Two-step mucosal competitive exclusion flora treatment to diminish salmonellae in commercial broiler chickens.

There is a need to control the intestinal colonization of broiler chickens by salmonellae in order to reduce the contamination of poultry products. A two-step treatment of broiler chicks with a mucosal competitive exclusion culture (MCE) was tested, in which the MCE was first sprayed on chicks in the hatchery followed by administration in the first drinking water. Three commercial flocks were treated and compared with parallel, untreated control flocks. Customary husbandry practices were employed. Environmental, hatchery, skin with feathers, and cecal samples were analyzed at 3 and 7 wk for the presence of salmonellae. Carcass rinse samples of fully processed birds were analyzed similarly. The results indicated that initial feed, water, and litter contamination was at a low frequency (< 10%). Eggshell fragments and chick paper pads were frequently contaminated (> 50%). After 3 wk growth, contamination of litter, skin with feathers, and ceca were significantly (P < .05) reduced in treated flocks as compared with control flocks. Salmonellae prevalence in ceca and in processed carcass rinses was also significantly (P < .05) reduced from 41% in control flocks to 10% in treated flocks. The study showed that treatment of chickens in a commercial setting with MCE cultures can serve as a useful means to reduce salmonellae contamination.

Characterization of Salmonella california and S. typhimurium strains with reduced ability to colonize the intestinal tract of broiler chicks.

This study determined the ability of eight strains of Salmonella and their agarsubcultured variants to colonize the intestinal tract of broiler chicks. Nalidixic-acid (NAL)-resistant and streptomycin-resistant subcultured strains (S. california 1989/A and S. typhimurium 3366/A) that persisted in the ceca of chicks in lower numbers than their NAL-resistant parent strains (1989/O and 3366/O) were selected for additional study S. typhimurium strain 3366/A was present in the ceca of chicks in lower numbers than the parent strain 3366/O when given concomitantly with the parent strain or when the two strains were given separately to different chicks. S. california 1989/A strain was present in the ceca in lower numbers than the parent strain after concomitant oral or intracloacal inoculation. Strains 3366/O and 3366/A of S. typhimurium differed in growth rates in BHI broth and cecal mucus. The lipopolysaccharide (LPS) profile indicated that LPS components present in S. california 1989/O were missing from strain 1989/A. A mutant of 1989/O--2095/R--was also LPS- and colonization-deficient.

Prevalence of Campylobacter spp. and Salmonella spp. on Pork Carcasses and the Reduction Effected by Spraying With Lactic Acid.

Two hundred and twenty-five pork carcasses were sampled immediately after slaughter and 24 h postmortem for the presence of Campylobacter spp. and Salmonella spp, Campylobacter spp. were present on 23 carcasses with 9 isolates from the shoulder area and 14 isolates from the ham. Salmonella spp. were isolated from 63 carcasses with 29 isolates from the shoulder and 34 isolates from the ham area. Spraying with a 2% solution of lactic acid reduced the numbers of both organisms that could be isolated immediately and 24 h after slaughter from the 75 carcasses sprayed. All of the Campylobacter spp. isolated in this study were confirmed to be Campylobacter coli .

Broiler Carcass Reprocessing, A Further Evaluation.

The microbiological quality of 745 conventionally processed and 745 reprocessed broiler carcasses was determined. Carcasses were taken from the processing line prior to entering the chiller in five commercial processing plants. Each plant was sampled twice during the winter, spring, and summer. Analyses included aerobic bacteria, Enterobacteriaceae , and Escherichia coli counts plus qualitative Salmonella (SAL) prevalence. Differences between overall mean log10 counts for aerobic bacteria, Enterobacteriaceae , and E. coli were not significant. The prevalence of SAL detected on conventionally processed and reprocessed carcasses also was not significantly different. Some variation was observed in microbiological quality of carcasses among processing plants. Although the SAL prevalence appeared to decline from winter to summer replications, no significant trend could be demonstrated. Continuation of the practice of reprocessing carcasses appears justified.

Research note: in ovo administration of a competitive exclusion culture treatment to broiler embryos.

Exposure of chicks to salmonellae in the hatchery and hatchery environment limits the effectiveness of a competitive exclusion (CE) culture treatment. Therefore, in an attempt to apply treatment before chicks are exposed to salmonellae, the CE culture was introduced in ovo to unhatched embryos. An undefined, anaerobically grown CE culture, derived from cecal contents of healthy adult chickens, was diluted 1:1,000 or 1:1,000,000 and inoculated either into the air cell or beneath the inner air cell membrane of 17-day-old incubating hatching eggs. The treated chicks were more resistant than untreated chicks to varying challenge levels of Salmonella typhimurium, indicating that it may be possible to initiate protection of chicks to salmonellae challenge prior to hatching into a contaminated environment.

Binding of Salmonella strains to immobilized intestinal mucosal preparations from broiler chickens.

The binding kinetics of radiolabeled Salmonella california 1989/O (mannose-sensitive hemagglutinin-positive [MSHA+]) to immobilized mucus or enterocytes isolated from broiler ceca and inhibition of binding by D-mannose and sodium metaperiodate were characteristic of adherence of mannose-sensitive type 1 fimbriae of bacteria to eukaryotic mannose-containing receptors. Binding by radiolabeled strains 1989/O (in the presence of D-mannose) and S. typhimurium S 7471 N (MSHA-, non-fimbriated) indicated non-specific binding that was characterized by less binding to enterocytes and mucus and lack of inhibition by carbohydrates or prior treatment with sodium metaperiodate. Inhibition of non-specific binding to enterocytes by pretreatment with various enzymes or by the presence of tetramethylurea or p-nitrophenol (known to disrupt hydrophobic interactions) indicate involvement of multiple sites and hydrophobic bonding. Strain-specific outer-membrane preparations inhibited non-specific binding to a greater extent than did lipopolysaccharide, Escherichia coli outer-membrane preparations, or bovine serum albumin.

Influence of coccidiosis on Salmonella colonization in broiler chickens under floor-pen conditions.

The influence of coccidiosis on colonization of Salmonella typhimurium in broiler chickens under floor pen conditions was studied by semiquantitative methods. Chickens of two groups, unmedicated and medicated with nicarbazin (125 ppm via the feed), were exposed to three species of Eimeria (Eimeria tenella, Eimeria maxima, and Eimeria acervulina) at 2, 3, and 4 wk of age and given S. typhimurium in the feed 2 days later. Salmonella typhimurium was isolated most often (100%) from ceca of chickens exposed at 3 wk of age. Birds in the unmedicated group were positive for S. typhimurium at a higher rate than those in the medicated group. Salmonella typhimurium was detected in livers only in a few unmedicated birds.

Effect of fructooligosaccharide on Salmonella colonization of the chicken intestine.

The influence of fructooligosaccharide (FOS) on the ability of Salmonella typhimurium to grow and colonize the gut of chickens was investigated. In vitro studies showed that Salmonella did not grow when FOS was the sole carbon source. When FOS was fed to chicks at the .375% level, little influence on Salmonella colonization was observed. At the .75% level, 12% fewer FOS-fed birds were colonized with Salmonella compared with control birds. When chicks given a partially protective competitive exclusion (CE) culture were fed diets supplemented with .75% FOS, only 4 of 21 (19%) chickens challenged with 10(9) Salmonella cells on Day 7 became colonized as compared with 14 of 23 (61%) chickens given CE alone. When chickens were stressed by feed and water deprivation on Day 13 and challenged with 10(9) Salmonella on Day 14, 33 of 36 (92%) chickens fed a control diet were colonized compared with only 9 of 36 (25%) chickens fed a .75% FOS diet. Chickens treated with FOS had a fourfold reduction in the level of Salmonella present in the ceca. Feeding FOS in the diet of chickens may lead to a shift in the intestinal gut microflora, and under some circumstances may result in reduced susceptibility to Salmonella colonization.

Methods for inoculation and recovery of Salmonella from chicken eggs.

Various methods of applying inoculum and recovering low numbers of artificially inoculated Salmonella typhimurium on eggs were evaluated. Inoculation methods tested were suspending cells in 1) .85% saline; 2) 1% peptone; 3) sterile chicken fecal paste; or 4) a 1:10 dilution of chicken feces in .85% saline. Sampling methods tested were 1) shell and membrane massage; and 2) mortar and pestle grinding of shells and membranes. The method that yielded the best recovery of low numbers of Salmonella was as follows: 1) apply cell suspension in 1% peptone to egg; 2) sample egg by a hand crush and massage of shell and membranes in 50 mL of buffered peptone; 3) incubate shell and membranes in buffered peptone overnight and then plate onto selective agar. Methods that did not improve sensitivity of recovery included varying the inoculum drying time, addition of FeSO4 or Cleland's reagent to the recovery medium, and varying the temperature of the inoculum to affect penetration.

Simultaneous colonization of Campylobacter jejuni and Salmonella typhimurium in day-old chicks.

Day-old chicks were challenged with Campylobacter jejuni or Salmonella typhimurium or both to assess the influence of these two bacteria upon the colonization of one another. Median colonization dose (CD50), a measure indicating the number of organisms required to colonize one-half of the challenged chickens, was used to assess the influence of these organisms upon the colonization of one another. Chicks were gavaged with serial dilutions of 1) C. jejuni, 2) S. typhimurium, 3) dilutions of C. jejuni and a fixed level of S. typhimurium, or 4) dilutions of S. typhimurium and a fixed level of C. jejuni. Six days postchallenge, cecal contents were quantitatively assayed for the challenge organisms. Birds challenged with only C. jejuni or birds challenged with both C. jejuni and S. typhimurium had a CD50 of 120 C. jejuni cells. The CD50 values for S. typhimurium alone (48 cells) and S. typhimurium simultaneously colonized with C. jejuni (180 cells) were not significantly different (P less than .05). The colonization levels of either organism were generally correlated with the challenge dose and ranged up to 63 million cells per gram. It was concluded that the CD50 for both C. jejuni and S. typhimurium in 24-h-old chicks were similar, and the presence of one of these particular co-colonizers does not influence the CD50 of the other.

Extent of salmonellae contamination in breeder hatcheries.

Egg fragments, paper pads from chick boxes, and fluff samples were obtained from six commercial broiler breeder hatcheries and analyzed for the presence and level of salmonellae. Overall, 42 of 380 samples (11.1%) from those hatcheries were contaminated with salmonellae. Salmonellae organisms were detected in 22 of 145 (15.2%), 5 of 100 (4.6%), and 15 of 125 (12%) samples of egg fragments, fluff, and paper pads, respectively. The percentage salmonellae-positive samples from each of the six hatcheries were 1.3, 5.0, 22.5, 11.4, 36.0, and 4.3% respectively. Of the 140 samples randomly selected for enumeration, salmonellae were found in 11 samples. Four of these 11 samples had greater than 10(3) salmonellae per sample, 3 others had greater than 10(2) but less than 10(3), and the remaining 4 had less than 10(2). Salmonella serotypes isolated were S. berta, S. california, S. give, S. hadar, S. mbandaka, S. senftenberg, and S. typhimurium, all of which have previously been isolated from poultry. The incidence and extent of salmonellae-positive samples found in the breeder hatcheries were much less than that previously found in broiler hatcheries. Many factors contribute to the lower incidence and level of salmonellae found in the breeder hatcheries; however both the breeder and broiler hatcheries present critical control points in the prevention of salmonellae contamination during commercial poultry production. The cycle of salmonellae contamination will not likely be broken until contamination at these critical points is eliminated.

A Comparison of an Enzyme Immunoassay, DNA Hybridization, Antibody Immobilization, and Conventional Methods for Recovery of Naturally Occurring Salmonellae from Processed Broiler Carcasses.

Three hundred and ninety raw processed broiler carcasses were obtained from four processing plants and evaluated for the presence of salmonellae by the whole bird rinse procedure. Enzyme immunoassay (Salmonella-Tek™), colorimetric DNA hybridization (GENE-TRAKR), and antibody immobilization (1-2 Test™) methods were compared to the Food Safety Inspection Service (FSIS) culture method for detection of salmonellae. All samples were preenriched in buffered peptone water incubated at 37°C then transferred to and enriched in TT broth (Difco, Detroit, MI) incubated at 42°C for 24 h. After enrichment, manufacturer's instructions were then followed for "rapid" procedures and the FSIS recovery and confirmation scheme was followed for the cultural method. For each sample, if discrepant results were observed between the four procedures, selective plates were streaked from both the TT selective broth and the GN postenrichment broth (Difco). All samples which were positive after initial sampling of TT broth plus additional positive samples from TT selective and GN postenrichment broths were called confirmed cultural positives. Salmonellae were detected on 71% of the carcasses using confirmed cultural procedures and 65% with FSIS culture. Presumptive salmonellae positive samples were found 66, 71, and 76% of the time with the 1-2 Test, GENE-TRAK, and Salmonella-Tek, respectively. As compared to confirmed culture, only 1.4% false positives were observed with the 1-2 Test and GENE-TRAK, whereas 6% false positives were obtained using Salmonella-Tek. When the false negatives for the four methods were compared to confirmed culture, Salmonella-Tek, GENE-TRAK, 1-2 Test, and FSIS culture had 0.4, 2.5, 7.2, and 8.3%, respectively. The close correlation of these rapid procedures to the conventional procedures warrants consideration of these procedures for detection of salmonellae from broiler chickens.

Fifty percent colonization dose for Salmonella typhimurium administered orally and intracloacally to young broiler chicks.

One and 3-day-old chicks were challenged with varying levels of Salmonella typhimurium by gavage or intracloacal administration. Chicks were killed 5 days postchallenge, and ceca were analyzed for the presence of S. typhimurium. About 100-fold fewer S. typhimurium cells were required to colonize young chicks by the intracloacal route than by gavage. It was hypothesized that the low pH of the upper gastrointestinal tract contributes to the higher levels of Salmonella required to colonize young chicks via the oral route. The pH measurements in the gizzard of freshly killed chicks were variable, but most were low enough to be bactericidal. Presence of salmonellae in the hatchery environment and the low level of cells (2 cfu) required to colonize young chicks via cloacal challenge suggest that day-of-hatch chicks may be at a high colonization risk from salmonellae in the hatchery.

Presence and impact of Salmonella contamination in commercial broiler hatcheries.

Egg fragments from hatching trays, swabs of belting material, and paper pads from three broiler hatcheries were sampled for the presence and level of salmonellae. Salmonella serotypes were recovered from 71, 80, and 74% of the egg fragments, belting material, and paper pads, respectively. Overall, salmonellae were found in 75, 91, and 67% of the samples taken at Hatchery 1,2, and 3, respectively. Thirty-eight of 40 randomly selected samples contained greater than 10(3) salmonellae cells per sample. All of the Salmonella serotypes encountered in the present study had previously been isolated from poultry. The presence and persistence of salmonellae contamination in the hatchery suggests that the vulnerable day-of-hatch chick may be at a greater colonization risk in the hatchery than during grow-out. Contamination and penetration of the shell of hatching eggs may constitute the most important link (or critical control point) in the transmission of salmonellae to young birds and eventually the consumer. An effective intervention method may have to be employed at this point to break the transmission link and significantly impact the overall problem of Salmonella colonization in poultry.

Influence of host lineage on cecal colonization by Campylobacter jejuni in chickens.

The resistance to cecal colonization by Campylobacter jejuni was assessed by challenging three crossbred stocks of commercially available broiler chickens. These three stocks, designated A, B, and C, were related as follows: Offspring from four pedigreed grandparent flocks were used as progenitors. Stock B was derived by cross-breeding grandparent 1 with grandparent 3. Stocks A and C were crossbreeds from grandparents 1 and 2 and grandparents 3 and 4, respectively. Campylobacter jejuni were gavaged into 48-hour-old chicks, using the same levels of challenge dose for each of the different chicken stocks. Six days post-challenge, the birds were sacrificed, and cecal contents were plated onto Campylobacter-selective media. Results from two replicate trials with three isolates of C. jejuni indicated that chicken stock A was colonized in only two of 60 ceca, stock B in six of 60, and stock C in 19 of 60 chicken ceca. Statistical analysis of these data indicate that resistance to cecal colonization by C. jejuni was significantly (P less than 0.05) influenced through chicken host lineage.

In vitro attachment of Salmonella typhimurium to chick ceca exposed to selected carbohydrates.

This investigation was designed to study the effect of selected carbohydrates on the in vitro attachment of Salmonella typhimurium to the ceca of chickens 1 and 2 weeks of age. Ceca were surgically removed from chickens immediately after euthanasia, inverted on glass rods, and then rinsed with sterile saline before being exposed to S. typhimurium in a solution of saline containing the carbohydrate to be evaluated. Attachment of S. typhimurium to ceca was reduced in 1-week-old chicks in the presence of N-acetyl-D-galactosamine, L-fucose, D-galactose, L+arabinose, and D+mannose. Minimal or no reduction in attachment of S. typhimurium was noted when ceca from 2-week-old chicks were exposed to the same compounds. Carbohydrates investigated and found to be ineffective for reduction of S. typhimurium attachment to chick ceca were D+fucose and N-acetyl-D-glucosamine.

Methods for Detecting Processing Temperatures of Previously Cooked Meat and Poultry Products - A Review.

Title 9 of the Code of Federal Regulations establishes prescribed thermal treatment for a variety of meat and poultry products. These requirements are to ensure the destruction of harmful microorganisms and viruses that cause diseases in humans and livestock. The information presented in this review provides information relative to the current procedures used by the Food Safety and Inspection Service (FSIS) for monitoring the adequacy of heat treatment of meat and poultry products; and the research activities that have been and are currently being conducted to develop new and/or improved methods for determining the maximum internal temperature of meat and poultry products. Currently, FSIS is using a protein "Coagulation Test" for monitoring the maximum internal temperature (MIT) of both beef and pork products heat processed to temperatures lower than 65°C; a residual "Acid Phosphatase Activity Method" for determining the MIT of canned hams, canned picnics and canned luncheon meat, and a third method, known as the "Bovine Catalase Test", for the determination of catalase which gives a pass/fail indication at a cooking temperature of 62.8°C for rare roast beef and cooked beef. Since 1957, several attempts have been made to develop new and/or improved methods. These include an evaluation of the enzyme systems and various physical techniques. The lack of new and/or improved methods is not due to the lack of research efforts in this area, as evidenced by this review. The challenge is the development of a method which can accurately determine within ± 1.0°C the endpoint temperature in the temperature range (67.8 - 70.0°C) that is of most interest to FSIS.

Growth of Clostridium perfringens in cooked chili during cooling.

U.S. Department of Agriculture regulations require that brick chili be cooled from 48.9 degrees C to 4.4 degrees C within 2 h of cooking, but processors may not always be able to comply. Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures. Inoculated samples were heated (75 degrees C for 20 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h. No growth was observed for C. perfringens in samples held at 26.7 degrees C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 degrees C and after 2 h in samples held at temperatures between 37.8 degrees C and 48.9 degrees C. Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions. The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase. It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C. perfringens in chili. Actual data agreed closely with predicted results. The results should be useful for evaluating the hazard potential for growth of C. perfringens in chili.

Colonization characteristics of Campylobacter jejuni in chick ceca.

We report our findings on several parameters influencing cecal colonization of chickens by Campylobacter jejuni. Thirty-five colony-forming units (CFU) of a composite culture of C. jejuni colonized the ceca of one-half of the newly hatched chicks challenged by oral gavage. A challenge dose of 3500 CFU/chick consistently colonized the ceca of all chicks challenged. Challenge doses of approximately 10(5) CFU of C. jejuni per chick resulted in consistent cecal colonization, regardless of whether the birds were challenged 1, 2, or 3 days post-hatch. Four isolates showed consistently strong cecal colonization abilities, whereas two isolates colonized the ceca in only 20 of 122 chicks when given levels of 10(5) CFU per chick. One of these poorly colonizing isolates was repeatedly transferred by fecal-oral passage through chicks; subsequently, this isolate was able to consistently colonize chicks. Competitive exclusion (CE) microflora did not diminish the colonization rates for C. jejuni. Birds treated with five different CE cultures were colonized at a rate of 81 of 84 chicks; control chicks were similarly consistently colonized (45 of 46 chicks).

Effect of anticoccidial and antimicrobial feed additives on prevention of Salmonella colonization of chicks treated with anaerobic cultures of chicken feces.

Chicks were treated orally on the day of hatch with either fresh or frozen competitive exclusion (CE) cultures (native gut microflora). Chicks were fed either unmedicated feed or one of five commercial broiler starter rations or nine experimental feed mixtures containing varying amounts and combinations of anticoccidial and antimicrobial medicaments. After 2 days, they were challenged with approximately 10(6) colony-forming units of a nalidixic-acid-resistant strain of Salmonella typhimurium. Six days later, chicks were sacrificed and ceca were analyzed for S. typhimurium. Colonization of 2-day-old chicks was prevented or at least greatly reduced in most instances by treatment of chicks with a CE culture, but the efficacy of CE broth cultures stored at -70 C diminished over time. Not all CE cultures tested gave equal protection against Salmonella colonization, and CE cultures were more susceptible to some feed additives than others. Of the commercial or experimental feed tested, only the feed containing the combination of nicarbazin and bacitracin interfered with the protective effect of the CE culture.