Progress 01/01/13 to 12/31/17
Outputs
Target Audience:The target audience for our work are physicians, veterinarians and public health officials interested in how food borne diseases cause long term chronic disease problems in humans. We mainly report to other scientists in these fields by publishing scientific manuscripts and by giving talks at scientific meetings. We also occasionally report directly to the public to explain risks to their health from food borne bacteria and antibiotic resistance. A main target audience are medical and veterinary medical students who Dr. Mansfield teaches. Here the main messages are targeting how bacteria cause food borne disease and how to recognize this disease in humans and animals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In a continuing process, several students were mentored in 2016-2017 for study of enteric diseases of food animals. Jean Brudvig, DVM (PhD candidate) will defend her PhD thesis in December 2017 and began a job at Michigan State University as a clinical pathologist/ diagnostician. Phillip Brooks successfully defended his doctoral defense in May 2016 with a focus on C. jejuni genomics and bioinformatics and the role of antibiotics in exacerbating the disease due to this bacterium. He has taken a postdoctoral position at the School of Veterinary Medicine, University of California at Davis and studying bioinformatics methods for study of bacterial organisms in ground water. Phillip is from a minority group (African American) underrepresented in the sciences. This shows the success of the Mansfield lab in mentoring students to stay in fields related to food safety. How have the results been disseminated to communities of interest?In the past year, we have disseminated our scientific results to communities of interest by publishing scientific papers, giving scientific talks at national and international meetings and presenting posters at scientific and international meetings. Three publications have been submitted or published in scientific journals, three talks have been given, and twelve abstracts were accepted and given as talks or posters at national or international meetings. Dr. Mansfield also gave instructional programs to medical students, veterinary students and practicing veterinarians designed to enhance their knowledge of microbiology and foodborne illness. Dr. Mansfield also gave instructional programs to veterinary residents or assistant professors designed to improve their ability to conduct scientific research and progress in their careers. What do you plan to do during the next reporting period to accomplish the goals?In the next project, our long term goal is to develop effective inhibitors to decrease viability and fitness of C. jejuni in humans and its natural hosts to prevent disease and transmission. Our overarching hypothesis is that particular C. jejuni strains, microbial communities, and host innate and adaptive immune responses as well as suites of antibiotic resistant elements in the pathogen and microbial community interact to determine disease outcomes. Our short term goal is to examine these interactions by applying a systems-based approach to discover mechanisms of pathogenesis in AR C. jejuni in animal models of tolerance, enteric disease, and acute disease that elicits autoimmunity. Bioinformatics and core support will serve as a major discovery platform for the entire project. Mathematical models will be explored to identify key genes, transcriptional networks, and metabolic mediators of the pathogen, host and microbial communities that define these interactions, which may serve as novel drug targets. Our approaches will uncover new knowledge regarding AR C. jejuni and will serve as a guide for other investigators studying AR pathogens and their hosts. Based on extensive preliminary data for each of our long term goals, the team will carry out the following Specific Aims: • (Specific Aim 1). Determine the synergistic role of host response, microbiome and antibiotic treatment in enhancing colonization, enteritis and risk of Guillain Barré syndrome after infection with C. jejuni. We will study animal models to determine the interaction pathways between the host response, microbiome and antibiotic treatment in enhancing colonization, enteritis and risk of Guillain Barré syndrome after infection with C. jejuni. We will develop mathematical models to explain these interactions and make predictions, which form the foundation for later discovery efforts in Aims 2 and 3. • (Specific Aim 2). Identify molecular pathways important for the emergence of resistance and evolution of virulence in vivo to guide the discovery of novel therapeutics. We will define conditions and pathways important for the emergence of antibiotic resistance in vivo and develop a catalog of mutations associated with resistance and enhanced fitness. Networks identified in Aim 2 will be validated using deep-sequencing based genome interrogation approaches to identify novel targets for therapeutic discovery. This will set the stage for identifying compounds to test as novel therapeutics that have the ability to block AR infections. • (Specific Aim 3): Determine the impact of antibiotic resistant C. jejuni infections on the intestinal microbiome in patients during acute infections and post recovery. We will use metagenomics to compare microbial communities among patients with AR and susceptible C. jejuni infections. We will also examine differences in the abundance of resistance genes, microbes, and viruses, and identify targets that have a negative impact on patient outcomes or disease severity. This work forms the foundation for iterative experimental testing in the animal models and for the retrospective screening of human intestinal metagenomes for therapeutic, resistance and fitness targets identified in Aims 1 and 2. Outcomes. We expect to identify molecular pathways in different pathogen-host-microbiome combinations that are important for disease caused by AR C. jejuni. We also expect to identify antimicrobial mechanisms that drive pathways involved in the emergence of AR C. jejuni. Information generated will inform antimicrobial practices to prevent severe disease and suggest microbiome amendments for testing as preventatives or treatments. These efforts also facilitate discovery of novel therapeutic compounds that can be applied to prevent and treat acute campylobacteriosis and the chronic autoimmune sequelae linked to it.
Impacts
What was accomplished under these goals?
Accomplishments Impact. Guillain-Barré syndrome (GBS) is a post infectious disorder in which the body's immune system attacks part of the peripheral nervous system. The first symptoms include varying degrees of weakness or tingling sensations in the legs that can spread to the arms and upper body. These symptoms can increase in intensity until certain muscles cannot be used at all and, when severe, the person is almost totally paralyzed. We have shown that C. jejuni strains from patients with GBS produce neurological disease in mice. The mechanisms controlling this type of autoimmunity can now be studied in these mouse models. We have shown that the main mechanism resulting in peripheral nerve dysfunction is molecular mimicry; some C. jejuni strains have sialylated outer core oligosaccharides that mimic host gangliosides (GM1/ GD1a) on the peripheral nerves. We have proofs that C. jejuni with a certain configuration of its lipooligosaccharide (LOS) on the outer surface can induce antiganglioside antibodies that attach to and damage peripheral nerves. Antibiotic treatment worsened the outcomes and intensified these autoimmune responses. Interestingly, we could make better mouse models of human disease for studying GBS by giving them a fecal transplant of human microbiota. AIM 1) is to determine whether differences in innate responses produce different evolutionary trajectories in C. jejuni 11168 populations infecting mice. Campylobacter jejuni induces autoimmune peripheral neuropathy via Siglec-1 and IL-4 axes Campylobacter jejuni is a spiral, gram-negative, microaerophilic bacterium that is the most common bacterial cause of gastroenteritis worldwide. C. jejuni infection has also been causally linked with development of the peripheral neuropathy called Guillain Barré Syndrome (GBS). We have previously shown that C. jejuni isolates from human enteritis patients induce a Type1/17 cytokine dependent colitis response in IL-10-/- mice. In contrast, isolates from human GBS patients colonize the IL-10-/- mice without inducing colitis but instead induce autoantibody elicitation targeted against peripheral nerve antigens. We show here that the autoantibody response is dependent upon blunted Type1/17 but enhanced Type2 cytokine production by T helper cells. Autoantibody elicitation also correlated with enhanced macrophage infiltration in the sciatic nerve and its dorsal root ganglia. Autoantibodies and these histological changes were significantly decreased in mice depleted of IL-4, without leading to colitis induction. Histological damage in the sciatic nerve was associated with abnormal gait and hind limb movements in the IL-10-/- mice, consistent with this syndrome's manifestation in humans. Furthermore, we show here that Siglec1 is a central antigen presenting cell receptor that mediates GBS but not colitogenic isolate uptake, T cell differentiation and autoantibody elicitation. Therefore, this is the first mouse model of an autoimmune disease induced directly by a bacterium and it is dependent upon Siglec1 and IL-4 axes. Aim 2 is to test the generality of the results by examining virulence evolution in phylogenetically and phenotypically distinct sequenced C. jejuni strains infecting the same mice. NOD B7-2-/- mice as a model for Acute Inflammatory Demyelinating Polyneuropathy (AIDP): a standardized approach to phenotyping reveals new features of Spontaneous Autoimmune Peripheral Polyneuropathy Guillain Barré Syndrome (GBS) is the world's leading cause of acute neuromuscular paralysis, while Acute Inflammatory Demyelinating Polyneuropathy (AIDP) is the most common form of GBS in the USA. Limited progress has been made in understanding the pathogenesis of GBS and in developing therapies because of lack of good, tractable animal models. It is known that NOD B7-2-/- mice develop spontaneous autoimmune peripheral neuropathy (SAPP) starting at 20 weeks of age with females experiencing higher rates and more severe disease than male mice. We hypothesized that NOD.B7-2-/- mice have disease and lesions that mimic the AIDP form of GBS. In a closed, specific pathogen free colony of NOD B7-2-/- mice, female mice had higher rates of inflammatory peripheral nerve disease and higher inflammation scores than male mice. Development of SAPP disease correlated with the presence of inflammation in the dorsal root ganglion, the brachial plexus, and the sciatic nerve, which in rare cases, occurred in mice younger than 20 weeks of age. Flow cytometry revealed distinct differences in cell numbers and composition of sciatic nerve inflammatory infiltrates between NOD B7-2-/- mice and NOD wild type negative controls. NOD B7-2-/- mice with SAPP had significant increases in the numbers of neutrophils (P= 0.0218), dendritic cells (P=0.0116), and myeloid cells (P=0.0258) in the sciatic nerves, compared to wild type mice. As SAPP developed, daily observation and weekly phenotyping showed first a disrupted gait pattern and reluctance to rear on the side of the cage, followed by decreased movement, increased respiratory rate with tremors and finally overgrown toenails, flipped hind limbs and an intermittent foot drag when walking. Mice did not progress beyond this stage due to humane endpoints. Taken together these phenotypic and pathologic characteristics show that SAPP in NOD.B7-2-/- mice most closely resembles the AIDP form of GBS. These phenotyping and lesion evaluation methods will aid study of AIDP pathogenesis. Janus-faced immunity to an enteric pathogen: How Campylobacter mediates colitis and autoimmunity. Campylobacter jejuni is a spiral, Gram-negative, microaerophilic bacterium that is the most common bacterial cause of gastroenteritis. Previous infection with C. jejuni has been linked with several chronic autoimmune disorders including development or flare-up of Inflammatory Bowel Disease,Reiter's Arthritis, Irritable Bowel Syndrome, and the peripheral neuropathy Guillain Barré Syndrome (GBS). More on IBD here. The occurrence of GBS is temporally associated with development of autoantibodies that target gangliosides on peripheral nerves. The C. jejuni strains isolated from GBS patients have modifications of the outer core of the lipooligosaccharide shown to mimic several peripheral nerve gangliosides such as GM1, GM2, GD1a and others. At present, plasmapheresis and intravenous immunoglobulin (IVIg) treatment are the only known treatments with beneficial effect, but only 60% of GBS patients improve. Thus, new therapeutics are critically needed, but drug development pipelines are limited by lack of appropriate animal models for efficacy testing. In an effort to develop a model to study enteric and systemic manifestations of this abcterium we showed that a number of isolates of C. jejuni from human enteritis patients induce colitis in the C57BL/6 IL-10-/- mouse model whereas isolates from human GBS patients colonize these mice with little or no colitis. The study presented here addresses the C. jejuni strain dependent immunological mechanisms behind induction of colitis by a gastroenteritis patient derived strain versus induction of an asymptomatic colonization by the GBS patient derived strains. Further, we show that infection of mice with C. jejuni strains from GBS patients, but not from colitis patients, elicits autoantibody production that react against several nerve gangliosides consistent with thoseseen in human GBS cases. Production of these autoantibodies correlate temporally also cause peripheral neuropathic phenotype and histological manifestation that is consistent with this syndrome's manifestations in humans.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Malik A., Gadsden BJ, Ethridge A, Brudvig J and Mansfield LS. 2017. Campylobacter jejuni induces autoimmune peripheral neuropathy via Siglec-1 and IL-4 axes, Submitted to Experimental Neurology.
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
Malik A. and Mansfield LS. 2017. Janus-faced immunity to an enteric pathogen: How Campylobacter mediates colitis and autoimmunity. In preparation as a review for Journal of Immunology.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Gadsden BJ, Malik A, Kim HY, Bell JA, Mansfield LS, 2017. NOD CD86-/- mice as a working model for Guillain-Barr� Syndrome: New clinical features of Spontaneous Peripheral Polyneuropathy and a standardized approach to phenotyping,In preparation for the Journal of Peripheral Neuropathies.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target Audience: The target audience for our work are physicians, veterinarians and public health officials interested in how food borne diseases cause long term chronic disease problems in humans. We mainly report to other scientists in these fields by publishing scientific manuscripts and by giving talks at scientific meetings. We also occasionally report directly to the public to explain risks to their health from food borne bacteria and antibiotic resistance. A main target audience are medical and veterinary medical students who Dr. Mansfield teaches. Here the main messages are targeting how bacteria cause food borne disease and how to recognize this disease in humans and animals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In a continuing process, several students were mentored in 2015-2016 for study of enteric diseases of food animals. Barbie Gadsden, DVM (PhD candidate) successfully defended her PhD thesis in December 2015 and began a job at MPI pharmaceuticals in Matawan, Michigan as an Immunologist/ Pathologist. Phillip Brooks passed his doctoral defense in May 2016 with a focus on C. jejuni genomics and bioinformatics and the role of antibiotics in exacerbating the disease due to this bacterium. He has taken a postdoctoral position at the School of Veterinary Medicine, University of California at Davis and will study bioinformatics methods for study of bacterial organisms in ground water. Both Phillip and Barbie are from minority groups (African American) underrepresented in the sciences. This shows the success of the Mansfield lab in mentoring students to stay in the field of food safety. Three other students are pursuing PhD degrees in food safety related to these enteric pathogens including Jean Brudvig, DVM, Daniel Claiborne and Hinako Terauchi. Jean Brudvig passed her preliminary exam in 2015. Five undergraduate students worked in the lab on these projects including Christopher Bejcek, Alexander Ethridge, James Chen, Zachary Jansen and Keenan Odea. Dr. Linda Mansfield continued to act as the Principal Investigator and the Administrative Core Leader of the Michigan State University, Enterics Research Investigational Network, Cooperative Research Center (MSU ERIN CRC) in 2015-2016. This is a multidisciplinary, highly integrated research center to study the enteric microbiome in health and disease with the long-term goal of understanding and alleviating one of the most prevalent and important global health problems, diarrheal illness. The central overarching theme of the center is to explore and elucidate the relationship of the enteric microbiome to acute diarrheal illness. Dr. Mansfield gave talks at the Conference of Research Workers on Animal Diseases, the Twenty-fifth Anniversary of the David Hyde Allergy and Asthma Research Centre in the United Kingdom, the Bach Endowed lectureship at Western University, Kalamazoo, MI and at the American College of Veterinary Internal Medicine Forum in Denver Colorado among other talks. Dr. Mansfield along with Dr. Susan Ewart organized a grantsmanship workshop for assistant professors and residents in Large Animal Clinical Sciences at the College of Veterinary Medicine at MSU where preharvest food safety was prominently featured. Dr. Mansfield also attended the NC1202 annual meeting (December 3-4) and the Conference of Research Workers on Animal Diseases (December 4-6) both in Chicago, Illinois in 2016. Two of the undergraduates presented their work at the Undergraduate Research and Arts Forum (UURAF) at Michigan State University. Each of the three graduate students presented their work at the Phi Zeta Research Day, American Society for Microbiology and the Society for Neurology meetings in 2016. How have the results been disseminated to communities of interest? In the past year, we have disseminated our scientific results to communities of interest by publishing scientific papers, giving scientific talks at national and international meetings and presenting posters at scientific and international meetings. Three publications have been submitted or published in scientific journals, four talks have been given, and ten abstracts were accepted and given as talks or posters at national or international meetings. Dr. Mansfield also gave instructional programs to medical students, veterinary students and practicing veterinarians designed to enhance their knowledge of microbiology and foodborne illness. Dr. Mansfield also gave instructional programs to veterinary residents or assistant professors designed to improve their ability to conduct scientific research and progress in their careers. What do you plan to do during the next reporting period to accomplish the goals?Our future goals are to define C. jejuni gene expression controlling the different pathotypes that we have defined in the reported murine models. We will continue this study on the genetic diversity of human and animal isolates of C. jejuni that we have acquired through the US Centers for Diseases Control and Prevention and from a collaboration with Dr. Shannon Manning from an epidemiologic study in Michigan. Methods will include Illumina sequencing, screening in mice with human microbiota given by fecal transplant for Campylobacter gene expression, and selected gene knockouts followed by challenge in the murine models. We are especially interested in evolutionary mechanisms of C. jejuni and how it initiates autoimmune disease in its host.
Impacts
What was accomplished under these goals?
BALB/c and C57BL/6 mice givenCampylobacter jejunidisplayed evidence of Guillain-Barré syndrome autoimmunity, with varied immune responses and disease outcomes Background: Campylobacter jejuniis a common bacterial cause of both human diarrhea and infection antecedent to Guillain-Barré syndrome (GBS), a rare sequela of various infections leading to debilitating peripheral neuropathy. Proposed mechanisms of GBS pathogenesis followingC. jejuniinfection involve molecular mimicry-induced nerve damage initiated by cross-reactive antibodies toC. jejunilipo-oligosaccharide and structurally similar peripheral nerve gangliosides. Both host genetic andC. jejunistrain differences may impact disease manifestations, including anti-ganglioside antibody production and GBS disease development. Methods: The current study investigatedin vivoimmune responses and disease manifestations of anti-inflammatory interleukin (IL)-10 deficient BALB/c and C57BL/6 mice infected with colitogenic C. jejuni11168 or GBS patient-derivedC. jejuni260.94 strains. We hypothesized that severity of colitis and character of immune response, evaluated by anti-C. jejuniand anti-ganglioside plasma IgG subtypes, would vary depending on both mouse andC. jejunistrain genetic backgrounds. Ten IL-10-/-mice of both backgrounds were orally inoculated withC. jejuni11168,C. jejuni260.94, or vehicle at 6 weeks of age (60 mice total). Results: BALB/c IL-10-/-mice infected withC. jejuni11168 exhibited the lowest survivorship, the most severe gross pathology at necropsy and highest grade histopathologic lesions in the ileocecocolic junction, and the highest mean plasma concentrations of anti-C. jejuniIgG1, IgG3, IgG2b, and IgG2a/c antibodies. Significant increases in GM1 and GD1a anti-ganglioside antibody isotypes varied between treatment groups, although no significant differences in macrophage infiltration into lumbar dorsal root ganglia were seen. Immunohistochemical assessment ofC. jejuni abundance and localization in the ileocecocolic junction and evaluation of IFN-γ, IL-4, and IL-17 cytokine production in colon tissue reflecting the local adaptive immune response are underway. Conclusions: These findings provide a new murine model of GBS followingC. jejuniinfection designed to examine differences in pathogenesis based on host genotype and infectingC. jejunistrain. Antimicrobial Susceptibility Profiles of Human Campylobacter jejuni Isolates and Association with Phylogenetic Lineages Background: Campylobacter jejuni is a zoonotic pathogen and the most common bacterial cause of human gastroenteritis worldwide. With the increase of antibiotic resistance to fluoroquinolones and macrolides, the drugs of choice for treatment, C. jejuni was recently classified as a serious antimicrobial resistant threat. Methods: Here, we characterized 94 C. jejuni isolates collected from patients at four Michigan hospitals in 2011 and 2012 to determine the frequency of resistance and association with phylogenetic lineages. Results: The prevalence of resistance to fluoroquinolones (19.1%) and macrolides (2.1%) in this subset of C. jejuni isolates from Michigan was similar to national reports. High frequencies of fluoroquinolone-resistant C. jejuni isolates, however, were recovered from patients with a history of foreign travel. A high proportion of these resistant isolates were classified as multilocus sequence type (ST)-464, a fluoroquinolone-resistant lineage that recently emerged in Europe. A significantly higher prevalence of tetracycline-resistant C. jejuni was also found in Michigan and resistant isolates were more likely to represent ST-982, which has been previously recovered from ruminants and the environment in the U.S. Notably, patients with tetracycline-resistant C. jejuni infections were more likely to have contact with cattle. Conclusions: These outcomes prompt the need to monitor the dissemination and diversification of imported fluoroquinolone-resistant C. jejuni strains and to investigate the molecular epidemiology of C. jejuni recovered from cattle and farm environments to guide mitigation strategies. .
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Cha W, Mosci R, Wengert SL, Singh P, Newton DW, Salimnia H, Lephart P, Khalife W, Mansfield LS, Rudrik JT, and Manning SD. 2016. Antimicrobial susceptibility profiles of human Campylobacter jejuni isolates in Michigan and the association with phylogenetic lineage and disease severity, Front Microbiol. 2016 Apr 26;7:589. doi: 10.3389/fmicb.2016.00589.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience for our work are physicians, veterinarians and public health officials interested in how food borne diseases cause long term chronic disease problems in humans. We mainly report to other scientists in these fields by publishing scientific manuscripts and by giving talks at scientific meetings. We also occasionally report directly to the public to explain risks to their health from food borne bacteria and antibiotic resistance. A main target audience are medical and veterinary medical students who Dr. Mansfield teaches. Here the main messages are targeting how bacteria cause food borne disease and how to recognize this disease in humans and animals. Changes/Problems:There were no changes to the project plan in 2014-2015. What opportunities for training and professional development has the project provided?In a continuing process, several students were mentored in 2014-2015 for study of enteric diseases of food animals. Ankit Malik (PhD candidate) successfully defended his PhD thesis in December 2014 and began a job at St. Jude's Children's Hospital as an Immunologist/ Microbiologist. He is a postdoctoral candidate and is focused on an immunology project related to food safety. This shows the success of the Mansfield lab in mentoring students to stay in the field of food safety. How have the results been disseminated to communities of interest?Dr. Linda Mansfield continued to act as the Principal Investigator and the Administrative Core Leader of the Michigan State University, Enterics Research Investigational Network, Cooperative Research Center (MSU ERIN CRC) in 2014-2015. This is a multidisciplinary, highly integrated research center to study the enteric microbiome in health and disease with the long-term goal of understanding and alleviating one of the most prevalent and important global health problems, diarrheal illness. The central overarching theme of the center is to explore and elucidate the relationship of the enteric microbiome to acute diarrheal illness. Dr. Mansfield gave talks at the Conference of Research Workers on Animal Diseases, the American Academy of Asthma, Allergy and Immunology, and at the Society for Neurobiology, Chicago, IL. Dr. Mansfield along with Dr. Bo Norby organized the Spring seminar series in Large Animal Clinical Sciences at the College of Veterinary Medicine at MSU where preharvest food safety was prominently featured. People attending came from the Agricultural, Veterinary Medicine, Microbiology and Food Science, and Human Nutrition departments. Dr. Mansfield also attended the NC1202 annual meeting in Chicago, Illinois on November December 2014. Two of the undergraduates presented their work at the Undergraduate Research and Arts Forum (UURAF) at Michigan State University. Each of the three graduate students presented their work at the American Society for Microbiology and the American Association of Immunologists meetings in 2015. What do you plan to do during the next reporting period to accomplish the goals?Our future goals are to define C. jejuni gene expression controlling the different pathotypes that we have defined in the reported murine models. We will continue this study on the genetic diversity of human and animal isolates of C. jejuni that we have acquired through the US Centers for Diseases Control and Prevention and from a collaboration with Dr. Shannon Manning from an epidemiologic study in Michigan. Methods will include Illumina sequencing, screening in mice with human microbiota given by fecal transplant for Campylobacter gene expression, and selected gene knockouts followed by challenge in the murine models. We are especially interested in evolutionary mechanisms of C. jejuni and how it initiates autoimmune disease in its host.
Impacts
What was accomplished under these goals?
Specific Aim (4). Determine whether innate responses and adaptive responses mediate GBS and MFS in murine models A Human Microbiome Enhanced Campylobacter jejuni Induced Autoantibodies and Th-2 Skewing of the Adaptive Immune Response after Fecal Transplant Rationale: Guillain-Barré Syndrome (GBS) is a common cause of acute generalized paralysis. Infection with the enteric pathogen C. jejunioften precedes GBS when bacterial lipo-oligosaccharide resembling host nerve gangliosides activates the immune system to produce autoantibodies. We showed that C. jejuni11168 from an enteritis patient produced T helper-1/17 responses in C57BL/6 IL-10-/- mice, while a C. jejuni GBS patient strain (260.94) induced blunted Th-1/17 but enhanced Th-2 responses. Only Th-2 antibodies cross-reacted with nerve gangliosides. We hypothesized that mice with human gut microbiota (Humicrobiota) would mount a stronger immune response to C. jejuni infection with elevatedC. jejuni-specific and autoantibodies. Methods: C57BL/6 germ-free mice were given a human fecal transplant, bred after gut microbiota stabilized, and their offspring used in a 30 day infection trial. Congenic Humicrobiota and mouse microbiota (Momicrobiota) mice were inoculated with C. jejuni enteritis strain 11168, GBS strain 260.94 or sham inoculated. Plasma was collected from all mice and levels of Th-1 and Th-2 antibody isotypes to nerve gangliosides andto C. jejunistrains were measured. IL-4 and IFNg responses were measured in gut lamina propria cells by RT-PCR. Results: Autoimmune responses were significantly elevated by the presence of Humicrobiota. Humicrobiota mice had significantly higher levels of IgG1 antibodies to C. jejuni 11168 and to GM1 and GD1a nerve gangliosides than infected Momicrobiota mice.Infected Humicrobiota mice had a 12-fold increase in IL-4 levels and decreased IFN-g levels. Conclusions: Humicrobiota enhanced C. jejuni induced autoantibodies to nerve gangliosides and Th-2 skewing of the adaptive immune response. NIH U19AI090872. Specific Aim (5). Determine whether autoantibody or autoreactive T cells transfer the response to naïve mice Campylobacter jejuni induces autoimmune peripheral neuropathy via Siglec-1 and IL-4 axes Campylobacter jejuni is a microaerophilic bacterium that is the most common bacterial cause of gastroenteritis worldwide. C. jejuni infection has also been causally linked with development of the peripheral neuropathy called Guillain Barré Syndrome (GBS). We have previously shown that C. jejuni isolates from human enteritis patients induce a Type1/17 cytokine dependent colitis response in IL-10-/- mice. In contrast, isolates from human GBS patients colonize the IL-10-/- mice without inducing colitis but instead induce autoantibody elicitation targeted against peripheral nerve antigens. We show here that the autoantibody response is dependent upon blunted Type1/17 but enhanced Type2 cytokine production by T helper cells. Autoantibody elicitation also correlated with enhanced macrophage infiltration in the sciatic nerve and its dorsal root ganglia. Autoantibodies and these histological changes were significantly decreased in mice depleted of IL-4, without leading to colitis induction. Histological damage in the sciatic nerve was associated with abnormal gait and hind limb movements in the IL-10-/- mice, consistent with this syndrome's manifestation in humans. By using blocking antibodies in ex vivo and in vivo models, we also show that Siglec1 is a central antigen presenting cell receptor that mediates GBS but not colitogenic isolate uptake, T cell differentiation and autoantibody elicitation. This data demonstrates autoimmune manifestations of GBS induced by oral infection with C. jejuni HB93-13 that is dependent upon Siglec1 and IL-4 axes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Flies AS, Maksimoski MT, Mansfield LS, Weldele ML and Holekamp KE. 2014. Characterization of toll-like receptors 1-10 in spotted hyenas. Veterinary Research Communications, Veterinary Research Communications Jun;38(2):165-70. doi: 10.1007/s11259-014-9592-3. Epub 2014 Feb 2.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Flies AS, Mansfield LS, Grant CK, Weldele ML, Holekamp KE. 2015. Markedly elevated antibody responses in wild versus captive spotted hyenas show that environmental and ecological factors are important modulators of immunity, PLOS ONE, in press.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kopper JA, Patterson JS and Mansfield LS. Metronidazolebut not IL-10 or prednisolonerescues Trichuris muris infected C57BL/6 IL-10 deficient mice from severe disease. Veterinary Parasitology, 2015 Aug 6. pii: S0304-4017(15)00380-5. doi: 10.1016/j.vetpar.2015.07.038..
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Flies, A. S., Mansfield, L. S., Johnston, E.S., Grant, C. K., and Holekamp, K. E. 2015. Socioecological predictors of immune defenses in a wild spotted hyenas. Functional Ecology, Revision under review.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Invited Talk. Mansfield, LS. Campylobacter jejuni induces acute and chronic disease leading to autoimmunity. Department of Microbiology and Molecular Genetics, Michigan State University, Dec 2015.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Talk. Mansfield LS, Brakel KA, Malik A, Bell JA. 2015. A Human Microbiome Enhanced Campylobacter jejuni Induced Autoantibodies and Th-2 Skewing of Adaptive Immunity after Fecal Transplant American Academy of Allergy, Asthma and Immunology Meeting, Houston TX, February 20-24.
- Type:
Other
Status:
Published
Year Published:
2014
Citation:
Mansfield, L.S. 2014. Transplanting human gut microbes into mice causes the animals to have increased potentially dangerous autoimmune responses. Press release, 114th meeting of the American Society for Microbiology, Boston, MA, May 17-20.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audience for our work are physicians, veterinarians and public health officials interested in how food borne diseases cause long term chronic disease problems in humans. We mainly report to other scientists in these fields by publishing scientific manuscripts and by giving talks at scientific meetings. We also occasionally report directly to the public to explain risks to their health from food borne bacteria and antibiotic resistance. A main target audience are medical and veterinary medical students who Dr. Mansfield teaches. Here the main messages are targeting how bacteria cause food borne disease and how to recognize this disease in humans and animals. Changes/Problems: Nothing to report What opportunities for training and professional development has the project provided? Once again, several students were mentored for study of enteric diseases of food animals. Jessica St. Charles (PhD candidate) successfully defended her PhD thesis in 2013 and began a job as a Immunologist/ Microbiologist for MPI. She is a supervisory microbiologist and leads their program in food safety. Ankit Malik graduated from the PhD program in October 2014 and has been hired as a postdoctoral candidate by St. Jude’s hospital in Memphis, TN. This shows the success of the Mansfield lab in mentoring students to stay in the field of food safety. Three other students are pursuing PhD degrees in food safety related to these enteric pathogens including Barbie Gadsden, DVM, Jean Brudvig, DVM, and Phillip Brooks. One of these students has passed their preliminary exam. Two of the graduate students are underrepresented minorities (African American). Four undergraduate students are working in the lab on these projects including Elizabeth Gensterbloom, Alexander Ethridge, James Chen, and Christopher Bejcek. How have the results been disseminated to communities of interest? Dr. Linda Mansfield continued to act as the Principal Investigator and the Administrative Core Leader of the Michigan State University, Enterics Research Investigational Network, Cooperative Research Center (MSU ERIN CRC) in 2013-2014. This is a multidisciplinary, highly integrated research center to study the enteric microbiome in health and disease with the long-term goal of understanding and alleviating one of the most prevalent and important global health problems, diarrheal illness. The central overarching theme of the center is to explore and elucidate the relationship of the enteric microbiome to acute diarrheal illness. During October 1, 2013 to September 30, 2014, two half day symposia were organized to discuss scientific progress. Dr. Mansfield also gave an invited talk to Merial-NIH Veterinary Scholars Symposium in 2013 where 400 veterinary students attended. Dr. Mansfield also participated in a round table at the Merial-NIH Scholars Symposium in 2013 entitled “Becoming Faculty: a short course on launching a scientific career”. Dr. Mansfield along with Dr. Paul Bartlett organized the Fall seminar series in Large Animal Clinical Sciences at the College of Veterinary Medicine at MSU where preharvest food safety was prominently featured. People attending came from the Agricultural, Veterinary Medicine, Microbiology and Food Science, and Human Nutrition departments. Also, Dr. Mansfield attended and presented at a scientific conference held by the National Institutes of Health in Ann Arbor, MI on enteric diseases. Dr. Mansfield also helped to organize and attended the NC1202 annual meeting in Chicago, Illinois on November 30tht and December 1st, 2013. What do you plan to do during the next reporting period to accomplish the goals? Our future goals are to define C. jejuni gene expression controlling the different pathotypes that we have defined in the reported murine models. We will continue this study on the genetic diversity of human and animal isolates of C. jejuni that we have acquired through the US Centers for Diseases Control and Prevention and from a collaboration with Dr. Shannon Manning from an epidemiologic study in Michigan. Methods will include Illumina sequencing, screening in mice with human microbiota given by fecal transplant for Campylobacter gene expression, and selected gene knockouts followed by challenge in the murine models. We are especially interested in evolutionary mechanisms of C. jejuni and how it initiates autoimmune disease in its host.
Impacts
What was accomplished under these goals?
Specific Aim (2). Determine whether autoimmune sequelae vary with differences in C. jejuni LOS profiles and differences in the LOS region Experiment #1. Reversible motility mutations and parallel σ54 loss during experimental evolution of Campylobacter jejuni Summary. Campylobacter jejuni is a foodborne bacterial pathogen that only replicates naturally within a host. When we experimentally evolved five independent host-adapted C. jejuni populations in rich broth medium, we observed the loss of flagellar motility—an essential function for efficient host colonization. At early time points during broth adaptation the motility defect was often reversible by selection for motility in semi-solid media, but after thirty-five days of laboratory culture, motility was lost irreversibly in the majority of cells in five independently evolved populations. Genome re-sequencing revealed numerous disruptive mutations in genes in the C. jejuni flagellar transcriptional cascade, including genes known to affect expression of the σ54 (RpoN) regulon and chromosomal deletion of rpoN in all evolved lines. Our work shows that a pathogenic C. jejuni strain is quickly attenuated by experimental laboratory evolution and provides insight into the role of genomic instability in C. jejuni evolvability. Based on this data we suggest a potential stepwise mechanism of C. jejuni genetic adaptation to a novel environment by high-rate, reversible mutations for rapid phenotypic selection, followed by irreversible mutations during prolonged selective pressure. Background. Motility has been long been known to be essential for host colonization and virulence in C. jejuni (1), but non-flagellated mutants have been shown to grow faster in broth culture than the flagellated parent strain (2). Regulation of flagellar biosynthesis has been shown to be phase variable (3). Our lab has previously shown that a single passage in C57BL/6 IL-10-/- mice results in increased virulence in C. jejuni 11168 that is accompanied by both changes in expression level and phase variation in “contingency” genes containing homonucleotide tracts subject to length variation due to slip-strand mutagenesis (4, 5). Furthermore, variation in among C. jejuni strains infecting C57BL/6 IL-10-/- mice in ability to colonize and cause enteritis in C57BL/6 IL-10-/- mice is correlated with possession of C. jejuni 11168 genes that are up-regulated in cells recovered from ceca of germ-free C57BL/6 IL-10-/- mice compared to broth-grown cells, including 11 genes. Materials and methods. In the current study we experimentally evolved five independent host-adapted C. jejuni populations in rich broth medium and examined flagellar motility ina time course design. Results. Motility was lost in all populations during evolution in broth (Figure 8). There was a higher frequency of reversibly non-motile cells at early time points during experimental evolution. Multiple ORF-disrupting reversible and irreversible motility-affecting mutations were detected in evolved populations after 35 passages, including homopolymeric tract length changes and parallel large deletions in rpoN (s54). Motility-affecting mutations present in the populations after 35 passages were common in loci involved in early events in the flagellar biosynthesis regulatory cascade. There are two main efforts that are still in progress to finish this project 1) competition experiments between non-motile and motile isolates to determine relative fitness based on motility and 2) documentation of homopolymeric tract length changes at particular motility-associated loci in reversibly non-motile isolates from early passages (Figures 9, 10, 11, 12). Discussion. Both the genome size (~1.6 Mb) and GC content (~30%) of C. jejuni approach those of obligate intracellular symbionts (7). C. jejuni also lacks a functional MMR DNA repair system, a deficiency thought to contribute to high rates of slip-strand mutagenesis in homopolymeric nucleotide tracts and thus to phase variation (8). C. jejuni strains possess varying number of pseudogenes: 19 in C. jejuni 11168 and 47 in RM1221 (9). These characteristics and our results documenting large deletions in a genomic region containing pseudogenes indicate that C. jejuni meets the conditions necessary for the stepwise scenario of genome reduction proposed for intracellular symbionts by Moran et al. (7). The observed increase in frequency of large deletions in vitro may have been driven by selection for loss of unnecessary genes as described by Koskiniemi et al. (10). Finally, C. jejuni strain 81-116 can survive and replicate in human monocytes for up to 7 days (11). While motility would be unlikely to be lost in a natural disease setting where motility within the host GI tract is required, we propose that C. jejuni has the evolutionary potential to become an intracellular enteric pathogen like Listeria monocytogenes. We have made significant progress in pathogenesis studies of the foodborne pathogen Campylobacter jejuni. We have developed several mouse models that will now permit study of C. jejuni strains with selected gene knockouts. We have developed a mouse model of autoimmune diseases arising after C. jejuni infection including Inflammatory Bowel Disease and Guillain Barré Syndrome that has yielded important new knowledge for the medical field and that has been published in a Nature Journal. We now know that the evolution of C. jejuni in the host can lead to changes in its surface coat that can trigger autoimmune disease in genetically susceptible hosts. We have started to characterize the disease process and the genes controlling virulence in this bacterium. In 2015, we will begin to screen drug treatments in the GBS mouse model to look for new treatments for this serious autoimmune disease.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Malik A., Sharma D., St. Charles J.L., Dybas L.A., Mansfield L.S. 2013. Contrasting immune responses mediate Campylobacter jejuni induced colitis and autoimmunity, Nature Mucosal immunology, 13 November 2013; doi: 10.1038/mi.2013.97.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Samuelson DR, Eucker TP, Bell JA, Dybas LA, Mansfield LS, Konkel ME. 2013. The Campylobacter jejuni CiaD effector protein activates MAP 1 kinase signaling pathways and is required for the development of acute disease. Cell Communication and Signaling 2013, 11:79, doi:10.1186/1478-811X-11-79.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Target audience - The target audience for this work are scientists in related fields. Our goals were to focus on disseminating knowledge and provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories. Graduate students: Once again, several students were mentored for study of enteric diseases of food animals. Jessica St. Charles successfully defended her PhD thesis and obtained a job in the pharmaceutical industry at MPI Research. Barbie Gadsden (DVM, PhD candidate) successfully passed her preliminary examination for the PhD. Three other students are pursuing PhD degrees in food safety related to these enteric pathogens including Ankit Malik, Phillip Brooks and Jean Brudvig, DVM. Two undergraduate students are working in the lab on these projects including Alexander Ethridge and Elizabeth Gensterbloom. Changes/Problems: There have been no major changes in our approach to the experimental plan for this project. What opportunities for training and professional development has the project provided? Participants - Principal Investigator: Linda S. Mansfield (Station Representative), Julia A. Bell, Ph.D., Research Assistant Professor; Graduate Students: Jessica L. St. Charles, Ph. D. candidate, Ankit Malik, Ph. D. candidate, Barbie Gadsden, DVM, Ph. D. candidate, Jean Brudvig, DVM, Ph. D. candidate and Phillip Brooks, Ph.D. candidate. The target audience for this work are scientists in related fields. Our goals were to focus on disseminating knowledge and provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories. Graduate students: Once again, several students were mentored for study of enteric diseases of food animals. Jessica St. Charles successfully defended her PhD thesis and obtained a job in the pharmaceutical industry at MPI Research. Barbie Gadsden (DVM, PhD candidate) successfully passed the preliminary examination for the PhD. Three other students are pursuing PhD degrees in food safety related to these enteric pathogens including Ankit Malik, Phillip Brooks and Jean Brudvig, DVM. Two undergraduate students are working in the lab on these projects including Alexander Ethridge and Elizabeth Gensterbloom. How have the results been disseminated to communities of interest? Seminars and talks - Dr. Mansfield organized a Fall seminar in enteric research. People attending came from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Cathy Robinson, PhD (Are hypervirulent strains more ecologically fit than other Clostridium difficile strains?), Ankit Malik, MS, PhD candidate (Contrasting immune responses mediate Campylobacter jejuni induced colitis andautoimmunity), and Brian Nohomovich, DO, PhD candidate (Protective phage populations of the gastrointestinal tract). Also, Dr. Mansfield attended and presented at a scientific conference held by the National Institutes of Health in Washington, D.C. on Enteric Diseases. Dr. Mansfield helped to organize and attended the NC1041 in Chicago, Illinois on December 1st and 2nd, 2012. She and her graduate student Jessica St. Charles also attended the Conference of Research Workers on Animal Diseases. Jessica presented a talk about her work entitled "Campylobacter jejuni isolates from calves have A, B and C lipooligosaccharide (LOS) biosynthetic locus classes similar to human Guillain Barré syndrome associated strains and Dr. Mansfield gave a talk entitled “Comparison of induced small animal models for Guillain Barré syndrome (GBS) as post infectious sequelae to Campylobacter jejuni infection”. Dr. Mansfield also assisted in organizing the Merial-NIH Veterinary Scholars Symposium: “Comparative Medicine: Meeting Global Needs” that was held in East Lansing, MI in August 2013 where she organized a session devoted to food borne pathogens. What do you plan to do during the next reporting period to accomplish the goals? We will continue to follow the stated objectives of our project.
Impacts
What was accomplished under these goals?
Genetic basis for Campylobacter jejuni induced pathotypes in the host Campylobacter jejuni mediated autoimmune neuropathies in Humicrobiota mouse models (Linda S. Mansfield, PI) Summary. In 2013, we completed 9 experiments infecting mice with C. jejuni strains from patients with Guillain Barré Syndrome (GBS) and 7 experiments exploring the mechanisms mediating colitis and autoimmunity. Top accomplishments include 1) identifying the best mouse model for studying GBS induced by C. jejuni, 2) Identifying neurological phenotypes and lesions in mouse models of GBS, 3) determining that colitis and GBS disease outcomes after C. jejuni infection have alternative T cell regulatory pathways in the C57BL/6 mouse model, and 4) determining that the outcome of infection with C. jejuni strains is correlated with genome content of pathogen loci that are up- and down-regulated in the host. Specific Aim (1) - Characterize definitively the neurological signs and disease lesions associated with Guillain Barré and Miller Fisher Syndromes in murine models using single blind methods Outcomes - Guillain Barré Syndrome (GBS) is an acute peripheral neuropathy characterized by limb weakness and loss of tendon reflex that can arise when infecting Campylobacter jejuni strains have lipo-oligosaccharide (LOS) structures that mimic gangliosides found on peripheral nerves (GM1, GD1a, GQ1b). We hypothesized that GBS can develop in NOD mice or their congenic IL-10 and B7-2 knockouts secondary to C. jejuni infection and that clinical signs and pathological lesions can be detected. Mice were gavaged orally with C. jejuni strains HB93-13 and 260.94 from patients with GBS and assessed for clinical neurological signs, anti-ganglioside antibodies, cellular infiltrates, and lesions in peripheral nervous tissue. Significant increases in anti-ganglioside antibodies against single gangliosides (GM1 and/or GD1a) occurred in infected mice of all genotypes. Infected mice of the NOD WT and the NOD IL-10-/- genotypes produced IgG1 isotype anti-ganglioside antibodies directed against a mixture of gangliosides GM1 and GQ1b. Infected mice of all genotypes showed significant differences on phenotypic testing for nerve impairment compared to uninfected controls. Peripheral nerve lesions with macrophage infiltrates were significantly different between the sham-inoculated mice and C. jejuni infected mice of both the NOD WT and NOD IL-10-/- genotypes. Based on these results, we conclude that NOD WT mice are the best mouse model of natural onset GBS. In preparation for submission to the Journal of Autoimmunity. Specific Aim (2) Determine whether innate responses and adaptive responses mediate GBS and MFS in murine models Outcomes - Contrasting immune responses mediate Campylobacter jejuni induced colitis and autoimmunity - Campylobacter jejuni is a leading cause of foodborne enteritis that has been linked to the autoimmune neuropathy, Guillain Barré Syndrome (GBS). C57BL/6 IL-10+/+ and congenic IL-10−/− mice serve as C. jejuni colonization and colitis models, respectively, but a mouse model for GBS is lacking. We demonstrate that C57BL/6 IL-10-/- mice infected with a C. jejuni colitogenic human isolate had significantly upregulated Type1 and 17 but not Type2 cytokines in the colon coincident with infiltration of phagocytes, T cells and Innate Lymphoid Cells (ILC’s). Both ILC and T cells participated in IFN-g, IL-17 and IL-22 upregulation but in a time- and organ-specific manner. Reciprocal regulation of ILC and T-cell infiltration was observed with ILC expanding early and returning to background levels during the subsequent expansion of T cells. T cells were necessary for colitis as mice depleted of Thy-1+ cells were protected while neither Rag1-/- nor IL-10R blocked Rag1-/- mice developed colitis after infection. Depleting IFN-g, IL-17 or both significantly ameliorated colitis and drove colonic responses towards Type 2 cytokine and antibody induction. In contrast, C. jejuni GBS patient strains induced mild colitis associated with blunted Type 1/17 but enhanced Type 2 responses. Moreover, Type2 but not Type1/17 antibodies cross-reacted with peripheral nerve gangliosides demonstrating autoimmunity. Accepted and In Press, Nature - Mucosal Immunology. Specific Aim (3) - Determine effects of Humicrobiota on murine host responses in the presence and absence of three pathotypes of Campylobacter jejuni Experimental design. Germ free C57BL/6 mice received fecal transplants from healthy human donors with no recent history of antibiotic or probiotic use. Their microbial communities were allowed to become stable over time within the germ free incubator. Second generation offspring were removed from the incubator, transported to a containment facility and kept under specific pathogen free or sterile barrier conditions. Mice were orally inoculated with C. jejuni enteric (11168) and GBS (260.94) strains or sham inoculated and observed for clinical signs for 30 days. After necropsy, gastrointestinal (GI) gross and histopathology were assessed, GI tissues were plated for isolation of C. jejuni, RNA was collected for RT-PCR of key immune response genes, and plasma was collected for ELISAs assays for C. jejuni strain-specific antibodies (11168 and 260.94 antigen) and for autoantibodies against mixed gangliosides (GM1/GQ1b, GM1/GD1a) and single gangliosides (GM1, GD1a) of several isotypes (IgG1, IgG2b, IgG2c, IgG3). Outcomes - C57BL/6 mice with Humicrobiota had significant elevations in autoimmune responses after infection with C. jejuni compared to congenic infected mice with Momicrobiota. In the colon, infected Humicrobiota mice had decreased IFN-g and a 12-fold increase in IL-4. These changes were accompanied by significant elevations in autoantibodies of the IgG1 and IgG3 isotypes directed against several peripheral nerve gangliosides. Thus, the humicrobiota, but not the Momicrobiota conferred a GBS immune response mediated by a Th2 dependent mechanism after infection with an enteric C. jejuni strain. This suggests that the pathogenesis of GBS includes factors mediated by the microbiome. Further, no significant differences in the humicrobiota were observed between the sham inoculated groups with Humicrobiota managed by SPF and sterile means demonstrating that Humicrobiota mouse work can be carried out under conditions practical for most investigators using these methods.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Samuelson D.R., Eucker T.P., Bell J.A., Dybas L.A., Mansfield L.S., Konkel M.E. 2013. The Campylobacter jejuni CiaD effector protein activates MAP 1 kinase signaling pathways and is required for the development of acute disease. Cell Communication and Signaling 2013, 11:79, doi:10.1186/1478-811X-11-79.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Malik A., Sharma D., St. Charles J.L., Dybas L.A., Mansfield L.S. 2013. Contrasting immune responses mediate Campylobacter jejuni induced colitis and autoimmunity, Nature Mucosal immunology, In press.
- Type:
Websites
Status:
Published
Year Published:
2013
Citation:
Mansfield, LS, 2013. Gut microbes may provide targets for food-borne diseases. AgBioResearch Legislative Report on Food Safety and Security, MSU AgBioresearch & MSU Extension Legislative Report 2012-13, p. 19.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
St. Charles, J.L. 2013. EXAMINATION OF CAMPYLOBACTER JEJUNI IN THE PATHOGENESIS OF GUILLAIN BARR� SYNDROME. Comparative Medicine and Integrative Biology Graduate Program, Michigan State University Thesis.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
A. Malik and L.S. Mansfield. Contrasting immune responses mediate Campylobacter jejuni induced colitis and autoimmunity, American Society for Microbiology, Denver, CO, May 2013.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: 1) Draft Genome Sequences of Two Campylobacter jejuni Clinical Isolates, NW and 2600. Whole-genome sequences of C. jejuni isolates NW and D2600 were determined by Illumina sequencing at the Genomics Technology Support Facility at Michigan State Univ. NW was isolated at Sparrow Hospital in Lansing, MI, from a patient returning from Kenya with acute gastroenteritis. Strain D2600 was also isolated from a patient with gastroenteritis provided to us by the Centers for Disease Control and Prevention. These isolates colonized the C57BL/6 interleukin 10 knockout mouse model stably for 30 days but did not produce enteritis (1). Three other C. jejuni strains, including NCTC11168, that were tested in parallel both produced enteritis during initial infection and increased in virulence after a single passage in mice, while strains NW and D2600 did not (1). Virulent strains (such as NCTC11168) induce an inflammatory response that results in diarrhea, enlarged ileocecocolic lymph node, thickened colon and cecum wall, and occasional hemorrhagic enteritis (1, 7). The increased virulence of NCTC11168 during serial passage in mice was associated with mutations in genes containing hypermutable homopolymeric tracts, or contingency loci (6). Paired-end, 75-cycle reads were assembled using Velvet v1.1.04 (12) to generate 1,622,415 bases in 56 contigs for D2600 and 1,652,701 bases in 60 contigs for NW. Paired-end, 75-cycle reads were assembled using Velvet v1.1.04 (12) to generate 1,622,415 bases in 56 contigs for D2600 and 1,652,701 bases in 60 contigs for NW. The average G_C contents were 30.31% and 30.46% for NW and D2600, respectively. Annotation of the contigs was done using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline and yielded 1,619 and 1,690 potential protein-coding regions in the D2600 and NW genomes, respectively. The NW genome is similar in gene content and order to those of the sequenced strains RM1221 and S3, which were both isolated from chickens in the US. RM1221 contains four C. jejuni insertion elements (CJIE1 through CJIE4) (3, 9), of which S3 contains parts of CJIE1 and CJIE4 (2). The NW genome contains a sequence from only one insertion element, CJIE3. Contingency loci are defined by homopolymeric guanine tracts greater than seven bases in length in the C. jejuni genome (6,10), and an initial search for these tracts showed that the genomes of NW and D2600 contain 21 and 26 contingency loci, respectively. Notably, strain D2600 contains the genes encoding a class B lipooligosaccharide (LOS) structure (4, 8), and a homopolymeric guanine tract is present within the 2,3-sialyltransferase gene cstII. This tract leads to the variable expression of sialylated LOS (5) and is intriguing considering the association of LOS sialylation and the development of post infection autoimmune neuropathy (5, 11). An analysis of changes to the NW and D2600 genomes resulting from mouse passage is ongoing. This whole-genome shotgun project was deposited at DDBJ/EMBL/GenBank. Illumina reads were deposited at the NCBI Sequence Read Archive. PARTICIPANTS: Several students were mentored for study of enteric diseases of food animals. John Paul Jerome (PhD candidate) successfully defended his PhD thesis and has gone on to a job as a postdoc with Professor Sheng Yang He who has an endowed chair to study food borne pathogens in plants. Four other students are pursuing PhD degrees in food safety related to these enteric pathogens including Jessica St. Charles, Ankit Malik, Barbie Gadsden, DVM, and Phillip Brooks. Two of these students have passed their preliminary exams. Dr. Gadsden is preparing for this exam. Two of the graduate students are underrepresented minorities (African American). Four undergraduate students are working in the lab on these projects including Hahyung Kim, Elizabeth Gensterbloom, Julian Yu, and Anthony Brooks. Anthony was accepted to medical school and will start in 2013. Dr. Mansfield organized a seminar in Food and Waterborne Diseases for the faculty and students of Michigan State University on October 19, 2012. People attending came from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Shannon D. Manning, PhD, MPH Michigan State University (Evolution and Pathogenesis of E. coli O104:H4 infections) and Kathryn Eaton DVM, PhD, University of Michigan (Host, pathogen, and microbiota contribute to disease severity in EHEC-infected mice). Dr. Mansfield helped to organize and attended the USDA Enteric Diseases Meeting NC1041 in Chicago, Illinois on December 1st and 2nd as part of the Conference of Research Workers on Animal Diseases. Her graduate student Jessica St. Charles gave a talk at CRWAD about her work entitled "Campylobacter jejuni isolates from calves have A, B and C lipooligosaccharide (LOS) biosynthetic locus classes similar to human Guillain Barre syndrome associated strains". For the same CRWAD meeting, Dr. Mansfield gave a talk at CRWAD entitled "Comparison of induced small animal models for Guillain Barre syndrome (GBS) as post infectious sequelae to Campylobacter jejuni infection". Dr. Mansfield gave an invited talk entitled "Murine models of the autoimmune neuropathy Guillain Barre Syndrome" for the Small Animal Models of Enteric Diseases, NIAID, National Institutes of Health, September 13-14, 2012. She also gave an invited talk entitled "Campylobacter jejuni induces mixed Type 1 and 17 responses in acute and chronic disease" for the Enterics Research Investigational Network, Cooperative Research Centers Annual Meeting, National Institutes of Health, Seattle, Washington, May 22-23, 2012. TARGET AUDIENCES: The main target audience is the scientific community studying food safety and medical interventions for food borne pathogens. We reach this audience with high quality research publications in scientific journals as well as talks to scientific meetings and producer groups for food animals. We also have a long term effect by teaching undergraduate, graduate students and postdoctoral candidates these advances and strategies for studying food borne pathogens. The ultimate goals of this work are to translate this information into prevnetatives and treatments for food borne diseases. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Study Impact: The C. jejuni human clinical isolates NW and D2600 colonized C57BL/6 interleukin 10 knockout mice without inducing a robust inflammatory response (J. A. Bell et al., BMC Microbiol. 9:57, 2009). We announce draft genome sequences of NW and D2600 to facilitate comparisons with strains that induce gastrointestinal inflammation in this mouse model. Whole genome sequences for two human clinical isolates of C. jejuni are provided as a resource to the scientific community. Important differences in the genomes illustrate the diversity of these strains.
Publications
- Jerome J.P., Klahn B.D., Bell J.A., Barrick J.E., Brown C.T., and Mansfield L.S. 2012. Draft Genome Sequences of Two Campylobacter jejuni Clinical Isolates, NW and D2600, Journal of Bacteriology, J Bacteriol. 2012 Oct;194(20):5707-8.
- Jerome J.P. , Barrick J.E. , Kim H.Y., Klahn B.D., Brown C.T., Mansfield L.S.. 2012. Reversible motility mutations and parallel sigma54 loss during experimental evolution of Campylobacter jejuni. In review at Molecular Biology and Evolution.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: 1)Examination of Miller Fisher Syndrome (MFS) in a Murine Model. Acute neuropathies Guillain Barre Syndrome (GBS) and MFS can follow Campylobacter jejuni infection. LOS of C. jejuni mimics host nerve gangliosides, eliciting autoimmune responses that cross react with gangliosides on host tissues. GBS is an ascending symmetrical paralysis affecting the PNS. MFS is a descending neuropathy affecting the CNS before affecting peripheral nerves. Antibodies to gangliosides GM1 and GD1a are associated with GBS and antibodies to GQ1b with MFS. We hypothesized that C. jejuni CF93-6 (MFS patient strain) infected mice would exhibit neurological signs, autoantibodies and lesions consistent with MFS. Orally infected mice were tested for neurological phenotypes and nerve and myelin damage. ELISA was used to detect the presence of autoantibodies. Phenotypic tests indicated that infected mice of all genotypes displayed a neurological phenotype with differences in clinical manifestation between the C. jejuni strains. ELISA data showed the presence of autoantibodies to GM1 (14/75 mice), GD1a (17/75 mice), and GQ1b (6/75 mice). 1/15 C57BL/6 mice infected with CF93-6 displayed both a MFS neurological phenotype and anti-GQ1b antibodies. Results suggest that there is a manifestation of MFS in C57BL/6 mice that is distinguishable from that of GBS in NOD mice. 2)Overview of Emerging Campylobacter Species. Our knowledge of Campylobacter genetic diversity continues to expand. At the genus level, novel Campylobacter spp. have been reported recently from various hosts: Campylobacter canadensis (whooping cranes), Campylobacter volucris (black-headed gulls), Campylobacter subantarcticus (albatrosses, penguins), Campylobacter avium (poultry), Campylobacter cuniculorum (rabbits), and Campylobacter insulaenigrae (seals, harbor porpoise). Campylobacter lari-like strains isolated from humans and mollusks included one cluster that was named Campylobacter peloridis and a second cluster that was named Campylobacter lari subsp. concheus. Phenotypic studies supported these organisms as members of the genus. Phylogenetic analyses of whole genome or 16S rDNA sequences indicated several of the new species are closely related to campylobacters known to cause human disease. The more distantly related C. concisus and Arcobacter spp. have been isolated from humans and animals with diarrheal disease. C. jejuni and C. coli, exhibit high genomic diversity. However, after sequencing more than forty strains from different hosts, Stanhope and colleagues showed that there are barriers to interspecies recombination in the core genomes of the two species. Additionally, phenotypic differences in disease produced can be discerned among strains of C. jejuni. We delineated five pathotypes of C. jejuni in C57BL/6 IL-10-/- mice: lack of colonization, colonization without disease, colonization with watery or hemorrhagic enteritis, and colonization with neurological signs with or without enteritis. These pathotypes correspond to the spectrum of human disease; yet, gene content did not predict pathotype: each strain possessed a unique set of present, absent or divergent virulence factors. PARTICIPANTS: Principal Investigator: Linda S. Mansfield (Station Representative), Julia A. Bell, Ph.D., Research Assistant Professor; Graduate Students: John Paul Jerome, Ph. D. candidate, Jessica L. St. Charles, Ph. D. candidate, Ankit Malik, Ph. D. candidate, Barbie Gadsden, DVM, Ph. D. candidate TARGET AUDIENCES: The target audience for this work are scientists in related feilds. Our goals were to focus on disseminating knowledge and provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories. Graduate students: Once again, several students were mentored for study of enteric diseases of food animals. Jamie Jennifer Kopper (DVM candidate) successfully defended her PhD thesis and will go on to finish two more years of veterinary school. Four other students are pursuing PhD degrees in food safety related to these enteric pathogens including John Paul Jerome, Jessica St. Charles, Ankit Malik and Barbie Gadsden, DVM. Three of these students have passed their preliminary exams, two during 2011. One is preparing for this exam. Four undergraduate students are working in the lab on these projects. One undergraduate was accepted to and started medical school during 2011. Seminars and talks: Dr. Mansfield organized a Spring seminar in Food and Waterborne Diseases for the faculty and students of Michigan State University. People attending have come from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Christopher Waters, PhD (Biofilms in foodborne bacteria) and John Paul Jerome (Contingency genes in Campylobacter jejuni). Also, Dr. Mansfield attended and presented at a scientific conference held by the National Institutes of Health in Baltimore, Maryland on Enteric Diseases. Dr. Mansfield helped to organize and attended the USDA Rushmore Enteric Diseases Meeting NC1041 in Chicago, Illinois on December 3rd and 4th. Her graduate student John Paul Jerome gave a talk about his work entitled "Contingency loci dynamics during Campylobacter jejuni infection". Dr. Mansfield gave a talk for the Department of Large Animal Clinical Sciences at Michigan State University entitled "Investigating the role of Campylobacter jejuni in acute and chronic disease: the genetic basis of pathotypes" on 02/01/2011 PROJECT MODIFICATIONS: none.
Impacts
Study Impact: Because host genetic background affects disease phenotype, we conclude that clinical presentation of C. jejuni infection is controlled by a complex interplay of factors. Where newly described species fall in the spectrum of disease and the genetic factors controlling disease expression remain to be determined. We have shown that only some C. jejuni strains cause neurological disease. We have developed mouse models to examine this type of disease. Contingency genes and LOS locus genes are responsible for this phenotype. This is important to study because this severe autoimmune disease happens after C. jejuni infection and could happen after C. jejuni vaccination.
Publications
- Mansfield LS, Kopper JJ, St. Charles JL, Bell JA, Malik A, Dybas L, Fried D. 2011. Addition of a Thelper-2 (Th2) immunomodulator to antibiotic treatment improves survival and decreases clinical signs of inflammatory bowel disease induced by Campylobacter jejuni infection in a murine model, Campylobacter, Helicobacter and Related Organisms Meeting, August 28-September 1, 2011.
- J. L. St. Charles, A. M. Zaleski, E. J. Smith, J. A. Bell, L. S. Mansfield. 2011. Examination of Miller Fisher Syndrome in a Murine Model, Campylobacter, Helicobacter and Related Organisms, Vancouver, British Columbia, August 28-September 1, 2011.
- J. L. St. Charles, J. A. Bell, L. S. Mansfield. 2011. Examination of Guillain Barre Syndrome following Campylobacter jejuni infection in a murine model, PhiZeta research Day, College of Veterinary medicine, Michigan State University. October 14, 2011.
- L. S. Mansfield. 2011. Overview of Emerging Campylobacter Species, Campylobacter, Helicobacter and Related Organisms, Vancouver, British Columbia, August 28-September 1, 2011.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Background: Campylobacter jejuni infection in humans usually causes watery and bloody diarrheas; infection can also result in an asymptomatic carrier state or autoimmune neurological sequelae (Miller Fisher and Guillain Barre Syndromes). We assessed the spectrum of pathology caused by 16 C. jejuni strains in C57BL/6 IL-10 knock out mice to relate differences in outcomes to genetic differences between strains; genome sequences are available for 10 strains. Methods: Ten individually caged mice were inoculated per os with ~10x10 cfu of each single C. jejuni strain. Mice were monitored for clinical signs for 35 days (or until clinical signs necessitated euthanasia) and then euthanized and necropsied. Presence of C. jejuni in tissues and feces was evaluated by culture and PCR; histopathology, in sections of the ileocecocolic junction; and antibody response, by ELISA. Mice inoculated with broth and C. jejuni 11168 served as controls. Results: Variation was observed in colonization proficiency, mouse survival after inoculation, and degree of clinical signs and pathology. Three strains were less proficient colonizers; five strains caused severe disease in the majority of mice. Seven strains caused bloody diarrhea in at least one mouse. One strain produced neurological signs. These results, combined with published data, distinguished five pathotypes of C. jejuni in C57BL/6 IL-10KO mice; no colonization, colonization with little or no disease, colonization with moderate or severe enteritis, colonization with hemorrhagic enteritis, and colonization with neurological sequelae either with or without enteritis. Preliminary comparisons of genome content (presence or absence of COGS) of the 10 sequenced strains did not reveal genome-wide differences that corresponded to differences in pathogenicity in mice. Conclusions: C. jejuni-infected C57BL/6 IL-10KO mice display a spectrum of disease outcomes similar to that of humans. Genetic differences related to differences in pathogenicity are likely to be found as point mutations, indels, or changes in gene expression. PARTICIPANTS: Collaborators included Michael Stanhope (Cornell University) and Michael Konkel (Washington State University). TARGET AUDIENCES: Focus on disseminating knowledge - Provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories. Several students were mentored for study of enteric diseases of food animals. Five students are pursuing PhD degrees in food safety related to these enteric pathogens including John Paul Jerome, Jessica St. Charles, Jamie Jennifer Kopper (DVM candidate), Ankit Malik and Barbie Gadsden, DVM. Three of these students have passed their preliminary exams. Two are preparing for this exam. Vijay Rathinam, successfully defended his Ph.D. thesis and obtained a postdoctoral position at the University of Massachusetts at Wooster, MA where he is studying the pathogenesis of inflammatory bowel disease due to bacterial pathogens. One undergraduate student working in the lab on these projects was accepted into veterinary schools (Alexander Adrian, University of Minnesota). Dr. Mansfield organized a Fall seminar in Food and Waterborne Diseases for the faculty and students of Michigan State University. People attending have come from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Shannon Manning, PhD, MPH (Shigatoxin producing E. coli) and Robert Britton, PhD (Probiotics for treating enteric diseases). Also, Dr. Mansfield attended and presented at a scientific conference held by the National Institutes of Health at Cambridge, Maryland on Food and Water borne Pathogens. Dr. Mansfield was an invited speaker for the Norman E. Borlaug International Agricultural Science and Technology Fellowship Program mentor at Polish Food Safety Meeting on 04/23/2010 and gave an invited keynote address at the American Society for Microbiology entitled "Examining the genetic basis of pathotypes in Campylobacter jejuni using murine models" on 05/28/2010. Dr. Mansfield organized and attended the USDA Multistate Research Project 2009 Meeting NC1041 in Chicago, Illinois on December 4th and 5th. During this reporting period we developed three tractable murine models for study of C. jejuni. This allowed for determination of both host genetic factors and bacterial genetic factors underlying disease caused by this pathogen. We also utilized these models to track C. jejuni in the live host and to examine innate and adaptive immune responses that control this pathogen. PROJECT MODIFICATIONS: Our future goals are to define C. jejuni gene expression controlling the different pathotypes that we have defined in the reported murine models. We will continue this study on the genetic diversity of human and animal isolates of C. jejuni that we have acquired through the US Centers for Diseases Control and Prevention and Dr. Al Lastovica, Bellville, South Africa. Methods will include microarray (using a supplemented whole open reading frame (ORF) array from the 7 sequenced strains of C. jejuni), Illumina sequencing, in vivo expression technology (IVET), and selected gene knockouts followed by challenge in the murine models.
Impacts
Study Impact: We showed that murine models can be used as surrogates for humans for study of Campylobacter jejuni pathogenesis. Using these models, we showed that infected mice display a spectrum of disease outcomes similar to that of humans when screened with 17 different strains from infected patients. Comparisons of genome content (presence or absence of COGS) of the 10 sequenced strains did not reveal genome-wide differences that corresponded to differences in pathogenicity in mice. Thus we conclude that genetic differences related to differences in pathogenicity are likely to be found as point mutations, indels, or changes in gene expression. These results demonstrate that virulence phenotypes/pathotypes vary with strain of C. jejuni but the underlying genetic mechanisms controlling these phenotypes is not due to gene content alone.
Publications
- Parthasarathy G and Mansfield LS. 2009. Recombinant IL-4 (rIL-4) enhances Campylobacter jejuni invasion of Intestinal Pig Epithelial Cells (IPEC-1), Microbial Pathogenesis, 2009 Jul;47(1):38-46. Epub 2009 May 3, PMID: 19409975.
- Wilson DL, Rathinam VK, Qi W, Wick LM, Landgraf J, Bell JA, Plovanich-Jones A, Parrish J, Finley RL, Mansfield LS and Linz JE. 2010. Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL-10 deficient mice. Microbiology, Papers in Press. Published April 1, 2010 as doi:10.1099/mic.0.035717-0.
- Jerome, J.P., J.A. Bell, A.E. Plovanich-Jones, J.E. Barrick, C.T. Brown, L.S. Mansfield, 2010. Standing genetic variation in contingency genes drives the adaptation of Campylobacter jejuni to a novel host. PloS One, Addressing critiques prior to resubmission after a positive review.
- Bell, J.A., J. R. Gettings, J. P. Jerome, J. J. Kopper, A. Plovanich-Jones, V. A. K. Rathinam, J. L. St. Charles, E.J. Smith, A. G. Staunton, and Mansfield, L.S. 2010. Campylobacter jejuni genome content and virulence phenotypes (pathotypes) in C57BL/6 IL-10KO mice, submitted to Infection and Immunity.
- J.P. Jerome, A.E. Plovanich-Jones, J.A. Bell, Linda S. Mansfield. 2009. Genetic changes during serial passage of Campylobacter jejuni in C57BL/6 IL-10KO mice are associated with enhanced virulence, Phi Zeta Research Day, College of Veterinary Medicine, Michigan State University, East Lansing, MI.
- Bianca A. Buffa, Julia A. Bell, Vijay A. K. Rathinam, Jamie J. Kopper, Alice J. Murphy, and Linda S. Mansfield. 2009. Responses of C57BL/6 interleukin-10 deficient and proficient mice to infection with low doses of Campylobacter jejuni 11168. Phi Zeta Research Day, College of Veterinary Medicine, Michigan State University, East Lansing, MI.
- Vijay A.K. Rathinam, Daniel M. Appledorn, Kathleen A. Hoag, Andrea Amalfitano and Linda S. Mansfield. 2009. TLR4-MyD88 and TLR4-TRIF signaling axes cooperate in activating dendritic cells in response to an intracellular enteric pathogen. Pattern Recognition Molecules and Immune Sensors, Keystone Meetings, Banff, Alberta CA, March 2009.
- J. A. Bell, A. Brooks, B. A. Buffa1, J. R. Gettings, J.P. Jerome, J. J. Kopper, T. Lefebure, A. E. Plovanich-Jones, V. A. Rathinam, J. St. Charles, E. Smith, A. Staunton, M. J. Stanhope, L. S. Mansfield. 2010. Phylogeny, gene content, and virulence phenotypes (pathotypes) of Campylobacter jejuni strains in C57BL/6 interleukin-10 deficient mice, National Institutes of Health, Food and Water borne Diseases Integrated Research Network, Cambridge,MD,June 1 to 4,2010.
- J. St. Charles, J. A. Bell, A. Brooks, B. A. Buffa, J.P. Jerome, J. J. Kopper, V. A. Rathinam, E. Smith, A. Staunton, L. S. Mansfield. Murine Models for Study of Guillain Barre Syndrome (GBS) and Other Autoimmune Neuropathies following Campylobacter infection, National Institutes of Health, Food and Water borne Diseases Integrated Research Network, Cambridge, MD, June 1 to 4, 2010.
- J.A. Bell, J.L. St. Charles, A.J. Murphy, V.A. Rathinam, A.E. Plovanich-Jones, E.L. Stanley, J.E. Wolf, J.R. Gettings, T.S. Whittam, Tristan Lefebure, Michael Stanhope, L.S. Mansfield. 2009. Pathogenicity of Campylobacter jejuni strains in C57BL/6 IL-10KO mice varies with strain genetic background, FWDIRN Annual Meeting, Stevenson, WA. March 30 to April 1, 2009.
- J.P. Jerome, A.E. Plovanich-Jones, J.A. Bell, Linda S. Mansfield. 2009. Genetic changes during serial passage of Campylobacter jejuni in C57BL/6 IL-10KO mice are associated with enhanced virulence, FWDIRN Annual Meeting, Stevenson, WA. March 30 to April 1, 2009.
- J.D. Olmstead, V.A.K. Rathinam, L.S. Mansfield. 2009. Determining organ specific recovery of Campylobacter jejuni in a murine disease model, FWDIRN Annual Meeting, Stevenson, WA. March 30 to April 1, 2009.
- J.L. St. Charles, J.A. Bell, and L.S. Mansfield. 2009. Development of murine models for study of Guillain-Barre Syndrome, FWDIRN Annual Meeting, Stevenson, WA. March 30 to April 1, 2009.
- L. S. Mansfield. Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10KO mice, NC1041 Enteric Diseases of Swine and Cattle: Prevention, Control and Food Safety Meeting, Chicago, IL; December 5 to 6, 2009.
- L. S. Mansfield. 2010. Developing murine models for the study of enteric pathotypes of Campylobacter jejuni. Pathology Seminar Series, Department of Pathology and Laboratory Medicine, University of Wisconsin, March 17, 2010.
- L.S. Mansfield, J.A. Bell, J.L. St. Charles, J.P. Jerome, A.E. Plovanich-Jones. 2010. Examining the genetic basis of pathotypes in Campylobacter jejuni using murine models, National Veterinary Institute (PIWet), Lublin, Poland, April 19 to 23, 2010.
- L. S. Mansfield, J. A. Bell, J. L. St. Charles, J. P. Jerome. 2010. Examining the Genetic Basis of Pathotypes in Campylobacter jejuni Using Murine Models, American Society for Microbiology General Meeting, San Diego, CA. May 23 to 27, 2010.
- J.A. Bell, J. R. Gettings, J. P. Jerome, J. J. Kopper, A. Plovanich-Jones, V. A. K. Rathinam, J. L. St. Charles, E.J. Smith, A. G. Staunton, L. S. Mansfield. 2010. Outcome of Campylobacter jejuni Infection of C57BL/6 IL-10KO Mice Varies with Strain, American Society for Microbiology General Meeting, San Diego, CA. May 23 to 27, 2010.
- E. J. Smith, J.D. Olmstead, L. S. Mansfield. 2010 Monitoring Campylobacter jejuni using Fluorescent in situ Hybridization, American Society for Microbiology General Meeting, San Diego, CA. May 23 to 27, 2010.
- J.P. Jerome, J.A. Bell, A.E. Plovanich-Jones, C.T. Brown, L.S. Mansfield. 2010. The Role of Contingency Genes in the Adaptation of Campylobacter jejuni to a Novel Host, American Society for Microbiology General Meeting, San Diego, CA, May 23-27, 2010.
- J. L. St. Charles, J. A. Bell, L. S. Mansfield. 2010. Development of Murine Model to Study Guillain Barre Syndrome Following Campylobacter jejuni Infection, American Society for Microbiology General Meeting, San Diego, CA, May 23 to 27, 2010.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Invited Speaker, National Institutes of Health, Food and Water borne Diseases Integrated Research Network, Michigan State University Microbiology Research Unit Progress, 2009 Annual Meeting, Stevenson, WA. 3/29 - 4/1/2009. Presented the following talks: 1) J.A. Bell, J.L. St. Charles, A.J. Murphy, V.A. Rathinam, A.E. Plovanich-Jones, E.L. Stanley, J.E. Wolf, J.R. Gettings, T.S. Whittam, Tristan Lefebure, Michael Stanhope, L.S. Mansfield. 2009. Pathogenicity of Campylobacter jejuni strains in C57BL/6 IL-10-/- mice varies with strain genetic background, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009. 2)J.P. Jerome, A.E. Plovanich-Jones, J.A. Bell, Linda S. Mansfield. 2009. Genetic changes during serial passage of Campylobacter jejuni in C57BL/6 IL-10-/- mice are associated with enhanced virulence, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009. 3)J.D. Olmstead, V.A.K. Rathinam, L.S. Mansfield. 2009. Determining organ specific recovery of Campylobacter jejuni in a murine disease model, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009. 4)JL St. Charles1,3, JA Bell1, and LS Mansfield. 2009. Development of murine models for study of Guillain-Barré Syndrome, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009. Speaker, Pattern Recognition Molecules and Immune Sensors, Keystone Meetings, Banff, Alberta CA, March 2009. Presented the following work: Vijay A.K. Rathinam, Daniel M. Appledorn, Kathleen A. Hoag, Andrea Amalfitano and Linda S. Mansfield. 2009. TLR4→MyD88 and TLR4→TRIF signaling axes cooperate in activating dendritic cells in response to an intracellular enteric pathogen. Speaker, World Association for the Advancement of Veterinary Parasitology. Presented the folowing work: Recombinant Interleukin-4 enhances Campylobacter jejuni invasion of Intestinal Pig Epithelial Cells (IPEC-1)by G. Parthasarathy and L.S. Mansfield Several students were mentored for study of enteric diseases of food animals. Five students are pursuing PhD degrees in food safety related to these enteric pathogens including John Paul Jerome, Jessica St. Charles, Jamie Jennifer Kopper, Ankit Malik and Vijay Rathinam. Three of these students have passed their preliminary exams. Two are preparing for this exam. One, Vijay Rathinam, successfully defended his Ph.D. thesis and obtained a postdoctoral position at the University of Massachusetts at Wooster, MA where he is studying the pathogenesis of inflammatory bowel disease due to bacterial pathogens. Two undergraduate students working in the lab on these projects were accepted into veterinary schools (Bianca Buffa, Michigan State University and Jenna Gettings, North Carolina State University). Jenna Gettings will also complete a M.Sc. degree in public health as part of her veterinary curriculum. Dr. Mansfield organized a summer seminars in Food and Waterborne Diseases for the faculty and students of Michigan State University. PARTICIPANTS: Several students were mentored for study of enteric diseases of food animals. Five students are pursuing PhD degrees in food safety related to these enteric pathogens including John Paul Jerome, Jessica St. Charles, Jamie Jennifer Kopper, Ankit Malik and Vijay Rathinam. Collaborations were carried out with Michael Stanhope, Cornell University and Andrea Amalfitano at Michigan State University. TARGET AUDIENCES: The target audience for this work is people working as scientists specializing in bacterial pathogenesis, food safety and immunology. This work is relevant to medical and veterinary medical practitioners. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Campylobacter jejuni-induced activation of dendritic cells involves cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes Campylobacter jejuni is an important cause of human enteritis and has been linked to the development of autoimmune diseases. Recently, we showed that infection of murine dendritic cells (DCs) with C. jejuni resulted in DC activation and induction of Campylobacter-specific Th1-effector responses. Toll-like receptor (TLR) signaling through myeloid differentiation factor-88 (MyD88) and/or Toll-IL-1R domain-containing adaptor-inducing IFN-gamma (TRIF) is critical in inducing immunity against pathogens. In this study, we investigated the role of TLR2, TLR4, MyD88, and TRIF signaling in C. jejuni-induced inflammatory activation of DCs. DC upregulation of MHC-II and costimulatory molecules after C. jejuni challenge was profoundly impaired by TLR2, TLR4, MyD88, and TRIF deficiencies. Similarly, C. jejuni-induced secretion of IL-12, IL-6, and TNF-alpha was significantly inhibited in TLR2-/-, TLR4-/-, MyD88-/-, and TRIF-/- DCs compared to wild-type DCs; however, the magnitude of inhibition was greater in MyD88-/-, TRIF-/-, and TLR4-/- DCs than in TLR2-/- DCs. Furthermore, C. jejuni induced interferon regulatory factor-3 (IRF-3) phosphorylation and IFN-gamma secretion by DCs in a TLR4-TRIF-dependent fashion, further demonstrating activation of this pathway by C. jejuni. Importantly, TLR2, TLR4, MyD88, and TRIF deficiencies all markedly impaired Th1-priming ability of C. jejuni-infected DCs. Thus, our results show that cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes represents a novel mechanism mediating C. jejuni-induced inflammatory responses of DCs. To our knowledge, such a mechanism has not been demonstrated previously for an intact bacterium.
Publications
- J.A. Bell, J.L. St. Charles, A.J. Murphy, V.A. Rathinam, A.E. Plovanich-Jones, E.L. Stanley, J.E. Wolf, J.R. Gettings, T.S. Whittam, Tristan Lefebure, Michael Stanhope, L.S. Mansfield. 2009. Pathogenicity of Campylobacter jejuni strains in C57BL/6 IL-10-/- mice varies with strain genetic background, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.
- J.P. Jerome, A.E. Plovanich-Jones, J.A. Bell, Linda S. Mansfield. 2009. Genetic changes during serial passage of Campylobacter jejuni in C57BL/6 IL-10-/- mice are associated with enhanced virulence, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.
- J.D. Olmstead, V.A.K. Rathinam, L.S. Mansfield. 2009. Determining organ specific recovery of Campylobacter jejuni in a murine disease model, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.
- JL St. Charles1,3, JA Bell1, and LS Mansfield. 2009. Development of murine models for study of Guillain-Barré Syndrome, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.
- Mansfield LS, Patterson JS, Fierro BR, Murphy AJ, Rathinam VA, Kopper JJ, Barbu NI, Onifade TJ, and JA Bell. 2008. Genetic background of IL-10-/- mice alters host-pathogen interactions with Campylobacter jejuni and influences disease phenotype, Microbial Pathogenesis 2008 Oct;45(4):241-57 Epub2008 Jun 11.
- Parthasarathy G and Mansfield LS. 2008. Recombinant IL-4 (rIL-4) enhances Campylobacter jejuni invasion of Intestinal Pig Epithelial Cells (IPEC-1), Microbial Pathogenesis, Accepted April 22, 2009. ISSN 0882-4010.
- Bell JA, St. Charles JL, Murphy AJ, Rathinam VA, Plovanich-Jones AE, Stanley EL, Wolf JE, Gettings JR, Whittam TS, Mansfield LS. 2009. Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10(-/-) mice, BMC Microbiology, 2009 Mar 18;9:57, PMID: 19296832.
- Rathinam VA, Appledorn DM, Hoag KA, Amalfitano A, Mansfield LS. 2009. Campylobacter jejuni-induced activation of dendritic cells involves cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes, Infection and Immunity, 2009 Mar 30, PMID: 19332531.
- Rathinam VA, Hoag KA, and Mansfield LS. 2008. Dendritic cells from C57BL/6 mice undergo activation and induce Th1-effector cell responses against Campylobacter jejuni. Microbes and Infection 2008 Oct;10(12-13):1316-24. Epub 2008 Aug 5, PMID: 18725315.
- Abstracts V.A.K. Rathinam, Daniel M. Appledorn, Kathleen A. Hoag, Andrea Amalfitano and Linda S. Mansfield. 2009. TLR4→MyD88 and TLR4→TRIF signaling axes cooperate in activating dendritic cells in response to an intracellular enteric pathogen. Pattern Recognition Molecules and Immune Sensors, Keystone Meetings, Banff, Alberta CA, March 2009.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: During this reporting period we published two manuscripts and two abstracts. We attended two scientific meetings and reported results of our projects. We also participated in identifying the causative agent of a food borne outbreak in Michigan. PARTICIPANTS: The members of the Mansfield Laboratory were the main workers on this project. We worked in collaboration with Dr. Michael Stanhope at Cornell University and Dr. Michael Konkel at Washington State University. TARGET AUDIENCES: The target audience for these projects are scientists interested in food and waterborne illnesses, physicians, veterinarians and food safety practitioners within the food industry. PROJECT MODIFICATIONS: There are no major changes in the aims reported previously. We have made excellent progress, published our major findings and are working on the next goals for the coming year.
Impacts
We hypothesized that particular genetic backgrounds enhance rates of colonization, increase severity of enteritis, and allow for extraintestinal spread when inbred IL-10 knockout mice are infected with pathogenic C. jejuni. Campylobacter jejuni stably colonized C57BL/6 and NOD mice, while congenic strains lacking IL-10 developed typhlocolitis following colonization that mimicked human campylobacteriosis. However, IL-10 deficiency alone was not necessary for presence of C. jejuni in extraintestinal sites. C3H/HeJtlr4 knockout mice that specifically express the Cdcs1 allele showed colonization and limited extraintestinal spread without enteritis implicating this interval in the clinical presentation of C. jejuni infection. Furthermore, when the IL-10 gene is inactivated as in C3Birtlr4 knockout IL-10 knockout mice, enteritis and intensive extraintestinal spread were observed, suggesting that clinical presentations of C. jejuni infection are controlled by a complex interplay of factors. These data demonstrate that lack of IL-10 had a greater effect on C. jejuni induced colitis than other immune elements such as TLR4 (C3H/HeJ, C3Bir IL-10 knockout), MHC H-2g7, diabetogenic genes, and CTLA-4 (NOD) and that host genetic background is in part responsible for disease phenotype. C3Bir IL-10 knockout mice where Cdcs1 impairs gut barrier function provide a new murine model of C. jejuni and can serve as surrogates for immunocompromised patients with extraintestinal spread. Food-borne Campylobacter jejuni (Cj) is an important cause of enteritis. We showed that C57BL/6 and congenic interleukin (IL)-10 knockout mice serve as models of Cj colonization and enteritis, respectively. Thus, C57BL/6 mice are resistant to Cj induced disease. Because dendritic cells (DCs) are central to regulating adaptive immune responses, we investigated the interaction of Cj with murine bone marrow-derived DCs (BM-DCs) to assess bacterial killing, DC activation, and the ability of Cj-infected BM-DCs to stimulate Campylobacter-specific T cell responses in vitro. BM-DCs challenged with Cj efficiently internalized and killed Cj 11168 and significantly upregulated surface MHC-II, CD40, CD80 and CD86 demonstrating a mature phenotype. Infected BM-DCs secreted significant amounts of tumor necrosis factor-alpha (TNF-alpha), IL-6 and IL-12p70. Formalin-killed Cj also induced maturation of BM-DCs with similar cytokine production but at a significantly lower magnitude than live bacteria. Maximal activation of murine BM-DCs required internalization of Cj; attachment alone was not sufficient to elicit significant responses. Also, various strains of Cj elicited different magnitudes of cytokine production from BM-DCs. Finally, in a coculture system, Cj-infected BM-DCs induced high level interferon-gamma (INF-gamma) production from CD4(+)T cells indicating Th1 polarization. Thus, DCs from resistant C57BL/6 mice initiate T cell responses against Cj.
Publications
- Mansfield LS, Patterson JS, Fierro BR, Murphy AJ, Rathinam VA, Kopper JJ, Barbu NI, Onifade TJ, and JA Bell. 2008. Genetic background of IL-10 knockout mice alters host-pathogen interactions with Campylobacter jejuni and influences disease phenotype, Microbial Pathogenesis 2008 Oct;45(4):241-57. Epub 2008 Jun 11.
- Rathinam VA, Hoag KA, and Mansfield LS. 2008. Dendritic cells from C57BL/6 mice undergo activation and induce Th1-effector cell responses against Campylobacter jejuni. Microbes and Infection 2008 Oct;10(12-13):1316-24. Epub 2008 Aug 5.
- Rathinam VA, Appledorn DM, Olmstead JD, Hoag KA, Amalfitano A, and Mansfield LS. 2008. Campylobacter jejuni-induced Activation of Murine Dendritic Cells Involves Cooperative Signaling through MyD88 and TRIF. American Society for Microbiology, Boston, MA.
- Rathinam VA, Appledorn DM,Hoag KA, Amalfitano A, and Mansfield LS. 2008. Campylobacter jejuni-induced Activation of Murine Dendritic Cells Involves Cooperative Signaling through MyD88 and TRIF. 15th International IRA Conference, Chantilly, VA
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Progress 01/01/07 to 12/31/07
Outputs
Activities The Mansfield laboratory conducted and analyzed a large number of experiments on food and waterborne pathogens in 2007. The main focus was Campylobacter jejuni, mainly those isolates of the bacterium received from human cases that were submitted to the US Centers for Disease Control. Using microarrays and mouse animal models, we analysed the ability of these bacteria to cause disease with the aim toward preventing or treating this disease. We also isolated bacteria from the well water on Bass Island OH after a large water borne outbreak of gastrointestinal disease in people visiting the island and studied the Campyloabcter and Arcobacter isolates that we got from the water samples. Dissemination Dr. Mansfield reported data from these studies for the American College of Laboratory Animal Medicine Regional Meeting, 2007. here she discussed enteric bacteria as causes of chronic inflammation in the gastrointestinal tract and the use of nematode parasite products
as potential treatments. Here the main target audience was practicing veterinarians in academia, the pharmaceutical industry or clinical practice. Dr. Mansfield also presented a poster at the American Society for Microbiology Meeting in Toronto, Canada entitled Campylobacter jejuni Induces a Spectrum of Pathotypes in Murine Models Dependent on Genotype of Host and Pathogen. Here the main target audience were scientists interested in infectious diseases and public health practitioners and administrators Both of these works were published as abstracts.
Impacts
In 2007, we developed a new murine model of Campylobacter jejuni colonization and enteritis. This tool provides a signficant step forward in the process to understand how food and water borne Campylobacter spp. cause disease. The mice can act as surrogates to examine what genes control the disease process. They can also be used as a surrogate for humans to test prevention and teratment strategies. During this period we also learned some of the main host immune system components that control resistance to this bacterium. These could possibly be used to target treatment to people suffering from gastroenteritis caused by Campylobacter.
Publications
- Mansfield LS, Schauer DB, Fox JG. 2007. Animal Models of Campylobacter jejuni infections, In: Campylobacter, I. Nachamkin and M.J. Blaser (eds), American Society for Microbiology Press, Washington, D.C., In press.
- Theng-Theng Fong, Linda S. Mansfield, David L. Wilson, David J. Schwab, Stephanie L. Molloy and Joan B. Rose. 2007. Massive Microbiological Groundwater Contamination Associated with a Waterborne Outbreak in Lake Erie, South Bass Island, OH, Journal of Environmental Health Perspective 115(6):856-864.
- Mansfield LS, Bell JA, Wilson DL, Murphy AJ, Elsheikha HM, Rathinam VA, Fierro BR, Linz JE, and Young VB. 2007. C57BL/6 and congenic interleukin-10 deficient mice can serve as models of Campylobacter jejuni colonization and enteritis, Infection and Immunity 75(3):1099-115. Epub 2006 Nov 27.
- J. A. Bell, J. L. St. Charles, A. J. Murphy, V. A. Rathinam, T. S. Whittam, L. S. Mansfield. 2007. Campylobacter jejuni Strain Adaptation and Diet Influence Virulence and Disease and Mediate Pathotype in the C57BL/6 IL-10-/- Murine Model, American Society for Microbiology, Toronto, CA.
- L. S. Mansfield, J. L. St. Charles, B. R. Fierro, A. J. Murphy, V. A. Rathinam, S. A. Apodaca, J. S. Patterson, T. S. Whittam, J. A. Bell. 2007. Campylobacter jejuni Induces a Spectrum of Pathotypes in Murine Models Dependent on Genotype of Host and Pathogen, American Society for Microbiology, Toronto, CA.
- Vijay A.K. Rathinam, Kathleen A. Hoag and Linda S. Mansfield. 2007. Murine dendritic cells produce IL-12 and induce Th1 polarization in response to Campylobacter jejuni infection. American Society for Microbiology, Toronto, CA.
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Progress 01/01/06 to 12/31/06
Outputs
We have developed a new murine model to act as a surrogate for study of Campylobacter jejuni virulence in the host. C557BL/6 mice and their IL-10 knockout develop colonization and enteritis, respectively, when they are infected with C. jejuni strain 11168. We have used these mice to screen 7 C. jejuni strains isolated from patients with various disease presentations. Some C. jejuni strains produced severe disease in the mice while others did not. Those producing disease belonged to three related clonal complexes as identified by Multilocus Sequence Typing (MLST). This work will serve as the basis for virotyping/pathotyping Campylobacters to determine which have genetic attributes capable of causing severe disease. We have also finished and the published all the past work on identifying prevalence and mechanisms of antibiotic resistance in campylobacters from dairy cattle.
Impacts
This work has the potential to provide an accurate screen to determine which Campylobacter jejuni strains have the potential to cause severe disease in humans. Once identified these strains can be genetically analyzed to determine the genes underlying the potential to cause disease.
Publications
- Mansfield LS, Bell JA, Wilson DL, Murphy AJ, Elsheikha HM, Rathinam VA, Fierro BR, Linz JE, and Young VB. Novel models of C. jejuni colonization and enteritis in C57BL/6 mice and their syngeneic IL-10 knockout, Infection and Immunity, in press 27 November 2006.
- Halbert LW, Kaneene JB, Linz JE, Mansfield LS, Wilson D, Ruegg PL, Warnick LD, et al. 2006. Genetic mechanisms contributing to reduced tetracycline susceptibility of Campylobacter isolated from organic and conventional dairy farms in the midwestern and northeastern United States, J. Food Protect. 69(3):482-8.
- Halbert LW, Kaneene JB, Ruegg PL, Warnick LD, Wells SJ, Mansfield LS, Fossler CP, Campbell AM, Geiger-Zwald AM. 2006. Evaluation of antimicrobial susceptibility patterns in Campylobacter spp isolated from dairy cattle and farms managed organically and conventionally in the midwestern and northeastern United States. J Am Vet Med Assoc. 2006 Apr 1;228(7):1074-81.
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Progress 01/01/05 to 12/31/05
Outputs
Current project completed in 2005: The comparison of automated microbroth dilution and agar dilution for antimicrobial susceptibility of Campylobacter jejuni isolated from dairy sources Lisa W. Halbert, John B. Kaneene, Linda S. Mansfield, Pamela L.Ruegg, Lorin D.Warnick, Scott J. Wells, Chuck P. Fossler, Amy M. Campbell BS; Angela M.Geiger-Zwald Abstract Utilizing commercially prepared antimicrobial panels, microbroth dilution was compared to agar dilution for determining antimicrobial susceptibility in Campylobacter isolates from dairy cattle. A group of 190 Campylobacter isolates were used to assess the performance of both techniques for ampicillin, and a second group of 172 Campylobacter isolates were used for ciprofloxacin, erythromycin, nalidixic acid and tetracycline. Our results indicate that microbroth dilution and agar dilution agree within +/- 1 log2 dilution for 86.7% of the isolates tested. Ciprofloxacin had the highest level of agreement for isolates
tested by both techniques, resulting in a kappa of 0.886 and 97.1% agreement +/- 1 log2 dilution. The most disagreement was observed in determining the susceptibility of isolates to ampicillin and erythromycin (82.1% and 79.7% agreement +/-1 log2 dilution), but kappa statistics were considered to have good agreement for these antimicrobials. There were no significant differences in the summary statistics for any of the five antimicrobials evaluated for the isolates analyzed by the percentage of resistant isolates, MIC50, MIC75 or MIC90 beyond +/- 1 log2 dilution. There was no association in the classification of resistance by the testing methods employed. The quality control strain of C. jejuni ATCC 33650 performed in a consistent manner for both agar dilution and microbroth dilution. We conclude that microbroth dilution may be an acceptable alternative to agar dilution for determining susceptibility in Campylobacter in research or surveillance where flow of samples, labor efficiency,
and cost may not be amendable to performing agar dilution.
Impacts
This study describes a new automated method for determing the susceptibility of the bacteria Campylobacter jejuni to common antibiotics. This is a useful new method that can be used in medical practice for both humans and animals for this extremely common food borne pathogen.
Publications
- Young VB and Mansfield LS. 2005. Campylobacter Infection-Clinical Context. In: Campylobacter jejuni: New perspectives in molecular and cellular biology, Horizon press, pp.1-12.
- Halbert LW, Kaneene JB, Mansfield LS, Ruegg PL, Warnick LD, Wells SJ, Fossler CP, Campbell AM, Geiger-Zwald AM.. 2005. Comparison of automated microbroth dilution and agar dilution for antimicrobial susceptibility of Campylobacter jejuni isolated from dairy sources, Journal of Antimicrobial Chemotherapy (2005) 56:686-691.
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Progress 01/01/04 to 12/31/04
Outputs
During the last period of this project, we constructed a microarray chip that represents at least 95% coverage of the open reading frames encoding genes of Campylobacter jejuni. This microarray has been tested and proved to be og high quality in terms of repeatability and representation of the genome. In the future, it will be used to compare Campylobacters that have passed through the digestive tract of food animals (and control Campylobacters that ahve not) in order to understand the genetic factors that allow this bacteria to cause disease. This information will provide the basis to control infection in animals (by vaccination or treatment) to prevent infection of humans.
Impacts
This work helps to understand how the food borne pathogen Campylobacter jejuni causes disease in a host that is similar to humans. It will form the basis for development of a vaccine against this pathogen.
Publications
- Wilson DL, Bell JA, Young VB, Wilder SR, Mansfield LS, and Linz JE. 2003. Variation of the natural transformation frequency of Campylobacter jejuni in liquid shake culture, Microbiology 149:3603-3615.
- Mansfield LS and Gauthier DT. 2004. Lymphoglandular complexes are important colonic sites for Immunoglobulin A induction against Campylobacter jejuni in a swine disease model. Comparative Medicine 54(5):514-523.
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Progress 01/01/03 to 12/31/03
Outputs
During the current period we have developed a swine animal disease model for use in testing Campylobacter jejuni isolates that have had major virulence genes knocked out. We conducted transposon mutagenesis in C. jejuni strain 11168 and 81-176 and successfully produced 2 libraries that each have over 1500 unique C. jejuni mutants. One experiment has been conducted to screen mutants in the animal model. In this project a C. jejuni cytolethal distending toxin mutant was screened compared to it's isogenic parent in the pig model. Pigs given the parent strain had marked disease and pathology in the small and large intestines. Pigs given the CDt mutant had milder disease and the pathology was different than in the parent strain. Most noteworthy was that there was still pathology present in the pigs given the mutant indicating that CDt toxin does not account for all of the damage done by C. jejuni These results were presented in September 2003 at the Campylobacter,
Helicobacter and Related Organisms Meeting in Aahrus Denmark.
Impacts
This work helps to understand how the food borne pathogen Campylobacter jejuni causes disease in a host that is similar to humans. It will form the basis for development of a vaccine against this pathogen.
Publications
- Mansfield LS, Gauthier DT, Abner SR, Jones KM, Wilder SR, and Urban JF. 2003. Enhancement of disease and pathology by synergy of Trichuris suis and Campylobacter jejuni in the colon of immunologically naive swine. American Journal of Tropical Medicine and Hygiene 68(1): 70-80.
- Wilson DL, Bell JA, Young VB, Wilder SR, Mansfield LS, and Linz JE. 2003. Genetic exchange by natural transformation between isogenic Campylobacter jejuni populations in liquid shake culture, Microbiology 149: 1-13.
- Mansfield LS, Kiupel M, and Pickett CL. 2003. Pigs given Campylobacter jejuni 81-176 cdt mutant had severe clinical signs and pathology different from those given the isogenic wild type parent. Campylobacter, helicobacter and Related Organisms Meeting, Aarhus, Denmark.
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Progress 01/01/02 to 12/31/02
Outputs
The long term goal of our research is to eliminate Campylobacter from the food chain. The long-term goal of this project is to develop proteomic methods that could serve as a resource for the Michigan State University, MSU, research community, the veterinary community, food animal producers, USDA, the medical community, and US bioterrorism research. Hypothesis. Protein expression profiles of pathogenic microorganisms can serve as a rapid typing scheme in animal and human outbreaks and sporadic cases. Specific Aims. 1.Standardize the passage and growth methodologies for C. jejuni isolates for analysis. 48 hour growth curves have been generated for 20 of the 2500 C. jejuni isolates from animals and humans with disease due to Campylobacter, as well as isolates from normal animals and the environment. This panel of isolates includes C. jejuni parent strains and their isogenic offspring with mutations in genes encoding potential virulence attributes. 2.Analyze isolates
using proteomic expression methodology. We are working with collaborators in the MSU Genomics Technology Support Facility and Wayne State University to establish methods for typing Campylobacter isolates using automated high throughput 2 dimensional electrophoresis methods. We have run 2 dimensional gel electrophoresis followed by LC Mass Spectroscopy on the 20 isolates mentioned. 3.Begin development of a database of protein expression profiles. The database is under development. 4.Begin development of a microarray of C. jejuni cDNA. We have developed a collaboration with members of the MSU Genome Support Facility and Wayne State University to print C. jejuni genome microarrays. 80 percent of the cDNAs have been cloned. Sequencing of these cDNAs is scheduled to commence soon. Once sequencing is complete, we will print the microarray and enter a testing phase to determine the reproducibility.
Impacts
The goal of this project is to develop proteomic methods that could serve as a means of rapid identification of pathogenic bacteria. It may be possible to use the protein-based fingerprint provided by proteomic expression profiling to rapidly identify bacteria (in this case Campylobacters) that cause disease, and distinguish them from those that do not cause disease. This could be used by many US and statewide laboratories or other medical professionals to quickly determine whether a Campylobacter isolate expressed proteins that function in virulence, in producing disease in the host.
Publications
- Jones KM, Wilder SR, Pickett C, and Mansfield LS. 2001. Swine in vivo rectal challenge model for rapid screening of virulence in Campylobacter. Infection and Immunity, in preparation.
- K.M. Jones, P. Padungtod, M. Kiupel, L.S. Mansfield. A swine rectal challenge model to evaluate immune responses to Campylobacter jejuni infection. Accepted as an abstract for presentation at the American Society for Microbiology Meeting, Washington D.C., May 2003.
- Wilson DL, Bell JA, Wilder SR, Young VB, Mansfield LS, and Linz JE. 2001. Genetic exchange by natural transformation between isogenic C. jejuni populations in liquid shake culture, Microbiology, accepted 2003.
- Mansfield LS, Gauthier DT, et al. 2002. Enhancement of disease and pathology by synergy of Trichuris suis and Campylobacter jejuni in the colon of immunologically naive swine.Amer.J.Trop.Med.Hyg. 68(1):70-80.
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