This chapter discusses Salmonella in eggs and explores a discussion of Salmonella enteritidis and the table egg, which have received so much publicity since 1988. It examines the Salmonella contamination of fertile hatching eggs and the impact of that on the contamination of raw poultry meat. Egg-associated outbreaks of salmonellosis, generally attributed to either bulk egg products or cracked shell eggs, occurred frequently before enactment of the 1970 Egg Products Inspection Act. A 1987 nosocomial outbreak at a New York hospital, traced to the use of raw eggs in the preparation of mayonnaise, was illustrative of the epidemiology of eggborne Salmonella enteritidis (SE) infections. Explaining the high rate of isolation of SE and the frequent association of some SE strains with eggs requires an understanding of the attributes that distinguish epidemiologically important strains from other SE strains and from other serotypes. Detectable antibodies to SE have been found in egg yolk by enzyme-linked immunosorbent assay and microantiglobulin methods.

Results obtained by using standard, conventional tube and plate tests to rapidly and accurately detect and identify pathogenic bacteria in foods present many inherent problems. Increased use of plastic made possible the development of disposable multichambered trays, plates, and dishes of various shapes and sizes which were utilized by the manufacturers of packaged identification systems or kits. The disadvantages of radioisotopes in immunoassays have led to the development of alternative procedures with other labels including enzymes, coenzymes, fluorescent dyes, fluorogenic substrates, chemilu-minescent precursors, bacteriophages, and metal ions. The primary advantage of DNA hybridization is that if the appropriate DNA sequences are selected for, the test should be highly selective with very low levels of false negatives and false positives. The age of rapid, miniaturized techniques for identification of bacteria received much impetus in the middle to late 1940s, when Bachman and Weaver added concentrated inocula to small tubes of different media.

J.P. Buts et al., 1993. Saccharomyces boulardii for Clor tridium difficile-associated enteropathies in infants. J. Pedi atric Gastroenterology and Nutrition 16:419–425. E.F. Caballero et al., 1993. Valor alimenticio de la levadura torula (Candida utilis) en dietas paraves. Vet. Mex. 24(2):145-147. F. Castex et al., 1990. Prevention of Clostridium difficile-in duced experimental pseudomembranous colitis by Saccha rmyces boulardii: a Scanning electron microscopic and microbiological study. J. Gen. Microbiol. 136:1085-1089. G.W. Elmer et al., 1987. Supression by Saccharomyces boulardii of toxigenic Clostridium difficle overgrowth after Vancomycin treatment in hamsters. Antimicrobial Agents and Chemotherapy 31(1): 129-131. F.T. Jones et al., 1991a. A Survey of Salmonella contami nation in modern broiler production. J. Food Protect. 54:502-507. F.T. Jones et al., 1991b. A survey of Campylobacter jejuni contamination in modern broiler production and processing systems. J. Food Protect. 54:259-262. J.E. Kvenberg et al., 1987. Economic impact of colonization control on foodborne disease. Food Technol. Jul. 1977-1981.

Clostridium difficile is commonly associated with a spectrum of disease in humans referred to as C. difficile-associated disease (CDAD) and use of antimicrobials is considered a risk factor for development of disease in humans. C. difficile can also inhabit healthy food animals and transmission to humans is possible. As a result of the complexity and cost of testing, C. difficile is rarely tested for antimicrobial susceptibility. A total of 376 C. difficile strains (94 each from swine and dairy feces, and 188 from beef cattle feces) were isolated from healthy food animals on farms during studies conducted by the National Animal Health Monitoring System. Using the Etest (AB Biodisk, Solna, Sweden), samples were tested for susceptibility to nine antimicrobials implicated as risk factors for CDAD (linezolid, amoxicillin-clavulanic acid, ampicillin, clindamycin, erythromycin, levofloxacin, metronidazole, rifampicin, and vancomycin). Vancomycin was active against all isolates of C. difficile (MIC90 = 3.0 μg/ml) while almost all isolates (n = 369; 98.1%) were resistant to levofloxacin. With the exception of vancomycin, resistance varied by animal species as follows: linezolid (8.5% resistance among swine versus 2.1 and 1.1% resistance among dairy and beef, respectively), clindamycin (56.4% resistance among swine versus 80% and 90.9% resistance among dairy and beef, respectively), and rifampicin (2.1% and 0% resistance among swine and dairy cattle isolates, respectively versus 14.3% resistance among beef isolates). Regardless of species, multiple drug resistance was observed most often to combinations of clindamycin and levofloxacin (n = 195; 51.9%) and ampicillin, clindamycin and levofloxacin (n = 41; 10.9%). The reason for the variability of resistance between animal species is unknown and requires further research.

Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by alcohol shock and isolation on tryptic soy agar supplemented with 5% sheep blood, and cycloserine-cefoxitin fructose agar. The double alcohol shock method (DS) used alcohol shock prior to and after selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by isolation on tryptic soy agar supplemented with 5% sheep blood and cycloserine-cefoxitin fructose agar. A total of 55 (15.9%, n = 345) swine fecal samples, 32 (2.4%, n = 1,325) dairy cattle fecal samples, and 188 (6.3%, n = 2,965) beef cattle fecal samples were positive for C. difficile by either method. However, the DS was significantly better than the SS for the recovery of C. difficile from swine feces, while the SS was significantly better than the DS for the recovery of C. difficile from beef cattle feces. There was no significant difference between methods for the recovery of C. difficile from dairy cattle feces. This study suggests that food animals might harbor C. difficile and it provides critical information that isolation methods might not have universal application across animal species.

To evaluate whether the number of Escherichia coli bacteria in carcass rinses from chicken slaughter establishments could be monitored for the purpose of microbial process control, we drew a random sample from 20 of 127 large USDA-inspected operations. In 2005, every 3 months, two sets of 10 carcass rinses, 100 ml each, were collected from establishments, netting 80 sample sets from the rehang and postchill stages. E. coli and Campylobacter numbers and Salmonella prevalence were measured. Mixed-effect models were used to estimate variance of mean log(10) E. coli cell numbers of 10-carcass rinse sample sets. Relationships between E. coli and Campylobacter and Salmonella were examined. For 10-carcass rinse sets, at both the rehang and postchill stages the mean log(10) E. coli CFU/ml fit the logistic distribution better than the normal distribution. The rehang overall mean log(10) E. coli was 3.3 CFU/ml, with a within- sample set standard deviation of 0.6 CFU/ml. The overall postchill mean log(10) E. coli was 0.8 CFU/ml, with 13 establishments having mean log(10) E. coli CFU/ml values of less than 1.0 and 7 having mean values of 1.2 or more. At the midpoint separating these establishments, a mean log(10) E. coli CFU/ml of 1.1, the within- sample set standard deviation was 0.5 CFU/ml, with smaller standard deviations as means increased. Postchill sample sets with mean log(10) E. coli counts less than or equal to 1.1 CFU/ml had lower overall prevalence of Salmonella and mean log(10) Campylobacter CFU/ml than sample sets with higher means. These findings regarding reductions in E. coli numbers provide insight relevant to microbial process control.

The purpose of this study was to compare a conventional culture broth method (Bolton enrichment), a newly developed proprietary broth method (TECRA Campylobacter enrichment), and direct plating for recovery of Campylobacter spp. from chicken carcass rinsates. Whole carcass rinses were taken from 140 carcasses at rehang (immediately after defeathering but before evisceration) and from 140 carcasses at postchill from eight different processing plants in the United States. The rinsate samples were packed in ice and shipped overnight to the laboratory. Aliquots of the rinsate were transferred into Bolton and TECRA enrichment broths and were direct plated. Standard laboratory procedures with Campy-cefex plates were followed for recovery of Campylobacter spp. For rehang carcasses, 94% were positive for Campylobacter spp. with the TECRA enrichment broth and 74% were positive with the Bolton enrichment broth. For postchill carcasses, 74% were positive for Campylobacter spp. with the TECRA enrichment broth and 71% were positive with the Bolton enrichment broth. Compared with the Bolton enrichment broth, TECRA enrichment broth significantly suppressed non-Campylobacter microflora (P < 0.05). Overall, TECRA enrichment broth yielded an 11% higher total number of Campylobacter-positive samples compared with the Bolton enrichment broth. Campylobacter spp. detection in postchill samples was significantly greater (P < 0.05) by enrichment (84%) than by direct plating (19%). The high number of Campylobacter-positive samples obtained with all procedures indicated that 99% of the carcass rinsates obtained at rehang and 84% obtained at postchill contained Campylobacter spp.

It is unclear how effective different types of broiler carcass wash steps are in lowering the presence or numbers of pathogenic bacteria. We tested for individual and combined effectiveness of 5 separate on-line wash steps applied between bleed-out and chilling in a commercial broiler processing plant. Carcasses were sampled directly before and after each wash step: pre-scald brush washer, post-feather pick (New York dressed) spray washer, inside/outside spray washer, postevisceration brush washer, and final prechill spray washer. Carcasses were examined for numbers of Campylobacter and Escherichia coli and presence of Salmonella using standard cultural methods. Overall, numbers of Campylobacter were lowered from log 2.58 to 1.15 cfu/mL of carcass rinse, but no single wash step caused a significant decrease. Overall, Salmonella prevalence was decreased from 80 to 24%; however, no wash step caused a significant decrease by itself. The 5 wash steps in series lowered E. coli numbers from log 4.60 to 2.69 cfu/mL; the New York-dressed spray wash and the postevisceration brush washer each had a significant effect on E. coli. When examined separately, the benefit of broiler carcass wash steps may not be evident. However, when combined with overall processing, wash steps can be effective to lessen bacterial contamination on carcasses and be useful for pathogen control. Additional studies are necessary to maximize the effectiveness of carcass washers.

The objective of this study was to measure the effect of broiler processing on the prevalence, serotype, and antimicrobial resistance profiles of salmonellae. Twenty U.S. commercial processing plants representing eight integrators in 13 states were included in the survey. In each of four replications, 10 carcasses from one flock were collected at rehang and 10 more carcasses were collected at postchill; each carcass was sampled by whole-carcass rinse. Salmonella organisms were isolated from carcass rinses by standard cultural techniques, serotypes were determined, and the resistance to 15 antimicrobials was measured. Overall, Salmonella was detected on 72% of carcasses at rehang (ranging from 35 to 97%) and on 20% of carcasses postchill (ranging from 2.5 to 60%). In every instance, a significant (P < 0.05) decrease in Salmonella prevalence was noted between rehang and postchill. The four most common serotypes, accounting for 64% of all Salmonella isolates, were Kentucky, Heidelberg, Typhimurium, and Typhimurium var. 5-; most isolates of Kentucky (52%), Heidelberg (79%), and Typhimurium (54%) serotypes were susceptible to all antimicrobial drugs tested. However, only 15% of the Typhimurium var. 5- isolates were pansusceptible; more than one-half of the isolates of this serotype were resistant to three or more drugs. No isolate of any serotype exhibited resistance to amikacin, ceftriaxone, ciprofloxacin, or trimethoprim-sulfamethoxazole. These data demonstrate that although processing lessens carcass contamination with Salmonella, antimicrobial-resistant isolates may still be present.

In 1999, the USDA-Food Safety and Inspection Service introduced an inspection system called the Hazard Analysis and Critical Control Point-Based Inspection Models Project (HIMP). The HIMP varies from standard inspection in that the emphasis of Food Safety and Inspection Service inspection program personnel is shifted. Each carcass is still visually inspected according to the Poultry Products Inspection Act, but some responsibility for food safety and identification and removal of defects is shifted from the regulatory agency to the processor, freeing up inspectors to more effectively verify the process and food safety system of the establishment. This survey was conducted in 2 stages: first to examine carcasses collected in HIMP and non-HIMP plants and then to test carcasses from all 20 volunteer plants currently operating under HIMP inspection. Carcasses were collected at rehang and postchill being careful to follow the same flock through processing. Postchill carcasses from HIMP plants were found to have equal bacterial contamination (numbers of Campylobacter and Escherichia coli and presence of Salmonella) as carcasses from standard HACCP plants. Overall, HIMP inspection, which places additional responsibility on the plant for process control, does not affect the microbiological quality of fully processed broiler carcasses.

The association of Campylobacter spp. with raw poultry products has been well established. Campy-Line agar (CLA) is a recently developed selective culture medium that allows very few non-Campylobacter colonies to grow. The few contaminants able to grow on CLA from typical broiler chicken carcass rinses were identified and found to be sensitive to the antimicrobial agent sulfamethoxazole (SMX). The purpose of our experiment was to determine the influence of SMX on recovery of C. jejuni from broiler carcass rinse samples when added to Campy-Cefex agar or CLA. Cefex and CLA were prepared with and without the addition of 25 mg SMX/L media. Broiler carcass rinse samples (post-pick, n = 80 and post-chill, n = 80) were obtained from eight different commercial processors across the United States and were surface plated on the selective agars. Both Cefex and CLA with and without SMX recovered similar populations of Campylobacter; however, significantly fewer contaminants were observed on the Cefex with added SMX and the CLA with or without SMX compared to normal Cefex. The more selective CLA with SMX had no contaminants present from this sample type, which simplified enumeration. Addition of SMX should be considered for increasing selectivity of Campylobacter-specific media.

Previous studies have demonstrated that when Campylobacter or Salmonella were either orally or intracloacally inoculated into day-old broiler chicks, within 1 h, these bacteria moved rapidly to the lymphoid organs. These bacteria were still present 1 wk after inoculation. Two different market-age (6 and 8 wk old) broilers were obtained from 2 commercial poultry operations and brought to the laboratory for analysis. Necropsy was limited to the removal of the spleen, liver and gallbladder (L-GB), and ceca using aseptic techniques. To reduce the possibility of cross-contamination between samples, the spleen and L-GB were aseptically removed before the ceca. Samples were individually bagged, and standard laboratory procedures for Campylobacter and Salmonella were carried out for all samples. Fifty-two 6-wk-old broilers were analyzed, and Campylobacter were found in 19 of 52 L-GB, 19 of 52 spleens, and 26 of 52 ceca. Salmonella were found in 5 of 52 L-GB, 8 of 52 spleen, and 4 of 52 ceca. Eighty 8-wk-old broilers were analyzed, and Campylobacter were found in 3 of 80 L-GB, 5 of 80 spleens, and 19 of 80 ceca. Salmonella were found in 41 of 80 L-GB, 38 of 80 spleens, and 52 of 80 ceca. The internal organs of the younger birds were more heavily contaminated with Campylobacter, whereas Salmonella was the predominant organism isolated in the older birds. All Campylobacter isolates were found to be Campylobacter jejuni. The predominant Salmonella serotype was Salmonella Typhimurium; however, 7 other serotypes were found. Overall, C. jejuni was found in 22 of 132 L-GB, 24 of 132 spleen, and 45 of 132 ceca, whereas Salmonella serovars were isolated from 46 of 132 L-GB, 46 of 132 spleen, and 56 of 132 ceca. There is no doubt that these bacteria are naturally present in these organs. The significance of these reservoirs in the internal organs of commercial broilers is yet to be determined but could play a role in the microbiology of the intestinal tract and hence the final food product.

In 1996 the Food and Drug Administration Center for Veterinary Medicine established the National Ant1microb1al Resistance Monitoring System Entenc Bacteria (NARMS) as a post-approval mon1tonng program From 1997 through 2005, 10,565 Salmonella ISolates originated from swme slaughter/processmg (n=3,848), d1agnoshc (n=4,579) and on-farm (n=2138) sources as part of the an1mal arm of NARMS Relat1ve to 2005, the top five Salmonella serotypes from slaughter/processing (in decreas1ng frequency) were S . Derby, S . Typh1munum var. 5-, S lnfant1s, S Anatum, and S Johannesburg wh1le diagnostiC serotypes were S Typh1murium var 5-, S CholeraesUis var kunzendorf, S Derby, S Typh1munum, and S He1delberg Increased ant1m1crob1al res1stance was most often observed for d1agnost1c versus slaughter/processing isolates although there were exceptions for some drug and serotype combinations For all years, greater than 55% of the slaughter/processing isolates were e1ther pan-susceptible or resistant to only one antimicrobial, which was most often tetracycline Since 1997, approximately 41% of the ISolates exhibited mu1t1-drug resistance, defined as res1stance to ~2 antimicrobials Of the 723 S Typh1munum DT104 ISolates from swme only 24% (n=176) ongmated from slaughter/processmg These data reaffirm that overall patterns of res1stance are h1ghly dependent on the Salmonella serotype distribution and IS variable when measured at different points along the farm to fork continuum.

3 Abstract: Salmonella vaccination programs using killed bacterins in breeders and live auxotrophic-strain vaccines early in the life of their progeny have gained popularity in today's poultry industry. In this study we evaluated the intestinal humoral immune response to a live auxotrophic vaccine used on hatchlings with and without maternal antibody and related this response to challenge with a blend of two antibiotic-resistant Salmonella marker strains. Forty wk-old ISA Brown (Institute de Selection Animale, France) breeders from ® a Salmonella-free flock were vaccinated twice at a three wk interval with commercially-prepared autogenous trivalent bacterin, serogroups B, C and D1 (Lohmann Animal Health International, Gainesville, GA), or a serovar Enteritidis bacterin (Fort Dodge Animal Health Inc, Overland Park, KS). Half of the progeny from these treatments (hatched from eggs laid 3 wks after second bacterin dose) were given a live Salmonella serovar Typhimurium (LiveST) mutant vaccine (Fort Dodge Animal Health Inc, Overland Park, KS), by coarse spray on arrival in the brooding premises. On days 3, 13 and 34, intestinal immunoglobulins (Ig) A and G were sampled and measured on enzyme-linked immunosorbent assay plates coated with Salmonella serovars Enteritidis (SELPS) or Typhimurium (STLPS) Lipopolysaccharide. On the same days, a second group of birds was challenged with a blend of antibiotic-resistant serovars Enteritidis and Typhimurium strains. Cecal and composite liver-heart-spleen samples obtained 7 days post-challenge were cultured and colonies enumerated. Maternal IgG observed up to 13 days had no effect on subsequent LiveST-stimulated antibody production. No protective effect of maternal antibody was demonstrated, except when combined with LiveST given to the progeny. Killed vaccines delivered to the breeders combined with a live vaccine delivered to the progeny resulted in reduced invasiveness after challenge, as shown by a reduction in liver-heart-s pleen Salmonella counts. One dose of LiveST enhanced intestinal IgG (Optical Densities (OD) >0.576) up to 34 days when measured on STLPS, but only to 13 days when measured on SELPS, with titers decreasing with age. Increased IgA was observed only at 13 days. Three and 13 but not 34 days bacterial counts were decreased by the live ST vaccine treatment, for both cecal (1.05 and 1.09 log ) and liver-heart-spleen (0.32 10 and 0.06 log ) samples, indicating that a second dose might be necessary for prolonged protection. The 10 protective effect of the live vaccine, but not of maternal IgG, leads us to hypothesize that protection might be due to stimulation of cell-mediated intestinal immunity and/or a competitive exclusion effect of the LiveST vaccine. Reduction but not elimination of Salmonella colonization by vaccination highlights the importance of vaccines as complementary tools and not substitutes of integral biosecurity programs to contro l Salmonella in poultry.

Campylobacter is a human pathogen associated with chicken and chicken meat products. This study was designed to examine the prevalence and number of Campylobacter on broiler chicken carcasses in commercial processing plants in the United States. Carcass samples were collected from each of 20 U.S. plants four times, roughly approximating the four seasons of 2005. At each plant on each sample day, 10 carcasses were collected at rehang (prior to evisceration), and 10 carcasses from the same flock were collected postchill. A total of 800 carcasses were collected at rehang and another 800 were collected postchill. All carcasses were subjected to a whole-carcass rinse, and the rinse diluent was cultured for Campylobacter. The overall mean number of Campylobacter detected on carcasses at rehang was 2.66 log CFU per ml of carcass rinse. In each plant, the Campylobacter numbers were significantly reduced by broiler processing; the mean concentration after chill was 0.43 log CFU/ml. Overall prevalence was also reduced by processing from a mean of > or =30 of 40 carcasses at rehang to > or =14 of 40 carcasses at postchill. Seven different on-line reprocessing techniques were applied in the test plants, and all techniques resulted in <1 log CFU/ml after chilling. Use of a chlorinated carcass wash before evisceration did not affect the postchill Campylobacter numbers. However, use of chlorine in the chill tank was related to lower numbers on postchill carcasses. Overall, U.S. commercial poultry slaughter operations are successful in significantly lowering the prevalence and number of Campylobacter on broiler carcasses during processing.

The Collaboration 1n Ammal Health and Food Safety Epidemiology (CAHFSE), a USDA JOint program of ARS , APHIS, and FSIS was established to track food borne pathogens and monitor animal health issues.Fecal samples (n=9020) were collected and cultured for Salmonella from pens of p1gs near slaughter we1ght (generally~ 22 weeks old) from swine farms 1n five U.S. states.A prevalence of 8.0, 10.1, and 8.5% was observed 1n 2003, 2004 , and 2005, respectively The top 10 serotypes accounted for 94% of the total Salmonella isolates with S. Derby (45%), S.Typhimurium var.5-(15%), and S Heidelberg (9%) comprising the top three serotypes each year.Salmonella G1ve was found in 8% of samples in 2003 , 3% of samples m 2004 , but was not found in the top 10% of ISolates m 2005.The percentage of Salmonella isolates that were susceptible to all of the 16 antimicrobials tested increased from 6% in 2003 to 15% in 2005.At the same time , the percentage of 1solates res1stant to 1 0 or more antim1crob1als increased from 1% to 15%.The mcrease 1n multiple drug res1stance was comc1dent with an mcrease 1n the percentage of S .Derby isolates.Overall, frequency of resistance to individual antimicrobials was relatively stable from 2003 to 2005 and observed differences were related to changes in serotypes over time, which highlights the Importance of reporting res1stance data by mdiv1dual serotype.CAHFSE prov1des a mechanism to monitor changes 1n serotypes of Salmonella as well as antimicrobial resistance patterns over t1me

3 Abstract: Resistance to Salmonella challenge of breeders under three vaccination programs and of their chicks with and without mucosal Competitive Exclusion (CE) (CHR Hansen) treatment was assessed. Vaccine treatments combined a live Aro-A Salmonella Typhimurium (ST) vaccine and an autogenou s commercially prepared (Lohmann Animal Health) trivalent killed vaccine (serogroups B, C and D). 2 1 Treatments combined: 2 live and 2 killed doses or 3 live and 1 killed dose delivered at 1, 21, 77 and 126 d of age; or 2 killed doses delivered at 77 and 126 of age; and a non-vaccinated control (C). At 3, 6, 11, 17 and 22 wks of age, a portion of breeder pullets was removed and challenged per os with 10 cells of a 3-strain 7 10 significant reductions between (live) vaccinates and non-vaccinates at 3 (0.82 log) and 6 wks (0.85 log) challenges. By 11 wks, there were no differences in Salmonella levels between vaccinates and controls, indicating that 1-d and 3-wk live vaccine protection had diminished with time. All vaccination treatments reduced breeder cecal counts (1.15-1.30 log) by wk 22. Passive immunity from breeder vaccination treatments was not effective in diminishing chick cecal counts as shown by comparable susceptibility of chicks from vaccinated and control breeders, regardless of breeder age. Chick CE treatment consistently diminished cecal (1.41 log) and LHS (0.306 log) counts. These results show that live Aro-A ST vaccination decreases counts during the first 6 wks of age, as do all programs by 22 wks of age and that competitive exclusion is the most effective treatment in reducing hatchling Salmonella counts.