Functional Genomics of Johne`s Disease: Deciphering Kinetics of Effective Immunization

FUNCTIONAL GENOMICS OF JOHNE`S DISEASE: DECIPHERING KINETICS OF EFFECTIVE IMMUNIZATION

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1015769

Grant No.

2018-67015-28243

Cumulative Award Amt.

$499,880.00

Proposal No.

2017-05798

Multistate No.

(N/A)

Project Start Date

Jun 15, 2018

Project End Date

Jun 14, 2022

Grant Year

2018

Program Code

[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
PATHOBIOLOGICAL SCIENCES

Non Technical Summary
Infection with Mycobacterium avium subspecies paratuberculosis (M. ap) causes severe economic losses to the dairy industry in the USA and worldwide. In the USA alone, every dairy herd loses $400/cow on average due to complications from Johne's disease (JD). With the support from the USDA/NIFA program, we were able to develop highly protective vaccine candidates against Johne's disease that require further development before use in national control programs. For example, little information is currently available about the performance of the live vaccine candidates (LAV) in the target host, cattle. Even when an effective vaccine is developed, we still don't have a strategy of how to employ such vaccine for a successful control program under field conditions. The ultimate goal of this project is to understand the dynamics of the development of JD protective immunity following LAV immunization, to help in developing an effective control strategy against JD. To achieve our goal we will analyze the persistence and immunity of live vaccine candidates (LAV) in calves, the target host for JD vaccine development. We will also examine how LAV generate an effective immune responses that can overcome JD. Finally, we will characterize how calves response to M. ap infection and vaccination. Overall, the outcomes of the proposed aims will allow us to have a better understanding of JD disease progression and could identify key markers used for the diagnosis of JD. More importantly, our study could establish an optimum protocol for effective immunization, desperately needed to control JD which will in support with the mission of the Animal Health and Disease program at the NIFA/AFRI.

Animal Health Component

50%

Research Effort Categories

Basic

25%

Applied

50%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3410	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology;

Keywords

Goals / Objectives
The ultimate goal of this project is to understand the dynamics of the development of JD protective immunity following LAV immunization, to help in developing an effective control strategy against JD. To achieve our goal we propose the following aims:Aim 1: Decipher the persistence and immunity of live vaccine candidates (LAV). We will use the bovine model of paratuberculosis and a leading LAV candidate (M.apDlipN) to analyze both localized (granuloma) and systemic (blood cells) immune responses to LAV in comparison to the inactivated vaccine, Mycopar. In addition, we will characterize the persistence and shedding of LAV from vaccinated animals.Aim 2: Examine kinetics of effective LAV immunization against JD. We will examine the dynamics of development of protective immunity in calves, when M. ap exposure at different times following immunization.Aim 3. Profile the host responses to M. ap infection and immunization. We have already collected samples from calves infected with M. ap or vaccinated with lipN vaccine and more samples will be collected during execution of above aims. We will use high-throughput DNA- and RNA-sequencing to compare the microbiomes and transcriptomes of samples from naïve and infected compared to immunized animals.

Project Methods
In this project, we will start our efforts by deciphering the safety and immunogenicity of LAV candidates in calves, the target host for JD vaccination. Such analysis will allow us to understand cellular and humoral responses associated with LAV that distinguish them from the licensed, inactivated vaccine (Mycopar). Second, we will examine the kinetics of developmentof protective immunity to LAV to identify mechanisms of and durability of immunity, the key factors for a successful national strategy to control JD in dairy cattle. Third, we will examine the host responses and microbiome-associated profiles in calves immunized with LAV to identify a set of markers that could be used to develop an assay that can differentiate infected from vaccinated animals (DIVA). The DIVA assay will be essential and complement the SICST to allow the use of LAV, especially in states where a program for testing against bovine tuberculosis is required or needed in the future. Because of the time and budget constraints, we will develop and test the performance of the DIVA assay in a later phase of this project, but will focus our efforts on the discovery of suitable DIVA markers. Overall, we believe that our set of experimental approaches addresses the central hypothesis of this project and is sufficient to tackle the overall goal. The outcomes of this project will provide novel insights into immunopathogenesis of JD at the molecular and cellular levels.

Progress 06/15/18 to 06/14/22

Outputs
Target Audience:researchers, veterinarians, and dairy herd managers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In this project, a graduate student, a scientist and research assistant are working on various aspects of paratuberculosis vaccine. I work with the team members to accomplish the project goals and at the same time develop their technical, professional and presentation skills. I supported participation of team members in both local and national meetings where they presented their research findings. When experiment conclude, I will work with the students to present the project findings through scientific reports and presentations in national and international conferences. In general, training is provided in different aspects of bacteriology and genetic manipulations (Basic bacteriology, genetics and genomics). In addition, more chances are given for training on different approaches to analyze host immune responses (Immunology). How have the results been disseminated to communities of interest?Publication of 4 reports already with 2 more reports under preparation. Three presentations at a scientific meeting. Persistence and virulence of M. paratuberculosis: a role for global gene regulators in disease and control. The 99th conference of Research Workers on Animal Diseases (CRWAD), December 1-5, 2018. Chicago, IL. Respiratory Models for non-tuberculous mycobacterial infections. The 99th Conference of Research Workers on Animal Diseases (CRWAD), December 1-5, 2018. Chicago, IL. A heterologous prime boost vaccination strategy against Johne's disease. The 101 conference of Research Workers on Animal Diseases (CRWAD), December 7, 2021. Chicago, IL. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? -To accomplish aims 1 and 2 of this project, we conducted safety and immunogenicity studies in goats and also in calves. To decipher the dynamics of the immune responses elicited by both live-attenuated and inactivated vaccines, we analyzed key immunological parameters of goats immunized through different routes. Within a few weeks, the inactivated vaccine triggered the formation of granulomas both at the site of inoculation and in regional lymph nodes, that increased in size over time and persisted until the end of the experiment. In contrast, granulomas induced by the live-attenuated vaccine were small and subsided during the study. Interestingly, in this vaccine group, both histological, and immunohistochemical analyses demonstrated an initial abundance of intra-histiocytic mycobacterial bacilli at the site of inoculation, with recruitment of very minimal T lymphocytes to poorly organized granulomas. Over time, granulomas became more organized, with recruitment of greater numbers of T and B lymphocytes, which coincided with a lack of mycobacteria. For the inactivated vaccine group, mycobacterial bacilli were identified extracellularly within the center of caseating granulomas of increasing size, with relatively equal proportions of B- and T-lymphocytes maintained across both early and late times. Despite the differences in granuloma-specific lymphocyte recruitment, markers for cell-mediated immunity (e.g. IFN-γ release) were robust in both injected live and inactivated vaccine groups. In contrast, the intranasal live-attenuated vaccine did not elicit any reaction at site of inoculation, nor cell-mediated immune responses. Finally, animals in the inactivated vaccine group significantly reacted to purified protein derivatives (PPD) from M. bovis, while animals receiving the live vaccine group remained unchanged, suggesting cross reactivity of the inactivated vaccine with testing for bovine tuberculosis. Overall, these results depict the cellular recruitment strategies driving immune responses elicited by both live-attenuated and inactivated vaccines that target Johne's disease. The results of this study is currently under consideration for publication in Frontiers In Veterinary Science. To further enhance the efficacy of the pgsN vaccine, we utilized a prime/boost (PB) vaccination strategy in combination with a novel nanoadjuvant system composed of Quil A and chitosan (QAC). In this study, we evaluated the safety, immunogenicity and protective efficacy of pgsN administered either as live or inactivated vaccine in a PB regimen in cattle. Calves were primed at 4 weeks of age and boosted 4 weeks following the initial priming. Calves were challenged 2 months post vaccination with a virulent bovine M. ap strain and followed for 6 months. Significant changes in body weight, temperature, general health conditions were not observed between sham- and pgsN- vaccinated calves suggesting an overall safety of the vaccines, as expected. Calves vaccinated with the PB regimen showed significant skin induration (compared to PBS group) in response to Johnin PPD indicating a robust cellular immune response and potentially enhanced protection. Importantly, the same animals did not respond to M. bovis antigens, suggesting its utility in bovine tuberculosis enzootic areas. Challenged animals showed bacterial shedding in their feces only up to 8 days post challenge. Histological analyses of collected samples indicated the overall protection in groups with the live-attenuated pgsN immunization followed by 2 doses of inactivated and live-attenuated vaccination strategy. Overall, results from this trial demonstrated that the pgsN vaccine formulations tested were safe and induced a potent cellular immunity and potential protective immunity in cattle, further paving the way for testing in the field. -Finally for Aim III, we analyzed the microbiome of intestinal tissues collected from vaccinated and challenged animals.Although no differences in alpha diversity were found between groups, our beta diversity analysis showed that the unchallenged group clustered separately from the other groups, with the Mycopar group varying slightly from the other groups (PBS, pgsN, and pgsN+QuilA). An analysis of the operational taxonomic units (OTUs) associated with these differences revealed key bacteria that were highly abundant within groups. Specifically, we found that OTUs belonging to the Candidatus Saccharimonas genus (phylum Patescibacteria) were found in high abundances (upwards of 6% of the entire microbiota) in our lipN group. Importantly, Candidatus Saccharimonas is thought to be a beneficial bacteria which is hypothesized to aid in the reduction of inflammation in Crohn's disease. Moreover, this finding agrees with our histology results, which showed that the lipN group not only had the least inflammatory response but also of mildest intensity amongst all vaccine groups. We also found a second bacterium, Eubacterium minutum, that was also found in high abundances in the lipN group. Eubacterium minutum is also found in high abundance in Crohn's disease patients, and antibody markers associated with this taxon have been used in diagnostic tests in the past for the disease. It is thought that this taxon is associated with the release of major mediators of inflammation as well as an increased turnover rate of neutrophils, worsening inflammation during the diseased state. Finally, we also identified an OTU belonging to a mucosal-associated Actinobacteria known to be associated with abomasal ulcers in higher abundances in our Mycopar and PBS groups. These findings perhaps reflect the fact that these two treatments are indicative of a more severe disease state. Currently, we are writing a manuscript to report on the findings of the last 2 studies.

Publications

Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Ali, Z. I., A. M. Saudi, R. Albrecht and A. M. Talaat (2019). The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall Journal of Dairy Science.
Type: Journal Articles Status: Published Year Published: 2021 Citation: 1. Ali, Z. I., M. Hanafy, C. Hansen, A. M. Saudi and A. M. Talaat (2021). Genotypic analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin Journal of Dairy Science 104(1): 211-220.
Type: Journal Articles Status: Published Year Published: 2022 Citation: 1. Abdelaal, H. F. M., T. C. Thacker, B. Wadie, M. V. Palmer and A. M. Talaat (2022). Transcriptional Profiling of Early and Late Phases of Bovine Tuberculosis. Infect Immun 90(2): e0031321.
Type: Journal Articles Status: Published Year Published: 2022 Citation: 1. Hildebrand, R. E., S. S. Chandrasekar, M. Riel, B. J. B. Touray, S. A. Aschenbroich and A. M. Talaat (2022). Superinfection with SARS-CoV-2 Has Deleterious Effects on Mycobacterium bovis BCG Immunity and Promotes Dissemination of Mycobacterium tuberculosis. Microbiology Spectrum 0(0): e03075-03022.
Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Mostafa Hanafy, Chungyi Hansen, Yashdeep Phanse, Chia-wei Wu, Kathryn Nelson, Sophie A Aschenbroich and Adel M Talaat. Characterization of Early Immune Responses Elicited by Live and Inactivated Vaccines against Johnes Disease in Goats.

Progress 06/15/20 to 06/14/21

Outputs
Target Audience:Veterinarians, farmers, dairy herd managers and other scientists working in the field. @font-face { panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; ; mso-fareast-}p {mso-style-priority:99; mso-margin-top-alt:auto; margin-right:0in; mso-margin-bottom-alt:auto; margin-left:0in; mso-pagination:widow-orphan; ; mso-fareast-}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSection1;} Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training continued for a graduate student, a scientist and research assistant working together on various aspects of this project. How have the results been disseminated to communities of interest?I am presenting our project finding in The 102th Conference of Research Workers on Animal Diseases (CRWAD), December 3-7, 2021. Oral presentation, in person. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete analysis of samples collected from the calf experiment to accomplish goals for Aims I and II. Also, we plan to complete the microbiome analysis Aim III in this project.

Impacts
What was accomplished under these goals? For Aims I and II, we sacrificed calves infected and vaccinated with Johne's disease vaccine (Total 20 calves). We were able to necropsy all animals and collect all of the needed tissues for culturing and histopathological analyses. Culturing of tissue samples are still pending, due to the long incubation period for M. paratuberculosis but fecal samples were positive for M. paratuberculosis up to 8 days post challenge. Finally, for Aim III, we obtained interesting data gleaned from the metagenomic analysis of 16SrRNA sequences from the gut of infected calves. We are working with our collaborator to decipher the conclusions from this analysis and plan next steps for testing.

Publications

Type: Journal Articles Status: Published Year Published: 2021 Citation: Ali, Z. I., M. Hanafy, C. Hansen, A. M. Saudi and A. M. Talaat (2021). Genotypic analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin. Journal of Dairy Science 104(1): 211-220.

Progress 06/15/19 to 06/14/20

Outputs
Target Audience:Veterinarians, farmers, dairy herd managers and other scientists working in the field. @font-face { panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; ; mso-fareast-}p {mso-style-priority:99; mso-margin-top-alt:auto; margin-right:0in; mso-margin-bottom-alt:auto; margin-left:0in; mso-pagination:widow-orphan; ; mso-fareast-}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSection1;} Changes/Problems:We faced several challenges related to the COVID-19 pandemics which delayed the start of our Johnes' disease vaccine evaluation in calves. However, we got the needed permissions to continue our work with our laboratory staff (instead of the campus provided staff) to care for the animals using strict rules for safe working on animal maintenance and sample collection. What opportunities for training and professional development has the project provided?Training continued for a graduate student, a scientist and research assistant working together on various aspects of this project. How have the results been disseminated to communities of interest?Oral presentation. Kinetics of ruminant immunity elicited by live attenuated vaccine against Johne's disease. The 101th Conference of Research Workers on Animal Diseases (CRWAD), December 4-8, 2020. Online. What do you plan to do during the next reporting period to accomplish the goals?We are working on tissue and blood samples collected from vaccinated animals in experiments related to Aims I and II. Also, we plan to complete the microbiome analysis for tissue samples collected for Aim III in this project.

Impacts
What was accomplished under these goals? For Aims I and II, we continued our year-long sampling of JD vaccines given to calves before 35 days of age. This experiment faced several challenges because of COVID-19 pandemic. However, we were able to initiate the experiments working with a limited staff capacity. We were able to collect all of our fecal and blood samples for further analyses (ELISA testing, IFN-g levels and culturing, among other tests we usually run on these samples). Several cultured samples are still pending, due to the long incubation period for M. paratuberculosis. Finally, for Aim III, we continued to work on optimizing PCR amplifications of 16SrRNA sequences from the gut of infected calves to analyze the microbiome of naïve vs. vaccinated or vaccinated and challenged animals.

Publications

Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Ali, Z. I., M. Hanafy, C. Hansen, A. M. Saudi and A. M. Talaat (Under review). Journal of Dairy Science.

Progress 06/15/18 to 06/14/19

Outputs
Target Audience:Veterinarians, farmers, dairy herd managers and other scientists working in the field. @font-face { panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face { panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:-536859905 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; ; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}p {mso-style-priority:99; mso-margin-top-alt:auto; margin-right:0in; mso-margin-bottom-alt:auto; margin-left:0in; mso-pagination:widow-orphan; ; mso-fareast-}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSection1;} Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In this project, a graduate student, a scientist and research assistant are working on various aspects of paratuberculosis vaccine. I work with the team members to accomplish the project goals and at the same time develop their technical, professional and presentation skills. I supported participation of team members in both local and national meetings where they presented their research findings. How have the results been disseminated to communities of interest?Three presentations at a scientific meeting in addition to the publication reported above. Persistence and virulence of M. paratuberculosis: a role for global gene regulators in disease and control. The 99th conference of Research Workers on Animal Diseases (CRWAD), December 1-5, 2018. Chicago, IL. Respiratory Models for non-tuberculous mycobacterial infections. The 99th Conference of Research Workers on Animal Diseases (CRWAD), December 1-5, 2018. Chicago, IL. A Protective Vaccine Against Johne's Disease in Cattle. The 100th Conference of Research Workers on Animal Diseases (CRWAD), November 2-5, 2019. Chicago, IL. What do you plan to do during the next reporting period to accomplish the goals?Will continue analysis of tissue samples and immunological parameters collected for experiments in Aims I and II. Also, we plan to run through samples we collected for the microbiome analysis for Aim III.

Impacts
What was accomplished under these goals? For Aims I and II, we initiated a vaccine testing proejct in calves using different vaccine formulas to decipher the type of immunity generated by each vaccine formula and vaccination route. This experiment build on an earlier experiment where goats were used to better decipher the the immune responses at the site of immunization and the kinetics of immunity in vaccinated only animals. We also started to work on the microbiome analysis of fecal and tissue samples for Aim III of this project.

Publications

Type: Journal Articles Status: Published Year Published: 2019 Citation: Ali, Z. I., A. M. Saudi, R. Albrecht and A. M. Talaat (2019). The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall. J Dairy Sci 102(6): 4935-4944.

Progress 06/15/18 to 05/15/19

Outputs
Target Audience:researchers, veterinarians, and dairy herd managers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In this project, a graduate student, a scientist and research assistant are working on various aspects of paratuberculosis vaccine. I work with the team members to accomplish the project goals and at the same time develop their technical, professional and presentation skills. I supported participation of team members in both local and national meetings where they presented their research findings. When experiment conclude, I will work with the students to present the project findings through scientific reports and presentations in national and international conferences. In general, training is provided in different aspects of bacteriology and genetic manipulations (Basic bacteriology, genetics and genomics). In addition, more chances are given for training on different approaches to analyze host immune responses (Immunology). How have the results been disseminated to communities of interest?Publication of 1 report already. Two presentations at a scientific meeting. Persistence and virulence of M. paratuberculosis: a role for global gene regulators in disease and control. The 99th conference of Research Workers on Animal Diseases (CRWAD), December 1-5, 2018. Chicago, IL. Respiratory Models for non-tuberculous mycobacterial infections. The 99th Conference of Research Workers on Animal Diseases (CRWAD), December 1-5, 2018. Chicago, IL. What do you plan to do during the next reporting period to accomplish the goals?Will continue analysis of tissue samples collected for experiments in Aim I. Will start a new immunization and challenge study for Aim II. Finally, we plan to run through tens of samples we collected for the microbiome analysis for Aim III.

Impacts
What was accomplished under these goals? -We initiated animal experiments to analyze the immunity of live attenuated vaccine (LAV) in goats compared to Mycopar and control groups (Aim I). Basically, we used the goat model of paratuberculosis to examine safety aspects of paratuberculosis vaccine candidates and the granuloma formation following immunization different vaccine constructs. At the end of the experiments, granuloma tissues were collected and currently under further analysis. -To examine kinetics of the development of immune responses (Aim II). We collected blood sampels from different goat groups immunization with LAV and inactivated vaccines. Our analysis indicated that unique immune profile (as determined by flow cytometry analysis) exists for immunization with LAV compared to inactivated vaccine (Mycopar). -For the microbiome analysis (Aim III), we started to extract genomic DNA from cow tissues vaccinated with different vaccine constructs or vaccinated and challenged with M. paratuberculosis.

Publications

Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Ali, Z. I., A. M. Saudi, R. Albrecht and A. M. Talaat (2019). The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall Journal of Dairy Science.