Performing Department
Veterinary Medicine
Non Technical Summary
Campylobacter jejuni is a major cause of sheep abortion worldwide, incurring a significant economic burden for producers. Historically Campylobacter fetus was the main species associated with sheep abortion; however, our recent work revealed a remarkable species shift because C. jejuni has replaced C. fetus in the etiology of sheep abortion. More surprisingly, a single C. jejuni clone (named clone SA for sheep abortion) has emerged as the predominant cause of abortion outbreaks in the United States. Clone SA is highly abortifacient in pregnant animals and resistant to tetracycline, the only class of antibiotics approved for treating sheep abortion in the U.S. How this clone causes abortion is unknown, but its ability to cause bacteremia and systemic infection is considered a key step in the pathogenesis. With support from ILHAC, we have developed an efficient mouse model to assess the systemic spread of clone SA. Results from the mouse model have indicated that bacteremia and liver infection develop readily following oral inoculation (the natural route for abortion in sheep) with clone SA. With the availability of this mouse model and the advance in high-throughput sequencing technology, we are ideally poised to identify the Campylobacter genes required for systemic infection. The specific objectives of this project are to: 1) Systematic identification of bacterial factors involved in intestinal invasion and systemic spread by clone SA, using mouse infection model and cutting-edge transposon mutagenesis coupled with high-throughput deep sequencing technology, and 2) Functional confirmation of the identified genes in inducing systemic infection by clone SA using gene-specific knockout and evaluation in the mouse model. Results from this project will allow us to identify key bacterial factors that are responsible for abortion induction by Campylobacter, which may eventually be used for development of protective vaccines.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our long-term goal is to develop practical and effective measures to control Campylobacter-associated abortion in sheep flocks. The specific goal of this application is to identify bacterial factors that are important in invasion and systemic infection by C. jejuni using the established mouse animal model. The specific objectives of this proposal are:1.Systematic identification of bacterial factors involved in intestinal invasion and systemic spread by C. jejuni, using mouse infection model and cutting-edge transposon mutagenesis coupled with high-throughput deep sequencing. A large saturating library of transposon mutants of the highly-pathogenic C. jejuni clone SA will be constructed. This mutant pool will be used in the mouse model to investigate the bacterial genes involved in bacteremia and liver infection. 2.Functional confirmation of the identified genes in inducing systemic infection by C. jejuni using gene-specific knockout and evaluation in the mouse model. Using traditional microbiological and molecular genetics methods, the function of selected bacterial genes identified in Objective 1 in inducing systemic infection will be further investigated in the mouse model. We expect that findings from the proposed study will significantly advance our understanding of Campylobacter pathogenesis and potentially identify novel candidates for the development of protective vaccines against Campylobacter abortion in sheep. This work will capitalize on a cost-effective mouse infection model that can be used to assess Campylobacter pathogenicity.
Project Methods
Specific Objective 1. Systematic identification of bacterial factors involved in intestinal invasion and systemic spread by C. jejuni, using mouse infection model and cutting-edge transposon mutagenesis coupled with high-throughput deep sequencing. A large saturating library of transposon mutants of IA3902 will be constructed. Our laboratory has used this transposon for random and efficient mutagenesis of C. jejuni. IA3902 isolate is genetically amenable and we have made a number of insertional mutants in this isolate. We plan to generate a high-density library with approximately 10,000 transposon mutants, which will give more than 5X coverage of the entire genome of IA3902 (1686 ORFs). The transposon mutant library (input pool) will be inoculated into a group of 10 mice via the oral route (ca. 109 CFU per animal). Blood and liver will be collected to culture Campylobacter at 12 hr post inoculation at necropsy. The recovered Campylobacter colonies will be pooled for each sample type. Genomic DNA will be prepared from the input library and each pooled output population. DNA samples will be processed using a transposon-specific primer and an adaptor to enrich the transposon-chromosome junction sequences, which are then subject to deep sequencing by the use Next-Gen DNA sequencing technology. Since the genomic sequence of IA3902 is known, the transposon insertion sites in each population (input and output) can be easily identified using the sequence data. By comparing the mutant population in each sample with the original input population we will identify those mutants that are negatively selected during systemic infection. Specific Objective 2. Functional confirmation of the identified genes in inducing systemic infection by C. jejuni using gene-specific knockout and evaluation in the mouse model. To verify the role of the negatively selected genes, we will specifically inactivate each of them and assess their contribution in inducing systemic infection in the mouse model. If a large number of mutants are identified from the genome-wide screening, we will selectively confirm a subset of the mutants, which will be selected on the basis of total absence in the output population and the predicted functions of the affected genes. We have full expertise for mutant construction in C. jejuni and have all necessary tools and reagents available in our laboratory. If a mutant shows a change in virulence compared to wild-type IA3902 in the mouse model, we will complement the mutant to confirm its specific role in bacteremia. In the mouse model, each group (n=10) will be inoculated orally with either a mutant strain or wild-type IA3902 (108 CFU/mouse). At 12 hr after challenge, blood and liver will be collected and processed for Campylobacter isolation. Following culture results, one-way analysis of variance (ANOVA) will be used to determine the statistical significance of differences between treatment groups in the occurrence and intensity of bacteremia and liver infection.