Aims: The objective of this study was to determine if survival of culturable Campylobacter jejuni outside the host was increased by entrapment in pre‐established biofilms. Methods and Results: Campylobacter jejuni was inoculated into four biofilm populations isolated from poultry environments and cultured at three temperatures. Survival of culturable Camp. jejuni in some pre‐established biofilms was extended vs survival of culturable Camp. jejuni in broth. But some biofilms were detrimental to survival of culturable Camp. jejuni. Denaturing gradient gel electrophoresis analysis indicated differences in bacterial profiles depending on initial source and temperature of culturing, which may have had impacts on survival of culturable Camp. jejuni. Further investigation showed no evidence of interspecies cell signalling indicating that secondary colonization was only physical. Conclusions: The results of this study show Camp. jejuni’s attachment to surfaces is facilitated by pre‐established biofilms and survival of culturable Camp. jejuni may be extended in some pre‐established biofilms, but these biofilms do not fully explain long‐term survival of culturable Camp. jejuni outside hosts. Significance and Impact of the Study: This study provides new information concerning survival of culturable Camp. jejuni outside the host and shows biofilms may be important in transmission and prevalence of Camp. jejuni.
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.
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.
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.
In this study, we evaluated the ability of different Campylobacter phenotypes (biofilm versus planktonic) to colonize young poultry. It has been suggested that a persistent Campylobacter biofilm reservoir may be involved in the initial contamination of poultry flocks. Campylobacter jejuni cultured adherent to agar was utilized as the biofilm model and C. jejuni cultured in broth was evaluated as the planktonic model. In 2 independent trials, 1-d-old broiler chicks were given 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). Cecal contents of all birds were evaluated by culturing 12 d after the initial challenge with C. jejuni. In both trials, birds challenged with C. jejuni cultured in broth had approximately 3 to 4 log higher cecal Campylobacter concentration than birds challenged with C. jejuni cultured adherent to agar. Using 2 cell lines (INT 407 and DF1), virulence of C. jejuni cultured in broth versus adherent to agar also was evaluated by challenging monolayers of eukaryotic cells with 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). The virulence study also showed differences of C. jejuni cultured in broth or agar in attachment and invasion abilities to tissue culture cells, but differences were not as consistent as with the chick colonization study. This study indicates that phenotype may play a role in colonization of chickens and virulence by C. jejuni.
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