Location: Ruminant Diseases and Immunology Research
2023 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
In support of Objective 1, we have utilized proprietary bacterial-vector system to generated isogenic Mannheimia haemolytica (M. haemolytica) putative adhesin mutants. Previously, we have generated M. haemolytica and Pasteurella multocida (P. multocida) isogenic filamentous hemagglutinin, sialic acid, and capsular mutants. The identity of isogenic mutants has been confirmed by polymerase chain reaction, Western blot, and/or sialic acid uptake assays. These mutants will be tested in animal studies to assess their role in colonization in the upper respiratory tract.
In support of Objective 1, we have established a collaboration with USDA-APHIS to sequence, assemble, and analyze the genomes of 81 Mycoplasma bovis (M. bovis) isolates from cattle and bison. We have developed methodology to perform comparative proteomic analysis on M. bovis isolates with the goal of identifying gene products contributing to virulence phenotypes. Genes involved in adhesion (vsps, p26, p48, alpha-enolase), host-immune evasion (mib-mip, mnuA), and glycerol metabolism are putative virulence factors of M. bovis. Once virulence factors have been identified, these genes can be mutated to confirm their contribution to virulence.
In support of Objective 2, we worked to develop a BRSV/bacterial co-infection model. The study design included four groups of calves: uninfected controls (n=2); BRSV challenge (n=4); Pasteurella multocida challenge (n=4); BRSV/P. multocida co-infection (n=4). For bacterial co-challenge, P. multocida was selected because it is commonly considered a highly opportunistic bacterial member of the normal respiratory tract microflora which typically does not cause primary lung infection on its own. Calves (n=8) were challenged with BRSV via aerosol. On day 7 post-BRSV challenge, one group of four calves received P. multocida via aerosol. Bacteria were more frequently isolated from lung lobes and bacterial counts were approximately 10-fold higher in co-infected calves compared to calves infected with P. multocida alone. We now have a challenge co-infection model which can be used to begin to understand how viral infection sets up the host for secondary bacterial infection by an opportunistic member of the normal respiratory tract microflora.
In support of Objective 2, we purified lipopolysaccharide (LPS) from both M. haemolytica and P. multocida wild-type and sialic acid mutants, confirmed the lack of sialic acid in mutant LPS preparations, and their immune effects were tested using peripheral blood mononuclear cells isolated from cattle. Toll-like receptor 4 has been identified as the major pattern recognition receptor involved in recognition of bacterial LPS. It is reported that in other bacterial species, sialylated LPS can shield conserved core oligosaccharide structures from recognition by the host immune system. We have already generated M. haemolytica (neuA) and P. multocida (nanPU) sialic acid uptake mutants (Objective 1). LPS from both wild-type and sialic acid mutants did not show detectable difference in pro- and anti-inflammatory cytokine responses. However, M. haemolytica sialic acid mutant was highly susceptible to phagocytic and complement-mediated (serum) killing as compared to wild-type isolate. Thus, the results confirm that LPS can shield these pathogens from recognition by the host immune system and provide information that can lead to new intervention strategies for bovine respiratory disease.
In support of Objective 2, we collected tissues from bison, which were inoculated with a modified live Mannheimia haemolytica vaccine expressing Mycoplasma bovis antigens. Animals were assigned to a control group or to a group vaccinated with M. haemolytica-expressing Mycoplasma bovis antigens. Bison were challenged with Bovines Herpes Virus-1, and 4 days later with M. bovis. Ribonucleic acid (RNA) will be extracted and sequenced from blood samples taken from each animal. Messenger and small non-coding RNA will be sent for sequencing. The goal is to understand, at the molecular level, how this vaccine is protecting cattle against pathogens producing respiratory disease.
In support of Objective 2, RNA was extracted from blood samples collected from animals inoculated with a vaccine platform based on Mannheimia haemolytica, expressing Mycoplasma mycoides antigens. Animals were assigned to a control group, to a group vaccinated with an inactive form of M. haemolytica, or to a group vaccinated with M. haemolytica expressing Mycoplasma mycoides antigens. Messenger and small non-coding ribonucleic acid will be sent for sequencing. The goal is to understand, at the molecular level, how this vaccine is protecting cattle against pathogens producing respiratory disease.
In support of Objective 3, we have demonstrated a vaccine with M. haemolytica expressing (and secreting) Mycoplasma bovis antigens exhibited highly significant protection against M. bovis lung challenge. Vaccinated calves had reduced clinical signs, lung lesions, lung bacterial load, and mortality. The same regions of M. bovis protective gene fragments were codon optimized to express in prokaryotic expression system (Escherichia coli), expressed and purified as recombinant His6-tagged proteins. Recombinant proteins were formulated in a commercially available adjuvant (Emulsigen D) to prepare injectable vaccine and tested initially in bison. Animals vaccinated with M. bovis recombinant proteins showed antigen-specific humoral (antibody) immune responses as compared to adjuvant only vaccinated group. The same immunogenic antigens genes of Mycoplasma mycoides subspecies mycoides small colony (MmmSC), the causative agent of contagious bovine pleuropneumonia, was targeted to generate modified-live M. haemolytica mucosal vaccine candidate. Regions of protective MmmSC proteins were codon optimized and cloned into M. haemolytica. M. haemolytica vaccine strains expressing MmmSC protective antigens have been generated and identity of the vaccine strains were confirmed by polymerase chain reaction and Western blot assays. These candidate vaccines for MmmSC will be tested in collaboration with researchers in Kenya.
Accomplishments
1. Counteracting antibiotic resistance to salmonella improve treatment options. Salmonella is a leading cause of human foodborne illness, and multidrug resistance (MDR; resistance to =3 antimicrobial classes) in Salmonella is a prevalent issue worldwide. In the United States, recent human outbreaks associated with consumption of food animal products have involved Salmonella isolates that are resistant to multiple antibiotics, including those that have been used in animal production, such as tetracycline. Because few new antibiotics are being developed, it is becoming increasingly necessary to improve the effectiveness of existing antibiotics. One mechanism that bacteria use to develop resistance to an antibiotic (like tetracycline) is to rapidly pump the antibiotic out of the bacterial cell before it can exert its antimicrobial effect. ARS researchers in Ames, Iowa, evaluated a pump inhibitor (efflux pump inhibitor, EPI) called 1-(1-Naphthylmethyl)-Piperazine (NMP) for its ability to recover tetracycline sensitivity in four MDR outbreak isolates of Salmonella. NMP reduced resistance in tetracycline-resistant Salmonella to levels that would no longer classify the isolates as ‘resistant’ to tetracycline, thereby restoring the effectiveness of the antibiotic against the MDR Salmonella isolates. Thus, efflux pump inhibitors, such as NMP, have the potential to contribute to novel treatment options available to livestock producers to aid in combatting antibiotic resistant bacteria.
2. Rapid test developed to differentiate between Mannheimia haemolytica genotype 1 and 2 to aid in bovine respiratory disease complex diagnosis. Mannheimia haemolytica is the primary bacterial pathogen involved in bovine respiratory disease complex (BRDC), commonly known as shipping fever, causing extensive economic losses to the beef and dairy cattle industries in the United States. It is an opportunistic bacterial pathogen that primarily resides as a commensal of the upper respiratory tracts of healthy cattle and leads to develop BRDC when the host's immune system is compromised. M. haemolytica isolated from North American cattle were classified into two genotypes, 1 and 2. Genotype 1 is commonly isolated from healthy animals while genotype 2 is largely isolated from lungs of animals affected with BRDC. However, isolation of both genotypes from samples collected from animals suffering from BRDC can occur and complicate BRDC diagnosis. Therefore, ARS researchers in Ames, Iowa, developed a rapid colorimetric assay (loop-mediated isothermal amplification) to distinguish M. haemolytica genotype 1 from genotype 2 strains, but further optimization under field conditions is needed. If approved for use in livestock, it could be beneficial to cattle producers and veterinarians as an aid to reduce BRDC.
3. Label-free detection of bacterial endotoxin using bovine NK-lysin antimicrobial peptide-functionalized nanoporous alumina membrane. Small antimicrobial proteins (NK-lysin) produced by cattle white blood cells show strong antimicrobial activity. The antimicrobial activity of small proteins is due to the selective and specific binding to bacterial membranes followed by pore formation in the membranes. ARS researchers in Ames, Iowa, explored whether this selective binding property of small antimicrobial proteins with bacterial membranes can be used to develop a biosensor system to label-free detection of bacterial products. The small antimicrobial protein functionalized biosensor was able to quickly bind nanogram amounts of bacterial products (lipopolysaccharides) in the samples. Further studies are needed to assess whether this novel detection system can be used to rapid detection of bacterial products (or bacteria) from blood samples.
4. Response of heifers vaccinated against bovine viral diarrhea virus with a combination of multivalent modified live and inactivated viral vaccines. Bovine viral vaccines, as either live or killed formulations, are widely used in the field. Moreover, they are often used in combination by vaccinating with one type and then re-vaccinating with the other. However, despite various reports that certain combinations are better than others, very few studies have actually evaluated how vaccinations with these different vaccines impacts the ability of the animal to respond to subsequent re-vaccination with the other type. ARS researchers in Ames, Iowa, completed a thorough analysis of the immune response of cattle following vaccination with a live or killed vaccine, followed by re-vaccination with the other type. This new information will be of interest to researchers, veterinarians and producers interested in developing and improving upon vaccination regimens against bovine viruses.
Review Publications
Dassanayake, R.P., Clawson, M.L., Tatum, F.M., Briggs, R.E., Kaplan, B.S., Casas, E. 2023. Differential identification of Mannheimia haemolytica genotypes 1 and 2 using colorimetric loop-mediated isothermal amplification. BMC Research Notes. 16. Article 4. https://doi.org/10.1186/s13104-023-06272-8.
Jiang, N., Shrotriya, P., Dassanayake, R.P. 2022. NK-lysin antimicrobial peptide-functionalized nanoporous alumina membranes as biosensors for label-free bacterial endotoxin detection. Biochemical and Biophysical Research Communications. 636:18-23. https://doi.org/10.1016/j.bbrc.2022.10.097.
Sacco, R.E., Mena, I., Palmer, M.V., Durbin, R.K., Garcia-Sastre, A., Durbin, J.E. 2022. An intranasal recombinant NDV-RSV F opt vaccine is safe and reduces lesion severity in a colostrum-deprived calf model of BRSV infection. Scientific Reports. 12(1). Article 22552. https://doi.org/10.1038/s41598-022-26938-w.
Paredes-Sanchez, F.A., Sifuentes-Rincon, A.M., Lara-Ramirez, E.E., Casas, E., Rodriguez-Almeida, F.A., Herrera-Mayorga, E.V., Randal, R.D. 2023. Identification of candidate genes and SNPs related to cattle temperament using a GWAS analysis coupled with an interacting network analysis. La Revista Mexicana de Ciencias Pecuarias. 14(1):1-22. https://doi.org/10.22319/rmcp.v14i1.6077.
Price, E.D., Dassanayake, R.P., Bearson, S.M. 2023. Increasing antimicrobial susceptibility of MDR Salmonella with the efflux pump inhibitor 1-(1-Naphthylmethyl)-piperazine. Biochemical and Biophysical Research Communications. 668:49-54. https://doi.org/10.1016/j.bbrc.2023.05.035.
Kaplan, B.S., Hofstetter, A.R., McGill, J.L., Lippolis, J.D., Norimine, J., Dassanayake, R.P., Sacco, R.E. 2023. Identification of a DRB3*011:01-restricted CD4+ T cell response against bovine respiratory syncytial virus fusion protein. Frontiers in Immunology. 14. Article 1040075.. https://doi.org/10.3389/fimmu.2023.1040075.
