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Project rationale: Our proposed research relating to construction of a bioluminescent C. jejuni strain and in vivo real-time imaging would provide an unabridged approach to understand intra-intestinal as well as extra-intestinal biology of C. jejuni in chickens. <P>
Our proposed work would address aspects of C. jejuni interactions with chickens that are poorly understood; 1) dynamics of C. jejuni interaction with the intestinal tissues. It is still unclear whether bacteria reside in the lumen or mucus layer of the crypts or in the epithelium of the intestinal tissues, and 2) C. jejuni colonization of extra-intestinal tissues. Our approach would provide visual representation of kinetics of C. jejuni colonization of extra-intestinal tissues and may identify novel tissues of C. jejuni replication. It is not known how long C. jejuni persists in these extra-intestinal tissues and if these tissues serve as reservoir for C. jejuni. Determining whether these C. jejuni colonized extra-intestinal tissues ultimately contribute to re-infection of the intestinal tract leading to its persistence and shedding, is highly critical and would help device effective control strategies. Therefore, we anticipate that the use of bioluminescent C. jejuni may bridge the gap in our knowledge in understanding the colonization process observed in chickens. <P>
The specific objectives of this proposal are: <oL> <LI> To generate a bioluminescent strain of C. jejuni for in vivo real-time pathogenesis. We will use the modified EZ::TN transposon containing promoter-less lux operon to isolate bioluminescent C. jejuni that constitutively expresses strong bioluminescence using an endogenous promoter. Further, we will identify a C. jejuni strain that constitutively exhibits strong bioluminescence, colonizes, persists and sheds similar to its parental strain in chickens. <LI>To determine C. jejuni pathogenesis temporally in real-time in chickens Firstly, we will determine the dynamics of C. jejuni interaction with intestinal tissues. We will kinetically determine the association of bioluminescent C. jejuni with different parts of the intestinal tissues and its impact on persistence and shedding. Secondly, we will determine the dynamics of C. jejuni localization in extra-intestinal tissues. We will kinetically determine the association of bioluminescent C. jejuni with extra-intestinal tissues and its impact on intestinal colonization, persistence and shedding of C. jejuni.
NON-TECHNICAL SUMMARY: Preharvest control and elimination of Campylobacter from poultry would constitute an important measure for controlling human campylobacteriosis. Currently, there are no vaccines available against Campylobacter for use in poultry and current bio-security measures are not effective. Understanding the pathophysiology of Campylobacter in chickens is an important first step towards developing rational control measures. There is a critical need for better understanding of the mechanisms underlying the intra-intestinal as well as extra-intestinal biology of C. jejuni in chickens. We hypothesize that use of bioluminescent C. jejuni coupled with real-time imaging will provide a rational and unabridged approach to study C. jejuni pathogenesis in chickens. Here, we propose using bioluminescent C. jejuni to visualize temporally in real-time the pathogen-host interaction in a greater detail.
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APPROACH: To generate a strain of bioluminescent C. jejuni for in vivo real-time pathogenesis. Bioluminescent C. jejuni will be generated by electroporating the modified EZ::TN transposome containing promoter-less luxCDABE genes. The transposome will be electroporated into C. jejuni cells, the resulting kanamycin resistant transposants will be screened for bioluminescence using In Vivo Imaging System (IVIS). To quantitate bioluminescence, transposon mutants will be grown in MH broth, cell density will be adjusted to a constant OD, and bioluminescence will be determined. To identify a C. jejuni strain that constitutively exhibit strong bioluminescence, colonize, persist and shed similar to its parental strain in chickens, one-day-old broiler chicks will be inoculated orally with C. jejuni mutants that are strongly bioluminescent in broth. The infected chickens will be imaged daily for 14 days using our IVIS to monitor colonization. We will determine the relative strength of bioluminescence as well as their ability to bioluminesce throughout the course of infection. To determine C. jejuni pathogenesis temporally in real-time in chickens. To understand the dynamics of C. jejuni interaction with intestinal tissues, one-day old broiler chicks will be inoculated orally with bioluminescent C. jejuni, infection and persistence will be monitored by imaging chicks daily for 2 weeks. Fecal samples will be imaged daily to assess fecal shedding. To study the dynamics of C. jejuni association with different parts of the intestinal tissues as well as its nature of survival within the intestinal tissues, at specified times 2 chicks will be killed, entire gastrointestinal tract including crop will be removed and the presence of bioluminescent C. jejuni will be determined by ex vivo imaging. To determine the exact location of C. jejuni replication within the intestinal tissues, those regions that bioluminesce will be further analyzed by image-guided dissection. To determine whether C. jejuni is inside the epithelial cells, we will inoculate separate group of chickens with bioluminescent C. jejuni containing constitutively expressing GFP protein from plasmid pWM1007. Through image-guided dissection we will isolate bioluminescent positive tissues, fix with paraformaldehyde and analyze by fluorescent microscopy. To understand the dynamics of Campylobacter association with different extra-intestinal tissues, one-day old broiler chicks will be inoculated orally with bioluminescent C. jejuni, bioluminescence will be monitored by imaging chicks daily. Since, bioluminescent imaging can reveal dynamics of the infection in individual chicken, indicating waves of growth and clearance we would be able to monitor the impact of extra-intestinal colonization on the dynamics of intra-intestinal colonization of bioluminescent C. jejuni. At specified times different extra-intestinal tissues from 2 chicks will be removed and the presence of bioluminescent C. jejuni will be determined by ex vivo imaging.
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PROGRESS: 2007/09 TO 2008/08 <BR>
OUTPUTS: Objectives of our study were 1) To generate a bioluminescent strain of C. jejuni for in vivo real-time pathogenesis, and 2) To determine C. jejuni pathogenesis temporally in real-time in chickens. Preharvest control and elimination of Campylobacter from poultry would constitute an important measure for controlling human campylobacteriosis, which is the most common form of bacterial food poisoning in the United States and world-wide. Currently there are no effective methods available to control Campylobacter in poultry. Understanding the pathophysiology of Campylobacter in chickens is an important first step towards developing rational control measures. We hypothesize that use of bioluminescent C. jejuni coupled with real-time imaging will provide a rational and unabridged approach to study C. jejuni colonization in chickens. <BR>
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The proposed work would enable us to visualize temporally in real-time the 1) dynamics of C. jejuni association with different parts of intestinal tissues in chickens; particularly, the nature of its survival within the intestinal tissues (lumen or mucus layer of crypts or epithelial cells) and 2) dynamics of C. jejuni colonization of extra-intestinal tissues; whether, colonization of C. jejuni in extra-intestinal tissues serves as a source for re-infection of the intestinal tract thus leading to its persistence and shedding. Towards this end we have generated number of plasmid constructs to deliver the bioluminescence encoding lux genes into campylobacter. This would enable us to isolate a C. jejuni strain that constitutively exhibits strong bioluminescence, colonizes, persists and sheds similar to its parental strain in chickens. <BR>
PARTICIPANTS: Dr. Gireesh Rajashekara, Principal Investigator, is responsible for the leadership of the project and Dr. Qijing Zhang, Co-Principal Investigator, is serving as an expert consultant and provide advice regarding C. jejuni pathogenesis studies in chickens. The project has provided oppotrunity for training staff as well as graduate and undergraduate students <BR>
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IMPACT: 2007/09 TO 2008/08<BR>
Real time imaging provides a novel visual representation of the spatio-temporal distribution of bioluminescent campylobacter and accelerates our understanding of the C. jejuni colonization in chickens. Real time imaging facilitates better understanding of the campylobacter adherence to food surfaces following various processing procedures. These studies facilitates designing effective control strategies to reduce campylobacter contamination in poultry and poultry products thereby ensuring safety of our food.