Location: Ruminant Diseases and Immunology Research
2024 Annual Report
Objectives
Objective 1: Define the virulence determinants and mechanisms used by Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis and Mycoplasma mycoides cluster agents to cause disease in ruminant species.
Subobjective 1.1: Identify microbial mechanisms used by commensal bacteria to become pathogens.
Subobjective 1.2: Identify the mechanisms of bacterial colonization of the host.
Objective 2: Determine the host-pathogen interactions associated with infection with Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis and Mycoplasma mycoides cluster agents, including development of animal models.
Subobjective 2.1: Continue the development of animal disease models to study respiratory disease complex.
Subobjective 2.2: Identify the host factors that drive the early innate immune response to bacterial infection.
Subobjective 2.3: Characterize functional genomics of the host associated with respiratory infection.
Objective 3: Develop intervention strategies to prevent or treat respiratory infections that minimizes the development of antibiotic resistant bacteria. This includes the development of easily administered vaccines and developing and evaluating immune-modulators to prevent and/or treat respiratory disease.
Subobjective 3.1: Develop and test vaccines that induce early immunity in young animals.
Subobjective 3.2: Develop and test vaccines that induce early mucosal immunity in young animals.
Objective 4: Following identification of virulence determinants, utilize synthetic genome and other approaches to engineer Mycoplasma mycoides cluster agents for enhancing the understanding of disease pathogenesis and for use as potential vaccines.
Subobjective 4.1: To determine if hydrogen peroxide (H2O2) is a virulence determinant in-vivo.
Subobjective 4.2: Identify MmmSC virulence determinants and vaccine targets through NextGen genomic sequencing and analysis using archived, newly obtained MmmSC field and experimental strains.
Subobjective 4.3: Identify MmmSC virulence determinants and vaccine targets through NextGen transcriptomic sequencing and analysis of bacteria and host during infection.
Subobjective 4.4: To develop a synthetic genomic live attenuated vaccine (LAV) approach.
Subobjective 4.5: Development of a subunit vaccine.
Objective 5: Determine the role of surface lipoproteins for vaccine enhancement of disease in Mycoplasma mycoides subsp. mycoides small colony.
Approach
Binding of bacteria to mucosal surfaces, and evasion of host innate, and adaptive immunity are critical to successful colonization and maintenance of infection. Identification of key molecular players in these interactions should enable potentially effective intervention strategies. We plan to utilize a coordinated, multipronged approach to characterize molecular mechanisms promoting respiratory bacterial colonization, adherence, and persistence in cattle. While much knowledge has been gained from studying individual pathogens, less is known concerning co-infections involving bacterial and viral pathogens. Given the expertise of our research team, we will focus on BHV-1 and BRSV as the viral pathogens, and Mannheimia haemolytica, Pasteurella multocida, and Mycoplasma bovis as the bacterial agents. Mycoplasma mycoides was added to this project by the USDA Animal Health NPLs in response to congressional appropriations. A research team from the University of Connecticut will carry out objectives related to M. mycoides cluster agents in collaboration with ARS researchers. We will continue the development of experimental animal models and specific mutants to describe molecular mechanisms enabling bacteria to colonize the respiratory tract and examine influences of primary viral infection on secondary bacterial infections. Bacterial genes or gene products so identified, will be used for developing and testing novel vaccines and/or immunomodulators. The overriding goal is to reduce or eliminate BRDC, which will substantially benefit producers. However, as specific pathogens involved in BRDC can cause significant disease in wild ruminants, there are aspects of this plan that include isolates from those species. For example, M. bovis has emerged in bison, causing substantial economic losses and threatening stability of heritage herds. Therefore, strategies to reduce respiratory disease in wildlife will be valuable to the public interest in sustaining these populations, as well as reduce economic losses to producers.
Progress Report
To address Objective 1, we generated isogenic Mannheimia haemolytica (M. haemolytica) capsular, sialic acid, filamentous haemagglutinin (FHA), and adhesin mutants. We previously generated isogenic Pasteurella multocida (P. multocida) capsular, sialic acid, and FHA mutants. To assess the role of M. haemolytica adhesins on colonization in the upper respiratory tract of animals, calves were ordered, and the animal trial is scheduled to start in July-August 2024. A cattle study assessing colonization of various isogenic P. multocida mutants was completed. To assess whether cell surface glycosylation (capsule) and large cell-surface protein (FHA) play a crucial role in pathogenesis, M. haemolytica isogenic capsular and FHA mutants will be tested in a separate animal study. Calves were ordered and the study is scheduled to start in June 2024.
To address Objective 1, we obtained 79 cattle isolates of Mycoplasma bovis (M. bovis) from veterinary diagnostic labs. We also isolated M. bovis from 23 bison clinical samples. These efforts gave us a sizable collection of contemporary isolates from multiple host species (cattle and bison) and tissue types (nasal swabs, lungs, etc.) to analyze M. bovis genetics. Through a collaboration with US-MARC, we have optimized M. bovis genomic sequencing and genome assembly/analysis pipelines. We have acquired one isolate of Mycoplasma mycoides subspecies mycoides (MmmSC) and are in the process of acquiring more isolates, as we have an agreement with Centre de cooperation internationale en recherche agronomique pour le developpement (CIRAD) to acquire six additional type strains.
To address Objective 2, we evaluated a bovine herpesevirs-1 (BHV-1)/ M. bovis co-infection model. The study design included four groups of calves: uninfected controls (n=6), BHV-1 challenge (n=8), M. bovis challenge (n=8) and BHV-1/M. bovis challenge (n=8). M. bovis was used as it is commonly associated with viral co-infections in field settings. Calves were challenged with BHV-1 via aerosol on day 0 then challenged with M. bovis via aerosol on day 4. Half of the controls (n=3), BHV-1 challenge (n=4), M. bovis challenge (n=4), and BHV-1/ M. bovis challenge (n=4) were euthanized on days 8/9 post-infection with the remaining animals euthanized on days 13/14 post-infection. Co-infection increased the number of affected lung lobes and lung tissue damage at both time- points compared to single infections. All co-infected calves shed M. bovis in the nasal swab samples through the end of study where calves challenged only with M. bovis had no detectable M. bovis in the nasal swabs at 13/14 days post-infection. Co-infection also resulted in a higher frequency of M. bovis positive tracheal and middle ear swabs showing co-infection results in increased tissue colonization and bacterial replication.
To address Objective 2, tissues were collected from calves challenged with BRSV and P. multocida co-infection study. Animals were allocated in a control group, a group challenged only with BRSV, a group challenged only with P. multocida, and a group challenged with both pathogens. Blood, lymph nodes, lung, spleen, and liver samples were collected. Ribonucleic acid (RNA) will be extracted from each tissue, and messenger and small non-coding RNA will be sent for sequencing. These studies will uncover, at the molecular level, how these pathogens establish respiratory disease in cattle.
To address Objective 2, we extracted RNA, and sequence data was collected from an experiment in bison, in which animals were vaccinated with adjuvanted recombinant antigenic proteins of M. bovis. Animals were allocated in a control group, and a group vaccinated M. bovis antigens (EFTu and HSP70). Bison was challenged with BHV- 1, and four days later with M. bovis. Messenger RNA was extracted and sequenced from the blood of each animal. Next is to do a bio-informatics analysis of the sequenced data. The goal is to understand, at the molecular level, how this vaccine is protecting cattle against M. bovis infection.
To address Objective 2, we generated bacterial expression plasmids encoding lipoproteins from Pasteurellaceae (PlpE), M. bovis (P48) and Mycoplasma mycoides subspecies mycoides (LppQ). All the proteins were expressed by an Escherichia coli (E. coli) expression system and recombinant proteins were purified. To assess Toll-like receptor 2 mediated immune effects, these lipoproteins will be tested using peripheral blood mononuclear cells prepared from cattle.
To address Objective 3, we generated M. haemolytica vaccine strains expressing Mycoplasma mycoides subspecies mycoides (MmmSC) antigens (HSP70 and EFTu) fused to and secreted by an inactive yet immunogenic leukotoxin (leukotoxoid). The correct orientation of the vaccine construct and expression of chimeric antigenic proteins were confirmed by polymerase chain reaction and Western blot assays. Additionally, identical MmmSC proteins were expressed and purified as recombinant proteins in E. coli expression system. In collaboration with researchers at Kenya Agriculture and Livestock Research Organization (KALRO) Kikuyu, Kenya, cattle studies will be conducted in late 2024 in Kenya. Live M. haemolytica vaccine strains will be given orally on feed to cattle then challenged with MmmSC, which is the causative agent of contagious bovine pleuropneumonia (CBPP). Like a previous bison study, two doses of adjuvanted recombinant MmmSC protein vaccine will be administered to cattle twice (three weeks apart) followed by MmmSC challenge. Animals will be assessed for vaccine induced protection against CBPP.
To address Objective 4, collaboration was established with the researchers at Nairobi, Kenya to make a Mycoplasma mycoides subspecies mycoides (MmmSC) transposon mutant library, and the mutant library was generated. We established a second collaborative agreement with researchers in Kenya to sequence the entire mutant library to identify various MmmSC mutants, including ones with transposon insertions into the genes involved in glycerol metabolism and the production of hydrogen peroxide (H2O2). Of the mutants sequenced to date, we have not identified any containing such insertions into glycerol metabolism genes, but work is continuing to identify such mutants. Once identified, these mutants will be utilized to perform both in vitro and in vivo assessments to determine whether H2O2 is a required in vivo virulence determinant. In the meantime, using a related, RG-2(BSL-2) subspecies of Mycoplasma mycoides (Mm) we have begun generating a transposon mutant library so that we may assess whether hydrogen peroxide production is a virulence determinant in this subspecies of MmmSC. The Nagoya protocol has resulted in significant delays in processing of export/import permits that makes receiving nucleic acids from newly isolated MmmSC field strains from Africa very difficult. In lieu of these obstacles, we have conducted comparative genomic analysis using publicly available data sets and have identified several putative genes and gene pathways that we believe to be involved in the virulence and host specificity of MmmSC. Following further progress in the sequencing of the MmmSC mutant library in Kenya, we will assess mutants with transposon insertions that disrupt these pathways using in vivo experiments to assess their role in virulence.
To address Objective 4, a collaborative subaward agreement was established with researchers at International Livestock Research Institute (ILRI) Nairobi, Kenya regarding the sequencing of the MmmSC transposon mutant library. We designed an animal challenge experiment utilizing the Wild Type and a MmmSC gene-specific mutant that we have reason to believe will be attenuated. This experiment will be conducted from October-November 2024. In addition to assessing whether the mutant of interest is attenuated, we will be collecting samples to perform comparative transcriptomics of the host and pathogen, which will allow us to further identify potential virulence determinants, as well as biomarkers that are associated with more severe or less severe disease. We have acquired a precision tissue slicer that will allow us to obtain Precision cut bovine and caprine lung slices to utilize in experiments with an RG-2/BSL-2 surrogate strain of Mm to identify putative virulence factors of interest using transcriptomic and functional approaches.
To address Objective 4, we finalized a collaborative agreement and subaward with a faculty member at Institut National de la Reserche Agronomique at the University of Bordeaux, Bordeaux, France to work on the development of attenuated strains of MmmSC using synthetic genomic approaches. Informed by the virulence factors that our functional studies will identify, they will utilize synthetic genomic approaches, and other novel genome recombineering techniques to develop live attenuated MmmSC vaccine candidates. We have begun working on two specific genes of interest that could serve as potential vaccine candidates. We selected candidate proteins to express recombinantly and that could serve as protective antigens in a subunit vaccine formulation. We are currently designing modified sequences based on our patented lipoprotein modifications (WO2021113433A1) and will be optimizing them for expression in E. coli. We are currently in the process of obtaining IACUC permission to develop a preclinical model of disease using a surrogate, RG2/BSL- 2, strain of Mm that will allow us to assess the immunogenicity and efficacy of these vaccine candidates in vivo prior to work in cattle.
To address objective 5, we are working with the team at NADC to obtain the permits and licenses to enable work with MmmSC at their NADC ABSL3 facility in Ames, Iowa. We expect to make progress on the milestone once MmmSC is permitted. We may also extend our agreement with researchers at ILRI to conduct work related to the milestone at their facility in Kenya.
Accomplishments
1. Mannheimia haemolytica serotype 1 mutant lacking sialic acid on the outer membrane is more sensitive to phagocytic and serum killing. Sialic acids are nine-carbon amino sugars found in both bacteria and higher organisms. Sialic acid on the bacterial outer membrane (lipopolysaccharides (LPS), endotoxin) can camouflage bacteria from the host immunological responses, but the significance of sialic acid in Mannheimia haemolytica infection is currently unknown. ARS scientists in Ames, Iowa, determined the role of sialic acid in M. haemolytica virulence by generating a mutant without sialic acid in Mannheimia LPS. LPS from both wild-type and sialic acid mutant strains showed similar cytokine responses when incubated with cattle white blood cells, suggesting sialic acid in LPS does not reduce cytokine response. However, the M. haemolytica sialic acid mutant was highly sensitive to components in the innate immune system that remove bacteria from the body. These observations suggest that the sialic acid on LPS can act as a stealth mechanism to protect M. haemolytica against the innate immune system and contributes to the ability of Mannheimia to cause disease. Therefore, newly constructed M. haemolytica sialic acid mutant could serve a potential vaccine candidate.
2. Gene expression of Mannheimia haemolytica in different living states. Bacterial biofilms are organized communities of bacterial cells which grow by attaching to surfaces, each other, and are covered by a film-like substance called matrix. Bacteria form biofilms to protect from the host immune response and also due to the limitation of nutrients. Bacteria in the biofilms showed increased resistance to antibiotics compared to free-living bacteria. Mannheimia haemolytica, the predominant cause of bovine respiratory disease complex (BRD), forms biofilms, but the consequence of this is not known. ARS scientists in Ames, Iowa, selected three M. haemolytica serotypes associated with BRD, and assessed deferential gene regulation of M. haemolytica between biofilm and non-biofilm (free-living) stages. Over 400 genes were differentially expressed between biofilm and free-living cells. Some of the differentially expressed genes identified in this study could potentially be used to design new vaccines. Some the genes expressed only in biofilm cells are currently under investigation to confirm their role in biofilm formation.
3. Serum immunoglobulin (Ig) G levels correlate with clearance of Mycoplasma bovis (M. bovis) in naturally infected North American bison (Bison bison). M. bovis is a bacterial pathogen of cattle and bison capable of causing severe respiratory disease. In bison, M. bovis is responsible for large outbreaks of fatal disease with mortality exceeding 25%. Despite this, little information is available regarding the natural course and immune response to infection in bison. ARS scientists in Ames, Iowa, created a cohort of bison that were naturally infected with M. bovis. Over a 12-month period, the cohort was sampled every 2-3 months. Nearly half of the 41 bison in the cohort tested positive for M. bovis DNA, indicating they were infected. A majority of bison also had M. bovis specific antibody in response to the infection. A group of M. bovis infected bison were determined to have high levels of M. bovis specific IgG that correlated with a decline in Mannheimia DNA positive nasal swabs. These data suggest that antibody is important for the recovery of bison from M. bovis infection. Additionally, different antibody isotypes and targets may be more effective in clearing M. bovis infection in American bison.
4. Long noncoding (lncRNA), and microRNAs (miRNA) are segments of messenger RNA that do not produce a protein. They are known to be important regulators of the immune system. To evaluate the interactions among differentially expressed genes, LncRNA, and miRNA, ARS scientists in Ames, Iowa, produced three variants of Pasteurella multocida by disrupting three genes encoding virulence factors, and gene expression in response to these modifications were compared to the unmodified bacteria. Blood, liver, and various immune tissues were collected and lncRNA, miRNA and mRNA expression were assessed. There were differences in expression of immune genes, lncRNA and miRNA in the host due to the different Pasteurella multocida variants, except for the expression of lncRNA in one of the variants. There was a high correlation in the expression of immune genes, lncRNA and miRNA in animals challenged with one of the three variants. The identification of lncRNA and miRNA, associated with the immune genes from the host are necessary to determine the pathway the pathogen uses to produce the disease in the animal. This information is necessary to develop an intervention strategy to control the disease produced by the pathogen.
Review Publications
Menghwar, H., Tatum, F.M., Briggs, R.E., Casas, E., Kaplan, B.S., Azadi, P., Dassanayake, R.P. 2023. Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion. Microbiology Spectrum. Article e0294423. https://doi.org/10.1128/spectrum.02944-23.
Ma, H., Alt, D.P., Falkenberg, S.M., Briggs, R.E., Tatum, F.M., Clawson, M.L., Casas, E., Dassanayake, R.P. 2024. Transcriptomic profiles of Mannheimia haemolytica planktonic and biofilm associated cells. PLOS ONE. 19(2). Article e0297692. https://doi.org/10.1371/journal.pone.0297692.
Biernbaum, E.N., Dassanayake, R.P., Nicholson, E.M., Kudva, I.T. 2023. Comparative evaluation of antimicrobial activity of human granulysin, bovine and porcine NK-lysins against Shiga toxin-producing Escherichia coli O157:H7. PLOS ONE. 18(9): e0292234. https://doi.org/10.1371/journal.pone.0292234.
Ruiz-De La Cruz, G., Sifuentes-Rincon, A.M., Paredes-Sanchez, F.A., Parra-Bracamontes, G., Casas, E., Welsh Jr, T.H., Riley, D., Perry, G., Randel, R.D. 2023. Characterization of intronic SNP located in candidate genes influencing cattle temperament. Brazilian Journal of Animal Science. 52. Article e20220057. https://doi.org/10.37496/rbz5220220057.
Maina, T.W., Grego, E.A., Broderick, S., Sacco, R.E., Narasimhan, B., Mcgill, J.L. 2023. Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects neonatal calves against BRSV infection. Frontiers in Immunology. 14. Article 1186184. https://doi.org/10.3389/fimmu.2023.1186184.
Hau, S.J., Nielsen, D.W., Brockmeier, S.L. 2023. Prior infection with Bordetella bronchiseptica enhanced colonization but not disease with Streptococcus suis. Veterinary Microbiology. 284:109841. https://doi.org/10.1016/j.vetmic.2023.109841.
Nielsen, D.W., Hau, S.J., Mou, K.T., Alt, D.P., Brockmeier, S.L. 2023. Shifts in the swine nasal microbiota following Bordetella bronchiseptica challenge in a longitudinal study. Frontiers in Microbiology. 14. Article 1260465. https://doi.org/10.3389/fmicb.2023.1260465.
Sivasankaran, S.K., Bearson, B.L., Trachsel, J.M., Nielsen, D.W., Looft, T.P., Bearson, S.M. 2024. Genomic and phenotypic characterization of multidrug-resistant salmonella enterica serovar reading isolates involved in a turkey-associated foodborne outbreak. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2023.1304029.
Ma, H., Menghwar, H., Lippolis, J.D., Sarlo Davila, K.M., Casas, E., Dassanayake, R.P. 2024. Draft genome sequence of a multidrug-resistant pseudomonas aeruginosa strain isolated from a dairy cow with chronic mastitis. Microbiology Resource Announcements. Article e0117323. https://doi.org/10.1128/mra.01173-23.
Sacco, R.E., Jensen, E.D., Sullivan, Y., Labresh, J., Davis, W.C. 2024. An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit. Veterinary Immunology and Immunopathology. Article 110769. https://doi.org/10.1016/j.vetimm.2024.110769.
Dassanayake, R.P., Ma, H., Casas, E., Lippolis, J.D. 2023. Genome sequence of a multidrug-resistant Pseudomonas aeruginosa strain isolated from a dairy cow that was nonresponsive to antibiotic treatment. Microbiology Resource Announcements. Article e002892. https://doi.org/10.1128/MRA.00289-23.
Mosena, A.S., Ma, H., Casas, E., Dassanayake, R.P., Canal, C., Neill, J.D., Falkenberg, S.M. 2023. Multivariate analysis reveals that BVDV field isolates do not show a close VN-based antigenic relationship to US vaccine strains. BMC Research Notes. 16(1):121. https://doi.org/10.1186/s13104-023-06410-2.
Falkenberg, S.M., Ma, H., Casas, E., Dassanayake, R.P., Bolton, M.W., Raithel, G., Silvas, S., Neill, J.D., Walz, P.H. 2023. Multivariate analysis as a method to evaluate antigenic relationships between bovine viral diarrhea virus 1b field isolates and vaccine strains. Viruses. 15(10). https://doi.org/10.3390/v15102085.
Ruiz-De La Cruz, G., Sifuentes-Rincon, A.M., Paredes-Sanchez, F.A., Parra-Bracamontes, G.M., Casas, E., Riley, D.G., Perry, G.A., Welsh Jr., T.H., Randel, R.D. 2024. Analysis of nonsynonymous SNPs in candidate genes that influence bovine temperament and evaluation of their effect in Brahman cattle. Molecular Biology Reports. 51(1). Article 285. https://doi.org/10.1007/s11033-024-09264-4.