Progress 01/01/13 to 12/31/18
Outputs
Target Audience:Researchers, veterinarians, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Seven undergraduate, 4 graduate students, and four postdocs/scientists were trained under this project. How have the results been disseminated to communities of interest?Results from the project were published in scientific journals and presented at conference meetings. What do you plan to do during the next reporting period to accomplish the goals?All the components of the project have been successfully completed, and this is the final report.
Impacts
What was accomplished under these goals?
Objective 1: Identify bacterial factors that are important for systemic infection of C. jejuni clone SA. Under this objective, firstly, a transposon library of about 37,000 mutants of IA3902 (a clone SA isolate) was generated using EZ-Tn5Transposome kit (Epicenter) for genome-wide identification of genes involved in systemic infection. This library of mutants was inoculated into bile (a natural environment for C. jejuni). Those mutants survived in bile were collected from different time points. The input mutant population and output (mutants that survived) populations were subjected to Tn-seq. Additionally, the mutant library was further used to identify genes required for producing bacteremia, a key step in abortion induction using a mice model of bacteremia. The mutant pool recovered from mouse blood was compared with the input population by Tn-seq. From comparative analysis of C. jejuni strains and Tn-seq analysis of the transposon mutant library following inoculation into bile and mice, several genes or gene variants were suspected to be involved in the pathogenesis of clone SA. Among these, the capsule locus (CPS) and CJSA_0039 (putative tyrosine phosphorylated regulator, TypA) were inactivated and found to be required for induction of systemic infection and abortion in pregnant guinea pigs by IA3902. Secondly, to determine how C. jejuni clone SA adapts in blood, we performed a time-course transcriptome analysis of its survival using an ex vivo model of sheep whole blood. We observed that C. jejuni clone SA altered the expression of ~36% (600/1666) of its ORFs. The major dynamic changes in expression were associated with genes involved in transcription and translation, amino acid transport and metabolism, inorganic ion transport and metabolism, chaperones, cell wall/membrane biogenesis, energy production and conversion, signal transduction mechanisms, and cell motility. Through mutagenesis studies of a subset of up-regulated genes, five novel virulence factors were identified to be important for survival in sheep blood. One of them, cjsa_0039, which was previously shown to play an important role in abortion induction in the guinea pig model. Finally, we developed a strategy ("directed genome evolution") by using by using hybridization between abortifacient (IA3902) and nonabortifacient (NCTC11168) C. jejuni strains followed by selection in an animal disease (pregnant guinea pig) model and whole-genome sequence analysis. This strategy successfully identified several SNPs in porA, encoding the major outer membrane protein (MOMP), that were responsible for the hypervirulence of clone SA in the induction of systemic infection and abortion. We further characterized the key role the mutations played in infection following the natural infection route in a guinea pig model. We found that the mutations were important for clone SA to translocate the gut barrier and enter the bloodstream. Furthermore, directed mutagenesis showed that only the mutations in the surface-exposed loop 4 of MOMP were required for the hypervirulence of clone SA. Altogether, these findings identified key virulence determinants responsible for abortion induction by C. jejuni clone SA. Objective 2: determine the mechanisms responsible for the emergence of C. jejuni clone SA in sheep abortion. Under this objective, next generation sequencing (NGS) was first used to analyze whole-genomes of 99 C. jejuni isolates (including clone SA and non-clone SA abortion strains from different time spans and geographical regions). All the genomes were assembled into draft sequences with genome sizes between ~1.6 - 1.8 Mb. Pan-genome analysis showed significant differences in gene contents among the isolates. Notably, the clone SA isolates had similar gene contents, while the non-clone SA abortion isolates were more divergent at the genome level. Interestingly, several non-clone SA abortion isolates had similar genomes to epidemiologically non-abortifacient isolates, providing additional clues for understanding the emergence and evolution of clone SA by the population genomics method. A whole-genome phylogeny confirmed the clonal expansion of clone SA, with a monophyletic origin close to MLST sequence type ST-50 C. jejuni strains in the ST-21 clonal complex, a reduced recombination subsequent to the emergence and a reduced haplotype diversity, and led to an estimate of its emergence in the 1970s, which is in agreement with its clinical appearance in the U.S. sheep production as a cause of abortion. However, the non-clone SA abortion isolates were found to be paraphyletic and genetically indistinguishable from other C. jejuni isolates from non-abortion sources. Further population genetics analysis showed that the jump of tetO (gene conferring resistance to tetracycline) from plasmid into chromosome of clone SA facilitated the population expansion. In addition, analysis also indicated that the highly virulent allele of porA of clone SA was positively selected, suggesting its association with clone SA expansion. Altogether, these results provided significant insights into the molecular genetic events underlying the emergence, evolutionary history, and increased fitness of C. jejuni clone SA. Objective 3: Evaluate protective immunity induced by commercial and experimental vaccines against C. jejuni clone SA. Under this objective, we first screened several commercial sheep flocks for Campylobacter infection status using ELISA and culture, and subsequent vaccination trials were carried out using flocks that were most appropriate for our purpose (the ones with the least level of specific antibody and culture positivity). We then performed 4 different pilot studies with sheep (first time pregnant) to determine the most appropriate route and challenge dose for inoculation with C. jejuni clone SA (oral, intravenous [IV], and intraperitoneal [IP] challenge routes were used). The findings from these experiments indicated that IV route of challenge with a high dose inoculum (~2x109 CFU) gave consistent abortion induction in pregnant ewes. We also noticed that removing/reducing the cell-free endotoxin from the culture supernatant and pre-medication of ewes with an anti-inflammatory drug (flunixin meglumine) were necessary to prevent the endotoxin induced non-specific abortion. Using the established model, we determined the efficacy of several vaccines for preventing clone SA induced abortion. The two commercial Campylobacter vaccines and an experimental bacterin (made of a clone SA strain IA3902) were administrated subcutaneously into pregnant ewes with two doses at two weeks apart. The animals were challenged via IV with ~2×109 CFUs of clone SA strain IA3902. All vaccines induced a high antibody response against C. jejuni, but the IA3902 bacterin induced more specific antibodies to clone SA. Notably, the IA3902 bacterin produced significant protection against the challenge compared with the sham-vaccinated controls, whereas the commercial vaccines did not significantly differ from the sham controls in abortion rates. The findings indicated that the homologous bacterin was protective, while the commercial vaccines were ineffective in protecting sheep from C. jejuni clone SA induced abortion. Altogether, these results strongly suggest that a homologous vaccine can be used to effectively control C. jejuni clone SA on sheep farms.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Xu, C., Z. Wu, O. Sahin, and Q. Zhang. 2018. Invasion of cell cultures doesnt predict the hypervirulence of Campylobacter jejuni in abortion induction. ASM Microbe. June 2018. Atlanta, GA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Dai, L., Z. Wu, and Q. Zhang (2018). The transcriptional terminator of porA enhances expression of the major outer membrane protein in Campylobacter jejuni. Annual Conference of ASM Microbe, June 8-11, 2018, Atlanta, GA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Zhang Q. 2018. Deciphering Bacterial Pathogenesis: Harnessing the Power of Genomics and Experimental Evolution (featured speech). Dec. 3, 2018. Annual Conference of Conference for Research Workers in Animal Diseases. Dec. 1-5, Chicago, IL
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Sahin O. 2018. Emergence and Fitness of a hypervirulent Campylobacter jejuni clone associated with sheep abortion and human disease in the United States. Oral presentation at College of Veterinary Medicine Biomedical Sciences, Sokoine University of Agriculture, Morogoro, Tanzania
|
Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Researchers, veterinarians, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Zuowei Wu, Assistant Scientist, plays a major role in various parts of the project including functional genomics and whole genome sequencing. Yizhi Tang, graduate student, investigated the molecular mechanisms of phenotype changes due to gene mutations. Jinji Pang, visiting undergraduate student, worked on the effect of tetracycline treatment on the emergence of tetracycline resistant Campylobacter in sheep. Jing Xia, visiting graduate student, worked on the effect of tetracycline treatment and tetracycline resistance on the emergence tetracycline resistance C. jejuni and C. coli in sheep. Nada Pavlovic, Research Associate, provides technical help on various aspects of the project involving genomic characterization and animal studies. How have the results been disseminated to communities of interest?Via publications and posters/presentations at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?The main task for the next period is to assess the protection of various vaccines against sheep abortion in pregnant ewes caused by Campylobacter jejuni.
Impacts
What was accomplished under these goals?
Objective 1: Identify bacterial factors that are important for systemic infection of C. jejuni clone SA. To determine how C. jejuni clone SA adapts in blood, we performed a time-course transcriptome analysis of its survival using an ex vivo model of sheep whole blood. We observed that C. jejuni clone SA altered the expression of ~36% (600/1666) of its ORFs. The major dynamic changes in expression were associated with genes involved in transcription and translation, amino acid transport and metabolism, inorganic ion transport and metabolism, chaperones, cell wall/membrane biogenesis, energy production and conversion, signal transduction mechanisms, and cell motility. Through mutagenesis studies of a subset of up-regulated genes, five novel virulence factors were identified to be important for survival in sheep blood. One of them, cjsa_0039, which encodes a GTP-binding protein (TypA), was demonstrated to play an important role in abortion induction in the guinea pig model. These results identified new virulence factors involved in pathogenesis and abortion induction, and provide new insights into how C. jejuni adapts in blood during systemic infection. This specific objective is approximately 100% complete. Objective 2: determine the mechanisms responsible for the emergence of C. jejuni clone SA in sheep abortion. In order to investigate the influence of tetracycline use and tetracycline resistance on the emergence and evolution of C. jejuni clone SA, a controlled laboratory treatment study using commercial sheep naturally infected with Campylobacter was conducted. In two separate trials, each involving 80 feeder lambs with 40 in medicated group (tetracycline 350 mg/hd/d in feed) and 40 in non-medicated group, fecal samples were collected weekly for 3 weeks and cultured for Campylobacter. The analysis showed that the overall prevalence of Campylobacter in sheep feces was 87% (401/461 samples), of which 68.6% (275/401) was C. jejuni and 37.4% (150/401) was C. coli. Although it was not statistically significant, the medicated group presented a higher proportion of C. coli and lower proportion of C. jejuni than the non-medicated groups. It was found that nearly all of the Campylobacter isolates were resistant to tetracycline, even those isolates collected prior to the treatment. Interestingly, clone SA was not present within the lambs examined in the trials. These findings indicated that tetracycline-resistant Campylobacter is now highly prevalent in sheep and suggest that the selective role played by tetracycline in the emergence of clone SA likely occurred in the early stage. This specific objective is 100% complete Objective 3: Evaluate protective immunity induced by commercial and experimental vaccines against C. jejuni clone SA. We conducted two more sheep trials to determine the optimal dose and conditions that can reproducibly induce clinical abortion in pregnant ewes using the intravenous route of inoculation. The trials successfully identified the optimal dose (~1 x 109 CFU/ml) and also revealed that a washing step helped to remove cell-free endotoxin present in culture medium. With the optimized conditions, clinical abortion was consistently induced by C. jejuni clone SA in pregnant ewes without causing non-specific endotoxin-mediated shock. Both the culture (including uterus, placenta and fetal tissues) and histopathology results performed on specimens collected at necropsy confirmed the finding from clinical observations. With these results, we have successfully established a model that can be used to evaluate the protective role of vaccines against Campylobacter-induced abortion. A major vaccine trail is currently underway using this established model. This specific objective is approximately 50% complete and the rest of work will be completed by the end of the no-cost extension period.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Sahin O. Evolution and virulence of an emergent hypervirulent Campylobacter jejuni clone in the United States. Seminar series in Veterinary Biomedical Sciences, Iowa State University. January 12, 2017 (invited oral presentation).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Pang J, Yizhi Tang, Jing Xia, Wu Z, Lei Dai, Singh K, Changyun Xu, Lining Xia, Xiaoping Ma, Sahin O, Plummer Z, and Zhang Q. Prevalence and antimicrobial susceptibility of Campylobacter in sheep. Poster presentation at Conference of Research Workers in Animal Diseases Meeting, December 3-5, 2017, Chicago, IL
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Wu, Z., Kurkiewicz, D., Sahin, O., Liu, P., Zhang, Q. 2017. Blood adaptation mechanisms of Campylobacter jejuni associated with systemic infection. 19th International Workshop on Campylobacter, Helicobacter & Related Organisms.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Qijing Zhang. 2017. Emergence of a Highly Pathogenic Campylobacter jejuni Clone in the United States. IAFP Symposium on Mechanisms of Hypervirulence in Selected Foodborne Pathogens. International Association for Food Protection 2017 annual conference, July 9 - 12, 2017, Tampa, Florida.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Sahin O, Samantha A. Terhorst, Eric R. Burrough, Zhangqi Shen, Zuowei Wu, Lei Dai, Paul J. Plummer, Michael J. Yaeger, and Qijing Zhang. 2017. Key role of capsular polysaccharide in the induction of systemic infection and abortion by hypervirulent Campylobacter jejuni. Infect Immun. 85 (6): e00001-17
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Sahin O, Yaeger M, Wu Z, and Zhang Q. Campylobacter-associated diseases in animals. 2017. Annu Rev Anim Biosci 8(5): 21-42. (Invited review article)
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Tang Y, Meinersmann RJ, Sahin O, Wu Z, Dai L, Carlson J, Plumblee J, Genzlinger L, LeJeune JT, Zhang Q. 2017. Wide but variable distribution of a hypervirulent Campylobacter jejuni clone in beef and dairy cattle in the United States. Appl Environ Microbiol. Sep 29. pii: AEM.01425-17
|
Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Researchers, veterinarians, producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Zuowei Wu, Assistant Scientist, plays a major role in various parts of the project including functional genomics and whole genome sequencing. Yizhi Tang, graduate student, is investigating the molecular mechanisms of phenotype changes due to gene mutations. Jinhu Huang, visiting graduate student, worked on the functional characterization of immunodominant antigens recognized by the aborted sheep sera and preparation of subunit vaccines. Victoria Lashley, graduate student and pathology resident, worked on animal studies and histopathological examination of lesions. Nada Pavlovic, Research Associate, provides technical help on various aspects of the project involving genomic characterization and animal studies. How have the results been disseminated to communities of interest?Via publications and posters/oral presentations at scientific conference meeting. What do you plan to do during the next reporting period to accomplish the goals?We will further define the specific mutations in Loop 4 of MOMP that modulate the virulence of C. jejuni clone SA. More detailed analysis of the genome sequences of clone SA and other C. jejuni strain will be conducted to gain insights into the emergence and virulence of clone SA. Major efforts will be devoted to assess the efficacy of various vaccines against sheep abortion in pregnant ewes.
Impacts
What was accomplished under these goals?
Objective 1: Identify bacterial factors that are important for systemic infection of C. jejuni clone SA. We developed a strategy ("directed genome evolution") by using by using hybridization between abortifacient (IA3902, a clone SA isolate) and nonabortifacient (NCTC11168) strains followed by selection in an animal disease (pregnant guinea pig) model and whole-genome sequence analysis. This strategy successfully identified SNPs in porA, encoding the major outer membrane protein (MOMP), are responsible for the hypervirulence of clone SA in the induction of systemic infection and abortion. We further characterized the key role the mutations played in infection following the natural infection route in a guinea pig model. We found that the mutations were important for clone SA to translocate the gut barrier and enter the bloodstream. Furthermore, directed mutagenesis showed that only the mutations in the surface-exposed loop 4 of MOMP were required for the hypervirulence of clone SA. Therefore, we identified a key virulence determinant responsible for abortion induction by C. jejuni. Studies are currently underway to determine how the sequence polymorphisms in the outer membrane protein influence the virulence of this hypervirulent C. jejuni clone. This specific objective is approximately 90% complete and will be finished in the 5th-year funding period. Objective 2: determine the mechanisms responsible for the emergence of C. jejuni clone SA in sheep abortion. To ascertain the evolution and the adaptive genetic changes responsible for the emergence and predominance of clone SA, we analyzed the whole genomes of 99 C. jejuni isolates from a historical collection encompassing clinical isolates from sheep abortion cases collected from geographically distinct regions since early 1990s. The analysis indicated clonal expansion of clone SA with a monophyletic origin within ST-21 complex, with reduced recombination rate since its emergence in the early 1970s. Population genetics analysis showed that the jump of tetO (gene conferring resistance to tetracycline) from plasmid into chromosome of clone SA facilitated the population expansion. In addition, analysis also indicated that the highly virulent allele of porA of clone SA has transferred to non-clone SA (both from abortion and commensal) strains. These results provided significant insights into the molecular genetic events underlying the emergence, evolutionary history, and increased fitness of C. jejuni clone SA. This specific objective is approximately 85% complete and expected to be finished in the 5th year funding period. Objective 3: Evaluate protective immunity induced by commercial and experimental vaccines against C. jejuni clone SA. We have performed two pilot studies to determine the most appropriate route and challenge dose for inoculation of pregnant ewes with C. jejuni clone SA (oral, intravenous [IV], and intraperitoneal [IP] challenge routes were used). The findings from these experiments indicated that IV route of challenge with a high dose inoculum dose (~1x109 CFU) is necessary to induce abortion in pregnant ewes. We also noticed that removing/reducing the cell-free endotoxin in the culture supernatant made a significant difference in regard to abortion induction, i.e., washing off the inoculum in PBS rendered the organism non-abortifacient while the endotoxin induced non-specific abortion in some ewes. Currently, we are planning to perform another study to determine the optimal dose and other conditions for consistent abortion-induction in pregnant ewes by C. jejuni clone SA. Once the model is successfully established, we will then use vaccines (including a bacterin, recombinant vaccine, and commercial vaccines) to determine their efficacy for preventing clone SA induced abortion. This specific objective is approximately 35% complete and it is possible a no cost extension beyond the 5th year funding period is needed to complete this objective.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Wu, Z., P. Periaswamy, O. Sahin, M. Yaeger, P. Plummer, W. Zhai, Z. Shen, L. Dai, S. L. Chen, and Q. Zhang. 2016. Point mutations in the major outer membrane protein drive hypervirulence of a rapidly expanding clone of Campylobacter jejuni. Proc. Natl. Acad. Sci. U. S. A. 113 (38):10690-95. http://www.pnas.org/content/113/38/10690.long
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Wu Z, Mou K, Sahin O, and Zhang Q. 2016. Genomic insights into Campylobacter jejuni virulence and Population Genetics. Inf. Dis. Trans. Med. (http://www.tran-med.com/EN/abstract/abstract31.shtml).
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Sahin O, Yaeger M, Wu Z, and Zhang Q. Campylobacter-associated diseases in animals. Annual Review of Animal Biosciences. 2016. In Press (Invited review article).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Lashley V, Yaeger M, Ji J, Sahin O, and Wu Z. 2016. Investigating the suitability of a laboratory mouse model to study the pathogenesis of abortifacient Campylobacter jejuni. Submitted to Veterinary Pathology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Sahin O, Plummer P, Wu Z, Yaeger M, and Zhang Q. Emergence and predominance of a hypervirulent, tetracycline-resistant Campylobacter jejuni clone as a major cause of sheep abortion in the United States. 120th United States Animal Health Association (USAHA) Meeting, October 13-19, 2016, Greensboro, North Carolina. (Invited talk for the Committee on Sheep and Goats).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Sahin O. 2016. Emergence and fitness of a hypervirulent Campylobacter jejuni clone in the United States. Seminar talk given to graduate students at Interdepartmental Microbiology program, Iowa State University, Ames, IA. September 22, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Wu Z, Periaswamy B, Sahin O, Chen S, and Zhang Q. Population genomics of Campylobacter jejuni associated with sheep abortion. ASM microbe-2016. June, 2016, Boston, MA, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Zhang, Q. 2016. Emergence and zoonotic transmission of hypervirulent Campylobacter jejuni: impact on One Health. Symposium on Veterinary Science and Public Health. Shanghai, China, September 23, 2016.
|
Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Researchers, veterinarians, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Zuowei Wu, Assistant Scientist, plays a major role in various parts of the project including functional genomics and whole genome sequencing. Yizhi Tang, graduate student, is investigating the molecular mechanisms of phenotype changes due to gene mutations.Jinhu Huang, visiting graduate student, is working on the functional characterization of immunodominant antigens recognized by the aborted sheep sera and preparation of subunit vaccines. Nada Pavlovic, Research Associate, provides technical help on various aspects of the project involving genomic characterization and animal studies. Zhangqi Shen, Postdoctoral Researcher, is involved in gene specific mutagenesis and animal studies. How have the results been disseminated to communities of interest?Via publications and posters/presentations at scientific conference meetings. What do you plan to do during the next reporting period to accomplish the goals?The underlying molecular and biological basis for the enhanced virulence associated with the sequence polymorphism in the outer membrane protein of this unique C. jejuni clone will be investigated. More detailed analysis of the genome sequences of clone SA and other C. jejuni strain will be analyzed to gain insights into the emergence and virulence of clone SA. Protective ability of commercial and the recombinant (based on the newely identified virulence factor of clone SA) vaccines against sheep abortion will be determined in pregnant ewe experiments.
Impacts
What was accomplished under these goals?
Under Objective 1, we have employed a novel strategy to identify specific genetic determinants of C. jejuni clone SA (SA for sheep abortion) for induction of systemic infection and abortion in pregnant animals by taking advantage of natural competence of C. jejuni, positive selection in an animal model, and next generation sequencing. A nonabortifacient C. jejuni strain (NCTC11168) was transformed with the genomic DNA of an abortifacient clone SA strain (IA3902). The transformed NCTC11168 strain was then found to be fully virulent in the pregnant guinea pig disease model. Deep sequencing and comparative genomics analyses of the isolates from the aborted animals indicated that transfer of several point mutations in a gene encoding an outer membrane protein was associated with gain of virulence. Directed genetic experiments including allelic exchanges proved that sequence polymorphisms in the outer membrane protein are both necessary and sufficient for inducing abortion in pregnant animals. Therefore, we identified a key virulence determinant responsible for abortion induction by C. jejuni. Studies are currently underway to determine the biological basis for enhanced virulence associated with the sequence polymorphism in the outer membrane protein of this unique C. jejuni clone. Under Objective 2, to ascertain the evolution and the adaptive genetic changes responsible for the emergence and predominance of clone SA, we analyzed the whole genomes of 99 C. jejuni isolates from a historical collection encompassing clinical isolates from sheep abortion cases collected from geographically distinct regions since early 1990s. A whole-genome phylogeny confirmed the clonal expansion of clone SA, with a monophyletic origin close to MLST sequence type ST-50 C. jejuni strains in the ST-21 clonal complex, a reduced recombination subsequent to the emergence and a reduced haplotype diversity, and led to an estimate of its emergence in the 1970s, which is in agreement with its clinical appearance in the U.S. sheep production as a cause of abortion. However, the non-clone SA abortion isolates were found to be paraphyletic and genetically indistinguishable from other C. jejuni isolates from non-abortion sources. These results provided significant insights into the molecular genetic events underlying the emergence, evolutionary history, and increased fitness of C. jejuni clone SA. Under Objective 3, we have performed a pilot study to determine the most appropriate route for inoculation of pregnant ewes with C. jejuni clone SA (we used oral, IV and IP challenge routes). Currently, we are set to carry out another study to assess the most appropriate dose of clone SA to induce abortion in pregnant ewes following the all three inoculation routes. A bacterin (made up of killed clone SA cells) and a recombinant vaccine are prepared. The recombinant vaccine is based on identified virulence factors. These vaccines along with commercial vaccines will be included in the vaccination trials.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Liu F, Wang C, Wu Z, Zhang Q and Liu P.2015. A Zero-Inflated Poisson Model for Insertion Tolerance Analysis of Genes Based on Tn-seq Data. Bioinformatics, 2016 Feb 1. pii: btw061
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Zuowei Wu, Orhan Sahin, Michael Yaeger, Balamurugan Periaswamy, Swaine Chen, Victoria Lashley, Paul Plummer, Zhangqi Shen, and Qijing Zhang. Mutations in an outer membrane protein enhance the virulence of Campylobacter jejuni. Oral presentation at CHRO Conference Meeting, November 1-5, 2015, Rotorua, New Zealand.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Sahin O, Terhorst S, Burrough E, Shen Z, Wu Z, Plummer P, Yaeger M, and Zhang Q. Key role of capsular polysaccharide in the induction of systemic infection and abortion by Campylobacter jejuni. Poster presentation at CHRO Conference Meeting, November 1-5, 2015, Rotorua, New Zealand.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Wu Z, Sahin O, Terhorst S, Periaswamy B, Burrough E, Shen Z, Plummer P, Yaeger M, and Zhang Q. Identification of two key factors associated with Campylobacter-induced abortion. In Proceedings of the 96th Annual Meeting of the CRWAD. Dec. 8-10, 2015. Chicago, IL.
- Type:
Other
Status:
Published
Year Published:
2015
Citation:
Qijing Zhang. 2015. Emergence of highly pathogenic Campylobacter jejuni in the United States: impact on One Health. University of California-Davis. Davis, California, July 29, 2015.
- Type:
Other
Status:
Published
Year Published:
2015
Citation:
Qijing Zhang. 2015. Emergence and pathogenesis of a hyper-virulent Campylobacter jejuni clone associated with zoonotic diseases. University of Tennessee, Knoxville, March 23, 2015.
|
Progress 01/01/14 to 12/31/14
Outputs
Target Audience: Researchers, veterinarians, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Zuowei Wu, Postdoctoral Researcher, plays a major role in various parts of the project including functional genomics and whole genome sequencing. Yizhi Tang, graduate student, is investigating the molecular mechanisms of phenotype changes due to gene mutations. Jinhu Huang, visiting graduate student, is working on the functional characterization of immunodominant antigens recognized by the aborted sheep sera and preparation of subunit vaccines. Nada Pavlovic, Research Associate, provides technical help on various aspects of the project involving genomic characterization and animal studies. Zhangqi Shen, Postdoctoral Researcher, is involved in gene specific mutagenesis and animal studies. How have the results been disseminated to communities of interest? Via publications in journals and presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? The molecular genetic mechanism of abortion induction by a non-abortifacient C. jejuni strain following transformation with the DNA of an abortifacient C. jejuni strain will be determined. Underlying mechanisms of the loss of abortion phenotype in specifif gene mutants of C. jejuni abortion clone will be investigated. Whole genome sequences of large numbers of C. jejuni strains will be analyzed to understand the evolution and pathogenesis of clone SA in sheep abortion. Sheep experiments will be carried out to study the role of certain gene mutants of C. jejuni clone SA in abortion process and to evaluate the efficacy of commercial vaccines against clone SA incuced abortion in pregnant sheep.
Impacts
What was accomplished under these goals?
Under Objective 1, from comparative analysis of C. jejuni strains and Tn-seq analysis of the transposon mutant library following inoculation into bile and mice, several genes or gene variants were suspected to be involved in the pathogenesis of clone SA. Among these, the capsule locus (CPS) and CJSA_0039 (putative tyrosine phosphorylated regulator) were inactivated and found to be absolutely required for induction of systemic infection and abortion in pregnant guinea pigs by IA3902 (a clone SA isolate). To further screen for the genes involved in systemic spread, the transposon library of IA3902 was also tested in the pregnant guinea pig model. The genes contributing to abortion are currently being identified by Tn-seq analysis. Additionally, a novel transformation strategy was utilized to determine the specific genetic determinants required for abortion. Transforming C. jejuni 11168, which is a nonabortifacient strain, with genomic DNA of IA3902 resulted in gain of full virulence in the pregnant guinea pig model. Studies are currently ongoing to find out the exact genetic determinants for conferring virulence on the nonabortifacient strain. Under Objective 2, Next-Gen sequencing was used to analyze whole-genomes of 99 C. jejuni isolates (including clone SA and non-clone SA abortion strains from different time spans and geographical regions). All the genomes were assembled into draft sequences with average N50 of 272 Kb and genome sizes between 1.6~1.8 Mb. Pan-genome analysis showed significant differences in gene contents among the isolates. Notably, the clone SA isolates had similar gene contents, while the non-clone SA abortion isolates were more divergent at the genome level. Interestingly, several non-clone SA abortion isolates had similar genomes to epidemiologically non-abortifacient isolates, which provides additional clues for understanding the emergence and evolution of clone SA by the population genomics method. For objective 3, immunoproteomics was completed to identify potential immunogens. Selection of suitable sheep flocks for immunization experiments is being performed. Both ELISA and culture are being used to determine the infection status of sheep flocks.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zuowei Wu, Orhan Sahin, Fei Wang, Qijing Zhang. 2014. Proteomic identification of immunodominant membrane-related antigens in Campylobacter jejuni associated with sheep abortion. Journal of Proteomics, 99:111-122
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Zuowei Wu, Dason Kurkiewicz, Orhan Sahin, Michael Yaeger, Peng Liu, Paul Plummer, Zhangqi Shen, and Qijing Zhang. Transcriptomic responses of a Campylobacter jejuni strain associated with sheep abortion to sheep whole blood and identification of a novel abortion-inducing factor. In Proceedings of the 95th Annual Meeting of the CRWAD. Dec. 7-9, 2014. Chicago, IL
- Type:
Other
Status:
Other
Year Published:
2014
Citation:
Qijing Zhang. 2014. Emergence of a zoonotic and highly virulent Campylobacter jejuni clone in the United States. 2014. Great Plains Emerging Infectious Diseases Conference, University of Iowa. April 18, 2014 (invited presentation).
- Type:
Other
Status:
Other
Year Published:
2014
Citation:
Qijing Zhang. 2014. Emergence and evolution of a highly virulent and antibiotic-resistant Campylobacter jejuni clone in the U.S. Department of Biological Science, The University of Tulsa, Tulsa, OK, August 29, 2014 (invited presentation).
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Progress 01/01/13 to 12/31/13
Outputs
Target Audience: Researchers, veterinarians, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training: Zuowei Wu, Postdoctoral Researcher, plays a major role in various parts of the project including functional genomics and whole genome sequencing. Fei Wang, graduate student, is working on the functional characterization of immunodominant antigens recognized by the aborted sheep sera. Nada Pavlovic, Research Associate, provides technical help on various aspects of the project involving genomic characterization and animal studies. Zhangqi Shen, Postdoctoral Researcher, is involved in gene specific mutagenesis and animal studies. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Specific gene-knockout mutants identified via Tn-Seq approach will be evaluated in the pregnant guinea pig model. Genome sequences of a large number of C. jejuni abortion isolates will be analyzed to understand the emergence and fitness of clone SA in the USA. Promising vaccine cantidates will be evaluated in animal challenge/vaccination experiments.
Impacts
What was accomplished under these goals?
IMPACT:The progress has laid a solid foundation for us to further dissect the virulent mechanisms of C. jejuni clone SA in abortion induction and the evolutionary events that led to its emergence and predominance. Additionally, the immunization work will likely yield a vaccine candidate for this important pathogen. The project will directly benefit sheep production in the U.S. by facilitating the development of effective strategies for the control of Campylobacter-associated abortion. The work will also have a significant impact on human health because control of this emergent pathogen in ruminants will block its transmission to the human hosts. ACCOMPLISHMENTS: Under Objective 1, a transposon library of about 37,000 mutants of IA3902 was generated using EZ-Tn5Transposome kit (Epicenter) for genome-wide identification of genes involved in systemic infection. This library of mutants was inoculated into bile (a natural environment for C. jejuni). Those mutants survived in bile were collected from different time points. The input mutant population and output (mutants that survived) populations were subjected to Tn-seq. A number of mutants that survived poorly in bile were identified and are currently under investigation. Additionally, the mutant library was further used to identify genes required for producing bacteremia, a key step in abortion induction. For this purpose, this library of mutants was inoculated into mice and their blood were collected 8 hours after inoculation for culturing Campylobacter. The mutant pool recovered from mouse blood was compared with the input population by Tn-seq. This screening identified a number of mutants that failed to cause bacteremia. These mutants are currently under characterization. Under Objective 2, Illumina sequencing is being used to analyze whole-genomes of C. jejuni strains (both clone SA and non-clone SA isolates) from sheep abortions occurring since 1990s. Using the previously sequenced complete genome of IA3902 as scaffold, the newly sequenced 7 clone SA genomes were assembled and comparative genomics were performed in-silico to reveal genetic changes in clone SA on a time scale. As determined by mapping of sequence reads to the reference genome, the numbers of SNPs in each genome showed a positive correlation with the year of isolation of corresponding strains relative to that of IA3902 (i.e. 2006). SNP-based phylogenetic tree separated the genomes based on isolation year, placing the historical clone SA genomes (early 1990s) in a distinct cluster from those of epidemic clone SA genomes (post-2003). Comparative genomics of the de novo assembled genomes (consisting of 62-84 contigs) indicated an overall high degree of genomic synteny and sequence homology among clone SA. Based on this success, a larger collection of isolates (78) of both clone SA and non-clone SA lineages from sheep abortions were sequenced. Initial analyses indicated the high quality and coverage depth of the genomic sequences. These genomic sequence data are being analyzed to understand the evolution of C. jejuni clone SA. For objective 3, experiments were initiated to assess the immunogenicity of several membrane-associated proteins that were identified by immunoproteomics. Recombinant proteins were made mice immunized with the proteins produced high titers of antibodies against the antigens. Challenge studies will be performed to assess if they induce protective immunity.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Orhan Sahin, Zuowei Wu, Swaine Chen, and Qijing Zhang. Whole-genome sequencing for elucidation of evolution and fitness of an emergent Campylobacter jejuni clone associated with sheep abortion in the USA. CHRO Meeting. Aberdeen, Scotland. September 22-25, 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Wu Z., Huang J., Sahin O., Zhang Q. 2013. Functional genomic analysis of survival mechanism of Campylobacter jejuni in physiological sheep bile. Conference of Research Worker in Animal Diseases. Chicago, IL, Dec. 8-10, 2013
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