 2015). Those positive samples were further tested which showed 33 out of 45 positive for Listeria monocytogenes (Kim et al., 2015). A second study published by Wang, showed that 5 out of 104 (4.8%) raw mutton samples were positive for L. monocytogenes (Wang et al., 2013). Mutton had the least prevalence out of the four meat types studied (Wang et al., 2013). Lastly, older research previously mentioned from 1990 by Johnson and others also tested 10 lamb roasts from local grocery stores (Johnson et al., 1990). Out of the 10 lamb samples there were zero contaminated with Listeria (Johnson et al., 1990). With small-ruminant consumption on the rise and so few studies having been performed, we should be mindful that a retail baseline is needed for small- ruminant meat. 25 25 E. coli in Beef
Processing facility studies. A study testing for E. coli in beef samples at several different stages of processing including pre-evisceration, post-evisceration, and post-intervention, resulted in 15 out of 149 (10.07%), 2 out of 144 (1.39%), and 1 out of 290 (0.34%) positive results, respectively (Ransom, 2004). An older study from 1999 collected pre-evisceration, post-evisceration, and post-processing samples from four different US Midwestern beef processing facilities (Elder et al., 2000). Positive samples from pre-evisceration, post-evisceration, and post- processing were found in 148 out of 341 (43.40%), 59 out of 332 (17.77%), and 6 out of 330 (1.82%) samples, respectively (Elder et al., 2000). These studies show that with post-intervention strategies present in the process are important for reduction and keeping the food supply safe.
Retail studies. A study by Xia collected 1,806 ground beef samples to be tested for E. coli (Xia et al., 2010). Samples were collected over a period of five years from grocery stores located in Georgia, Maryland, Oregon, and Tennessee (Xia et al., 2010). Through PCR analysis 16 out of 1,806 (0.89%) samples were positive for E. coli and after Xia’s further evaluation of the positive E. coli samples none were found to belong to the serotype O157 (Xia et al., 2010). In 2014, Liao and others gathered 1,129 retail samples to be tested for E. coli from 24 different states (Liao et al., 2014). Using PCR analysis 9 of the samples were considered positive for E. coli however, when further analyzed none of these samples were serotype O157 (Liao et al., 2014). 26 26 E. coli in Pork
Processing facility studies. In 2011, baseline data were collected on market hogs from 253 establishments by the microbiological division of the USDA (United States Department of Agriculture, 2011b). A total of 3,920 sponge samples, 1,960 from each process point assessed, pre-evisceration and post-chill, with 95.81% and 11.78% positive, respectively (United States Department of Agriculture, 2011b). An earlier nationwide study of generic E. coli was performed by the USDA microbiological division, Food Safety Inspection Service (FSIS) in 1998. Samples were collected at various points including, “after chilling, the end point in slaughter, and dressing,” (United States Department of Agriculture, 1998). Presence of generic E. coli was sampled for on 2,127 carcasses where 937 (44.1%) were positive (United States Department of Agriculture, 1998). Among the samples with a presence for generic E. coli 96.2% had 100 or fewer and 91.5% had 10 or fewer cfu/cm2 (United States Department of Agriculture, 1998).
Retail studies. Xia also tested for E. coli in pork samples and found that 1 out of 1,167 (0.09%) samples were positive for E. coli. When further checked, the serotype was not found to be O157 (Xia et al., 2010).
E. coli in Small-Ruminants (Goats and Sheep)
Processing facility studies. A study performed at Kansas State University in 2013 collected 299 goat carcass samples from a slaughter facility on nine different days over the course of three months (Jacob, Foster, Rogers, Balcomb, & Sanderson, 2013). This study found that eight of the carcass samples were positive (2.7%) (Jacob et al., 2013). A 2001 study performed in South Yorkshire, UK collected beef and lamb samples from the carcass and at retail to test for E. coli 27 27
(Chapman, Cerdán Malo, Ellin, Ashton, & Harkin, 2001). Lamb meat carcasses samples were taken from the neck and the MPN method was used to determine that 10 out of 1500 (0.70%) samples were positive for E. coli (Chapman et al., 2001).
Retail studies. A study by Kim not only tested for Salmonella and Listeria, but also E. coli (Kim et al., 2015). Internet-purchased and retail small-ruminant samples positive for E. coli totaled 34 out of 134 (25.37%) (Kim et al., 2015). When further looking at the data positive goat and lamb samples totaled 14 out of 36 (38.89%) and 20 out of 98 (20.40%), respectively (Kim et al., 2015). The 2001 study performed in South Yorkshire, UK previously mentioned, also collected beef and lamb samples from local butcher shops to test for E. coli (Chapman et al., 2001). Using the MPN method types of lamb retail meat samples tested consisted of minced, burger, and sausage and was found to have 9 out of 1,144 (0.79%) positive (Chapman et al., 2001).
Processing Methods There are several different slaughter methods that could be implemented when processing small-ruminants. The methods chosen will depend on the facilities ability, cultural traditions, religious practices, age and size of the animal. Typically facilities in the United States have the following basic processing methods for small-ruminants: stunning, bleeding, pelt removal, evisceration, inspection by USDA-FSIS inspectors, then kept in a chiller until fabrication steps (Savell & Smith, 2009). A facility in Australia processes goats similar to hogs by stunning, bleeding, passing through a scalding tank, tumbling, singeing off any remaining hair and then placing the carcass into a chiller (Duffy et al., 2009). Again, methods of slaughter will change depending upon many influences, but 28 28 each process will have several main points where the carcass is controlled to prevent microbial contamination and ensure proper processing techniques. Fabrication of all meats should be done in a clean environment to help prevent microbial growth. Depending on the facilities abilities and beliefs, either a cold or warm room will be used to fabricate the meat. Both fabrication techniques have their advantages. Deboning the carcass has traditionally been done in a cold environment. When deboning and fabrication takes place in a refrigerated atmosphere, the advantages are centered around controlling microbial growth. Facilities that perform deboning and fabrication in a warmer environment have strong beliefs that it is better for the quality of their product. Although some advantages of hot boning are documented, others are assumed. A few reasons why manufactures believe that hot boning has advantages are: energy cost savings, higher yield, tenderization, water holding capacity, and less strenuous effort resulting in fewer injuries and quicker fabrication time (Pinto Neto, Beraquet, & Cardoso, 2013; Thomas, Anjaneyulu, & Kondaiah, 2008). Although more research could be done on hot-boning, both traditional and hot-boning are practiced throughout the world. In a retail environment, it is important that butchers and consumers both follow good practices. If a butcher cuts meat, they should have kept the meat at a cold temperature of 40°F or lower, washed their hands, worn gloves while performing tasks such as cutting or preparing, and should not handle money or other contaminated items before cutting the meat. Consumers should get product home as quickly as possible and back down to a temperature of 40°F or lower (United States Department of Agriculture, 2011a). If a consumer pulls their meat selection from a refrigerated case, a disposable plastic bag should be used to cover the packaging in the event it leaks (United States Department of Agriculture, 29 29
2011a). If leaking occurs it could soil fresh or cooked produce (United States Department of Agriculture, 2011a). And again, consumers should get the product back down to a temperature of 40°F or lower as soon as possible (United States Department of Agriculture, 2011a).
Controlling the Processing Methods There are several steps in the slaughter process that need to be controlled and monitored. Areas that need to be addressed are good food safety practices, preventing cross contamination, temperature control, and intervention strategies. While each of these controls are common knowledge, they provide a safety and ethical standard for the facility and to assure the consumers that product is handled in a safe manner.
Good Food Safety Practices Good food safety practices can include a number of plans and ideas to incorporate the laws set forth by the code of federal regulations (CFR). One of the most important plans to implement in a food facility to control a process is the Hazard Analysis Critical Control Point (HACCP). This plan will discuss each step of the process in detail and determine what the points of concern are and how they will be monitored and controlled. Having these control points helps prevent biological, chemical, and physical contamination. Generally, a small-ruminant HACCP plan will consist of three critical control points (CCPs). The first CCP is to check for visible fecal matter, milk, and ingesta. This CCP is actually a requirement set forth by FSIS Directive 6420.2 and is known as a zero tolerance standard (United States Department of Agriculture, 2011d). The second CCP is usually a lactic acid wash that is considered a biological pathogen hurdle. This wash is usually applied just before moving the carcass into the chiller. The last 30 30
CCP for a typical facilities plan is to check the carcass temperature the morning after slaughter to ensure it has reached a temperature of 44.6°F or below. This temperature will retard microbiological growth.
Prevention of Cross Contamination When processing meat carcasses the internal portion of a ruminant muscle is considered sterile (Erin Stafford Dormedy, 1999). Microbiological contamination of muscles foods primarily comes from cross-contamination with the contents of the intestinal tract of the animal, unsanitary equipment, and poor personal hygiene practices of employees. Prevention of microbiological cross contamination is a major factor that must be controlled in all facilities. If this process becomes out of control, a recall could be implemented if the problem is severe. In order to prevent cross contamination during processing, the possible sources must be understood. Cross contamination can occur in many different areas including the hide (Gill, McGinnis, & Bryant, 1998), the stick knife, gastrointestinal tract removal, lymph nodes, hands and clothes of the handlers, processing tools, and factory equipment (McEvoy et al., 2000) (Jay et al., 2005). Having good manufacturing practices (GMP) in place and following HACCP procedures should help prevent contamination.
Temperature Control “Keep it cold, keep it clean, and keep it moving” has long been the mantra of the meat processing industry. Having the ability to control the temperature of the process and processing room are other key factor in controlling microbial growth during the slaughter process. Meat should continue to be held at refrigeration temperatures in further processing steps after slaughter, as well as 31 31 during storage and transportation. This will retard growth of any microbiological pathogens.
Intervention Strategies Intervention strategies are additional strategies to temperature and cleanliness to control microbial growth in food products. Although many intervention strategies have been studied for application in the slaughter process, each come with their own challenges. Some intervention strategies include hot water washes, organic acid washes, steam pasteurization, and irradiation.
Hot water wash. Hot water wash methods have been accepted in the United States by the FSIS, however this method has not been embraced by the European Union (Hauge, Wahlgren, Røtterud, & Nesbakken, 2011). Patterson’s study showed hot water surface decontamination methods consisting of a temperature of 80- 96°C for 2 minutes. Unfortunately this long exposure time will result in carcass surface discoloration (Ellebracht, Castillo, Lucia, Miller, & Acuff, 1999) (Hauge et al., 2011). The European Union poses concerns of the cleanliness and quality of the recycled water used in the process and that this pasteurization step could help mask unhygienic slaughter process methods (Hauge et al., 2011). A study presented by Hauge showed that hot water pasteurization for lamb carcasses at 82°C for 8 seconds showed a reduction of 1.85 log CFU per 4500 cm2 (Hauge et
2 al., 2011). Smith showed an initial Salmonella count of 6.76 log10 CFU/cm and after 10 seconds of hot water applied to the carcass the microbial load of
2 Salmonella had been reduced to 3.41 log10 CFU/cm (Smith, 1992). A study presented by Ellebracht showed that control ground beef chubs had 4.7 log10 CFU/g present and with a hot water treatment application the Salmonella microbial load was reduced to 4.0 log10 CFU/g (Ellebracht et al., 1999). Ellebracht 32 32 also used a combination of hot water and lactic acid for a carcass treatment on ground beef chubs and showed a 2.9 log10 CFU/g (Ellebracht et al., 1999). Although these studies showed a decent microbial load reduction there are other treatments available that will give carcass surfaces an even greater reduction.
Organic acid wash. Organic acid washes consist of three types: acetic, citric, and lactic acid (Jay et al., 2005). In the meat industry lactic and acetic acid washes are typically used at 2 to 5% concentration and are crucial steps for the decontamination of bacteria on carcasses (Jay et al., 2005). Not only do the washes help prevent biological growth but it also preserves the shelf life (E. S. Dormedy, Brashears, Cutter, & Burson, 2000). These washes have been found to aid in controlling typical biological problems found in meat such as: Salmonella (Ellebracht et al., 1999), E. coli (Berry & Cutter, 2000), and psychotropic bacteria like Listeria (Jay et al., 2005). The acid washes are so important that many facilities place them as one of the CCPs in the HACCP program. The process of rinsing the meat carcass with acid will move the original pH range to one of a lower scale where the microorganism that we do not want present will have a much harder time growing (Erin Stafford Dormedy, 1999). Several studies have shown that when some acids are used the pH will return to original values after approximately 5 hours (Erin Stafford Dormedy, 1999). This is one factor as to why there is little time permitted after the acid wash to get the carcass into a blast chiller.
Steam pasteurization. Steam pasteurization processing is where steam will be in contact with the meat at 82°C to 97°C inside a chamber with atmospheric pressure for 6 to 12 seconds (Aymerich, Picouet, & Monfort, 2008). Typically a steam treatment will include water removal, steam pasteurization, and rapid 33 33 chilling (Aymerich et al., 2008). This process was approved in 1995 by the FDA for whole carcasses and parts of carcasses (Aymerich et al., 2008). Aymerich summarizes other articles who tested steam pasteurization treatment on beef, chicken, and pork (Aymerich et al., 2008). Beef carcasses held at 82.2°C for 6.5 seconds showed a reduction of 1.0 log CFU/cm2 (Nutsch, 1998). Another study on beef showed greater than 1.0 log CFU/cm2 reduction for E. coli, Salmonella, and Listeria, when steam treated at 93.3°C for 6 seconds (Retzlaff et al., 2004). Pork steam treated for 30 seconds had a 4.38 log CFU/cm2 Listeria reduction (Trivedi, Reynolds, & Chen, 2008). If steam pasteurization is used by a facility great care must be taken to not give the carcass a cooked look and to ensure that the entire surface has been steamed properly including areas which are difficult to reach or which may contain a higher amount of microbes (McCann, Sheridan, McDowell, & Blair, 2006). Being that there was a reduction of microbes in red meats, beef and pork, this could be an excellent way to reduce similar microbial problems that may occur in small-ruminants.
Irradiation. Irradiation is a non-thermal technology that exposes a food product to ionizing irradiation. In 1985 the USDA published the first irradiation rules on meat to inactivate Trichinella spiralis in pork, in 1997 irradiation of red ground meat was authorized (Aymerich et al., 2008). There are several ways a food product could be exposed to ionizing radiation and deemed as safe as defined by 21 CFR 179.26. “Gamma rays from sealed units of the radionuclides cobalt-60 and cesium-137, electrons generated from machine sources at energies not to exceed 10 million electron volts (MeV), x rays generated from machine sources at energies not to exceed 5 MeV except when x rays generated from machine sources using tanalum or fold as the target material using energies not to exceed 7.5 MeV” 34 34
(U. S. Food and Drug Administration, 2016b). In 21 CFR 179.26 b and c, limitations and labeling, respectively, requirements are set. Henriques tested the elimination of E. coli and Salmonella in sheep meat by gamma irradiation (Henriques et al., 2013). Three groups were tested: a control group which had a non-irradiated treatment, a group with 3 kiloGray (kGy) irradiation and a group with 5 kGy irradiation (Henriques et al., 2013). Results showed that gamma irradiation was effective at 3 and 5 kGy and that sanitation processes needed to be improved (Henriques et al., 2013).
Analysis Methods Available There are several different methods to sense, enumerate, and identify the microorganisms present in a food matrix. Test types will fall under one of the three categories: biological, chemical, or physical (Jay et al., 2005). Understanding various types of testing is critical to determine which test will work best for your sample type, the accuracy, the cost of testing, and the results to be achieved, qualitative or quantitative.
Biological Methods
Serotyping. While serotyping is typically used to distinguish pathogens that are gram-negative such as Salmonella and Escherichia, it can also help to distinguish some gram-positive bacteria such as Listeria (Jay et al., 2005). To identify pathogens by serotyping the method will distinguish what polysaccharide side chain is present. “Typical serotyping scheme is the use of specific antibodies (antiserum) to identify homologous antigens,” (Jay et al., 2005). There are two main types of antigens, O and H. The O antigens are made up of the O polysaccharide side chains on the outer surface of the bacteria and are identified 35 35 by their chemical make-up (Centers for Disease Control and Prevention, 2015e; Jay et al., 2005). H antigens contain protein structures and are threadlike shaped that make up part of the flagella (Centers for Disease Control and Prevention, 2015e). If a serotype contains a soluble antigen gel diffusion will be used, otherwise agglutination methods are used for antigen particulates (Jay et al., 2005).
Traditional enumeration. Traditional enumeration methods have been used for many years. As we progress into the future, technology progresses as well. PCR (polymerase chain reaction) machines can now detect for presence of microorganisms in less than an hour. Although this is a wonderful tool that can be used in industry to release products sooner traditional methods are still extremely important for learning, accuracy, and a second analysis for confirmation if a rapid method is being used. There are many different medias for various pathogens that can be used when performing traditional isolation methods to determine if a food product has been contaminated. Testing usually will consist of a non-selective pre- enrichment medium to help repair cells and grow pathogenic cells (U. S. Food and Drug Administration, 2014). In many cases when searching for a specific pathogen more than one type of enrichment broth needs to be used to increase the chances of finding multiple serotypes (U. S. Food and Drug Administration, 2014). After enrichment period, an allotted incubation time will correspond with the media used. Once media have been incubated, an isolation of colonies will be done on a selective agar. To determine further what strain or serotype the pathogen is, the BAM can be used for determination of medias and directions (W. H. Andrews, 1998). 36 36 Molecular Genetic Methods
PCR. Polymerase chain reaction (PCR) has become popular in the food industry due to quick result times and the ability to move forward with shipping product. PCR copies DNA by choosing a target segment of the DNA coding for a specific pathogen of interest, then the segment DNA is amplified. In simple terms, DNA is denatured by heating and unwound, each parent strand is used as a template to make the complimentary daughter strand (McPherson & Moller, 2000). Primers will attach to their complementary target sequences in the denatured DNA template and the polymerase will add corresponding pairs of free nucleotides to the parent strand (McPherson & Moller, 2000). As discovered by Watson and Crick, the nucleotide adenosine (A) always pairs with thymine (T), and cytosine (C) always pairs with guanine (G), and the making of the daughter cell follows these rules (McPherson & Moller, 2000). After performing the heating process again, which is usually around 94-95°C, it now has enough DNA to find the target sequence and detect for specific pathogen (McPherson & Moller, 2000).
Chemical Methods Adenosine triphosphate (ATP) is the energy within a living cell which dissipates after 2 hours of cell death (Jay et al., 2005). By using simple strategies, such as the luciferase system, to search for ATP an estimation of the microbial load that is present can be obtained. By using a luminometer the detection of cells can be seen due to emitting light when luciferase is in the presence of ATP (Jay et al., 2005). “The amount of light produced by firefly luciferase is directly proportional to the amount of ATP added,” (Jay et al., 2005). When this method was first being developed specifically for meat the problem of nonmicrobal ATP was faced (Jay et al., 2005). By use of the centrifuge, cation exchange resin, and 37 37 filtration, bacteria was able to be collected and amounts analyzed (Jay et al., 2005; Wood, 1983).
Physical Methods
Fiber optics. Using optical fiber made from glass or polymeric material is one of many physical methods for detecting microorganisms. Jay explains the concept of fiber optics as follows, “A fiber optic biosensor uses electronic or optical transduction to monitor a biological reaction, and reports it as an optical signal,” (Jay et al., 2005). The basic procedure for this type of analysis is to have an optical fiber probe covered with an antibody of concern, a laser light will move throughout the fiber and emerge as an evanescent wave (Jay et al., 2005). When the antigen binds with the antibody a fluorescent signal will travel up to the detection system (Jay et al., 2005). Light sources will typically consist of dyes (Jay et al., 2005). With a 20 minute detection time S. typhimurium colonies were able to be recovered from the waveguides (Jay et al., 2005). With small-ruminant consumption on the rise understanding the prevalence of microorganisms such as Salmonella and Listeria at a retail level helps contribute to baseline data and can allow for the food industry to find areas of improvement from farm to fork. The chosen microorganisms for this project are known to be unfavorable to our health and are two of the leading pathogens for illness and death.
METHODOLOGY
Sample Collection In order to determine a local and national prevalence of Salmonella, Listeria, generic E. coli, and coliforms, whole muscle and ground goat and lamb meat samples were collected. Local samples were defined as those purchased in California, and other U.S. retail samples identified online and sourced from businesses within the United States. Local retail samples were purchased, kept chilled below 4°F in a transport cooler, and delivered directly to the lab for analysis. Online samples were delivered within one to two business days from time of purchase. Samples were kept frozen during delivery. All purchased samples were then kept in a freezer at or below 0°C/32°F until subsequent analysis. A total of 100 samples were collected from both local and online markets.
Sample Processing Sample processing including microbial analysis, confirmation and isolation for Salmonella and Listeria both followed the Bacteriological Analytical Manual (BAM) procedures and used a PCR instrument, BAX System Q7 (DuPont; Wilmington, DE) for rapid detection (U. S. Food and Drug Administration, 2016a, 2017a). Sample processing for generic E. coli, coliforms and APC followed the 3M petri film guidelines (3M, 2015, 2017). 3M petri film methods, calculations and interpretation methods used are recognized in the BAM (U. S. Food and Drug Administration, 2001, 2017b). 39 39 Microbial Analysis for Salmonella Buffered peptone water (BPW; Remel, Lenexa, KS) was prepared and put into an incubator to be warmed to 35°C. Twenty-five grams of the thawed meat sample was aseptically transferred from its original packaging into a sterile blender™ bag (Nasco, Fort Atkinson, Wisconsin) with a mesh screen. The warmed 225 mLs of BPW was added to the same sterile blender bag as the meat. The bag was then massaged for 120 seconds. The sample was then incubated at 35°C for 20 – 24 hours (DuPont, 2005). After incubation was complete, a rack file was prepared using the PCR instrument software, BAX System Q7 (DuPont; Wilmington, DE). Labels, cluster tubes, and rack files were placed into corresponding positions with samples. The lysis reagent was made by adding 150 µL of the protease to one 12- mL bottle of lysis buffer provided by the BAX kit. Lysis reagent was aseptically transferred to each tube in 200 µL increments. Once the lysis reagent was in the cluster tubes 5 µL of each enriched sample was transferred to the corresponding lysis tube, using a new pipet for each sample. Caps were aseptically placed onto the lysis tubes. The DuPont™ Thermal Block (DuPont; Wilmington, DE) was set to the gram-negative program and samples began lysis. The thermal block brought the samples to 37°C for 20 minutes, then to 95°C for 10 minutes, and lastly held at 4°C for a minimum of 5 minutes or until removed from the chilling block. Prompts from the BAX instrument were then followed to ensure readiness for Salmonella testing. Caps were removed from lysis tubes, 50 µL of lysate was transferred into PCR tubes, and tubes were aseptically capped with optical lenses. The PCR tubes were loaded into the instrument, the remainder of prompts were followed, and analysis began. After a four-hour period of DNA amplification, results were read and recorded. 40 40 Positive Confirmation of Salmonella Samples Positive confirmation for Salmonella was performed using the following steps. One mL of original sample incubated in BPW was transferred into 9 ml of Rappaport-Vassiliadis soya peptone broth (RV; 3M, St. Paul, MN). These tubes were incubated for 18-20 hours at 42°C. After incubation of RV, the samples were streaked onto petri dishes with Xylose Lysine Desoxycholate (XLD; Remel, Lenexa, KS) and Brilliant Green Sulfur (BGS; Remel, Lenexa, KS) agars. Incubation of confirmation plates occurred for 18-24 hours at 37°C. After 24 hours, plates were analyzed for positive Salmonella growth and confirmed. Salmonella on XLD plates appear as red colonies with black centers, Salmonella colonies on BGS appear as red to pink-white. If unsuccessful, this step would be repeated up to four times to isolate positives.
Isolation of Salmonella Positives After incubation, typical Salmonella colonies were selected, added to 9 ml of TSB, and grown for 18-24 hours at 37°C. Once the final incubation period passed, 1 ml of the enrichment was added to cryo-tubes containing 100 µL of glycerol. The cryo-tubes were then frozen at -80°C.
Microbial Analysis for Listeria To test qualitatively for Listeria, the BAM methodology from Chapter 10 was followed (U. S. Food and Drug Administration, 2017a). Retail meat samples were aseptically weighed out to 25 grams and placed into 225 mL of buffered Listeria enrichment broth (BLEB; BD Difco, Sparks, MD) with pyruvate. The bag was then massaged for 120 seconds. Incubation took place for 4 hours at 30°C. After incubation of BLEB, 5 ml of 30% ethanol was added to each bottle of Listeria selective supplement, modified oxford (Oxoid, Hampshire, England). 41 41
After the powder in the bottle was dissolved, 2.25 ml of the supplement was added to each sample. Incubation continued for 24 to 48 hours at 30°C. The rapid screening method, AOAC Official Method 2003.12, referenced in the BAM manual was used to determine if there was any Listeria present (U. S. Food and Drug Administration, 2017a). Similarly, to the Salmonella analysis, the lab equipment for the PCR BAX machine was prepared. Labels, cluster tubes, and rack files were placed into corresponding positions with samples. The lysis reagent was made by adding 150 µL of protease to a 12 mL bottle of lysis buffer. The lysis reagent and sample were then added to the cluster tubes in 200 and 5 µL increments, respectively. New pipet tips were used for each sample and cluster tube caps were secured when transfers were completed. The cluster tubes were placed on the DuPont™ Thermal Block and set to run the gram positive program. Cluster tubes were heated to 55°C for 60 minutes, then to 95°C for 10 minutes and lastly held at 4°C for at least 5 minutes or until removed from the chilling block. Prompts from the BAX were then followed to ensure machine was ready for Listeria testing. Caps were removed from lysis tubes, 50 µL of lysate was transferred into PCR tubes, and tubes were aseptically capped using optical lenses. The PCR tubes were loaded into the instrument, the remainder of prompts were followed, and analysis began. After a 4-hour period of DNA amplification, results were read and recorded.
Positive Confirmation of Listeria Samples If the Listeria PCR results were positive a confirmation test was performed. Agar plates were premade by filling them with 15 ml of Tryptic Soy Agar (TSA; BD Difco, Sparks, MD). Plates were set under the fume hood to harden and were labeled and refrigerated until ready to use. Once ready to use 1 ml of the original 42 42
BLEB enriched sample was pipetted onto the agar. A sterile spreader was used to cover the entire plate with the sample. The plate rested for 2-4 hours. While sample plates were resting the modified oxford was prepared and poured onto the plates when ready. Agar was placed into a 35°C incubator and plates were checked for growth at 24 and 48 hours. At 24 hours the appearance of colonies were 1 mm in diameter, with gray or black colonies surrounded by a black halo. At 48 hours size of colonies were 2-3 mm in diameter. The color of the colonies at 48 hours were black with a black halo and a sunken center.
Isolation of Listeria Positives After incubation, five typical Listeria colonies were selected and streak onto TSA for purity. Incubation took place for 24 to 48 hours at 30°C. Once the 24 or 48-hour period passed a colony was added to a cryo-tube containing 100 µL of glycerol. The cryo-tubes were frozen at -80°C.
Microbial Analysis for E. coli, coliforms and APC Retail meat samples were added at 11 grams with 99 ml of BPW to a mesh sterile blender™ bag (Nasco, Fort Atkinson, Wisconsin). The bag was placed into a lab stomacher (Fisher Scientific, Hampton, NH) for 2 minutes at 300 RPM. One ml of the sample was transferred from the bag onto each 3M petri film. Then 11 ml of the sample was transferred from the original sample bag into a bottle of 99 ml of BPW. The bottle was then mixed for 25 seconds by arching the bottle in a 180° angle. Again, one ml of the sample was transferred from the bottle onto each 3M petri film. Then four more 1:10 dilutions were made in the same fashion stopping at 10-6. A total of two APC plates and two generic E.coli/coliform plates were made per dilution starting from 10-1 to 10-4. Dilutions 10-5 and 10-6 had two 43 43
APC plates each. The plates were stacked 20 high and placed into the incubator at 37°C for 48 hours. Petri films were recorded once the incubation periods were completed.
Statistical Analysis Positive frequency was calculated for Salmonella and Listeria. All samples collected, 100, were able to be tested. Mean, median, lowest number, highest number and standard error was calculated for APC, generic E. coli, and coliform. All samples collected, 100 were tested for mesophilic aerobic bacteria, however only 86 were within readable limits on the petri film. All 100 samples were also tested for generic E. coli and coliforms but due to error in the lab only 29 of the plates were able to be read.
RESULTS AND DISCUSSION
As reported in Table 1, there were 100 retail meat samples collected in total, 72 goat, 26 lamb, and 2 mix tested for the presence of Salmonella and Listeria. When asking retailers for goat meat a tray labeled mutton mix was sampled from. When asked if this was goat or lamb retailers were unsure and therefore will be labeled as mix. The data in Table 1 show that 0 out of 100 of the small-ruminant samples were positive for Salmonella and that 1 out of 100 (1.00%) samples were confirmed positive for Listeria. As reported in Table 1, the positive Listeria sample was whole, frozen, goat muscle from a local market in California.
Table 1. Presence of Salmonella and Listeria, in all samples tested from the U.S. # Organism Specie Location n Presence positive Online 0 5 0.00% Goats Local 0 67 0.00% Online 0 3 0.00% Salmonella Lambs Local 0 23 0.00% Online 0 0 0.00% Mix Local 0 2 0.00% Total 0 100 0.00%
Online 0 5 0.00% Goats Local 1 67 1.49% Online 0 3 0.00% Listeria Lambs Local 0 23 0.00% Online 0 0 0.00% Mix Local 0 2 0.00% Total 1 100 1.00%
All 100 samples were analyzed for mesohilic aerobic bacteria. The average mesophilic aerobic mesophilic bacteria count was 4.60 log10 cfu/g of meat 45 45
(standard error = 0; see figure 1). Fourteen out of 100 samples plated were found to be greater than 250 x 6 log cfu/gram of meat.
Figure 1. Mesophilic aerobic plate counts for 86 retail and online small-ruminant samples. Reported numbers are the mean log and error bars on the sample mean represent standard error.
Unfortunately, due to error in the lab only 29 of the 100 samples were tested for generic E. coli. The average generic E. coli count was 0.10 log10 cfu/g (standard error = 0.058). Results from the 3M petri film generic E. coli testing showed that there was a presence in both species, goat and lamb. Testing showed that 2 out of 20 goat meat samples and 1 out of 9 lamb meat samples were hosting an exceptional environment for potential enteric pathogen presence. Generic E. coli indication was present in one online goat meat sample and two locally sourced samples purchased within California, one goat meat and one lamb meat. Similarly, to generic E. coli, coliforms were not tested on all 100 samples collected due to error in the lab. The average coliform count was 0.49 log10 cfu/g (standard error = 0.198; see figure 2). When testing the 29 samples for coliforms both species, goat and lamb, showed indication of fecal contamination. 46 46
Figure 2. Generic E. coli and coliform counts for 29 retail and online small- ruminant samples. Reported numbers are the mean log and error bars on the sample mean represent standard error.
CONCLUSION
Small-ruminant consumption is on the rise in the U. S. and has the potential to be contaminated with harmful food pathogens. Listeria was present in 1% of small-ruminants samples that were evaluated. Although Salmonella was not detected in any of the samples, the potential for enteric pathogens was identified by testing for coliforms and generic E. coli, indicators of fecal contamination. Other studies confirm this in red meat. Salmonella and Listeria are important microorganisms for the food industry to continue monitoring. Salmonella is the second most occurring pathogen for illnesses and the first most occurring pathogen for illnesses resulting in hospitalization and death. Listeria follows behind in third for leading causes of pathogens contributing to death. There is no question that these pathogens are deadly and finding preventative measures to reduce and eliminate them are key to ensuring the safety of the food supply. Understanding the importance of food safety from farm to fork is a collective job that all participants should be aware of. Controlling the environment and handling from the farm through processing to the retail level is key to a safer food supply. Overall, further research should focus on testing more retail samples. Understanding where contamination occurs during processing, as well as the prevalence in retail food products, is vital to a safer food supply.
REFERENCES
3M. (2015). Petrifilm e. Coli/coliform count plates. Retrieved from https://multimedia.3m.com/mws/media/701951O/product-instructions-3m- petrifilm-e-coli-coliform-count-plate.pdf
3M. (2017). Petrifilm aerobic count plates. Retrieved from https://multimedia.3m.com/mws/media/695832O/product-instructions-3m- petrifilm-aerobic-count-plate.pdf
Ajmone-Marsan, P., Colli, L., Han, J. L., Achilli, A., Lancioni, H., Joost, S., . . . Lenstra, J. A. (2014). The characterization of goat genetic diversity: Towards a genomic approach. Small Ruminant Research, 121(1), 58-72. doi:http://dx.doi.org/10.1016/j.smallrumres.2014.06.010
Algino, R. J., Badtram, G. A., Ingham, B. H., & Ingham, S. C. (2009). Factors associated with Salmonella prevalence on pork carcasses in very small abattoirs in Wisconsin. Journal of Food Protection, 72(4), 714-721.
Aslam, M., Checkley, S., Avery, B., Chalmers, G., Bohaychuk, V., Gensler, G., . . . Boerlin, P. (2012). Phenotypic and genetic characterization of antimicrobial resistance in Salmonella serovars isolated from retail meats in Alberta, Canada. Food Microbiology, 32(1), 110-117. doi:https://doi.org/10.1016/j.fm.2012.04.017
Aymerich, T., Picouet, P. A., & Monfort, J. M. (2008). Decontamination technologies for meat products. Meat Science, 78, 114-129.
Berry, E. D., & Cutter, C. N. (2000). Effects of acid adaptation of Escherichia coli O157:H7 on efficacy of acetic acid spray washes to decontaminate beef carcass tissue. Applied and Environmental Microbiology, 66(4), 1493-1498. doi:10.1128/aem.66.4.1493-1498.2000
Bonke, R., Wacheck, S., Bumann, C., Thum, C., Stüber, E., König, M., . . . Fredriksson-Ahomaa, M. (2012). High prevalence of Salmonella enterica subsp. Diarizonae in tonsils of sheep at slaughter. Food Research International, 45(2), 880-884. doi:10.1016/j.foodres.2011.01.050
Centers for Disease Control and Prevention. (2014). Estimates of foodborne illness in the United States: CDC 2011 estimates: Findings. 49 49 Centers for Disease Control and Prevention. (2015a, 11/6/15). E. Coli (Escherichia coli) general information. Retrieved from https://www.cdc.gov/ecoli/general/index.html
Centers for Disease Control and Prevention. (2015b, 4/17/14). Estimates of foodborne illness in the United States. Retrieved from http://www.cdc.gov/foodborneburden/estimates-overview.html
Centers for Disease Control and Prevention. (2015c). Foodborne germs and illnesses. Retrieved from http://www.cdc.gov/foodsafety/foodborne- germs.html
Centers for Disease Control and Prevention. (2015d, 6/10/15). Listeria (listeriosis) clinical features/signs and symptoms. Retrieved from https://www.cdc.gov/listeria/outbreaks/ice-cream-03-15/signs- symptoms.html
Centers for Disease Control and Prevention. (2015e). Serotypes and the importance of serotyping Salmonella. Retrieved from http://www.cdc.gov/salmonella/reportspubs/salmonella-atlas/serotyping- importance.html
Centers for Disease Control and Prevention. (2016a, 12/12/2016). Listeria (listeriosis). Retrieved from https://www.cdc.gov/listeria/
Centers for Disease Control and Prevention. (2016b). Listeria (listeriosis) people at risk. Retrieved from https://www.cdc.gov/listeria/risk.html
Centers for Disease Control and Prevention. (2016c). Listeria (listeriosis) prevention. Retrieved from https://www.cdc.gov/listeria/prevention.html
Centers for Disease Control and Prevention. (2016d). Salmonella: Reports of selected Salmonella outbreak investogations. Retrieved from http://www.cdc.gov/salmonella/outbreaks.html
Centers for Disease Control and Prevention. (2017). Listeria outbreaks. Retrieved from https://www.cdc.gov/listeria/outbreaks/
Centers for Disease Control and Prevention. (2018, 3/1/18). Listeria. Retrieved from https://www.cdc.gov/listeria/faq.html
50 50 Chapman, P. A., Cerdán Malo, A. T., Ellin, M., Ashton, R., & Harkin, M. A. (2001). Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb carcasses and in raw beef and lamb products in south Yorkshire, UK. International Journal of Food Microbiology, 64(1), 139-150. doi:https://doi.org/10.1016/S0168-1605(00)00453-0
Crum, N. F. (2002). Update on Listeria monocytogenes infection. Current Gastroenterology Reports, 4(4), 287-296.
Da Rocha, L., Gunathilaka, G., & Zhang, Y. (2012). Antimicrobial-resistant Listeria species from retail meat in metro Detroit. Journal of Food Protection, 75(12), 2136-2141. doi:10.4315/0362-028X.JFP-12-774
Donnelly, C. W. (2001). Listeria monocytogenes: A continuing challenge. Nutrition Reviews, 59(6), 183-194. doi:doi:10.1111/j.1753- 4887.2001.tb07011.x
Dormedy, E. S. (1999). Implementation and microbial verification of HACCP (hazard analysis and critical control points) systems and HACCP intervention methods in meat processing establishments for the reduction of foodborne pathogens.
Dormedy, E. S., Brashears, M. M., Cutter, C. N., & Burson, D. E. (2000). Validation of acid washes as critical control points in hazard analysis and critical control point systems. Journal of Food Protection, 63(12), 1676- 1680. doi:10.4315/0362-028x-63.12.1676
Duffy, L., Barlow, R., Fegan, N., & Vanderlinde, P. (2009). Prevalence and serotypes of Salmonella associated with goats at two australian abattoirs. Letters in Applied Microbiology, 48(2), 193-197. doi:10.1111/j.1472- 765X.2008.02501.x
DuPont. (2005). Bax system q7 user guide (Vol. 3.6, pp. 1-194).
Elder, R. O., Keen, J. E., Siragusa, G. R., Barkocy-Gallagher, G. A., Koohmaraie, M., & Laegreid, W. W. (2000). Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing. Proceedings of the National Academy of Sciences of the United States of America, 97(7), 2999-3003.
Ellebracht, E. A., Castillo, A., Lucia, L. M., Miller, R. K., & Acuff, G. R. (1999). Reduction of pathogens using hot water and lactic acid on beef trimmings. Journal of Food Science, 64(6), 1094-1099. doi:10.1111/j.1365- 2621.1999.tb12289.x 51 51 Food and Agriculture Organization of the United Nations. (2014). Sources of meat. Meat and Meat Products. Retrieved from http://www.fao.org/ag/againfo/themes/en/meat/backgr_sources.html
Gill, C. O., McGinnis, J. C., & Bryant, J. (1998). Microbial contamination of meat during the skinning of beef carcass hindquarters at three slaughtering plants. International Journal of Food Microbiology, 42(3), 175-184. doi:http://dx.doi.org/10.1016/S0168-1605(98)00074-9
Goetsch, A. L., Merkel, R. C., & Gipson, T. A. (2011). Factors affecting goat meat production and quality. Small Ruminant Research, 101(1–3), 173-181. doi:http://dx.doi.org/10.1016/j.smallrumres.2011.09.037
Gordon Luikart, L. G., Laurent Excoffier, Jean-Denis Vigne, Jean Bouvet, and Pierre Taberlet. (2001). Multiple maternal origins and weak phylogeographic structure in domestic goats. Proceedings of the National Academy of Sciences of the United States of America, 98(10), 5927-5932.
Haiping Li, H. w., Jean-Yves D'Aoust, and John Maurer. (2013). Food microbiology: Fundamentals and frontiers (Vol. 4).
Hanlon, K. (2015). Presence of Salmonella, Escherichia coli O157 and campylobacter in small-ruminants.
Hauge, S. J., Wahlgren, M., Røtterud, O.-J., & Nesbakken, T. (2011). Hot water surface pasteurisation of lamb carcasses: Microbial effects and cost-benefit considerations. International Journal of Food Microbiology, 146(1), 69-75. doi:http://dx.doi.org/10.1016/j.ijfoodmicro.2011.02.003
Henriques, L. S. V., da Costa Henry, F., Barbosa, J. B., Ladeira, S. A., de Faria Pereira, S. M., da Silva Antonio, I. M., . . . dos Reis, E. M. F. (2013). Elimination of coliforms and Salmonella spp. In sheep meat by gamma irradiation treatment. Brazilian Journal of Microbiology, 44(4), 1147-1153. doi:10.1590/S1517-83822014005000003
Hunt, J. M. (2010). Shiga toxin-producing Escherichia coli (stec). Clin Lab Med, 30(1), 21-45. doi:10.1016/j.cll.2009.11.001
Hurd, H. S., Enøe, C., Sørensen, L., Wachman, H., Corns, S. M., Bryden, K. m., & Grenier, M. (2008). Risk-based analysis of the Danish pork Salmonella program: Past and future. Risk Analysis: An International Journal, 28(2), 341-351. 52 52 Jacob, M. E., Foster, D. M., Rogers, A. T., Balcomb, C. C., & Sanderson, M. W. (2013). Prevalence and relatedness of Escherichia coli O157:H7 strains in the feces and on the hides and carcasses of U.S. Meat goats at slaughter. Applied and Environmental Microbiology, 79(13), 4154-4158. doi:10.1128/aem.00772-13
Jay, J. M., Loessner, M. J., & Golden, D. A. (2005). Modern food microbiology (Vol. 7th). New York: Springer.
Johnson, J. L., Doyle, M. P., & Cassens, R. G. (1990). Incidence of Listeria ssp. In retail meat roasts. Journal of Food Science, 55(2), 572-572. doi:10.1111/j.1365-2621.1990.tb06818.x
Karakolev, R. (2009). Incidence of Listeria monocytogenes in beef, pork, raw- dried and raw-smoked sausages in Bulgaria. Food Control, 20(10), 953- 955. doi:https://doi.org/10.1016/j.foodcont.2009.02.013
Kim, C., Stein, R. A., & Pao, S. (2015). Comparison of the microbial quality of lamb and goat meat acquired from internet and local retail markets. Journal of Food Protection, 78(11), 1980-1987.
Lawley, R. (2013). Salmonella. Food Safety Watch. Retrieved from http://www.foodsafetywatch.org/factsheets/salmonella/
Liao, Y.-T., Miller, M., Loneragan, G., Brooks, J., Echeverry, A., & Brashears, M. (2014). Non-O157 shiga toxin-producing Escherichia coli in U.S. Retail ground beef. Journal of Food Protection, 77(7), 1188-1192. doi:10.4315/0362-028X.JFP-13-518
Maharjan, M., Joshi, V., Joshi, D. D., & Manandhar, P. (2006). Prevalence of Salmonella species in various raw meat samples of a local market in Kathmandu. Ann New York Academy of Sciences, 1081, 249-256. doi:10.1196/annals.1373.031
Maradiaga, M., Miller, M., Thompson, L., Pond, A., Gragg, S., Echeverry, A., . . . Brashears, M. (2015). Salmonella in beef and produce from Honduras. Journal of Food Protection, 78(3), 498-502. doi:10.4315/0362-028X.JFP- 14-450
McCann, M. S., Sheridan, J. J., McDowell, D. A., & Blair, I. S. (2006). Effects of steam pasteurisation on Salmonella typhimurium dt104 and Escherichia coli O157:H7 surface inoculated onto beef, pork and chicken. Journal of Food Engineering, 76(1), 32-40. doi:10.1016/j.jfoodeng.2005.05.024 53 53 McEvoy, J. M. (2003). The prevalence of Salmonella spp. In bovine faecal, rumen and carcass samples at a commercial abattoir. Journal of Applied Microbiology, 94(4), 693-700. McEvoy, J. M., Doherty, A. M., Finnerty, M., Sheridan, J. J., McGuire, L., Blair, I. S., . . . Harrington, D. (2000). The relationship between hide cleanliness and bacterial numbers on beef carcasses at a commercial abattoir. Letters in Applied Microbiology, 30(5), 390-395. doi:10.1046/j.1472- 765x.2000.00739.x
McPherson, M. J., & Moller, S. (2000). Pcr: BIOS Scientific Publishers.
Meyer, C., Fredriksson-Ahomaa, M., Sperner, B., & Märtlbauer, E. (2011). Detection of Listeria monocytogenes in pork and beef using the vidas® LMO2 automated enzyme linked immunoassay method. Meat Science, 88(3), 594-596. doi:https://doi.org/10.1016/j.meatsci.2011.01.035
Morand-Fehr, P., & Boyazoglu, J. (1999). Present state and future outlook of the small ruminant sector. Small Ruminant Research, 34(3), 175-188. doi:http://dx.doi.org/10.1016/S0921-4488(99)00071-1
Nakyinsige, K., Man, Y. B. C., & Sazili, A. Q. (2012). Halal authenticity issues in meat and meat products. Meat Science, 91(3), 207-214. doi:http://dx.doi.org/10.1016/j.meatsci.2012.02.015
Neusely de Silva, M. H. T., Valeria C. A. Junqueira, Neliane F. A. Silveira, Maristela S. do Nascimento, and Renato A. R. Gomes. (2013). Microbiological examination methods of food and water: CRC Press/Balkema.
Nomura, K., Yonezawa, T., Mano, S., Kawakami, S., Shedlock, A. M., Hasegawa, M., & Amano, T. (2013). Domestication process of the goat revealed by an analysis of the nearly complete mitochondrial protein-encoding genes. PLoS ONE, 8(8), 1-15. doi:10.1371/journal.pone.0067775
Nutsch, A. L. (1998). Bacterial decontamination of meat surfaces through the application of a steam pasteurization process. (9914220 Ph.D.), Kansas State University, Ann Arbor. Retrieved from http://search.proquest.com/docview/304422206?accountid=10349 Pinto Neto, M., Beraquet, N. J., & Cardoso, S. (2013). Effects of chilling methods and hot-boning on quality parameters of m. Longissimus lumborum from bos indicus nelore steer. Meat Science, 93(2), 201-206. doi:http://dx.doi.org/10.1016/j.meatsci.2012.08.024 54 54 Pollott, G., & Wilson, R. T. (2009). Sheep and goats for diverse products and profits. Rome Retrieved from http://www.fao.org/docrep/ 011/i0524e/i0524e00.htm.
Ransom, J. (2004). Preharvest and postharvest intervention strategies to reduce prevalence of pathogens in beef and beef products. In K. Belk & J. Sofos (Eds.): ProQuest Dissertations Publishing.
ReligionFacts.com. (2015). Islam. Retrieved from http://www.religionfacts.com/islam
Retzlaff, D., Phebus, R., Nutsch, A., Riemann, J., Kastner, C., & Marsden, J. (2004). Effectiveness of a laboratory-scale vertical tower static chamber steam pasteurization unit against Escherichia coli O157:H7, Salmonella typhimurium, and Listeria innocua on prerigor beef tissue. Journal of Food Protection, 67(8), 1630-1633. doi:10.4315/0362-028x-67.8.1630
Savell, J. W., & Smith, G. C. (2009). Meat science: Laboratory manual. [Lamb Carcass Evaluation]. (Eight Edition). American Press.
Smith, M. G. (1992). Destruction of bacteria on fresh meat by hot water. Epidemiology and Infection, 109(3), 491-496.
Thomas, R., Anjaneyulu, A. S. R., & Kondaiah, N. (2008). Effect of hot-boned pork on the quality of hurdle treated pork sausages during ambient temperature (37 ± 1 °c) storage. Food Chemistry, 107(2), 804-812. doi:http://dx.doi.org/10.1016/j.foodchem.2007.08.079
Tortora, G. J., Funke, B. R., & Case, C. L. (2013). Microbiology an introduction (Vol. 11). Boston, Massachuesetts: Pearson.
Trivedi, S., Reynolds, A. E., & Chen, J. (2008). Effectiveness of commercial household steam cleaning systems in reducing the populations of Listeria monocytogenes and spoilage bacteria on inoculated pork skin surfaces. LWT - Food Science and Technology, 41(2), 295-302. doi:10.1016/j.lwt.2007.02.017
U. S. Department of Health & Human Services. (2018, 4/21/18). Listeria. Retrieved from https://www.foodsafety.gov/poisoning/causes/ bacteriaviruses/listeria/index.html
U. S. Food and Drug Administration. (2001). Bacteriological analytical manual chapter 3 aerobic plate count. 55 55 U. S. Food and Drug Administration. (2014). Ora laboratory manual. Retrieved from https://www.fda.gov/downloads/ScienceResearch/ FieldScience/LaboratoryManual/UCM092226.pdf
U. S. Food and Drug Administration. (2016a). Bacteriological analytical manual chapter 5 Salmonella. Irradiation in the production, processing and handling of food, 21 C.F.R. § 179 (2016b).
U. S. Food and Drug Administration. (2017a). Bacteriological analytical manual chapter 10 detection of Listeria monocytogenes in foods and environmental samples, and enumeration of Listeria monocytogenes in foods. Retrieved from https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ ucm071400.htm
U. S. Food and Drug Administration. (2017b). Bam 4: Enumeration of Escherichia coli and the coliform bacteria.
United States Department of Agriculture. (2013). Salmonella questions and answers. Retrieved from http://www.fsis.usda.gov/wps/portal/fsis/ topics/food-safety-education/get-answers/food-safety-fact- sheets/foodborne-illness-and-disease/salmonella-questions-and-answers/
United States Department of Agriculture. The goat industry: Structure, concentration, demand and growth (pp. 25).
United States Department of Agriculture. (1998). Nationwide sponge microbiological baseline data collection program: Swine. Retrieved from https://www.fsis.usda.gov/wps/wcm/connect/cc3fe72c-a5cf-4b55-bbff- 3e0e8a396d46/Baseline_Data_Swine.pdf?MOD=AJPERES
United States Department of Agriculture. (2010). FSIS strategic plan for year 2011-2016.
United States Department of Agriculture. (2011a). Goat from farm to table. Retrieved from http://www.fsis.usda.gov/wps/wcm/connect/a8dd3e40- 5b49-42ff-86a5-138c708fa5b3/Goat_from_Farm_to_Table. pdf?MOD=AJPERES
United States Department of Agriculture. (2011b). The nationwide microbiological baseline data collection program: Market hogs survey. Retrieved from http://www.fsis.usda.gov/wps/wcm/connect/d5c7c1d6- 09b5-4dcc-93ae-f3e67ff045bb/Baseline_Data_Market_Hogs_2010- 2011.pdf?MOD=AJPERES 56 56 United States Department of Agriculture. (2011c). Small-scale U.S. Goat operations (pp. 1-41). USDA.
United States Department of Agriculture. (2011d). Verification of procedures for controlling fecal material, ingesta, and milk in slaughter operations. In Food Safety and Inspection Service (Ed.).
United States Department of Agriculture. (2013). Fresh pork from farm to table. Retrieved from https://www.fsis.usda.gov/wps/portal/fsis/topics/food- safety-education/get-answers/food-safety-fact-sheets/meat- preparation/fresh-pork-from-farm-to-table/CT_Index
United States Department of Agriculture. (2015). The nationwide microbiological baseline data collection program: Beef-veal carcass survey. Retrieved from https://www.fsis.usda.gov/wps/wcm/connect/b03963cc-0845-4cfe-b94e- 2c955ee5e2ef/Beef-Veal-Carcass-Baseline-Study- Report.pdf?MOD=AJPERES
United States Department of Agriculture. (2016a, February 29, 2016). Ground beef and food safety. Retrieved from http://www.fsis.usda.gov/ wps/portal/fsis/topics/food-safety-education/get-answers/food-safety-fact- sheets/meat-preparation/ground-beef-and-food-safety/CT_Index
United States Department of Agriculture. (2016b). Serotypes profile of Salmonella isolates from meat and poultry products January 1998 through December 2014. Retrieved from https://www.fsis.usda.gov/wps/portal/fsis/topics/ food-safety-education/
United States Department of Agriculture. (2018, 1/31/2018). Baseline data. Retrieved from https://www.fsis.usda.gov/wps/portal/fsis/topics/data- collection-and-reports/microbiology/baseline/baseline
Valerie M. Bohaychuk, G. E. G., Pablo Romero Barrios. (2011). Microbiological baseline study of beef and pork carcasses from provincially inspected abattoirs in Alberta, Canada. The Canadian Veterinary Journal (CVJ), 52(10), 1095-1100.
Vipham, J. L., Brashears, M. M., Loneragan, G. H., Echeverry, A., Brooks, J. C., Chaney, W. E., & Miller, M. F. (2012). Salmonella and campylobacter baseline in retail ground beef and whole-muscle cuts purchased during 2010 in the United States. Journal of Food Protection, 75(12), 2110-2115. doi:10.4315/0362-028x.jfp-12-077
57 57 W. H. Andrews, A. J., and T. S. Hammack. (1998). Bacteriological analytical manual (bam). Retrieved from http://www.fda.gov/Food/ FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.
Wang, X.-M., Lü, X.-F., Yin, L., Liu, H.-F., Zhang, W.-J., Si, W., . . . Liu, S.-G. (2013). Occurrence and antimicrobial susceptibility of Listeria monocytogenes isolates from retail raw foods. Food Control, 32(1), 153- 158. doi:https://doi.org/10.1016/j.foodcont.2012.11.032
Wong, T. L., Nicol, C., Cook, R., & MacDiarmid, S. (2007). Salmonella in uncooked retail meats in new zealand. Journal of Food Protection, 70(6), 1360-1365. doi:10.4315/0362-028x-70.6.1360
Wood, C. J. S. a. J. M. (1983). The rapid estimation of microbial contamination of raw meat by measurement of adenosine triphosphate (atp). Journal ofApplied Bacteriology, 55(3), 429-438.
Xia, X., Meng, J., McDermott, P. F., Ayers, S., Blickenstaff, K., Tran, T.-T., . . . Zhao, S. (2010). Presence and characterization of shiga toxin-producing Escherichia coli and other potentially diarrheagenic e. Coli strains in retail meats. Applied and Environmental Microbiology, 76(6), 1709-1717. doi:10.1128/aem.01968-09
Yang, D., Xiaolei, Z., Min, X., Arefnezhad, B., Zongji, W., Wenliang, W., . . . Yu, J. (2015). Reference genome of wild goat (capra aegagrus) and sequencing of goat breeds provide insight into genic basis of goat domestication. BMC Genomics, 16(1), 1-11. doi:10.1186/s12864-015-1606-1
Zhao, C., Ge, B., De Villena, J., Sudler, R., Yeh, E., Zhao, S., . . . Meng, J. (2001). Prevalence of campylobacter spp., Escherichia coli, and Salmonella serovars in retail chicken, turkey, pork, and beef from the greater washington, d.C., area. Applied and Environmental Microbiology, 67(12), 5431-5436. doi:10.1128/AEM.67.12.5431-5436.2001