INFLUENCE OF METHODOLOGY ON THE RECOVERY OF SALMONELLA FROM

RETAIL CHICKEN

by

MUSTAFA SIMMONS

(Under the direction of Daniel L. Fletcher)

ABSTRACT

Two experiments were conducted to determine the influence of methodology on the

recovery of Salmonella on retail chicken. The first experiment was designed to determine if

there was a difference in the incidence of Salmonella-positive carcasses reported by USDA-FSIS

and the incidence of Salmonella-positive carcasses at the retail level using an exhaustive

procedure. The second experiment was conducted to determine the effects of sampling

methodology on the recovery of Salmonella from whole carcasses.

In the first study 85 of the 251, or 33.9 %, of the retail carcasses were Salmonella-

positive. This is higher than both the 20 % from the 1994-1995 baseline and the 10.4 % reported

by USDA-FSIS in 1998. The incidence determined in the first study may have been higher due

to methodology, different sampling location (retail level versus processing plant), post-process

contamination, or the presence of giblets.

In the second study the significantly more (p<0.0001) carcasses were found Salmonella-

positive using whole carcass enrichment method than using the 30ml aliquot method

recommended by USDA-FSIS. Also the incidence of Salmonella-positive carcasses determined

by the whole carcass enrichment method (38 %) is comparable to the incidence determined in the

first experiment (33.9 %), and the incidence determined by the Aliquot method (13%) was

comparable to the incidence reported by USDA-FSIS in 1998 (10.4 %). Thus showing

methodology does have an effect on Salmonella recovery when low numbers of Salmonella per

carcass are expected.

INDEX WORDS: Salmonella, Broilers, Whole Carcass Enrichment, Methodology

INFLUENCE OF METHODOLOGY ON THE RECOVERY OF SALMONELLA FROM

RETAIL CHICKEN

by

MUSTAFA SIMMONS

B.S., University of Florida, 2000

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

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2002

© 2002

Mustafa Simmons

All Rights Reserved

INFLUENCE OF METHODOLOGY ON THE RECOVERY OF SALMONELLA FROM

RETAIL CHICKEN

by

MUSTAFA SIMMONS

Approved:

Major Professor: Daniel L. Fletcher

Committee: Nelson A. Cox

Mark E. Berrang

Electronic Version Approved:

Gordhan L. PatelDean of the Graduate SchoolThe University of GeorgiaAugust 2002

iv

ACKNOWLEDGMENTS

I would like to thank the following people for their support and guidance throughout my

time here.

My mother, for her continuous support and encouragement. She always entertained my

non-stop questioning as a child and told me I would be a scientist before I knew what one was.

Dr. Daniel L. Fletcher, for sharing his knowledge and experience, and helping me build a

strong foundation as a future scientist. He taught me to be more confident in my work and

abilities.

Dr. Berrang and Dr. Cason, for their training, laboratory space, intellectual input, and the

advice given concerning future plans.

Dr. Cox, for serving on my committee despite his busy schedule, and for doing the work

that served as a foundation for my projects.

Nicole Bartenfeld, David McNeal, and Lynda Jones, for their assistance with my projects

and for making my time in the lab more enjoyable.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ................................................................................................. iv

INTRODUCTION ...............................................................................................................1

LITERATURE.....................................................................................................................3

References.....................................................................................................................22

CHAPTER 1: RECOVERY OF SALMONELLA FROM RETAIL BROILERS USING A WHOLE CARCASS ENRICHMENT PROCEDURE......................................................52 Abstract .........................................................................................................................53

Materials and Methods..................................................................................................55

Results and Discussion .................................................................................................57

Acknowledgments.........................................................................................................61

References.....................................................................................................................62

CHAPTER 2: COMPARISON OF SAMPLING METHODOLOGY FOR THE DETECTION OF SALMONELLA ON WHOLE BROILER CARCASSES.....................74 Abstract .........................................................................................................................75

Materials and Methods..................................................................................................77

Results and Discussion .................................................................................................78

Acknowledgments.........................................................................................................80

References.....................................................................................................................81

SUMMARY.......................................................................................................................84

1

INTRODUCTION

Salmonellosis is the second most frequently reported cause of bacterial foodborne illness.

Each year 8000-39,000 cases are reported. However, estimates are as high as 2 million cases a

year (CDC). The Economic Research Service (ERS) estimates that the annual economic costs

due to Salmonella infections are $2.4 billion. This estimate includes the medical costs due to

acute infection, the value of time lost from work, and the economic value of premature deaths.

Poultry has historically been linked to Salmonella and has consistently received intensive

media and political attention. In 1996, the United States Department of Agriculture implemented

the Hazard Analysis Critical Control Point Systems ;Final Rule, also known as the Mega Reg.

The primary purpose of the Mega Reg is to reduce foodborne illness. One of the requirements of

the Mega Reg is that meat and poultry plants implement HACCP plans. HACCP plans identify

points where implementation of intervention steps can reduce potential hazards, such as

pathogenic microorganisms. To verify the effectiveness of HACCP plans microbiological testing

is required. In the case of poultry plants, tests are performed for generic E. coli and Salmonella.

Since the implementation of the Mega Reg the Food Safety and Inspection Service (FSIS)

reported a decrease in the percentage of Salmonella-positive broiler carcasses from a baseline of

20% to approximately 10.4%. This reduction is based on aliquot sampling in processing plants.

To date no research has been published to determine if this reduction has influenced Salmonella

incidence in fresh retail poultry products.

2

The purpose of this research was to determine the incidence of Salmonella positive

broiler carcasses at the retail level using an exhaustive methodology. The second part is to

determine the effects of sampling methodology on the recovery of Salmonella from broiler

carcasses.

3

LITERATURE REVIEW

The genus Salmonella has been widely researched and is of historical importance, as is

evident by the numerous books and review articles pertaining to the organism such as those by

van Oye (168), Cabello et al. (28), Neidhart et al. (115), Saeed et al. (138),Wray and Wray (176),

and Bell and Kyriakides (16). This review will cover only the basics of Salmonella in general

and its importance in poultry with an emphasis of current methodolgy and occurences.

SALMONELLA

Salmonella was first observed by Eberth in 1880 in spleen sections and mesenteric lymph

nodes from patients who died from typhoid. At that time the organism was known as typhoid

bacillus (64). In 1886 Salmon and Smith isolated Bacillus cholerae-suis from swine suffering

from hog cholerae (46). Other similar organisms were isolated from outbreaks of foodborne

disease and infected animals. The genus Salmonella, in honor of Salmon, was created in 1900 by

Lignieres to accommodate these organisms (36).

The genus Salmonella include Gram-negative, facultatively anaerobic, rod-shaped

bacteria belonging to the family Enterobacteriaceae. They are distinguished biochemically by

their inability to ferment lactose, production of hydrogen sulphide, and decarboxylation of amino

acids lysine and ornithine. And serologically they are distinguished by 3 major antigens: H or

flagellar antigen; O or somatic antigen; and Vi antigen, a superficial antigen overlying the O

antigen (71, 81,179).

4

The principal habitat of Salmonella is the intestinal tract of man and animals. Salmonella

serotypes can be host-adapted, or non-host-adapted or ubiquitous ( 95). Host adapted serotypes

usually cause serious systemic diseases. For example S. typhi, S. paratyphi-A, and S. sendai are

strictly human serotypes, whereas S. abortus-ovis, S. gallinarum, and S. typhi-suis are ovine,

avian, and porcine adapted serotypes, respectively (95). These host-adapted serotypes cannot

grow on minimal medium without growth factors, unlike ubiquitous Salmonella serotypes (95).

Ubiquitous Salmonella, such as S. typhimurium, can cause a wide range of clinical symptoms,

from asymptomatic infection to typhoid-like illnesses in immunocomprimised individuals or

highly susceptible animals such as mice (23).

Most non-typhoidal Salmonella enter the body via food contamination. However, person-

to-person spread of Salmonella may also occur (71). Symptoms usually begin 6-48 hours after

exposure and include nausea, vomiting, fever, diarrhea and abdominal pain. The duration of

symptoms vary but is usually 2 to 7 days ( 45, 71, 179).

If infection is to occur, Salmonella must survive several of the body’s basic defenses.

The first defense being the acidic environment of the stomach. Exposure to such low pH induces

the expression of genes involved in pH homeostasis and stabilization of macromolecules (56).

Those that survive the stomach must then contend with bactericidal compounds, such as bile

salts, of the small intestines (71). However, like other members of the family Enterobacteriaceae,

Salmonella copes well with these stress conditions (15). Finally, Salmonella must resist removal

by peristalisis. Carter and Collins (31) reported that 80% of the S. enteriditis that survive

passage through the stomach of mice are passed with the feces within 6-10 hours post-infection.

5

Salmonella that survive the defenses of the alimentary tract may then attach and invade

the intestinal mucosa (55, 83). Invasion causes the epithelial cells to release various

proinflammatory cytokines. They envoke an acute inflammatory response and may be

responsible for damage to the intestine (59).

Enterotoxin is also produced by many strains of Salmonella (83, 92, 140, 143). The

Salmonella enterotoxin acts by stimulating host adenylate cyclase. This leads to increased levels

of cellular cyclic AMP, which causes an increase in the concentration of sodium and chloride

ions, and consequently fluid accumulation in the intestinal lumen, which leads to diarrheal

symptoms (60).

Cytotoxin production has also been reported in some strains of Salmonella (9,119).

Reitmeyer et al. (131) report that Salmonella cytotoxin is an outer membrane component, and

thus is in direct contact with host cells, possibly having a role in cell damage or invasion. Koo et

al. (88) report Salmonella cytotoxin inhibits protein synthesis.

Salmonella specifically invade lymph follicles that are located in the intestinal wall of the

ileum. From there, the pathogens drain to the regional lymph nodes, where macrophages may

prevent further spread, causing the infection to remain localized in the intestine. If the

macrophages are unable to limit the spread, Salmonella can cause a systemic infection (15).

SALMONELLA IN POULTRY

In the early 1900’s Salmonella pullorum, caused one of the most important diseases in

commercial poultry (128). The disease was first described by Rettger in 1900 as a septicemia of

young chicks as reported by Snoeyenbos (150). With up to an 80% mortality rate in baby chicks,

6

pullorum disease is considered the greatest single factor that limited the early expansion of the

U.S. poultry industry (150). Another important poultry disease during this period was fowl

typhoid, caused by Salmonella gallinarum. It was first described by Klein in 1889 (128). Signs

of both diseases are dead embryos in the incubator or early chick mortality, anorexia,

dehydration, labored breathing, diarrhea, ruffled feathers, weakness, and adherence of feces to

the vent. The diseases also cause decreased egg production, reduced fertility and lower

hatchability (147).

In 1935 the National Poultry Improvement Program (NPIP) was established in the United

States (150). The goal of NPIP was to prevent the spread of Salmonella pullorum and

Salmonella gallinarum by preventing disease transmission to progeny by testing breeder flocks

(150). The testing programs and control measures implemented by NPIP resulted in a lower

incidence of the diseases in most developing countries during the 1950s and 1960s (126, 150,

154). The NPIP and Auxiliary Provisions is still currently active (3).

Since the 1940s there has been a rapid increase in the isolation of non-host specific

Salmonella serovars in humans and animals (57, 65). Reports have shown that poultry and

poultry products have been the main sources of non-host specific Salmonella infecting humans

(57, 77, 94, 109,144, 156).

Broiler chickens can harbor Salmonella from hatch through grow-out, however Millner

and Shaffer (110) reported that newly hatched chicks are most susceptible to intestinal

colonization. They showed colonization was dose dependent and varied with day of challenge.

Day old chicks could be colonized with as few as 5 Salmonella cells, and later colonization

required higher doses.

7

Transmission of Salmonella from hen to chick may occur as a result of infection of the

ovary and oviduct. Timoney et al. (163) inoculated the crop of adult hens with 106 Salmonella.

The organisms were found in the yolk or albumen of eggs of about 10% of the hens, indicating

transovarian transmission. During incubation, contaminated eggs containing gas-forming

bacteria may explode thereby contaminating other eggs. The chicks hatching from these eggs will

then be contaminated (136). Bailey et al (5) found that only one contaminated egg could lead to

substantial Salmonella spread throughout the hatching cabinet. Cason et al. (32) showed

horizontal spread of Salmonella also occurs during hatching of chicks. Cox et al (43) reported

that 75% of all hatchery samples were positive for Salmonella, and 38 of 40 randomly selected

samples contained greater than 103 Salmonella cells per salmple. However, Lahellec and Colin

(93) reported that Salmonella seotypes originating in the hatchery were less important in the final

product than those present in the grow-out house or those introduced into the house by vectors

during rearing.

There are numerous potential sources of Salmonella contamination throughout an

integrated poultry system, including feed, rodents, insects, litter, wild birds, humans, transport

coops, and the processing plant environment (82, 129, 135). Poultry feed is a recognized source

of Salmonella. Salmonella can be isolated regularly from feed ingredients, not only from

ingredients of animal origin but from plant origin as well, such as soy, rape, palm kernal, rice

bran and cottonseed (174). Davies et al. (48) reported the Salmonella serovars which were

isolated in the feed mill were also most widely distributed throughout the broiler flock. However,

Bailey et al. (8) identified ten different serotypes from feed samples, and only on one occasion

was the same serotype found in the feed also found on the fully processed broiler carcass.

8

Several methods are used to eliminate Salmonella from poultry feed, including heating,

irradiation and addition of chemical additives. The heat routinely used to manufacture feed often

can only reduce coliforms and Salmonella by 2 to 3 logs (42). Thus Salmonella may survive

after pelleting of highly contaminated rations. Leeson and Marcotte (96) showed finished feed

can be treated with doses of gamma irradiation up to 10kGy without affecting nutrional quality.

However, this method of decontamination is unlikely to be adopted by the industry, due to the

high cost of installing irradiation facilities in a feed mill. Nevertheless, neither heat nor

irradiation will protect feed from recontamination during storage and distribution. Hinton and

Linton (69), however, reported the addition of antimicrobial compounds, such as organic acids,

to poultry feed were effective in reducing the prevalence of Salmonella infections in poultry.

Insects, rodents and wild animals may also serve as vectors for Salmonella in poultry

houses (44, 67, 121). Flies that breed in broiler and layer houses are a potential reservoir of

Salmonella (4, 63). Olsen and Hammack (121) isolated Salmonella enteritidis from 2 of 15

pools of houseflies present in caged layer facilities. They also isolated Salmonella infantis from

house flies and dump flies, and Salmonella heidelberg from houseflies.

Rodents also play an important role in the epidemiology of Salmonella infection in

poultry. Henzler and Opitz (67) reported that on 5 farms where no Salmonella enteritidis was

isolated 6% of 232 mice were positive for Salmonella, while on 5 farms where Salmonella

enteritidis was present 31.8% of 483 mice were positive for Salmonella, 24% for Salmonella

enteritidis. A bacterial count from the mouse feces yielded more than 105 Salmonella enteritidis

cells per fecal pellet.

9

Wild birds may be a source of Salmonella in poultry and other domestic animals (47, 72,

78). Craven et al. (44) found that up to 33% of wild bird samples (intestinal and fecal) obtained

near poultry houses were positive for Salmonella.

Farm workers may also contribute to the spread of Salmonella. Bailey et al (8) reported

recovery rates of Salmonella from boot swabs at 12%, and from the outside dirt near the entrance

doors to houses at 6.1%, thus demonstrating how easily human movements and cross-

contamination can occur.

Poultry litter has also been shown to be a reservoir for Salmonella (19, 53, 165). Poultry

may ingest large numbers of Salmonella by picking at fecal or cecal droppings of littermates.

Snoeyenbos et al. (151) reported Salmonella was spread rapidly from infected day old chicks to

penmates reared on litter. These infected chickens will also shed Salmonella cells in their feces

throughout grow out. Tucker (165) reported that S. pullorum and S. gallinarum persisted for 11

weeks on new litter, but for only 3 weeks in built-up litter. Decreased moisture and high pH due

to dissolved ammonia, were shown to be the cause of the higher bactericidal activity of old built-

up litter compared with new litter (166). Carr et al (30) demonstrated that water activity (Aw) is a

critical parameter in the growth of Salmonella on the poultry litter surface. Hayes et al (66)

suggest that a low Aw environment (<.84), achieved via drying of the litter by ventilation or other

means, could deter the establishment or continuation of Salmonella contamination in a

commercial poultry house. Studies by Mallison et al (105) and Himathongkham et al (68)

support a reduction in Salmonella achieved by lowering the Aw in both laboratory and field

studies.

10

Limawongpranee et al (102) reported S. blockley persisted on a farm for more than a year

even after all-in-all-out cleansing and disinfection of the poultry house had been applied. This

suggest ordinary sanitation and control measures may fail to prevent infection or reinfection with

Salmonella.

One approach to eliminate Salmonella from the live bird environment is competitive

exclusion. Nurmi and Rantala (118) showed that Salmonella infections could be prevented by

feeding chicks anareobic cultures of normal intestinal flora of adult birds. Competitive exclusion

inhibits proliferation or reduces the number of Salmonella by the following mechanisms; creation

of a restrictive physiological environment, competition for bacterial receptor cites, elaboration of

antibiotic type substances, or the depletion of or competition for essential substrates (134).

Large-scale commercial field trials were conducted in Puerto Rico and Georgia to test the

efficacy of mucosal competitive exclusion (MCE) to protect broiler chickens against natural

Salmonella colonization of the intestinal tract and subsequent contamination of the processed

chickens from these treated flocks. In Puerto Rico, incidence rates of intestinal colonization with

Salmonella were reduced from 11% to 2%, and from 41% to 10% in processed carcasses (18). In

Georgia, incidence rates of intestinal colonization were reduced from 2% to undetectable and

from 9.5% to 4.5% in processed carcasses (6). However, Bailey et al (7) showed when

Salmonella contamination is present before the competitive exclusion microflora, significant

protection from Salmonella colonization is difficult to achieve. Despite the great amount of

research in developing competitive exclusion cultures and techniques, this strategy is not totally

effective in eliminating Salmonella from live birds.

11

Studies have also been conducted to develop vaccines to eliminate Salmonella from live

birds (104, 148, 149, 157). Killed Salmonella vaccines have not produced convincing levels of

protection against ubiquitous Salmonella challenge (12, 58, 164). However, poultry can be

successfully immunized against host specific species of Salmonella, such as S. gallinarum, using

live attenuated vaccines (149). These vaccines have been used extensively among breeding

flocks (14). In contrast, vaccines for Salmonella serovars associated with food poisoning , until

recently, were used only experimentally in poultry.

An inactivated S. enteriditis vaccine, created by iron restriction, has been licensed in

European countries, and shown to reduce the level of flock infection (14). Plasmid-cured strains

are avirulent, or of greatly reduced virulence, and are immunogenic and protective in the cases of

S. enteriditis (113) and S. gallinarum (10). However, the plasmids of some sreotypes, such as S.

typhimurium (13), are not associated with virulence and plasmid-cured strains of such bacteria

would not have the same effect. Thus far, no study has demonstrated good cross-protection

between different Salmonella serovars for any significant duration after vaccination (14).

A standard management practice in commercial broiler production is the removal of feed

immediately prior to transportation to slaughter and processing. Feed-withdrawal programs have

been shown to reduce intestinal contents, thus leaving less ingesta and feces available for

potential carcass contamination during transport and processing (26, 107, 122, 123, 170).

Rigby and Pettit (132) reported that 8hr feed withdrawal prior to cooping reduced the

amount of feces deposited in the coops by one fourth, but did not influence the incidence of

Salmonella-positive cloacal or cecal contents or the level of Salmonella within feces. However,

Ramirez et al (130) found that feed withdrawal increased the incidence of Salmonella isolation

12

from crops of experimentally and naturally infected broilers. Feed withdrawal has been reported

to cause an increase in broiler crop pH and a decrease in lactic acid concentration (34, 74). These

conditions may provide an improved environment for the proliferation of Salmonella. Durant et

al. (52) reported the crop contents from hens deprived from feed can cause increased virulence of

Salmonella by increasing the expression of genes necessary for intestinal invasion. Byrd et al.

(27) reported that the use of lactic acid or formic acid in drinking water during feed withdrawal

caused a significant reduction in the recovery of Salmonella typhimurium from broiler crops.

During transport of birds to the processing plant, birds defecate and consequently step or

lie in fecal matter. This leads to an increase in contamination with organisms of fecal origin

(125, 145). Unclean coops can also lead to contamination with Salmonella. Rigby et al. (133)

reported that the incidence of Salmonella-positive feathered carcasses increased from 42%

pretransport to 93% following transport in contaminated plastic coops. Bailey et al. (8) reported

Salmonella molade was isolated from broiler carcasses, and on pretransport coops, but not from

any on-farm samples, thus its likely that the coops were contaminated from a previous flock.

Wing flapping during unloading and also during hanging and bleeding creates aerosols and

dust that are deposited on the feathers and skin of nearby birds. Zottola et al. (180) reported isolating

Salmonella from the air in poultry plant unloading zones.

Despite efforts to control Salmonella, processing plants may actually contribute to the

increase in the incidence of Salmonella positive carcasses. Lillard (98) reported 5% of broilers

entering processing plants harbored Salmonella, and the incidence increased to 36% for

processed carcasses from the same flock. This is not only due to cross-contamination but also the

processing procedure itself changes the bacterial population on the bird’s skin. Thomas and

13

McMeekin (161) report that during processing, the predominantly Gram-positive microflora of

the skin of non-processed poultry carcasses is removed and replaced by heterogeneous

population consisting of mostly gram negative bacteria.

Defeathering in commercial poultry operations is achieved by dipping carcasses in hot

water immediately before feathers are picked by automatic equipment (124). Scalding affects

feather follicles and allows removal of feathers with as much as 80% less force (50). Scalding

and plucking of carcasses have been implicated as major sites of cross-contamination by

Salmonella (112, 116, 175). Humphrey and Lanning (75) reported that feces from the chicken

during scalding will dissociate to form uric acid and ammonium urate in the water, and this

provides buffering that maintains scald water at pH 6.0, which maximizes the heat tolerance of

Salmonella. Humphrey (73) reported that at pH 5.9-6.0 Salmonella typhimurium had a thermal

death value at 52 C (time required to destroy 90% of organisms initially present at a given

temperature, D 52 C) of 34.5 minutes. Yang et. al (177) reported destruction of Salmonella

typhimurium on chicken skin was observed only at a scalding temperature of 55 C or higher.

However, studies have shown that scalding at a higher temperature causes increased toughness in

cooked chicken meat (85, 127, 146).

Several researchers looked at the effect of scald water pH on the bactericidal activity (73,

100, 106 ). Humphrey et al. (76) reported the adjustment of chicken scald water to 9.0 ± 0.2

lowered the D 52 C value of Salmonella typhimurium from 34.5 to 1.25 min. However,

Humphrey and Lanning (75) found scalding at pH 9.0 had no effect on the incidence of

Salmonella contamination on broiler carcasses. Similarly, Lillard et al. (100) reported that the use

14

of 0.5% acetic acid scald water would kill bacteria in the scald water but did not significantly

reduce the bacteria on the broiler carcass.

Studies report Salmonella are isolated more frequently after picking than any other

operation (24, 111). Mulder et al. (112) reported picking resulted in cross-contamination when a

marker strain of E. coli was used to artificially contaminate broilers both internally and

externally.

Birds subjected to a sub-scald, above 56 C, have the epidermis removed during picking,

and as a result the microorganisms colonizing the stratum corneum are removed. The exposed

dermal skin is covered with capillary-sized channels and crevices and provides a new surface for

attachment and colonization (161).

The attachment of bacteria to poultry skin is well documented (117, 162, 97). Bacteria

attach to carcasses and form biofilms. Biofilms are adherent microcolonies enveloped with an

extensive polysaccharide matrix. Costerton et al. (35) state that bacteria within biofilms are more

firmly attached to surfaces than individually attached cells. In order to colonize new surfaces,

individual cells must be able to disperse from mature biofilms and reattach elsewhere (1).

Chilling is often thought to be key step in reducing bacteria on chicken carcasses (20, 22,

86, 90, 108). However, Surkiewicz et al. (158) found no reduction in the incidence of

Salmonella contamination after chilling. Morris and Wells (111) found a higher incidence of

Salmonella on carcasses after chilling than on carcasses prior to chilling. Campbell et al. (29)

reported that in federally inspected processing plants 5.5% of the carcasses entered the chiller

contaminated with Salmonella and 11.6% exited the chiller contaminated with Salmonella.

15

Immersion of the carcass causes a fluid film to cover the skin. Thomas (160) showed this

film contains several serum proteins, amino acids and other soluble or suspended compounds.

The presence of organic matter in this fluid film may explain why all skin bacteria are not

destroyed by chlorine. Yang et. al (177) showed that there was less than a 1 log reduction of

Salmonella typhimurium when carcasses spent 50 minutes in the chiller with 20 to 30ppm

residual free chlorine present. And how long the chilling water is used significantly reduced the

bactericidal activity.

Lillard and Thomson (101) reported hydrogen peroxide at 6,600ppm or higher in chiller

water was effective in reducing populations of aerobic microorganisms by 95-99.5%. However,

the reaction of H2O2 with catalase from broiler carcasses causes discoloration and swelling.

Fletcher et al. (54) reported that a sodium bicarbonate and H2O2 spray had little or no effect

following immediate use, but reduced bacterial loads by 0.3 log after 7 days storage.

Thomas and McMeekin (162) reported immersion chilling also changes the

microtopography of the skin causing capillary sized channels to form, which can be colonized by

bacteria. Skin swelling, associated with the uptake of water during immersion, opens and

exposes channels to contaminants present in the water. Lillard (97) reported an increase in the

number of attached bacteria with increased immersion time.

Microbial loads on meats are typically reduced by spraying carcasses with high pressure

jets of water or dipping carcasses in solutions containing various antimicrobial agents (e.g.,

hydrogen peroxide, ozone, chlorine dioxide, lactic acid, or trisodium phophate [TSP]) (21, 33,

79, 178). TSP is commonly used in the poultry industry (84, 139, 173). Proposed by Bender and

Brotsky (17), the process involves immersing whole birds for 15 minutes in a 10% solution of

16

Av-Gard™ trisodium phosphates and then allowing excess TSP to drip from the bird. Giese (61)

suggested that this process works partly by removing a fat coating on poultry skin, allowing the

bacteria to be washed from the carcass more effectively.

Coopen et al. (33) reported Salmonella incidence was reduced from 74.0% to 9.4% after

Av-Gard™ treatment of immersion-chilled broiler carcasses. MPN analysis of the positive

samples before and after treatment showed a decrease from an average 115 organisms per carcass

to 0.6 organisms per carcass. Salvat et al. (139) reported similar results, obtaining close to a 2 log

reduction measured by a miniature MPN procedure. Kim et al. (84) saw a reduction in the

number of inoculated Salmonella cells on TSP treated broiler skin using a scanning electron

microscope. However, it was difficult to see the difference in the number of attached bacteria for

non-inoculated skins due to the low number of bacteria present.

Despite these results, the efficacy of TSP in reducing attached Salmonella has been

questioned. Lillard (99) stated that TSP may not reduce the number Salmonella but residual TSP

may cause a lower recovery rate due to an increase in pH which causes lethal or sub-lethal injury

to Salmonella cells. Scantling et al. (142) found that TSP treated turkeys did not have a lower

levels of Salmonella and Listeria at retail level. In fact 56% and 88% of TSP treated turkeys

were positive for Salmonella and Listeria, while 31% and 6% of non-TSP turkeys were positive

for the same organisms.

Surface roughness and laminar flow velocity have an effect on efficacy of TSP treatment.

The time and temperature dependent changes that occur in the microtopography of poultry skin

as a result of water uptake during immersion chilling may hinder the penetration of antimicrobial

agents or high-pressure washes, thereby protecting bacteria (89). In a study by Korber et al. (89)

17

TSP treatment resulted in nearly 100% killing of control biofilms (no crevices on surface), while

the viability of remaining cells at the biofilm base was 83 +/- 12% when a crevice width of

.15mm was present. Bacteria within biofilms are also generally more resistant to biocides and

antibiotics than their planktonic (not in present in biofilm) counterparts. Dhir and Dodd (49)

showed that planktonic Salmonella enteritidis cells were more susceptible to biocide challenge

(phenol, chlorohexidine, and Virkon) than attached cells.

Breen et al. (21) reported quaternary ammonia compounds (QACs), especially

cetylpyridinium chloride and hexadecyltrimethylammonium bromide, were effective in both

reducing and inhibiting the attachment of Salmonella typhimurium. The QACs produced these

effects at concentrations lower than the concentration of TSP required to produce the same

effects.

Nayak et. al (114) reported zinc chloride at 25 and 50mM concentrations was effective in

detaching firmly attached Salmonella on chicken skin, and also interfered with the attachment of

Salmonella to chicken skin. They postulated this is due to electropositive zinc ions selectively

binding to negative charges on the cell membrane, and compete for specific receptor sites on the

skin surface. Zinc chloride would change the conformation of surface proteins so that receptor

sites on the skin surface are no longer exposed.

RECOVERY OF SALMONELLA FROM POULTRY CARCASSES

To verify measures taken to control Salmonella are effective, it is necessary to have

standard methods for the recovery an isolation of Salmonella from poultry carcasses. Unlike

clinical specimens, direct plating cannot be used to recover Salmonella from poultry carcasses.

18

The physiological state of Salmonella can greatly affect its culturability (36). Therefore methods

for recovery and isolation from a food product, where Salmonella is present in low numbers and

may be in a debilitated state due to chilling, heating or chemical treatments, are different from

methods used for clinical samples, where Salmonella is often in high numbers and in a vegetative

state (171).

Methods for the recovery and isolation of Salmonella from food products generally

include the following steps: sample collection and preparation; nonselective incubation, which

allows cells in a debilitated state to recover; selective enrichment, which inhibits other bacteria

allowing Salmonella to multiply to detectable levels; inoculation of plating media; screening of

suspect colonies; and biochemical and serological confirmation (2). The initial step of sample

collection and preparation is important in sampling poultry carcasses, because the non-

homogeneous nature of the carcass makes getting a representative sample difficult (137).

Therefore several sampling techniques have been developed. Some of the most common are

tissue swabbing, tissue maceration, and the whole carcass rinse.

Swabbing is a relatively easy and convenient method of sampling and is non-destructive.

However, Kotula and Pandya (91) reported Salmonella counts were higher on the breast of

carcasses than on the thighs or drums. Thus swabbing an arbritrary area of the carcass may

produce an inaccurate estimate of bacterial numbers or whether or not they are even present.

Cox et al (39) showed this inaccuracy was magnified if carcasses sampled were hard chilled or

crust frozen.

Tissue excision and maceration may be more effective than swabbing for recovery of

bacteria from whole broiler carcasses. Cox et. al (39) reported maceration yielded significantly

19

higher APC and enterobacteriaceae counts than swabbing on whole birds. However, again the

selection area is arbitrary and may not be a true representation of the carcass. Also this method is

destructive and carcasses must be downgraded after samples are taken.

Based on the uneven distribution, and relatively low numbers of Salmonella per carcass,

the whole carcass rinse method is considered the most appropriate for recovering bacteria from

fresh poultry carcasses (40, 169). Cox et al. (40) reported the number of Salmonella-positive

carcasses detected by the whole carcass rinse procedure was significantly higher (p = 0.001) than

for rinsing or maceration using neck-skin. Sarlin et al. (141) reported the number of Salmonella-

positive samples obtained by carcass rinse or skin sampling were significantly higher (p<0.05)

than with the swab method. The researchers suggest the sensitivity of the skin sampling was due

to using skin directly associated with the thoracic inlet and crop removal as opposed to neck skin.

There are several variations of the rinse procedure, based on the volume of rinse media used, and

the aliquot of rinse solution incubated for preenrichment.

These variations in volumes and aliquots can affect the ability to recover Salmonella.

Surkiwiez et al. (158) who reported that incubation of 270ml out of 300ml lactose broth used to

rinse poultry, as opposed to a 10 ml aliquot was four times more effective in recovering

Salmonella. Cox et.al (41) rinsed carcasses in 100ml of sterile water, incubated all of the

recoverable rinse, and reported as few as 20 inoculated cells could be recovered from carcasses,

even if the carcasses had been stored at –23 C for 3 or 6 months. To eliminate this variation Cox

and Blankenship (38) rinsed carcasses in 500ml of lactose broth and then incubated the entire

carcass in the rinse solution for 24h at 37 C. They reported as few as 8 inoculated Salmonella

cells per carcass could be recovered.

20

IDENTIFICATION OF SALMONELLA

There are several methods for confirming the identity of Salmonella including

biochemical, serological, and nucleic acid-based assays ( 36, 167, 171). The primary

biochemical methods employ triple sugar iron (TSI) and lysine iron agar (LIA) slants, which are

based on carbohydrate fermentation (TSI), lysine decarboxylation (LIA), and H2S production

(51). However, these tests alone should not be used for ultimate confirmation; as there are

atypical strains of Salmonella which can for example ferment lactose or not produce H2S, thus

giving false negative results (25). Another disadvantage of biochemical test is they require

isolated colonies, and the isolation procedure is time consuming (167).

There are several serological assays available for the detection of salmoenlla, including

agglutination-based serology and enzyme-linked immunosorbant assay (ELISA) (11).

Advantages of using serological techniques is they are less time consuming, and have a high

specificity (167). In terms of reliability, the false positive rates for environmental samples

ranged from 2.3 to 5.8% and the false negative rates from 0.4 to 22% (129, 159). Some non-

Salmonella such as citrobacters may cross-react with some somatic antisera, causing false

positives (171). Also, Hoorfar et al. (70) reported some proportion of presumptive Salmonella

isolates identified by biochemical testing lack either O-antigen or H-antigen or both, thus cannot

be identified by conventional serological procedures.

Nucleic-acid based assays would detect Salmonella that lack antigens (70). One

commercial assay available, based on DNA probes, is the colormetric Gene Trak Salmonella

assay (37). The assay employs Salmonella-specific DNA probes that hybridize with the

21

ribosomal RNA of the bacteria and uses a colormetric system for detection (167). Giese (62)

reports the probe assay requires the presence 103-105 cells thus pre-enrichment would most likely

be necessary. False-negative rates for this assay of 1.25 to13.4% and false-positive of 0.57 to

4.9% have been reported (129, 155, 159) for raw chicken, poultry feed, and environmental

samples. One disadvantage is this assay is more labor-intensive than commercially available

ELISA test, and cost for the instruments are required (167).

Polymerase chain reaction (PCR) can be used with the rfb gene cluster, which contains

the genes responsible for the biosynthesis and assembly of the O-antigen repeat unit. This

amplified DNA can then be tested for hybridization with a labeled DNA probe. (87, 70). The

entire process, in which an amount of DNA equivalent to an overnight culture is obtained, can be

completed in 2 hrs, and requires at least 102-103 cells per ml (167).

SALMONELLA IN RETAIL POULTRY

A search of the Web of Science Database (80) in May, 2002 for references containing

both Salmonella and poultry resulted in 2,969 hits. However, less than 50 of these references

report the incidence of Salmonella on products at the retail level. Several of these retail

references report a high incidence of Salmonella (>50%). While some more recent papers report

relatively low incidences (<10%).

22

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52

CHAPTER 1

RECOVERY OF SALMONELLA FROM RETAIL BROILERS USING A WHOLE

CARCASS ENRICHMENT PROCEDURE1

53

ABSTRACT

Whole, fresh broiler carcasses were purchased from grocery stores over a 20-week period.

Carcasses were selected based on having intact packages, and a unique USDA plant number and

sell-by-date, such that each bird represented a single processing plant and processing day

combination. Carcasses were tested for Salmonella using a rinse aliquot obtained after whole bird

incubation in the rinse media for 24 h. Based on the number of unique processing plants (based

on USDA plant number) and expiration dates, the number of birds available each week ranged

from 6 to 17. Over the 20 week period, 251 independent carcasses from 14 processing plants

were tested. Salmonella-positive carcasses ranged from 0 (for one week) to over 60% (for three

weeks). Only four of the 20 weeks had an incidence of less than 20% positive carcasses. For the

entire 20-week study, 85 of the 251 total carcasses tested, or 33.9 %, were found to be

Salmonella-positive. For those processing plants from which more than 10 carcasses were

obtained, Salmonella-positive carcasses ranged from less than 20 % (two plants) to over 40 %

(four plants). These results indicate that a whole carcass enrichment may be more sensitive for

detecting Salmonella-positive carcasses when low numbers of Salmonella per carcass are

expected.

Keywords: salmonella, broilers, whole carcass enrichment

54

The incidence of Salmonella on poultry carcasses has been well documented and is of

international importance. Although there are numerous references reporting Salmonella

isolations from live poultry, production environments, and processing plants, fewer references

are available which report Salmonella incidence on carcasses, parts, meat, or products at the

retail level. Thirty-nine reports of Salmonella incidence in retail poultry products from 17

countries during the period from 1961 through 2001 are listed in Table 1. Recent interest in

Salmonella in retail poultry is shown by more than half of the reports during this 40 year period

being conducted since 1990. Sampling and microbiological methods are not consistent and

reported Salmonella incidence is extremely variable (1.2 to 76.6 %). However, it is interesting

that there has been no obvious change in the retail incidence of Salmonella in the period from

1961 through 1990, and since 1990 when approximately half of the studies were conducted.

Because of the association between food borne illness and meat and poultry, the United

States government mandated that all meat and poultry plants in the United States comply with the

Pathogen Reduction and Hazard Analysis Critical Control Point Rule, commonly referred to as

the Mega-Reg (3). Part of this rule requires Salmonella testing conducted by the Food Safety and

Inspection Service (FSIS) and compliance with a baseline performance standard. Since the

implementation of the Mega-Reg, FSIS reports that the percentage of Salmonella-positive poultry

carcasses has decreased from the baseline incidence of 20.0 % to 10.4 % when sampled

immediately after chilling (6).

A rinse sampling method was used in about a third of the studies shown in Table 1, but

rinse volumes and the volumes of liquid that were cultured have been quite variable. The official

FSIS procedure requires that a whole carcass be taken immediately after chilling and placed in a

55

bag to which 400 ml of sterile buffered peptone water is added. The bag is shaken for

approximately 1 minute, and a 30 ml aliquot is removed for subsequent analysis (3).

Although widely used, rinse sampling does have some limitations. Lillard (28) reported

that only a small percentage of bacteria are recovered by rinsing, while the majority of organisms

remain firmly attached to the carcass. Treatment with chlorine, trisodium phosphate (TSP), or

other antimicrobial agents used during processing may also hinder recovery of Salmonella.

Lillard (29) stated that TSP may not reduce the number of viable Salmonella, but residual TSP

may have a negative effect on Salmonella detection due to sub-lethal injury to Salmonella cells.

Given the opportunity to resusitate, however, these cells can be detected in samples taken at a

later time, such as at the retail level.

Differences in rinse sampling procedures can also cause variation in results. However,

this variation can be minimized by enrichment and incubation of the whole carcass at 37 C for 24

h as described by Cox and Blankenship (16). In that study, as few as 8 inoculated Salmonella

cells per carcass could be recovered when a carcass was rinsed in 500ml of lactose broth and the

whole carcass was incubated with the rinse. The purpose of the present experiment was to

determine the incidence of Salmonella-positive poultry carcasses at the retail level using the

whole carcass enrichment procedure.

MATERIALS AND METHODS

Sample Collection

Whole, fresh broiler carcasses were purchased, at weekly intervals from grocery stores in

the Northeast Georgia area over a 20-week period. All carcasses were purchased on a Monday or

2Becton Dickinson, Sparks, Maryland 21152

56

Tuesday to ensure that birds were at least two to three days post-processing. Carcass selection

was based solely on intact packaging and a unique processing plant (USDA establishment

number) and sell-by-date combination. Thus each carcass represented a unique plant and

processing day.

Since the purpose of the experiment was to sample carcasses obtainable at the retail level,

no effort was made to balance retail outlets or processing establishments. Therefore, once the

samples were identified as having a unique plant and day identification, the samples were

randomly reassigned to arbitrary plant identifications and original source information relative to

retail or processing plant identification was purged from the data set.

Salmonella Analysis

On the day of purchase, the exterior of each package was swabbed with 100% ethanol and

opened with a sterile scalpel. Using a fresh pair of sterile gloves for each carcass, the giblets

were removed and discarded. The entire carcass and package exudate were transferred to a

sterile 16x16 polyethylene bag containing 500ml of buffered peptone water2. The carcasses

within the bags were then vigorously shaken for 1 minute. The bags containing the whole bird

and rinse solution were incubated for 24h at 37 C similar to the method described by Cox and

Blankenship (16), but using the 1% buffered peptone water instead of lactose broth.

After incubation, 0.5ml of incubated rinse solution was transferred to 10ml Rappaport-

Vassiliadis broth2 and also to 10ml TT2 (Hajna) and incubated at 42 C for 24h. Each broth was

3Oxoid, Basinstoke, Hampshire RG24 8PW, UK

4Microgen, Camberley, Surrey GU15 3DT, UK

57

then streaked onto BG sulfa agar2 and modified lysine iron agar3 plates, and incubated for 24hr at

35 C. Suspect Salmonella colonies were picked and used to inoculate triple sugar iron2 and

lysine iron agar2 slants and incubated for 24h at 35 C. Presumptive positives were confirmed

using Poly O2 and Poly H4 serological agglutination tests.

Statistical Analysis

To test sampling integrity against the possibility of cross contamination, the order of

samples was analyzed using linear regression in the General Linear Models (GLM) procedure of

SAS (36) and by calculating Chi-square and Fisher’s Exact Test values using PROC FREQ of

SAS to test the independence of adjacent pairs of carcasses sampled each day. The last carcass

sampled on each of the 20 sampling days was not included in this statistical test because it had no

following carcass, so 231 pairs of carcasses were tested .

RESULTS AND DISCUSSION

The results for Salmonella incidence for the 251 carcasses sampled over the 20-week

period are presented by week and total in Table 1.2. Salmonella incidence was variable over the

20-week period, ranging from 0 % (week 8) to over 60 % (weeks 11, 14, and 15). For the entire

20-week period, there were 85 Salmonella-positive carcasses out of the total of 251 carcasses

tested (33.9%).

58

For those plants from which more than 10 carcasses were sampled, the incidence of

Salmonella positive carcasses varied from less than 20 % (Plants B and H) to over 40 % (Plants

C, D, E, and G) (Table 3). The plant data is presented only to illustrate that the higher than

expected incidence of Salmonella noted in Table 1.3 was not entirely due to a few plants.

Although all of the carcasses were from USDA-inspected facilities, only two of the 8 plants were

below the 20 % Salmonella standard when sampled at the retail establishment and tested using a

more stringent recovery methodology.

To test whether cross contamination of samples occurred during the experiment, the data

were subjected to both linear regression and frequency response analyses. If cross contamination

occurred during sampling the incidence of Salmonella would increase as birds were individually

sampled each week (thus would result in a positive slope) or that the occurrence of a Salmonella

positive carcass would result in a higher frequency of subsequent positive as opposed to negative

carcasses. The incidence of Salmonella-positive carcasses by sampling order (1 = first carcass

sampled each week, 2 = second carcass sampled each week, and so on through those weeks that

had 17 carcasses sampled) are presented in Table 1.4. Linear regression of the incidence of

positive carcasses by sample position showed that the slope was not significantly different from

zero (P = .915). Frequency analysis of samples indicated that the incidence of Salmonella-

positive carcasses was 36.2% following a Salmonella-negative carcass and 34.2% following a

Salmonella-positive carcass. Chi-square (P= .762) and Fisher’s Exact Test (P = .439) both

indicated that the incidence of Salmonella on a carcass was independent of the incidence on the

previous carcass. Therefore, there was no statistical indication of serial cross-contamination

between samples.

59

A Salmonella incidence of 33.9 % in whole chicken carcasses at retail would be

substantially higher than the FSIS reports of 20.0 % from 1994-1995 baseline (4) and the 10.4 %

reported in 1998 (6) for carcasses sampled immediately after chilling. The higher incidence of

Salmonella in the current study may be due to several differences from FSIS surveys: different

sampling methodology (incubating a 30 ml aliquot from a 400 ml rinse versus whole carcass

incubation in a 500 ml rinse), different sampling location (processing plant versus retail meat

case), possibility of post-processing contamination, presence of giblets in retail carcasses (as

opposed to in-plant sampling of carcasses without giblets), sampling retail whole carcasses which

represent only about 10% of broiler production as opposed to USDA sampling of all carcasses

being processed, regional versus national sampling, and seasonal versus year-round sampling.

The overall incidence of Salmonella-positive carcasses reported in this study is consistent,

however, with relatively recent work that also used rinse sampling of retail chicken in the United

States. A survey in Ohio in 1990 found 43.0 % of chicken carcasses and parts were Salmonella

positive (12) and one in Arkansas in 1991 found that 33.3 % of whole carcasses were Salmonella

positive (25).

An exhaustive recovery method should be expected to find more positives than a partial

aliquot method if relatively few bacteria are expected on the carcasses. In a study of the

effectiveness of rinse sampling, Cox et al. (17) wrote, “...When numbers of cells are low,

incubation of a small aliquot...after rinsing is not adequate and could result in a gross

underestimation of the incidence of Salmonella-contaminated carcasses among processed

broilers.” The Salmonella testing procedure used by FSIS as described in the Mega-Reg is

similar to the method used in the baseline study, in which aliquots of the rinse liquid were taken

60

for culturing of several different types of bacteria, rather than using the entire rinse volume to test

for Salmonella. The value of the FSIS baseline is that it established a consistent methodology for

later comparisons, but that methodology may underestimate the incidence of Salmonella.

Waiting a few days before sampling may also allow injured bacteria to recover, as in the

case of bacteria on carcasses that are bought in supermarkets several days after processing.

Jerngklinchan et al. (26) reported that carcasses from processing plants had a lower incidence of

Salmonella than carcasses sampled at the retail level.

It is possible that HACCP-related changes in poultry processing reduced the number of

Salmonella cells on positive carcasses. In the Arkansas study of retail chicken (25), the number

of cells in rinses of positive carcasses ranged from 5 to 34 MPN per 100 ml of rinse liquid or

approximately 1.5 to 10.2 MPN in a 30 ml aliquot. A recent study in the Netherlands (19)

reported Salmonella MPNs of 10 or fewer on 89 % of fresh chicken parts and whole carcasses

that were sampled at the retail level. The FSIS baseline study (4) of broiler chickens found that

87.3 % of carcasses that were Salmonella positive (20.0 % of all carcasses) had MPNs of less

than 0.3 per ml of rinse, and the Canadian baseline study (7) found that 60.7 % of carcasses that

were Salmonella positive by the qualitative method (21.1 % of all carcasses) were below the

level of detection by the MPN method. If Salmonella bacteria are usually found on positive

carcasses at relatively low levels, then any reduction in numbers caused by HACCP-related

activities such as more vigorous washing, increased chlorine, antimicrobial rinses, TSP, or fewer

opportunities for cross-contamination might reduce the number of Salmonella to the point where

the size of the aliquot removed from a rinse can influence the probability of recovering the

organism. Further research is needed to determine the incidence of Salmonella in retail poultry

61

and the relative contributions of various differences in sampling and methodology to the

incidence level reported in the present study.

ACKNOWLEDGMENTS

This study was supported in part by state and Hatch funds allocated to the Georgia

Agricultural Experiment Station. The authors express their appreciation to Nicole Bartenfeld,

Mark Freeman, David McNeal and Lynda Jones for technical assistance.

62

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Manchester area: 1981- 1985. Epidem. Inf. 98:277-284.

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foodborne pathogens on retail broilers. Int. J. Food Microbiol. 3:273-285.

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12. Bokanyi, R. P. Jr., J. F. Stephens, and D. N. Foster. 1990. Isolation and characterization

of Salmonella from broiler carcasses or parts. Poult. Sci. 69:592-598.

13. Boonmar, S., A. Bangtrakulnonth, S. Pornrunangwong, N. Marnrim, K. Kaneko, and M.

Ogawa. 1998.Salmonella in broiler chicken in Thailand with special reference to

contamination of retail meat with Salmonella enteriditis. J. Vet. Med. Sci. 60:1233-1236.

14. Castillo-Ayala, A., M.G. Salas-Ubiarco, M.L Marquez-Padilla, M.D. Osorio-Hernandez.

1993. Campylobacter spp. and Salmonella spp. incidence in fresh and roast chicken in

Guadalajara, Mexico. Rev. Lat.-Amer. Microbiol. 35:371-375.

15. Chang, Y. H. 2000. Prevalence of Salmonella spp. in poultry broilers and shell eggs in

Korea. J. Food Prot. 63:655-658.

16. Cox, N. A. and L.C. Blankenship. 1975. Comparison of rinse sampling methods for

detection of salmonellae on eviscerated broiler carcasses. J. Food Sci 40:1333-1334.

17. Cox, N. A., A. J. Mercuri, D. A. Tanner, M. O. Carson, J. E. Thomson, and J. S. Bailey.

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Campylobacter and other bacterial contaminants of pre-market chicken and retail poultry

and meats, King County, Washington. Am. J. Public Health 76:401-406.

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Campylobacter and Salmonella isolated from retail chicken and associated packaging in

South Wales. Lett. Appl. Microbiol. 33:450-454.

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24. Hood, A. M., A. D. Pearson, and M. Shahamat. 1988. The extent of surface

contamination of retailed chickens with campylobacter jejuni serogroups. Epidem. Inf.

100:17-25.

25. Izat, A. L., J. M. Kopek, and J. D. McGinnis. 1991. Incidence of Salmonella on frozen

broiler chickens at retail. Poult. Sci. 70:1438-1440.

26. Jerngklingchan, J., C. Koowatananukul, K. Daengprom, and K. Saitanu. 1994.

Occurrence of Salmonella in raw broilers and their products in Thailand. J. Food Prot.

57:808-810.

27. Ladiges, W. C. and J. F. Foster. 1974. Incidence of Salmonella in beef and chicken. J.

Milk Food Technol. 37:213-214.

28. Lillard, H.S. 1988. Comparison of sampling methods and implications for bacterial

decontamination of poultry carcasses by rinsing. J Food Prot. 51:405-408.

29. Lillard, H.S. 1994. Effect of trisodium phosphate on Salmonella attached to chicken skin.

J Food Prot. 57:465-469.

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30. Mercuri, A. J., N. A. Cox, M. O. Carson, and D. A. Tanner. 1978. Relation of

enterobacteriaceae counts to Salmonella contamination on market broilers. J. Food Prot.

41:427-428.

31. Murakami, K., K. Horikawa, T. Ito, and K. Otsuki. 2001. Environmental survey of

Salmonella and comparison of genotypic character with human isolates in Western Japan.

Epidemiol. Infect. 126:159-171.

32. Norberg, P. 1981. Enteropathogenic bacteria in frozen chicken. Appl. Environ.

Microbiol. 42:32-34.

33. Plummer, R., S. J. Blissett, and C. E. R. Dodd. 1995. Salmonella contamination of retail

chicken products sold in the UK. J. Food Prot. 58:843-846.

34. Roberts, D. 1991. Salmonella in chilled and frozen chicken. Lancet. 337:984-985.

35. Rusul, G., J. Khair, S. Radu, C.T. Cheah, and R. Md. Yassin. 1996. Prevalence of

Salmonella in broilers at retail outlets, processing plants and farms in Malaysia. Int. J.

Food Microbiol. 33:183-194.

36. SAS Institute, 1988. SAS ® / STAT Guide for Personal Computers. Version 6.03 Edition.

SAS Institute Inc., Cary, NC.

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37. Scantling, M., A. Waldroup, J. Marcy, and M. Doyle. 1995. Antimicrobial treatments - is

the consumer getting false assurance? Poult. Sci. 74(Suppl. 1):81.

38. Swaminathan, B., M. A. B. Link, and J. C. Ayres. 1978. Incidence of salmonellae in raw

meat and poultry samples in retail stores. J. Food Prot. 41:518-520.

39. Uyttendaele, M., P. De Troy, and J. Debevere. 1999. Incidence of Salmonella,

Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry

carcasses and different types of poultry products for sale on the Belgian retail market. J.

Food Prot. 62:735-740.

40. Van Schothorst, M. , M. D. Northolt, and E. H. Kampelmacher. 1976. Studies on the

estimation of the hygienic condition of frozen broiler chickens. J. Hyg. Camb. 76:57-63.

41. Vorster, S. M., R. Greebe, G. L. Norttje, and M. L. van der Walt. 1991. Incidence of

foodborne bacterial pathogens in meat in the Pretoria area. S. Afr. J. Food Sci. Nutri.

3:51-54.

42. White, D.G., S. Zhao, R. Sudler, S. Ayers, S. Friedman, S. Chen, P. F. McDermot, S.

McDermott, D. D. Wagner, and J. Meng. 2001. The isolation of antibiotic-resistant

Salmonella from retail ground meats. New Eng. J. Med. 345:1147- 1154.

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43. Wilder, A. N. and R. A. MacCready. 1966. Isolation of Salmonella from poultry. New

Eng. J. Med. 274:1453-1460.

44. Wilson, E., R. S. Paffenbarger, Jr., M. J. Foter and K. H. Lewis. 1961. Prevalence of

Salmonella in meat and poultry products. J. Infect. Diseases. 109:166-171

45. Wilson, I.G., T.S. Wilson, and S.T.C. Weatherup. 1996. Salmonella in retail poultry in

Northern Ireland. Communicable Disease Report. 6:R64-66.

46. Woodburn, M. 1964. Incidence of Salmonella in dressed broiler-fryer chickens. Appl.

Microbiol. 6:492-495.

47. Zhao, C., B. Ge, J. De Villena, R. Sudler, E. Yeh, S. Zhao, D. G. White, D. Wagner, and

J. Meng. 2001. Prevalence of Campylobacter spp., Escherichia coli, and Salmonella

serovars in retail chicken, turkey, pork, and beef from the greater Washington, D.C., area

Appl. Environ. Microbiol. 67:5431-5436.

70

TABLE 1.1: Salmonella recovered from poultry and poultry meat products sampled at the retail level, 1961-2001Reference (reference number) Country Type of Sample Sampling Method Number of samples % Salmonella positiveWilson et al., 1961 (44) USA meat, skin, and organs maceration 525 16.8Woodburn, 1964 (46) USA meat, skin, and organs maceration 264 27.3Wilder and MacCready, 1966 (43) USA meat, skin, and organs maceration 237 50.2Harris et al., 1968 (22) USA whole carcass swab 862 4.3Unpub., 1970 (Anonymous, 1974) (2) USA unreported unreported 30 46.7Unpub., 1973 (Anonymous, 1974) (2) USA unreported unreported 61 21.3Ladiges and Foster, 1974 (27) USA whole carcass swab 36 8.2Van Schothorst et al., 1976 (40) Netherlands whole carcass swab 99 73.7Duitschaever, 1977 (20) Canada cut-up parts rinse 69 34.8Swaminathan et al., 1978 (38) USA meat maceration 41 7.3Mercuri et al., 1978 (30) USA whole carcass swab 240 51.2Norberg, 1981 (32) Sweden frozen carcass rinse 82 1.2Barrell, 1982 (9) UK flesh swab 82 36.6Bok et al., 1986 (11) South Africa whole carcass rinse 718 18.9Barrell, 1987 (10) UK flesh swab 78 28.2Fukushima et al., 1987 (21) Japan tissue maceration 120 49.2Hood et al., 1988 (24) UK whole carcass rinse 69 2.9De Boer and Hahne, 1990 (18) Netherlands meat, skin, and organs maceration 81 54.3Bokanyi et al., 1990 (12) USA carcass and parts rinse 142 43.0Izat et al., 1991 (25) USA whole carcass rinse 72 33.3Roberts, 1991 (34) UK fresh/ frozen unreported 496 52.8Vorster et al., 1991 (41) South Africa whole carcass rinse 26 19.2Castillo-Ayala et al., 1993 (14) Mexico whole carcass rinse 70 68.6Jerngklinchan et al., 1994 (26) Thailand skin, meat maceration 188 76.6Scantling et al., 1995 (37) USA turkey parts rinse 72 50.0Plummer et al., 1995 (33) UK tissue maceration 325 22.8Arroyo and Arroyo, 1995 (8) Spain liver maceration 40 45.0Wilson et al., 1996 (45) Northern Ireland skin maceration 140 7.1Rusul et al., 1996 (35) Malaysia whole carcass rinse 445 35.5Boonmar et al., 1998 (13) Thailand tissue maceration 50 64.0Consumer Reports, 1998 (5) USA unreported unreported ~1000 16.0Uyttendaele et al, 1999 (39) Belgium skin maceration 279 43.4 “ France skin maceration 434 32.9 “ Italy skin maceration 13 30.8 “ UK skin maceration 44 31.8Alexandre et al., 2000 (1) Chile meat, organs maceration 1524 9.4Chang, 2000 (15) Korea tissue maceration 27 25.9DuFrenne, 2001 (19) Netherlands whole carcass rinse, maceration 89 13.5a

White et al., 2001 (42) USA ground chicken maceration 51 35.0 “ USA ground turkey maceration 50 24.0 Harrison et al., 2001 (23) UK whole carcass rinse 95 53.0Zhao et al., 2001 (47) USA whole carcass rinse 212 4.2Murakami et al., 2001 (31) Japan parts rinse 90 37.8

aPercentage of samples from which Salmonella was isolated. Results from MPN tables were reported in the published paper (J. DuFrenne, personal communication, 2001).

71

TABLE 1.2: Total number of carcasses sampled, number of processing plantsrepresented, number and percentage of Salmonella by week and total for the 20 week testperiod.

Week Total number ofcarcassessampled

Number of plantssampled

Total number ofSalmonella

positive

% Salmonella positive

carcasses

1 6 6 1 16.7

2 10 5 5 50.0

3 10 6 4 40.0

4 17 8 6 35.3

5 11 6 3 27.3

6 14 11 4 28.6

7 13 8 2 15.4

8 13 7 0 0

9 12 7 2 16.7

10 14 8 6 42.9

11 14 8 9 64.3

12 13 8 6 46.2

13 14 7 4 28.6

14 11 8 7 63.6

15 11 7 7 63.6

16 13 7 4 30.8

17 15 7 4 26.7

18 13 8 4 30.8

19 10 7 3 30.0

20 17 9 4 23.5

Totals 251 85 33.9

72

TABLE 1.3: Incidence of Salmonella contamination on retail broilers by processing plant1

over a 20-week period.

Processing Plant Total number of carcassessampled

% Salmonella positivecarcasses

A 49 26.5

B 37 18.9

C 35 40.0

D 32 43.8

E 31 45.2

F 22 31.8

G 19 42.1

H 16 18.8

1Only plants from which greater than 10 carcasses were sampled are included in this table.

73

TABLE 1.4: Incidence of Salmonella on retail broilers by sampling position (e.g. 1 = firstcarcass sampled, 2 = second carcass sampled, and so on) over the 20-week period.

Sample Position Total number of carcasses atsample position

% Salmonella positivecarcasses

1 20 15.0

2 20 35.0

3 20 40.0

4 20 35.0

5 20 30.0

6 20 40.0

7 19 52.6

8 19 31.6

9 19 36.8

10 19 36.8

11 16 31.3

12 13 30.8

13 12 25.0

14 7 28.6

15 3 0.0

16 2 50.0

17 2 50.0

5Simmons, M. S., D. L.Fletcher, M. E. Berrang, and J. A. Cason. To be submitted toJournal of Food Protection

74

CHAPTER 2

COMPARISON OF SAMPLING METHODOLOGY FOR THE DETECTION OF

SALMONELLA ON WHOLE BROILER CARCASSES5

75

ABSTRACT

An experiment was conducted to compare two Salmonella recovery methods from the same

carcass. One hundred fresh, whole broiler chickens were purchased from retail outlets over a 5-

week period (20 carcasses per week). After carcasses were aseptically removed from the

packages and giblets removed, the carcasses were placed in sterile bags containing 400 ml of

buffered peptone, shaken for 60 s, and then a 30 ml aliquot was removed and incubated for 24h

at 37 C (aliquot sample). Immediately, an additional 130ml of buffered peptone was added to the

bag with the same carcass, bringing the volume to 500 ml, after which the carcass was reshaken

and the whole carcass and rinse were incubated for 24h at 37C (whole carcass enrichment

sample). Following incubation, a 0.5 ml sample from each method was placed into 10 ml each of

Rappaport-Vassiliadis and tetrathionate (Hajna) broth and incubated at 42 C for 24 h. Each broth

was then streaked onto BG Sulfa agar and modified lysine iron agar, and incubated for 24 h at 35

C. Suspect Salmonella colonies were inoculated on triple sugar iron and lysine iron agar slants

and incubated at 35 C for 24 h. Presumptive positives were confirmed using Poly O and Poly H

agglutination tests. Over the five week period, the Aliquot sample had 13 % Salmonella-positive

carcasses compared to 38 % for the whole carcass enrichment (WCE) sample, from the same

carcasses. Recovery ranged from 0/20 to 4/20 for Aliquot method, and 4/20 to 10/20 for WCE

method over the 5 week period. These results indicate that when low numbers of Salmonella are

expected, sampling methodology has a major influence on the identification of Salmonella-

positive carcasses.

Keywords: Salmonella, recovery methodology whole broilers, Salmonella incidence

76

Simmons et al. (7) reported that 34% of retail broilers sampled using a whole carcass

enrichment method were Salmonella-positive. This incidence was higher than the Salmonella

incidence of 10.4% from broiler carcasses sampled in processing plants reported by United States

Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) (3). However, the

results were consistent with several other references where Salmonella incidence was determined

in retail poultry products (7). Simmons et al (7) suggested that the difference in results with

USDA-FSIS may have been due to sampling methodology, or postmortem sampling time and

location (in-plant sampling versus retail sampling).

In the baseline study reported by USDA-FSIS (1) the Salmonella positive carcasses,

approximately 20% of those sampled, were subjected to an MPN estimate of Salmonella

numbers. FSIS reported approximately 42% of the Salmonella-positive carcasses had less than 12

cells per carcass and that 45% had less than 120 cells per carcass. Thus approximately 87% of

the Salmonella-positive carcasses had less than 120 cells per carcass.

It has been previously reported that samonellae ocuuring in low numbers are difficult to

detect (5, 8). The official USDA-FSIS Salmonella sampling procedure is based on rinsing the

whole carcass with 400ml of water, from which a 30ml aliquot is used for Salmonella recovery

(2). Based on the aliquot dilution (30/400 or 7.5%) and the expected low number of Salmonella

cells per carcass, the current USDA-FSIS sampling may well be underestimating the true

incidence of Salmonella-positive carcasses.

The purpose of this experiment was to determine the effect of sampling method on

Salmonella recovery from the same carcass. This experiment is based on the paired recovery of

6Becton Dickinson, Sparks, Maryland 21152

77

Salmonella from indiviual carcasses using both the official FSIS aliquot method and the whole

carcass enrichment procedure reported by Simmons et. al (7).

MATERIALS AND METHODS

Sample Collection

Twenty whole, fresh broiler carcasses were purchased each week from grocery stores in

Northeast Georgia over a 5-week period. Only carcasses with intact packages were selected. All

carcasses were purchased on a Monday to ensure that birds were at least 3 days post slaughter.

Carcasses were selected at random from four grocery stores without regard to plant, brand or sell-

by-date.

Isolation Procedures

On the day of purchase, the exterior of each package was swabbed with 100% ethanol

and opened with a sterile scalpel. Using a fresh pair of sterile gloves for each carcass, the giblets

were removed and discarded. The entire carcasses and package exudate were transferred to a

sterile 16x16 polyethylene bags containing 400ml of buffered peptone water6. The carcasses

within the bags were then vigorously shaken for 1 minute, and then a 30 ml aliquot was removed

(Aliquot). Immediately, an additional 130ml of buffered peptone was added to the bag with the

same carcass, bringing the volume to 500 ml, after which the carcass was again shaken and the

carcass and rinse solution kept together in the rinse bag as described by Simmons et al. 2002 .

Both the Aliquot and whole carcass enrichment (WCE) samples were incubated for 24 h at 37 C.

7Oxoid, Basinstoke, Hampshire RG24 8PW , UK

8Microgen, Camberley, Surrey GU15 3DT, UK

78

After incubation, from both th Aliquot and WCE sample, 0.5ml of incubated rinse

solution was transferred to 10ml Rappaprt-Vassiliadis broth6 and also to 10ml TT (Hajna)6 and

incubated at 42 C for 24h. Each broth was then streaked onto BG sulfa agar (BGS)6 and

modified lysine iron agar (MLIA)7 plates, and incubated for 24hr at 35 C. Suspect Salmonella

colonies were picked and used to inoculate triple sugar iron (TSI)6 and lysine iron agar (LIA)6

slants and incubated for 24h at 35 C. Presumptive positives were confirmed using Poly O6 and

Poly H8 serological agglutination tests.

Statistical Analyses

To test for differences in frequency of Salmonella-positive carcasses between the two

methods, a chi-square test was performed using the GENMOD procedure of SAS, with a

binomial distribution and logit link function (6).

RESULTS AND DISCUSSION

Weekly recovery ranged from 0/20 to 4/20 for the Aliquot method, and 4/20 to 10/20 for

WCE method (TABLE 2.1). Over the five week period, the Aliquot samples had 13 %

Salmonella-positive carcasses compared to 38 % for WCE samples, from the same carcasses.

This difference was significant (p-value <.0001).

All of the carcasses found to be positive using the Aliquot sampling method were found

to be positive using the whole carcass enrichment method, except for 1 carcass in week 4. It is

79

concievable that the 30ml aliquot removed the small number of Salmonella that may have been

on the carcass.

These results are consistent with those obtained by Surkiwiez et al. (9) who reported that

incubation of 270ml out of 300ml lactose broth used to rinse poultry, as opposed to a 10 ml

aliquot was four times more effective in recovering Salmonella. The sensitivity of the whole

carcass enrichment procedure was reported in a study by Cox and Blnakenship (4) in which as

few as 8 inoculated cells could be recovered from a carcass.

The results from the whole carcass enrichment method are consistent with those obtained

by Simmons et al. 2001(34%). Also the results from the aliquot method are closer to those

reported by FSIS (10.4%). The sampling location (retail level vs plant) may cause these results

to be slightly higher than those reported by FSIS.

This study showed that methodology has an effect on Salmonella recovery when expected

numbers of Salmonella per carcass may be low. It also showed that the differece in the results of

Simmons et al. (7) and FSIS (3) is in part be due to sampling methodology. The current carcass

sampling methodolgy recommended by FSIS may be underestimating the incidence of

Salmonella on poultry, especially if current industry practices are reducing the number of

Salmonella per carcass. Cox and Blankenship (4) reported that there was no difference in

Salmonella incidence when comparing the incubation of a 500ml rinse to a whole carcass

incubation in 500ml of lactose broth. Therefore, one solution, not as extreme as whole carcass

enrichment, would be to incubate all of the rinse solution instead of just the 30ml aliquot.

80

Acknowledgements

This study was supported in part by state and Hatch funds allocated to the Georgia

Agricultural Experiment Station. The authors express their appreciation to Nicole Bartenfeld,

Mark Freeman, David McNeal and Lynda Jones for technical assistance.

81

REFERENCES

1. Anonymous. 1996. Nationwide broiler chicken microbiological baseline data collection

program, July 1994-June 1995. Food Safety and Inspection Service, United States

Department of Agriculture. [Internet, WWW], ADDRESS: http://www.fsis.usda.gov/

OPHS/baseline/broiler1.pdf.

2. Anonymous. 1996. Pathogen Reduction; Hazard Analysis Critical Control Point

(HACCP) Systems; Final Rule. Code of Federal Regulations 144:38806-38944. Office of

the Federal Register, U. S. Government Printing Office, Washington, D. C.

3. Anonymous. 1998. First progress report on Salmonella testing for raw meat and poultry

products. Food Safety and Inspection Service, United States Department of Agriculture,

Washington, D.C.

4. Cox, N. A. and L.C. Blankenship. 1975. Comparison of rinse sampling methods for

detection of Salmonellae on eviscerated broiler carcasses. J. Food Sci 40: 1333-1334.

5. Montford, J. and F.S. Thatcher. 1961. Comparison of four methods of isolating

salmonellae from foods, and elaboration of preferred procedure. J. Food Sci. 26: 510.

6. SAS Institute, 1997. SAS / STAT ® Software: Changes and enhancements through relase

6.12. SAS Institute Inc. , Cary, NC.

82

7. Simmons, M. , D.L. Fletcher, J.A. Cason, and M. E. Berrang. 2002. Recovery of

Salmonella from retail broilers using a whole carcass enrichment procedure. (submitted to

J. Food Prot.)

8. Smyser, C. F. and G. H. Snoeyenbos. 1969. Evaluation of several methods of isolating

salmonellae from poultry litter and animal feeding stuffs. Avian Dis. 13:134.

9. Surkiewieez, B.F., R.W. Johnston, A.B. Moran, and G.W. Krum. 1969. A bacteriological

survey of chicken eviscerating plants. Food Technol. 23:1066-1069.

83

TABLE 2.1: Recovery of Salmonella from whole broiler carcasses using a 30ml aliquot

following a 400 ml buffered peptone rinse (Aliquot) or a 500ml buffered peptone rinse

and incubation of carcass and rinse together (WCE) over a 5-week period, analyzed

using Chi-square (P2) and p by week and for the entire experiment.

Number of Salmonella-positive carcasses

Week Aliquot WCE P2 p-value

1 4/20 9/20 2.9 0.0914

2 3/20 10/20 5.6 0.0181

3 0/20 8/20 10.0 0.0016

4 4/20 7/20 1.1 0.2881

5 2/20 4/20 0.8 0.3758

Total 13/100 38/100 21.2 <0.0001

84

SUMMARY

Two experiments were conducted to determine the influence of methodology on the

recovery of Salmonella on retail chicken. The first experiment was designed to determine if

there was a difference in the incidence of Salmonella-positive carcasses reported by USDA-FSIS

and the incidence of Salmonella-positive carcasses at the retail level using an exhaustive

procedure. The second experiment was conducted to determine the effects of sampling

methodology on the recovery of Salmonella from whole carcasses.

In the first study 85 of the 251, or 33.9 %, of the retail carcasses were Salmonella-

positive. This is higher than both the 20 % from the 1994-1995 baseline and the 10.4 % reported

by USDA-FSIS in 1998. The incidence determined in the first study may have been higher due

to methodology, different sampling location (retail level versus processing plant), post-process

contamination, or the presence of giblets. The results do indicate that a whole carcass

enrichment may be more sensitive for detecting Salmonella-positive when low numbers of

Salmonella per carcass are expected.

In the second study the significantly more (p<0.0001) carcasses were found Salmonella-

positive using whole carcass enrichment method than using the 30ml aliquot method

recommended by USDA-FSIS. Also the incidence of Salmonella-positive carcasses determined

by the whole carcass enrichment method (38 %) is comparable to the incidence determined in the

first experiment (33.9 %), and the incidence determined by the Aliquot method (13%) was

comparable to the incidence reported by USDA-FSIS in 1998 (10.4 %). Thus showing

85

methodology does have an effect on Salmonella recovery when low numbers of Salmonella per

carcass are expected.

The results from these two experiments indicate further research is necessary to determine

the effects of sampling methodology on Salmonella recovery. Also future research should be

conducted to determine the actual number of Salmonella cells present per carcass.