Progress 07/01/24 to 06/30/25
Outputs
Target Audience:Our target audience were the scientists and researchers involved in the study of mycobacterial diseases of animals. We were able to reach this audience at the International Association for Paratuberculosis 16th International Colloquium on Paratuberculosis in India (October 2024) and theannual meeting of the Conference of Workers in Animal Diseases (January 2025). Changes/Problems:The major problem we had to deal with was that Dr. John Bannantine left the National Animal Disease Center (NADC; Ames, IA) unexpectantly. However, Dr. Maria Alejandra Colombatti Olivieri (a Post-Doctorate at NADC) was instrumental in sending samples to the University of Nebraska, recording data, andcompiling results that we used to write the extension request and this progress report. What opportunities for training and professional development has the project provided?In this reporting period, Dr. Maria A. Colombatti Olivieri participated in the Conference of Research Workers in Animal Diseases meeting to fulfill the manditory grant report. In addition, she presented an update on this research at the International Association for Paratuberculosis 16th International Colloquium on Paratuberculosis. We continued to train the PhD Graduate Student and the Post-Doctorate. How have the results been disseminated to communities of interest?1) Oral presentation entitled "Evaluation of Mycobacterium avium subsp. paratuberculosisdeltaMap_1152 mutant as a live attenuated vaccine in Holstein's calves" was delivered at the 2024 International Association for Paratuberculosis 16th International Colloquium on Paratuberculosis. 2) Oral presentation entitled "Evaluation of Mycobacterium avium subsp. paratuberculosis DIVA vaccine in Holstein calves" was delivered at the 2025 Conference of Research Workers in Animal Diseases annual meeting. Both of these presentations were given in person by Dr. Maria A. Colombatti Olivieri and both are archived in the meeting proceedings. What do you plan to do during the next reporting period to accomplish the goals?These are the studies to be completed in the final reporting period. Genomic sequencing: All WT, complemented and mutant strains will be sequenced to determine the genomic stability of these strains in the context of a lack of complementation with the WT gene observed in some of our previous experiments. Serological tests: We will perform serological tests with the recombinant proteins and the samples from infected cattle that we received from the National Animal Disease Center in Ames, Iowa. If needed, we will purify additional amounts of proteins to test Johne's disease positive and negative cattle and goats stored in our veterinary diagnostic laboratories. Lipidomic and proteomic analyses: These assays will be conducted to determine changes in protein profiles in WT and mutant strains. Apoptosis: We will extend our apoptosis and necrosis assays previously published to the unmarked mutants and complemented strains. MAP survival: We will test invasion and survival using various MOIs in PBMC to determine if the MOI affects the relative microbial burdens.
Impacts
What was accomplished under these goals?
Previously, we successfully generated live-attenuated strains that can differentiate vaccinated from infected animals (DIVA) and demonstratedthat DMAP52 and DMAP56 marked and unmarked mutants are pro-apoptotic in macrophages. We conducted experiments for MAP K-10 wild-type (WT) and all mutants. Gene expression: We conducted PCR and RT-PCR assays to determine the expression of the MAP_1152 and MAP_1156 genes in the mutant strains. DNA and RNA was extracted using the MagMAX Total Nucleic Acid Isolation and the Direct-zol MiniPrep Plus Kits, respectively. As expected, DMAP52 and DMAP56 marked and unmarked mutants were negative for their respective genes.In addition, we confirmed the presence and expression at the transcriptional levels of the respective genes in the complemented mutants.No quantification of gene expression was performed at the protein level. Oxidative stress and pH: For in-vitro sensitivity to oxidative stress, strains were cultivated for 3 hours in 5, 20 or 50 mM hydrogen peroxide or 48 hours in 50 or 100 mM hydrogen peroxide followed by plating colony counts. There were significant differences at 3 hours to hydrogen peroxide at 5 mM (DMAP56 unmarked; 76.53%) and 20 mM (DMAP52 marked, 87.45%; DMAP52 unmarked, 86.08%; and DMAP56 unmarked 72.98%) compared to the WT (103.06% 5 mM and 105.59% 20 mM). No significant differences were observed for the 48 hour incubation. To evaluate sensitivity to low pH, the strains were incubated at pH levels (4.5 and 6.5) for 30 days taking OD readings and plating colony counts at Day 0 and every 10 days after. After 10 days, all mutants were susceptible to the low pH of 4.5 and displayed significant differencesfor optical density compared to the WT (3.17 x 106CFU/ml): DMAP52 marked (6.33 x 104 CFU/ml), DMAP56 marked (9.0 x 104 CFU/ml), DMAP52 unmarked (6.0 x 104 CFU/ml) and DMAP56 unmarked (6.0 x 104 CFU/ml). After counting colonies to determine survival, the WT was 14.19% compared toDMAP52 marked (1.63%), DMAP56 marked (0.97%), DMAP52 unmarked (0.72%) and DMAP56 unmarked (0.54%). The WT and all mutant strains grew similarly at pH 6.5. Cellular trafficking and colocalization:We previously reported that colony counts were used to determine intracellular survival. The results showed that all mutants displayed lower survival compared to the WT indicating that the unmarking did not alter the attenuated phenotype. For this reporting period, we performed confocal microscopy to determine the fate of MAP strains in bovine monocyte derived macrophages (bMDM). Blood was collected from healthy cows, negative for paratuberculosis infection, to isolate peripheral blood mononuclear cells (PBMC) that were resuspened in complete RPMI-1640 medium.After 48 hours, adherent monocytes were cultured in RPMI-1640 with 12% fetal bovine serum (FBS) for 7-10 days to obtain bMDM.Macrophages were infected at a multiplicity of infection (MOI) of20:1 bacilli per cell and incubated for 4 hours, fixing the cells with a 4% paraformaldehyde solution. The percent of colocalization within endosomes at early (Rab5), transition (Rab5 and Rab7), and late (Rab7) stages was assessed, respectively: WT (15.4%, 3.5% and 22.1%), DMAP52 marked (28.2%, 9.1% and 29.7%) and unmarked (26.8%, 9.3% and 31.1%), and DMAP56 marked (11.4%, 2.59% and 21.98%) and unmarked (14.2%, 2.9% and 14.7%). These results show that DMAP52 marked and unmarked had significantly more endosome colocalization at early, transition, and late stages as compared to the WT, a characteristic of attenuated strains. Cytokine production: To determine cytokine responses we collected PBMC from healthy cows as stated above. The cells were resuspended in RPMI-1640 overnight followed by a 24 hourinfection at a MOI of20:1 bacilli per cell. Each sample was split in half for cytokine measurements: 1) cell culture supernatants using the Milliplex® Bovine Cytokine/Chemokin Magnetic Bead Multiplex Assay (IFNg(interferon gamma), IL-17a (interleukin-17a), IL-1a, IL-1b, TNFa (tumor necrosis factor alpha), MCP-1 (monocyte chemoattractant protein-1), IL-4, and IL-10) and 2)RNA extraction (IFNg, IL-12a, IL-1a, TNFa, iNOS (inducible nitric oxide synthase), IL-17a, IL-23, RANTES (chemokine ligand 5, CCL5), TGFb (transforming growth factor beta), IL-10, FoxP3 (scurfin), IL-4, IL-13, IL-22, DEFB7(defensin beta 7)). For both tests, there were non-stimulated (negative) and Pokeweed Mitogen (positive) controls. There were no significant differences between the WT strain and both of the mutants, therefore the MAP mutants are immunogenic. Double mutant construction: We are constructing two double mutants (DMAP52 unmarked DMAP56 marked and DMAP52 marked DMAP56 unmarked) that we plan to test for further attenuation, unmarking, and DIVA capabilities to serve as a vaccine candidates. In conclusion, we have demonstrated the attenuation of DMAP52 unmarked in broth cultures and primary mononuclear cells. Protein purification: We purified recombinant MAP_1152 and MAP_1156 from E. coli extracts, so that we can assess the serum samples from the experimentally infected calves and additional samples from natural or experimentally infected cattle and goats (see future plan section below).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
M.A. Colombatti Olivieri, M. Hanafy, D.K. Zinniel, J.P. Bannantine and R.G. Barletta. Evaluation of Mycobacterium avium subsp. paratuberculosis deltaMap_1152 mutant as a live attenuated vaccine in Holsteins calves. Oral presentation and proceedings to the International Association for Paratuberculosis 16th International Colloquium on Paratuberculosis in India; October 2024.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
M.A. Colombatti Olivieri, M. Hanafy, D.K. Zinniel, J.P. Bannantine and R.G. Barletta. Evaluation of Mycobacterium avium subsp. paratuberculosis DIVA vaccine in Holstein calves. Oral presentation and proceedings to the Conference of Research Workers in Animal Diseases; January 2025.
- Type:
Other Journal Articles
Status:
Other
Year Published:
2025
Citation:
Faysal, M.A., M. Hanafy, D.K. Zinniel, F.Y. Tanni, E. Muthukrishnan, G. Rathnaiah and R.G. Barletta*. 2025. Cell death pathways in response to Mycobacterium tuberculosis and other mycobacterial infections. In preparation for submission to Infection and Immunity.
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Progress 07/01/23 to 06/30/24
Outputs
Target Audience:We targeted investigators in animal disease by discussing our results by an abstract and poster presentationat the Conference of Research Workers in Animal Diseases 2024 meeting for our funded USDA-NIFA AFRI Project. Changes/Problems:The main issues with the calf challenge study were the following: 1) The original wild type K-10 inoculum was contaiminated. 2) The complemented unmarked deletion mutant did not restore the wild type phenotype. 3) Calves died due to rotavirusoutbreak and pneumonia infections in the barns. What opportunities for training and professional development has the project provided?Dr. Mostafa Hanafy, in lieu of the PD Dr. Raul G. Barletta,participated inthe January 2024 Conference of Research Workers in Animal Diseases annual meeting to fullfil the mandatory grant report requirement. Mostafa assisted in training our PhD Graduate Student Md Atik Faysal in the manipulation of Mycobacterium aviumsubsp. paratuberculosis and molecular biology procedures. Training was further complemented by Dr. Barletta and the Laboratory Manager Denise Zinniel. How have the results been disseminated to communities of interest?1) Dr. Mostafa Hanafy presented a posterentitled "Development and Testing ofMycobacterium aviumsubsp.paratuberculosisDIVA Vaccines in Ruminants" at the January 2024 Conference of Research Workers in Animal Diseases annual meeting that is archived on the CRWAD website. What do you plan to do during the next reporting period to accomplish the goals?1) We will perform mRNA RT-PCR analysis to analyze the expression of the MAP_1152 and MAP_1156 genes. If needed, we will perform proteomic analysis to determine the expression at the protein level and generate new complementation constructs to be delivered into the mutant strains or newly reconstructed mutant strains. 2) We will perform genomic sequencing for the best vaccine candidate. 3) We will study the production of toxic oxygen radicals during macrophage infection in vitro by measuring the production of nitric oxide and reactive oxygen species. 4) We will purify large amounts of recombinant MAP_1152 and MAP_1156 from E. coli extracts to conduct serological test with JD positive and negative animalsto expand our previous DIVA tests. 5) We will perform confocal microscope analysis with the stored sample plates to better assess autophagy. 6) Submit abstracts and present research results to the 2024 International Colloquium on Paratuberculosis and the 2025 Conference of Research Workers in Animal Diseases meetings. 7) We will submit a manuscript to a microbiology journal with research results.
Impacts
What was accomplished under these goals?
The major activities during this reporting period were to complete experiments and the analysis of MAP growth properties, in vitro infection assays and a calf challenge study. Specifically, we performed the following experiments with the MAP strains listed below: A) determination of growth properties by obtaining optical density readings and colony forming units (AIM 1); B) in vitro infection of bovine monocyte-derived macrophages (bMDM) to evaluate intracellular survival, cell phenotyping and proliferation, cytokine production, and autophagy (AIM 1); C) analysis of samples collected during the calf challenge study started during the previous reporting period (AIM 3). Strain Name Genotype Description K-10 Wild type Parent strain DMAP52-mrk DMAP_1152::hyg Marked deletion mutant DMAP52-unm DMAP_1152 Unmarked deletion mutant DMAP52-mrk-comp DMAP_1152::hyg(pMV361::MAP_1152) Complemented marked deletion mutant DMAP52-unm-comp DMAP_1152(pMV361::MAP_1152) Complemented unmarked deletion mutant DMAP56-mrk DMAP_1156::hyg Marked deletion mutant DMAP56-unm DMAP_1156 Unmarked deletion mutant DMAP56-mrk-comp DMAP_1156::hyg(pMV361::MAP_1156) Complemented marked deletion mutant DMAP56-unm-comp DMAP_1156(pMV361::MAP_1156) Complemented unmarked deletion mutant A) Growth properties were previously determined for K-10, DMAP52-mrk and DMAP56-mrk. Results showed that the growth rate based on both the OD600 slope from Day 0 to 14 and CFU/mL vs. time for each marked deletion mutant was not significantly different compared with K-10. Thus, the strains grew at approximately the same rate. In this current study, we tested the growth rates of DMAP52-unm, DMAP52-mrk-comp, DMAP52-unm-comp, DMAP56-unm, DMAP56-mrk-comp, and DMAP56-unm-comp. Results showed that all of these strains have similar growth properties in broth cultures to each other and K-10. This is an important result showing that all marked, unmarked and complemented strains have these growth characteristics, which is a desirable property for candidate vaccine strains. This justifies the use of any of theseunmarked deletion mutants forthe calf challenge study as candidate vaccine strains. B) For the in vitro assays, bMDM were obtained from healthy control and JD infected cows, infected with all 9 MAP strains listed in the table above. Activation markers CD25+ for lymphocytes (CD4+, CD8+ and gdTCR+) and CD86+/MHCII+ for monocytes (CD14+) were measured for cell phenotyping and survival in bMDM by confocal microscopy at 24 and 72h post-infection. A non-stimulated/infected (NS) and a positive mitogen control (lipopolysaccharide) were included. For cytokine assays, we measured IL-12, TNF-α, IL-23, IL-10 and TGF-β, and the induced nitric oxide synthase (iNOS). For the preliminary results, we observed a significant decrease in cell viability, and no significant differences in cytokine production, cell populations and MAP survival in macrophages between the groups. Thus, no positive conclusion could be made from this early study. We repeated these experiments using CFU counting instead of confocal microscopyandmaximized bMDM viability obtaining valid results. For intracellular survival, all marked and unmarked deletion mutants displayed lower survival as compared to the wild type. This is consistent with our previous results with the marked deletion mutants alone. This is an important result to show that the unmarking of the deletion mutants did not alter the attenuated phenotype. However, we had difficulties in the complementation assays as the complemented marked and unmarked deletion mutant strains appear to have lower survival than the corresponding mutants, except for DMAP56-unm-comp, which showed restoration of the wild type phenotype. For cell phenotyping and proliferation, differences were identified in the innate immune cells. All MAP deletion mutants and the wild type induced more CD14+ CD86+ and CD14+ MHCII+ proliferation in bMDM from uninfected animals. In contrast, significant differences were found for CD14+CD86+ from both healthy and subclinical infected animals. Subclinical animals showed CD4+ activation after infection (CD4+CD25+), but this was not observed in the healthy cows. In contrast, the subclinical animals had a higher proportion of CD4+, gdTCR+, and CD14+ lymphocytes than the healthy animals. We determined the cytokine response by RT-qPCR. All deletion mutants except DMAP52-mrk-comp induced more pro-inflammatory cytokine responses compared to the wild type K-10. The significance of this finding is that the attenuated mutants displayed the immunogenicity patterns of a good vaccine candidate. For autophagy experiments, bMDM from three control cows were used in triplicate. The treatments were for stimulation (Earle's balanced salt solution, EBSS and rapamycin) and inhibition (3-methyladenine, chloroquine and bafilomycin) of autophagy. Autophagy induction led to decreased survival of DMAP52-mrk and DMAP52-unm, while only starvation (EBSS) and the 50uM concentration of rapamycin affected the survival of the wild type strain. Treatment with autophagy inhibitors did not increase survival compared to the negative control. Bafilomycin had the opposite effect, contrary to its expected role of increasing survival. Further evaluation is needed to determine whether the deletion mutant strains induce a higher percentage of autophagy than the wild type. We plan to perform confocal microscopy with the stored sample plates to better assess this process. C) For the calf challenge study, sixteenmale Holstein calves were used in two experimental groups of 5 animals infected at 2 to 3 weeks of age with three doses of 2 x 1011 CFU/mL of K-10, DMAP52-unm, and DMAP52-unm-comp while an uninfected animal was used as a control. The choice of using the MAP_1152 unmarked deletion mutant was based on the results obtained with the authophagy experiments, as well as a budget consideration with calf numbers which didn't allow us to test both unmarked deletion mutants.The immune response was evaluated at pre-infection, two weeks, and at 1, 2, 3, 6, and 8.5 months post-infection (mpi) by antibody and IFN-γ production, lymphocyte/monocyte populations, and a skin test. MAP detection was performed using feces and tissues through culture and qPCR. Due to a rotavirus outbreak and pneumonia, some calves died, resulting in 3-4 calves remaining in the deletion mutant and wild type groups. The infectious dose administered to the wild type group was significantly lower than that used in the DMAP52-unm group. Although the results between the two groups are not directly comparable, infection was confirmed in the wild type group. At 8.5 mpi, no significant humoral or cellular immune response was detected in the DMAP52-unm group. One animal each infected with DMAP52-unm and DMAP52-unm-comp tested positive on the skin test at 8.5 mpi, and no false positives for PPDb were detected. DMAP52-unm was detected in the spleen and liver from one animal by qPCR (39.1 and 39.8 Ct values respectively), but all feces and tissue cultures were negative. Similar results were obtained with the complemented unmarked deletion mutant obtaining Ct values forthe ileocecal valve (38.3), middle ileum (38.8), and proximal ileum lymph node (39). This suggeststhat the complementation of this strain did not fully restore the wild type phenotype but still persists longer in intestinal tissue than the unmarked deletion mutant. These overall findings indicatethat the DMAP52-unmunmarked deletion mutant strain is attenuated. However, further studies involving more animals and a longer observation period are essential to determine the potential use of this strain as a vaccine.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Hanafy, M., Maria Alejandra Colombatti Olivieri, John P. Bannantine, Denise K. Zinniel and Raul G. Barletta. Development and Testing of Mycobacterium avium subsp. paratuberculosis DIVA Vaccines in Ruminants. Proceedings Conference of Research Workers in Animal Diseases, January 2024.
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:The primary target audience were the scientists involved in the study of mycobacterial diseases of animals. These scientists were reached by an abstract and virtual poster of the 2022 USDA-NIFA AFRI Projectat the annual meetingof the Conference of Research Workers in Animal Diseases (January 2023 CRWAD). Changes/Problems:The most important change was the recruitment of a Postdoctoral Fellow (Dr. Mostafa Hanafy) that was instrumental in advancing the studies proposed. At the NADC, one of our Co-PIs (Stabel) retired on December 31, 2022. This has resulted in staffing issues since this Co-PI was to play a major role in the AIM 3 animal studies. Fortunately, we were able to hire a visiting scholar who has a DVM and PhD to compensate for this unexpected deficiency in staffing. Another problem we encountered is attrition of some animals that broke with Rota virus and respiratory disease. Some calves had to be put down because of this issue. What opportunities for training and professional development has the project provided?In this reporting period, the PD (Dr. Raul G. Barletta) participated in the 2023 Conference of Research Workers in Animal Diseases to fulfill the manditory grant report. Wewere able to recruit a new Postdoctoral Fellow (Dr. Mostafa Hanafy) that recently finished at the University of Wisconsin in a mycobacterial laboratory. Thus, his training was straighforward and he has demonstrating proficiency in performing assays and was instrumental in data analysis that was essential to accelerate the manuscript reported herin. How have the results been disseminated to communities of interest?1) Dr. Raul Barletta posted a virtual oralpresentation entitled "Development and Testing of Mycobacterium avium subsp. paratuberculosis DIVA Vaccines in Ruminants" at the 2023 Conference of Research Workers in Animal Diseases annual meeting which is archived on the CRWAD website. 2) Manuscriptentitled "Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains are Pro-apoptotic in RAW 264.7 Murine Macrophages" was published in the Vaccines journal. . What do you plan to do during the next reporting period to accomplish the goals?1) We will complete collecting growth curve optical density and colony forming unitdata and analyze the results. 2) We will further characterize the unmarked and comlemented strains by generating expression data(mRNA determinations by RT-PCR). 3) We will perform further bovinemacrophage infection and survival experiments to complement the experiments already completed with RAW 264.7 murine macrophages and correlate the data. 4) We will continue the calf challenge studies that are just underway since we completed the infections using milk replacer by the end of this reporting period on June 30, 2023. Therefore, we will continue to monitor and take samples from the animals as the trial proceeds. These samples will be assayed for cytokine and cell proliferation as well as PCR and culturing to determine infection status. 5) We will perform genomic sequencing for the best vaccine candidates.
Impacts
What was accomplished under these goals?
IMPACT: Johne's Disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most significant problems in animal health. Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. We propose a unique approach to develop MAP live-attenuated vaccines that can differentiate vaccinated from infected animals (DIVA). At the end of all proposed studies, we expect to have determined the best candidate vaccine strain out of two promising mutants that may display the potential to provide DIVA capabilities. AIM 1)For completingthe complementation of the MAP strains, we were able to comfirm by PCR that strain GPM669 carries the recombinant plasmidpBUN462 that was constructed in the backbone of pMV261 which is a multicopy replicating plasmid carrying the MAP_1152 wild type gene. These transformants have been stocked. As stated in the next reporting goals section for the previous report, we started the characterization of the attenuated MAP unmarked deletion mutants and complemented strains. Wild type (K-10), unmarked (GPM663 and 667) and complemented (GPM722, GPM727, GPM728 and GPM733) MAP cultures were started in order to perform growth curve analysis. The main activities during this reporting period were to complete and re-analyze all data related to growth curves,cell morphology, real-time apoptosis and necrosis assays, and apoptosis assays (DAPI staining andflow cytometry). To accomplish this analysis, we consulted withDirk K. Anderson the Manager of theUniversity of Nebraska-Lincoln (UNL) Flow Cytometry Core Facility. He provided the additional data analysis by creatingfluorescence-activated cell sortingdot plots using the FlowJo™ software. These dot plots displayed uninfected RAW 264.7 cells and those infected with each MAP strain in various stages at 6 hours post-infection: live, early apoptosis, late apoptosisand necrosis. In general, the MAP strains could be divided into two groups with low (wild types and transposon mutant 4H2) and high (deletion mutants DMAP52 and DMAP56) levels of early, lateand total apoptosis. We also consulted withEnakshy Dutta andVamsi Manthena from the UNL Department of StatisticsCross-disciplinary Collaboration and Consulting Lab toprovide SAS statistical support. They informed us of the correct model to use for each experiment, when to remove data outliers and check for normality.A manuscript was published with the following conclusions. The combined analysis of all methods confirmed the hypothesis that candidate vaccine strains DMAP52 and DMAP56 are pro-apoptotic in RAW 264.7 murine macrophages. None of these tests identified any significant differences between these two deletion mutants. The real-time assay indicated early induction of apoptosis for all MAP strains followed by secondary necrosis. DAPI staining was the quickest method to determine apoptosis that can be used to screen large mutant collections. Moreover, we determined that attenuation and immunogenicity in bovine macrophages could be well correlated with apoptosis in RAW 264.7 provided appropriate kinetic measurements are performed to choose the appropriate post-infection times to assess parameters. In addition, apoptosis always preceded the necrotic process that occurred at later time points. Overall, DMAP52 and DMAP56 were found to be excellent vaccine candidates against JD. For the in vitro experiments at the National Animal Disease Center (NADC), preliminary results were obtained in June at the end of the reporting period. For the cytokine stimulation experiment, we assayed 2 healthy control cows and 2 cows with subclinical JD. Ten strains served as the stimulants for cytokine proliferation, including all those described in the project narrative for this grant. We conducted the assay at 24 and 72 hours. Analysis is in progress. AIM2) Nothing new to report. AIM 3) Since in AIM 1 we confirmed the MAPtransformants carried the constructed complementation plasmids, these in addition to the wild type and deletion mutantswere sent to our collaborators at the NADC for cattle studies. Sixteen Holstein calves have been purchased, allowed to acclimate in containment and pre-inoculation blood and fecal samples were taken. The animals were then challenged with the wild type K-10, GPM663 (unmarked mutant carrying MAP_1152 deletion), and the corresponding complemented GPM728 mutant strain. At the end of the reporting period, the 16-day samples were collected. No statistically significant results were obtained at this early time point. This includes results for cell proliferation and cytokines. However, one animal from the mutant group was positive by fecal PCR at the 16-day time point taken on June 27th.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Barletta, R.G., J.P. Bannantine, J.R. Stabel, E. Muthukrishnan, D.K. Anderson, E. Dutta, V. Manthena, M. Hanafy and D.K. Zinniel. 2023 Mycobacterium avium subsp. paratuberculosis candidate vaccine strains are pro-apoptotic in RAW 264.7 murine macrophages. Vaccines. 11:1085. doi: 10.3390/vaccines11061085.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Barletta, R.G., D.K. Zinniel, E. Muthukrishnan, A. Turner, J.R. Stabel, J.P. Bannantine. Development and testing of Mycobacterium avium subsp. paratuberculosis DIVA vaccines in ruminants. Proceedings Conference of Research Workers in Animal Diseases; January 2023.
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The primary target audience were the scientists involved in the study of mycobacterial diseases of animals. These scientists were reached in person atthe annual meetings of the 2021Conference of Research Workers in Animal Diseases (CRWAD) when we presented the2022USDA-NIFA AFRI Project. In addition, we presented atthe 2022American Society for Microbiology annual meeting in Washington D.C. Changes/Problems:Dr. Ezhumalai Muthukrishnan unexpectantlyleft my laboratory in early May 2022 leaving his part of the research unfinished. We took over the analysis of the results, the writing of the manuscript that is still in progress,and the final recombinant strain confirmations and stocking. What opportunities for training and professional development has the project provided?In this reporting period, the PD (Dr. Raul G. Barletta) participated in the 2021 Conference of Research Workers in Animal Diseases to fulfill the manditory grant report. He also presented an update on this research at the 2022 American Society of Microbiolgy annual meeting. He attended the 2022 National Institute of Antimicrobial Resistance Research and Education (NIAMRRE) meeting in Lincoln, NE (May).We continued to train the 3 UCARE undergraduate students andthe post-doctoral student. How have the results been disseminated to communities of interest?1) Oral presentation entitled "Development and Testing of Mycobacterium avium subsp. paratuberculosis DIVA Vaccines in Ruminants" was delivered at the 2021 Conference of Research Workers in Animal Diseases annual meeting. 2) Poster presentation entitled "Mycobacterium avium subsp. paratuberculosis Attenuated Mutants Induce Apoptosis and Secondary Necrosis in Raw 264.7 Macrophage Cells" was delivered atthe 2022 American Society of Microbiolgy annaul meeting. Both of these presentations were given in person by Dr. Raul Barletta and the CRWAD meeting is archived in the meeting proceedings. The ASM meeting are being archived. What do you plan to do during the next reporting period to accomplish the goals?The priority for the next reporting period will focus on the characterization of the attenuated MAP unmarked in-frame deletion mutants now that the complementation experiments are close to be finished. This includes testing the mutants in MAP_1152 and MAP_1156 for invasion and intracellular survival in monocyte-derived macrophages. For this study, I will be assisted by the new post-doctoral researcher to be recruited. In addition, we will test antigens for DIVA capabilities and assess the mutants immunogenicity and pathogenicity in tissue cultures/calves in collaboration with the National Animal Disease Center.
Impacts
What was accomplished under these goals?
IMPACT: Johne's Disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most significant problems in animal health. Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. We propose a unique approach to develop MAP live-attenuated vaccines that can differentiate vaccinated from infected animals (DIVA). At the end of all proposed studies, we expect to have determined the best candidate vaccine strain out of two promising mutants that may display the potential to provide DIVA capabilities. AIM 1) As mentioned in the previous report period, MAP mutants were unmarked by first selecting on KanR to obtain pYUB870 transformants that have lost HygR, followed by counter selection on sucrose to remove the helper plasmid to obtain KanS, HygS, SucR transformants. Mutants were confirmed by PCR in order to stock these recombinant strains. During this reporting period, primers were created and ordered to conduct sequencing analysis for wildtype (K-10), marked (DMAP52 and 56) and unmarked (GPM663 and 664 is MAP1152; GPM667 and 669 is MAP1156) MAP mutants. DNA extractions for all strains were conducted and sent along with the primers to Eurofins Genomics (Louisville, KY) to obtain approximately 500 bp segments by primer walking that were then assembled into SnapGene version 6.0.2 for genes MAP_1152 and MAP_1156. This was important to verify that our strains are consistent with the published genomic MAP sequence. The next experiment was the complementation of these strains. The first steps were to order primers with restriction enzyme sites added to construct plasmids with the 2 genes listed above using the replicating plasmid pMV261 and the integrating plasmid pMV361 as a base. Next DNA was collected from these recombinant plasmids (pBUN462/pBUN463 with MAP_1152 and pBUN464/pBUN465 with MAP_1156) to verify that the genes inserted properly by PCR. In addition, Eurofins sequencing of these two genes by primer walking was conducted and analyzed via SnapGene software. Finally, 35 transformations were performed with the four plasmids and all seven MAP strains (optical density of 0.5 to 0.7) listed above. The entire sample was plated on MOADC agar supplemented with kanamycin 50 ug/ml. After two months of growth, colonies were counted and randomly picked to grow in liquid media. After another month of growth, PCRs were done to verify true recombinants with the kanamycin gene present. All transformation combinations except GPM669 with pBUN462 (still have a few samples growing to test) have been stocked. A second transformation with the unmarked MAP mutants and plasmid pYUB870 was also done to see if an additional electroporation would remove the leftover recombination junction. After 3 months of growth, all recombinants were confirmed by PCR for the kanamycin gene and stocked the new transformants. Another main experiment was to assay the unmarked deletion mutant strains for the induction of apoptosis using cell cultures of RAW 264.7 macrophages. The percent of apoptosis was quantified by characteristic nuclear morphology and visualized by treatment with the fluorescent DNA-binding dye, DAPI (4',6-diamidino-2-phenylindole). Macrophage cells were seeded into 24-well plates at a density of 5.0 x 104 cells/well, grown overnight to a final density of 2.0 x 105 macrophage cells/well (ca. 18 hours) and infected with MAP (MOI of 10). After a 6 hour incubation, cells were stained with 0.005 μg of DAPI for 15-30 min at 37°C. The percent of apoptotic nuclei was determined using a fluorescence microscope (Nikon ECLIPSE TE2000-U). Fluorescent micrographs were also collected. DMAP52 and DMAP56, as well as their unmarked derivatives (GPM663 and GPM667), showed a significant increase in apoptotic nuclei cell death. Finally, cell morphology experiments were conducted for K-10, DMAP52, DMAP56, GPM663 and GPM667. These MAP strains were grown to an OD600 of about 1.0. Bacteria were harvested and washed twice with 2X PBS with 0.05% Tween® 80 and resuspended in double distilled water (ddH2O) to a final concentration of 8.0 x 108 CFU/ml. Bacterial cells were transferred to a 300 mesh Formvar Carbon Type B copper grid using a nebulizer and fixed with 2% phosphotungstic acid pH 6.5 and 0.01% bovine serum albumin. Grids were dried overnight and visualized using a Hitachi H7500 transmission electron microscope (TEM) with a magnification range of 3,000 to 10,000X (HC mode 80 KV). The attenuated deletion mutants (DMAP52 and DMAP56), and their unmarked counterparts (GPM663 and GPM667) showed significant elongation compared to the wild type strain (UNL K-10). Aim 2) We are gathering samples for the DIVA test. Aim 3) Nothing to report in this period. Now that the complementation experiments are almost completed, these MAP cultures will be sent to the National Animal Disease Center.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Barletta, R.G., D.K. Zinniel, E. Muthukrishnan, A. Turner, J.R. Stabel and J.P. Bannantine. Development and testing of Mycobacterium avium subsp. paratuberculosis DIVA vaccines in ruminants. Poster presentation at the Conference of Research Workers in Animal Diseases Annual Meeting. December 2021. Symposium proceedings were published after this date.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Muthukrishnan, E., D.K. Zinniel, V. Manthena and R.G. Barletta. Mycobacterium avium subsp. paratuberculosis attenuated mutants induce apoptosis and secondary necrosis in RAW 264.7 macrophage cells. Poster presentation at the American Society of Microbiology Microbe Annual Meeting. June 2022.
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Progress 07/01/20 to 06/30/21
Outputs
Target Audience:The primary target audience were the scientists involved in the study of mycobacterial diseases of animals. These scientists were reached virtually in the annual meetings of the 2020 Conference of Research Workers in Animal Diseases (CRWAD), the 2020 USDA-NIFA AFRI Project Director Workshop and the 2021 American Society for Microbiology World Microbe Forum. In addition, we had several tele/video conferences with collaborators on the grant. We continued to submit annual abstracts to relevant scientific meetings (2021 CRWAD). Finally a virtual seminar entitled "Development of DIVA and Autolytic Attenuated Vaccines against Johne's Disease and Other Mycobacterial Infections" was presented at the University of Georgia. Changes/Problems:There were no difficulties in performing the scientific research. However, there were considerable negative impacts regarding the COVID-19 pandemic during this reporting period. Impacts of the pandemic included lockdowns (April 8-June 8) that prevented experimental work needed to publish two manuscripts. Additional time (June 9-August 8) was spent in developing laboratory SOPs, organizing sanitary stations, and starting cell lines and microbial cultures. Experiments were also delayed because of supplies (cameras and computer supplies, gloves, masks, pipettes, reagents). Undergraduate experiments were interrupted due to positive COVID-19 contacts and cases. Collaborations were affected by delays in mailing strains and materials, their lockdowns and pandemic planning. Filling frozen post-doc position was also delayed work for several weeks. What opportunities for training and professional development has the project provided?In this reporting period, the PD (Dr. Raul G. Barletta) participated and contributed the required abstract for this project to the USDA-NIFA AFRI Project Director Workshop Annual Meeting and the Conference of Research Workers in Animal Diseases Main Program. The PD also was able to give virtual seminars to the University of Georgia and the National Institute of Antimicrobial Resistance Research and Education (NIAMRRE). Three new UCARE undergraduate students were able to receive training in basic microbiological and molecular techniques, including PCR techniques. How have the results been disseminated to communities of interest?1) Gave a remote presentation entitled "Development and testing of Mycobacterium avium subsp. paratuberculosis DIVA vaccines in ruminants" for the Conference of Research Workers in Animal Diseases Main Program and the USDA-NIFA AFRI Project Director Workshop. 2) A virtual seminar entitled "Development of DIVA and Autolytic Attenuated Vaccines against Johne's Disease and Other Mycobacterial Infections" was presented at the University of Georgia. 3) Abstract entitled "Development and Testing of Mycobacterium avium subsp. paratuberculosis DIVA Vaccines in Ruminants" was accepted for the upcoming 2021 Conference of Research Workers in Animal Diseases. What do you plan to do during the next reporting period to accomplish the goals?Aim 1) We plan to complete the verification and characterize of the attenuated unmarked in-frame deletion mutants. Aim 2) We plan to test antigens for DIVA capabilities. Aim 3) We plan to transfer the verified unmarked mutants to our collaborators at NADC to start the assessment of immunogenicity and pathogenicity of unmarked mutants in calves.
Impacts
What was accomplished under these goals?
Impact: Annual losses due to JD are estimated in the range of $250 million to $1.5 billion for the United States cattle industry. We expect this project will result in the development of a second-generation live-attenuated vaccine against JD with commercial and agrosecurity potential. The overall impact is the embodiment of a JD vaccine with Differentiating Infected and Vaccinated Animals (DIVA) capabilities. Achievement of this goal would be of significant importance in ameliorating the negative impact of JD on national/international trade and the security of agricultural assets. Aim 1) Generate attenuated unmarked in-frame deletion mutants with DIVA capabilities:Wild type and marked deletion mutant strains were assayed for the induction of apoptosis and necrosis using cell cultures of RAW 264.7 macrophages. MAP strains were grown to mid-exponential phase in Middlebrook media, and used to infect (MOI, 10) macrophages for 1, 3, 6,12, 24 or 48 h. RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay (Promega) reagents were added, measuring the output with a multi-mode detection platform. To unmark the deletion mutants, the marked (HygR) strains were transformed with pYUB870 (KanR) that carries the sacB gene and gamma-delta resolvase. We observed activation of apoptosis at 3 and 6 h followed by secondary necrosis at 12, 24 and 48 h. The mutant strains DMAP52 and DMAP56 displayed apoptotic and necrotic trends compared to wild type NADC K-10, UNL K-10 and wild type like transposon mutant 4H2. These assays were confirmed by flow cytometry, apoptotic nuclear morphology and Caspase-Glo® 3/7 (Promega). Mutant unmarking proceeded by first selecting on KanR to obtain pYUB870 transformants that have lost HygR, followed by counter selection on sucrose to remove the helper plasmid to obtain KanS, HygS, SucR transformants. The marked mutants were clearly apoptotic, a landmark of good vaccine candidates. Steps for unmarking were completed. Aim 2) Test novel antigens for DIVA capabilities:We are gathering samples for the DIVA test. Aim 3) Assess the immunogenicity and pathogenicity of unmarked MAP mutants and their complemented strains in calves:Nothing to report in this period. Contingent upon completion of Aim 1.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
R.G. Barletta, D.K. Zinniel, E. Muthukrishnan, A. Turner, J.R. Stabel, and JP Bannantine. Development and Testing of Mycobacterium avium subsp. paratuberculosis DIVA Vaccines in Ruminants. Abstract for 2021 Conference of Research Workers in Animal Diseases.
- Type:
Other
Status:
Other
Year Published:
2021
Citation:
Ra�l G. Barletta. Development of DIVA and Autolytic Attenuated Vaccines against Johnes Disease and Other Mycobacterial Infections. Invited presentation given by videoconference to the University of Georgia.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Judith R. Stabel, John P. Bannantine, Denise K. Zinniel, Ezhumalai Muthukrishnan, Amy Turner and Raul G. Barletta. Development and testing of Mycobacterium avium subsp. paratuberculosis DIVA vaccines in ruminants. 2020 Conference of Research Workers in Animal Diseases Abstract ID 59692 Main Program Remote Presentation and USDA-NIFA AFRI Project Director Workshop Annual Meeting.
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