Botanical alternatives to antibiotics for use in organic poultry production.

The development of antibiotic resistant pathogens has resulted from the use of sub-therapeutic concentrations of antibiotics delivered in poultry feed. Furthermore, there are a number of consumer concerns regarding the use of antibiotics in food animals including residue contamination of poultry products and antibiotic resistant bacterial pathogens. These issues have resulted in recommendations to reduce the use of antibiotics as growth promoters in livestock in the United States. Unlike conventional production, organic systems are not permitted to use antibiotics. Thus, both conventional and organic poultry production need alternative methods to improve growth and performance of poultry. Herbs, spices, and various other plant extracts are being evaluated as alternatives to antibiotics and some do have growth promoting effects, antimicrobial properties, and other health-related benefits. This review aims to provide an overview of herbs, spices, and plant extracts, currently defined as phytobiotics as potential feed additives.

Survival of Salmonella enterica in poultry feed is strain dependent.

Feed components have low water activity, making bacterial survival difficult. The mechanisms of Salmonella survival in feed and subsequent colonization of poultry are unknown. The purpose of this research was to compare the ability of Salmonella serovars and strains to survive in broiler feed and to evaluate molecular mechanisms associated with survival and colonization by measuring the expression of genes associated with colonization (hilA, invA) and survival via fatty acid synthesis (cfa, fabA, fabB, fabD). Feed was inoculated with 1 of 15 strains of Salmonella enterica consisting of 11 serovars (Typhimurium, Enteriditis, Kentucky, Seftenburg, Heidelberg, Mbandanka, Newport, Bairely, Javiana, Montevideo, and Infantis). To inoculate feed, cultures were suspended in PBS and survival was evaluated by plating samples onto XLT4 agar plates at specific time points (0 h, 4 h, 8 h, 24 h, 4 d, and 7 d). To evaluate gene expression, RNA was extracted from the samples at the specific time points (0, 4, 8, and 24 h) and gene expression measured with real-time PCR. The largest reduction in Salmonella occurred at the first and third sampling time points (4 h and 4 d) with the average reductions being 1.9 and 1.6 log cfu per g, respectively. For the remaining time points (8 h, 24 h, and 7 d), the average reduction was less than 1 log cfu per g (0.6, 0.4, and 0.6, respectively). Most strains upregulated cfa (cyclopropane fatty acid synthesis) within 8 h, which would modify the fluidity of the cell wall to aid in survival. There was a weak negative correlation between survival and virulence gene expression indicating downregulation to focus energy on other gene expression efforts such as survival-related genes. These data indicate the ability of strains to survive over time in poultry feed was strain dependent and that upregulation of cyclopropane fatty acid synthesis and downregulation of virulence genes were associated with a response to desiccation stress.

Yerba mate enhances probiotic bacteria growth in vitro but as a feed additive does not reduce Salmonella Enteritidis colonization in vivo.

Yerba mate (Ilex paraguariensis) is a tea known to have beneficial effects on human health and antimicrobial activity against some foodborne pathogens. Thus, the application of yerba mate as a feed additive for broiler chickens to reduce Salmonella colonization was evaluated. The first in vitro evaluation was conducted by suspending Salmonella Enteritidis and lactic acid bacteria (LAB) in yerba mate extract. The in vivo evaluations were conducted using preventative and horizontal transmission experiments. In all experiments, day-of-hatch chicks were treated with one of the following 1) no treatment (control); 2) ground yerba mate in feed; 3) probiotic treatment (Lactobacillus acidophilus and Pediococcus; 9:1 administered once on day of hatch by gavage); or 4) both yerba mate and probiotic treatments. At d 3, all chicks were challenged with Salmonella Enteritidis (preventative experiment) or 5 of 20 chicks (horizontal transmission experiment). At d 10, all birds were euthanized, weighed, and cecal contents enumerated for Salmonella. For the in vitro evaluation, antimicrobial activity was observed against Salmonella and the same treatment enhanced growth of LAB. For in vivo evaluations, none of the yerba mate treatments significantly reduced Salmonella Enteritidis colonization, whereas the probiotic treatment significantly reduced Salmonella colonization in the horizontal transmission experiment. Yerba mate decreased chicken BW and decreased the performance of the probiotic treatment when used in combination. In conclusion, yerba mate had antimicrobial activity against foodborne pathogens and enhanced the growth of LAB in vitro, but in vivo yerba mate did not decrease Salmonella Enteritidis colonization.

Influence of acid-adaptation of Campylobacter jejuni on adhesion and invasion of INT 407 cells.

The aim of this study was to determine the influence of acid-adaptation on the survival as well as adhesion and invasion of human intestinal cells by nine Campylobacter jejuni strains after exposure to different stress conditions. Acid-adapted and nonadapted C. jejuni were exposed to different secondary stress conditions such as acid (pH 4.5), starvation (phosphate-buffered saline, pH 7.2), or salt (3% wt/vol NaCl). After exposure to the secondary stress, the adhesion and invasion abilities of the strains were evaluated in vitro in tissue culture using the human intestinal cell line INT 407. The survival rates of acid-adapted cells of some strains of C. jejuni exposed to different secondary stresses were found to be significantly higher than the non-acid-adapted cells. Similarly, some strains also showed an increase in adhesion and invasion (p<0.05) when acid-adapted C. jejuni were exposed to stresses such as acid, starvation, or salt as compared to non-acid-adapted C. jejuni. We found that adaptation to acid stress can enhance the survival of C. jejuni when exposed to secondary stresses and, thus, result in increased adhesion and invasion of human intestinal cells in vitro. However, the survival rates as well as the degree of adhesion and invasion were found to vary with the strain of C. jejuni, the time of adaptation to acid, the type of the secondary stress and exposure time to the secondary stress. These results show that adaptation to stresses could influence virulence of C. jejuni. Understanding the conditions by which C. jejuni adapts to stresses will provide information concerning how this organism is able to survive inside and outside the host. This, in turn, could offer methods to reduce or eliminate C. jejuni in the environment.

Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production.

Fresh and processed poultry have been frequently implicated in cases of human salmonellosis. Furthermore, increased consumption of meat and poultry has increased the potential for exposure to Salmonella enterica. While advances have been made in reducing the prevalence and frequency of Salmonella contamination in processed poultry, there is mounting pressure on commercial growers to prevent and/or eliminate these human pathogens in preharvest production facilities. Several factors contribute to Salmonella colonization in commercial poultry, including the serovar and the infectious dose. In the early 1900s, Salmonella enterica serovars Pullorum and Gallinarum caused widespread diseases in poultry, but vaccination and other voluntary programs helped eradicate pullorum disease and fowl typhoid from commercial flocks. However, the niche created by the eradication of these serovars was likely filled by S. Enteritidis, which proliferated in the bird populations. While this pathogen remains a significant problem in commercial egg and poultry production, its prevalence among poultry has been declining since the 1990s. Coinciding with the decrease of S. Enteritidis, S. Heidelberg and S. Kentucky have emerged as the predominant serovars in commercial broilers. In this review, we have highlighted bacterial genetic and host-related factors that may contribute to such shifts in Salmonella populations in commercial poultry and intervention strategies that could limit their colonization.

Alternative antimicrobial compounds to control potential Lactobacillus contamination in bioethanol fermentations.

Antibiotics are commonly used to control microbial contaminants in yeast-based bioethanol fermentation. Given the increase in antibiotic-resistant bacteria, alternative natural antimicrobials were evaluated against the potential contaminant, Lactobacillus. The effects of nisin, ϵ-polylysine, chitosan (CS) and lysozyme were screened against 5 Lactobacillus strains. A standard broth- microdilution method was used in 96-well plates to assess the minimal inhibitory concentration (MIC). L. delbrueckii subsp lactis ATCC479 exhibited maximal MICs with CS, ϵ-polylysine and nisin (1.87, 0.3125 and 0.05 mg/mL, respectively). Nisin reduced most Lactobacillus strains by 6 log CFU/mL after 48 hours with the exception of L. casei. Synergism occurred when ethylenediaminetetraacetic acid (EDTA) was added with nisin. An MIC of 0.4 mg/mL of nisin combined with the EDTA at an MIC of 1 mg/ml markedly suppressed L .casei by 6 log CFU/mL. In conclusion, alternative antimicrobials proved to be a potential candidate for controlling bacterial contamination in the fermentation process. Synergistic effect of nisin with EDTA successfully inhibited the nisin-resistant contaminant, L. casei.

Caprylic Acid reduces enteric campylobacter colonization in market-aged broiler chickens but does not appear to alter cecal microbial populations.

Campylobacter is a leading cause of foodborne illness in the United States, and epidemiological evidence indicates poultry products to be a significant source of human Campylobacter infections. Caprylic acid, an eight-carbon medium-chain fatty acid, reduces Campylobacter colonization in chickens. How caprylic acid reduces Campylobacter carriage may be related to changes in intestinal microflora. To evaluate this possibility, cecal microbial populations were evaluated with denaturing gradient gel electrophoresis from market-age broiler chickens fed caprylic acid. In the first trial, chicks (n = 40 per trial) were assigned to four treatment groups (n = 10 birds per treatment group): positive controls (Campylobacter, no caprylic acid), with or without a 12-h feed withdrawal before slaughter; and 0.7% caprylic acid supplemented in feed for the last 3 days of the trial, with or without a 12-h feed withdrawal before slaughter. Treatments were similar for trial 2, except caprylic acid was supplemented for the last 7 days of the trial. At age 14 days, chicks were orally challenged with Campylobacter jejuni, and on day 42, ceca were collected for denaturing gradient gel electrophoresis and Campylobacter analysis. Caprylic acid supplemented for 3 or 7 days at 0.7% reduced Campylobacter compared with the positive controls, except for the 7-day treatment with a 12-h feed withdrawal period. Denaturing gradient gel electrophoresis profiles of the cecal content showed very limited differences in microbial populations. The results of this study indicate that caprylic acid's ability to reduce Campylobacter does not appear to be due to changes in cecal microflora.

Presence of arsenic resistance in Salmonella enterica serovar Kentucky and other serovars isolated from poultry.

A collection of 125 Salmonella enterica poultry isolates (71 serovar Kentucky isolates, and the remainder belonging to serovars Alachua, Enteritidis, Hadar, Heidelberg, Montevideo, Mbandaka, Senftenberg, Typhimurium, and Worthington) were tested for the ability to grow on tryptic soy agar containing sodium arsenite [As(III)] or arsenate [As(V)]. All serovar Kentucky isolates and 18 of the non-Kentucky isolates were able to grow in the presence of 0.1 mM As(III), and 69 grew in the presence of 1 mM As(V). Thirty of the non-Kentucky isolates did not grow at these As(III) and As(V) concentrations, but seven grew at 1 mM As(III) and 10 mM As(V). PCR-based analysis demonstrated the presence of arsB and arsD sequences in all Kentucky isolates, whereas one or both of these sequences were present in only 30 of the other isolates. It remains to be determined if these arsenic-resistance determinants benefit Salmonella exposed to man-made arsenic-containing compounds in poultry environments.

Transmission of antimicrobial resistant non-O157 Escherichia coli at the interface of animal-fresh produce in sustainable farming environments.

The interaction of typical host adapted enteric bacterial pathogens with fresh produce grown in fields is complex. These interactions can be more pronounced in co-managed or sustainable farms where animal operations are, by design, close to fresh produce, and growers frequently move between the two production environments. The primary objectives of this study were to 1) determine the transmission of STEC or enteric pathogens from small and large animal herds or operations to fresh produce on sustainable farms in TN and NC, 2) identify the possible sources that impact transmission of AMR E. coli, specifically STEC on these systems, and 3) WGS to characterize recovered E. coli from these sources. Samples were collected from raw and composted manure, environment, and produce sources. The serotype, virulence, and genotypic resistance profile were determined using the assembled genome sequences sequenced by Illumina technology. Broth microdilution was used to determine the antimicrobial susceptibility of each isolate against a panel of fourteen antimicrobials. The prevalence of E. coli increased during the summer season for all sources tested. ParSNP trees generated demonstrated that the transmission of AMR E. coli is occurring between animal feeding operations and fresh produce. Ten isolates were identified as serotype O45, a serotype that is associated with the "Big Six" group that is frequently linked with foodborne outbreaks caused by non-O157 E. coli. However, these isolates did not possess the stx gene. The highest frequency of resistance was detected against streptomycin (n = 225), ampicillin (n = 190) and sulfisoxazole FIS (n = 140). A total of 35 (13.7%) isolates from two TN farms were positive for the blaCMY (n = 5) and blaTEM (n = 32) genes. The results of this study show the potential of AMR E. coli transmission between animal feeding operations and fresh produce, and more studies are recommended to study this interaction and prevent dissemination in sustainable farming systems.

Development of a real-time polymerase chain reaction assay for the simultaneous detection of Mycoplasma gallisepticum and Mycoplasma synoviae under industry conditions.

In this research we developed a real-time SYBR green assay to detect both Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in a single reaction. A total of 30,000 samples from broiler breeder flocks were screened using traditional serology (plate agglutination, enzyme-linked immunosorbent assay, hemagglutination inhibition) and polymerase chain reaction (PCR; traditional and real-time). It was determined that the real-time SYBR green PCR assay developed in this research was more rapid than all three methods tested and more sensitive and specific than culturing or serology. The SYBR green assay was optimized and could detect as few as 30 template copies of DNA per sample. In addition, the SYBR green assay was less expensive than traditional culturing and serology. MG and MS are infectious bacteria that can rapidly spread and infect commercial chicken flocks. These diseases can cause a significant loss to the poultry industry and especially to broiler breeders because infected flocks are destroyed under the National Poultry Improvement Plan MG and MS clean programs. The real-time SYBR green assay developed in this research has the potential to reduce the time it takes to reach a correct diagnosis and to arrest outbreaks of MG and MS.

Salmonellosis outbreaks in the United States due to fresh produce: sources and potential intervention measures.

Foodborne Salmonella spp. is a leading cause of foodborne illness in the United States each year. Traditionally, most cases of salmonellosis were thought to originate from meat and poultry products. However, an increasing number of salmonellosis outbreaks are occurring as a result of contaminated produce. Several produce items specifically have been identified in outbreaks, and the ability of Salmonella to attach or internalize into vegetables and fruits may be factors that make these produce items more likely to be sources of Salmonella. In addition, environmental factors including contaminated water sources used to irrigate and wash produce crops have been implicated in a large number of outbreaks. Salmonella is carried by both domesticated and wild animals and can contaminate freshwater by direct or indirect contact. In some cases, direct contact of produce or seeds with contaminated manure or animal wastes can lead to contaminated crops. This review examines outbreaks of Salmonella due to contaminated produce, the potential sources of Salmonella, and possible control measures to prevent contamination of produce.

Development of rapid detection and genetic characterization of salmonella in poultry breeder feeds.

Salmonella is a leading cause of foodborne illness in the United States, with poultry and poultry products being a primary source of infection to humans. Poultry may carry some Salmonella serovars without any signs or symptoms of disease and without causing any adverse effects to the health of the bird. Salmonella may be introduced to a flock by multiple environmental sources, but poultry feed is suspected to be a leading source. Detecting Salmonella in feed can be challenging because low levels of the bacteria may not be recovered using traditional culturing techniques. Numerous detection methodologies have been examined over the years for quantifying Salmonella in feeds and many have proven to be effective for Salmonella isolation and detection in a variety of feeds. However, given the potential need for increased detection sensitivity, molecular detection technologies may the best candidate for developing rapid sensitive methods for identifying small numbers of Salmonella in the background of large volumes of feed. Several studies have been done using polymerase chain reaction (PCR) assays and commercial kits to detect Salmonella spp. in a wide variety of feed sources. In addition, DNA array technology has recently been utilized to track the dissemination of a specific Salmonella serotype in feed mills. This review will discuss the processing of feeds and potential points in the process that may introduce Salmonella contamination to the feed. Detection methods currently used and the need for advances in these methods also will be discussed. Finally, implementation of rapid detection for optimizing control methods to prevent and remove any Salmonella contamination of feeds will be considered.

Using fecal microbiota as biomarkers for predictions of performance in the selective breeding process of pedigree broiler breeders.

Much work has been dedicated to identifying members of the microbial gut community that have potential to augment the growth rate of agricultural animals including chickens. Here, we assessed any correlations between the fecal microbiome, a proxy for the gut microbiome, and feed efficiency or weight gain at the pedigree chicken level, the highest tier of the production process. Because selective breeding is conducted at the pedigree level, our aim was to determine if microbiome profiles could be used to predict feed conversion or weight gain in order to improve selective breeding. Using 16s rRNA amplicon sequencing, we profiled the microbiomes of high and low weight gain (WG) birds and good and poor feed efficient (FE) birds in two pedigree lineages of broiler chickens. We also aimed to understand the dynamics of the microbiome with respect to maturation. A time series experiment was conducted, where fecal samples of chickens were collected at 6 points of the rearing process and the microbiome of these samples profiled. We identified OTUs differences at different taxonomic levels in the fecal community between high and low performing birds within each genetic line, indicating a specificity of the microbial community profiles correlated to performance factors. Using machine-learning methods, we built a classification model that could predict feed conversion performance from the fecal microbial community. With respect to maturation, we found that the fecal microbiome is dynamic in early life but stabilizes after 3 weeks of age independent of lineage. Our results indicate that the fecal microbiome profile can be used to predict feed conversion, but not weight gain in these pedigree lines. From the time series experiments, it appears that these predictions can be evaluated as early as 20 days of age. Our data also indicates that there is a genetic factor for the microbiome profile.

Precut prepackaged lettuce: a risk for listeriosis?

The most recent outbreaks of listeriosis have been traced back to contaminated ready-to-eat (RTE) poultry and meat products. However, Listeria monocytogenes can be isolated from every food group, including fresh vegetables. This review is focused on one of the most popular RTE vegetable products, precut prepackaged lettuce. The available literature concerning Listeria contamination of vegetables is reviewed, and possible reasons why no recent outbreaks or sporadic cases of listeriosis due to contaminated precut prepackaged lettuce are explored.

The functionality of the gastrointestinal microbiome in non-human animals.

Due to the significance of the microbiome on human health, much of the current data available regarding microbiome functionality is centered on human medicine. For agriculturally important taxa, the functionality of gastrointestinal bacteria has been studied with the primary goals of improving animal health and production performance. With respect to cattle, the digestive functions of bacteria in cattle are unarguably critical to digestion and positively impact production performance. Conversely, some research suggests that the gastrointestinal microbiome in chickens competes with the host for nutrients and produces toxins that can harm the host resulting in decreased growth efficiency. Concerning many other species including reptiles and cetaceans, some cataloging of fecal bacteria has been conducted, but the functionality within the host remains ambiguous. These taxa could provide interesting gastrointestinal insight into functionality and symbiosis considering the extreme feeding regimes (snakes), highly specialized diets (vampire bats), and living environments (polar bears), which warrants further exploration.

Impact of rearing conditions on the microbiological quality of raw retail poultry meat.

There is a gap in knowledge of microbiological quality in raw chicken products produced by nonconventional methods and no studies have reported the microbiological quality of turkeys produced under different rearing environments. Thus, the aim of this study was to compare the microbiological quality of conventionally and organically reared whole chicken and turkey carcasses purchased from 3 retail outlets in Knoxville, Tenn., U.S.A. A total of 100 raw broiler chickens organically (n = 50) and 50 raw turkey carcasses consisting of 3 brands reared either conventionally (n = 25) or organically (n = 25) were evaluated. The FDA BAM protocol for rinsing poultry carcasses was used to enumerate of aerobic bacteria, Campylobacter, and Staphylococcus spp., and for qualitative analysis of Salmonella. Organic chickens from one brand had the highest average counts of aerobic bacteria, Staphylococcus spp. and Campylobacter (4.8, 4.8, and 4.7 Log10 CFU/mL rinsate, respectively) while the other organic brand had the lowest average counts (3.4, 3.3, and 3.1, respectively) of all 4 brands evaluated. The organic turkeys had the highest average counts of these same bacteria (4, 3.9, and 3.8, respectively) compared to the 2 brands of conventional turkeys evaluated. Salmonella (5% prevalence) was isolated only from organic chickens and turkeys. From these data, it appears that the microbiological quality of the raw product was not dependent on rearing conditions and, thus, it cannot be assumed that organic raw poultry is safer than conventionally raised poultry in terms of microbiological quality.

Expression of hilA in response to mild acid stress in Salmonella enterica is serovar and strain dependent.

Salmonella enterica is the leading cause of foodborne illness with poultry and poultry products being primary sources of infection. The 2 most common S. enterica serovars associated with human infection are Typhimurium and Enteritidis. However, Kentucky and Heidelburg and the 2 most prevalent serovars isolated from poultry environments. Given the prevalence of other serovars in poultry products and environments, research is needed to understand virulence modulation in response to stress in serovars other than Typhimurium and Enteritidis. Thus, the objective of this research was to compare hilA gene expression (a master regulator of the virulence pathogenicity island) in response to acid stress among different strains and serovars of Salmonella. A total of 11 serovars consisting of 15 strains of S. enterica were utilized for these experiments. Cultures were suspended in tryptic soy broth (TSB) adjusted to pH 7.2, 6.2, or 5.5 with HCl or acetic acid. Total RNA was extracted from cultures at specific time points (0, 2, 4, and 24 h). Gene expression of hilA was measured with quantitative reverse transcriptase real time PCR (qRT-PCR). Growth and pH were measured throughout the 24 h time frame. Regulation of hilA in response to acid stress varied by serovar and strain and type of acid. The results of these experiments indicate that hilA regulation may have some impact on virulence and colonization of S. enterica. However, these results warrant further research to more fully understand the significance of hilA regulation in response to mild acid stress in S. enterica.

Characterization of Staphylococcus aureus isolates from retail chicken carcasses and pet workers in Northwest Arkansas.

Staphylococcus aureus can be carried on the skin and nasal passages of humans and animals as a commensal. A case of human methicillin-resistant S. aureus infection resulting from contact with pork has been reported. Poultry carcasses are sold at retail with the skin intact, but pork and beef typically are not. Thus, the risk of methicillin-resistant S. aureus human infection from whole raw poultry carcasses may be greater than that of exposure from pork or beef. The objective of this study was to isolate and characterize S. aureus from whole retail poultry carcasses and compare the isolates to S. aureus isolates from humans. A total of 25 S. aureus isolates were collected from 222 whole poultry carcasses. The isolates were characterized phenotypically with antibiotic resistance disc diffusion assays and genotypically using multilocus sequence typing. A total of 17 S. aureus isolates obtained from healthy humans were included and characterized in the same way as the poultry isolates. Staphylococcus spp. were recovered from all poultry carcasses. Only 25 poultry carcasses (11.2%) were contaminated with S. aureus. Of these 25 isolates, 36% were resistant to at least one of the antibiotics tested and 20% were resistant to two or more antibiotics tested. However, 100% of the human isolates were resistant to at least one of the antibiotics and 94% were resistant to two or more antibiotics. The results of the multilocus sequence typing indicate that most of the isolates grouped according to source. These results indicate a low prevalence of S. aureus present in poultry, and the isolates were not phenotypically similar to human isolates. The low number of S. aureus isolates from this study indicates that chicken carcasses would appear to not be a significant source of this bacterium.

Prescreening of microbial populations for the assessment of sequencing potential.

Next-generation sequencing (NGS) is a powerful tool that can be utilized to profile and compare microbial populations. By amplifying a target gene present in all bacteria and subsequently sequencing amplicons, the bacteria genera present in the populations can be identified and compared. In some scenarios, little to no difference may exist among microbial populations being compared in which case a prescreening method would be practical to determine which microbial populations would be suitable for further analysis by NGS. Denaturing density-gradient electrophoresis (DGGE) is relatively cheaper than NGS and the data comparing microbial populations are ready to be viewed immediately after electrophoresis. DGGE follows essentially the same initial methodology as NGS by targeting and amplifying the 16S rRNA gene. However, as opposed to sequencing amplicons, DGGE amplicons are analyzed by electrophoresis. By prescreening microbial populations with DGGE, more efficient use of NGS methods can be accomplished. In this chapter, we outline the protocol for DGGE targeting the same gene (16S rRNA) that would be targeted for NGS to compare and determine differences in microbial populations from a wide range of ecosystems.

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples.

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.