Project : USDA ARS

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Research Project: Host-pathogen Interactions and Control Strategies for Bacterial Respiratory Pathogens in Cattle

Location: Ruminant Diseases and Immunology Research

2025 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 identified putative virulent genes of bovine respiratory bacterial pathogens associated with adhesion, colonization, and pneumonia development. Mannheimia haemolytica is one of the main pathogens associated with bovine respiratory disease complex, causing billions of dollars in losses to the U.S. cattle industry. Isogenic Mannheimia haemolytica capsular, adhesin, and lipopolysaccharide sialylation-deficient mutants as well as Pasteurella multocida capsular, sialic acid uptake, and filamentous hemagglutinin (FHA) mutants were generated. The roles of Mannheimia haemolytica capsule and adhesin in the upper respiratory tract colonization was evaluated in a calf colonization model. To assess whether Mannheimia haemolytica capsule and sialic acid are involved in pneumonia development, a calf lung challenge study was completed. A calf study assessing the colonization of isogenic Pasteurella multocida mutants was completed. To assess whether Pasteurella multocida capsule, sialic acid, and FHA involved in pneumonia development, calves were ordered, and a lung challenge study will expect to start in mid-July 2025. In support of Objective 1, we continued to sequence Mycoplasma bovis genomes with the goal of identifying determinants of virulence and mechanisms necessary for the organism to cause disease in large ruminants. This information can be leveraged towards the development of novel interventions for use in cattle and bison and potentially have utility for other Mycoplasma species that negatively impact animal health. To this end we have sequenced an additional 12 genomes from Mycoplasma bovis isolates collected from bison and cattle and utilized a cutting-edge computational method to improve the analysis of Mycoplasma bovis genomes. This analysis has identified genes that perform critical functions in essential metabolic pathways. In support of Objective 2, genes encoding three important proteins, one from Mannheimia haemolytica (called PlpE), one from M. bovis (called P48), and one from Mycoplasma mycoides subspecies mycoides Small Colony (called LppQ) were cloned into an Escherichia coli expression system. The recombinant proteins were successfully expressed and purified for downstream applications. To assess whether these proteins are involved in Toll-like receptor 2 mediated innate immune cell activation and upregulation of cytokine genes expression, peripheral blood mononuclear cells prepared from cows were stimulated with corresponding proteins. Total cellular RNA was extracted, and real-time quantitative polymerase chain assay was performed. A significant upregulation of proinflammatory cytokines such as interferon, interleukin-1ß, and interleukin-6 transcripts expressions were observed in the cells stimulated with P48 as compared to PlpE and LppQ. Since P48 can induce TLR2-mediate inflammatory response and thus virulence, inactivation of P48 gene might reduce the severity of lung lesions. In support of Objective 2, tissues were collected from an experiment in cattle, in which animals were challenged with Bovine Herpes Virus-1 (BHV1) and Mycoplasma bovis. The goal of this study was to understand the molecular mechanisms driving the enhanced respiratory disease caused by the simultaneous infection with multiple pathogens, a system exemplified by bovine respiratory disease. An increased understanding of the genes and genetic pathways involved in disease pathogenesis is critical for the development of novel interventions to better protect cattle and other large ruminants against respiratory disease. Mycoplasma mycoides subspecies mycoides causes contagious bovine pleuropneumonia, a serious cattle disease in Sub-Saharan Africa. This disease harms cattle health and greatly affects the farmers and communities who rely on livestock for income and food. Therefore, in support of Objective 3, we developed a modified live-attenuated M. haemolytica vaccine strains expressing Mycoplasma mycoides subspecies mycoides (MmmSC) protective antigens (heat shock protein 70 and elongation factor Tu). To assess the immunogenicity of the selected antigens, calves were intranasally and orally vaccinated with vaccine strains. Blood samples were collected, and humoral immune response was evaluated. A strong humoral (antibody) immune response against vaccine antigens and M. haemolytica vaccine strains were observed within three weeks post-vaccination. These preliminary data suggest that M. haemolytica vaccine strains expressing MmmSC antigens can be used as a potential vaccine candidate to control MmmSC infections. This vaccine will be used to vaccinate calves followed by challenge with Mycoplasma mycoides in 2026. In support of Objective 3, we focused on development of non-antibiotic intervention strategies to prevent or treat respiratory infections. This includes the development of easily administered vaccines and developing and evaluating immune-modulators to prevent and/or treat respiratory disease. Bovine RSV causes respiratory disease in young calves and is a major viral pathogen in the bovine respiratory disease complex (BRDC), the leading cause of mortality and mortality in feedlot cattle. BRDC is estimated to cost farmers and ranchers over $1 billion annually. Two independent trials were conducted in which ARS scientists in Ames, Iowa, evaluated short-term and long-term duration of immunity of a protein-based nanovaccine in the neonatal calf model. The researchers compared the nanovaccine performance to a commercial modified live vaccine, and to nonvaccinated control calves. Experimentally challenged calves receiving nanovaccine treatment exhibited less viral replication in nasal samples compared to nonvaccinated calves at 6 months post-vaccination, suggesting this is a good vaccine candidate. Currently, lung damage and viral lung loads are being evaluated. This new nanovaccine has the potential to reduce the BRSV disease burden in cattle and these results will be of interest to scientists, veterinarians, and cattle producers. In support of Objective 4, UConn scientists collaborated with the International Livestock Research Institute (ILRI) in Nairobi, Kenya, to better understand the virulent genes associated with Mycoplasma mycoides subspecies mycoides small colony phenotype (MmmSC), which causes contagious bovine pleuropneumonia (CBPP). CBPP is a devastating disease with catastrophic economic impact, but very little is known about how MmmSC causes disease which has prevented development of safe and efficacious vaccines that can safeguard the U.S. agriculture from a potential introduction. To continue understanding the mechanisms by which MmmSC causes disease, we generated a large collection of mutant libraries, where each mutant has one gene disrupted. So far, we have made about four thousand (4000) mutant strains (or clones) and have now finalized sequencing them to find out what each mutation affects. From this, we have found eighteen (18) genes of high interest, and several others that may be implicated in virulence. One of our already sequenced mutants has a disruption in one of those key genes. We also found a mutant with a disrupted dihydrolipoamide dehydrogenase (lpd) gene. The UConn team has previously characterized this gene to serve as a virulence determinant in the poultry pathogen Mycoplasma gallisepticum (MG), and its disruption led to the development of a highly efficacious live attenuated MG vaccine. Given our previous findings, we wanted to assess whether lpd is also a virulence determinant in MmmSC. We conducted an animal trial using 24 Small African Zebu cattle to assess whether the lpd mutant strain of MmmSC was attenuated, as well as collect infected lung tissue samples to conduct downstream transcriptomic (gene expression) analysis of both host and pathogen. The UConn team has now obtained sequence data from one third of the samples to be sequenced and are currently analyzing gene expression patterns. This data will help us understand which genes are active during infection, revealing how both the pathogen and host respond at the molecular level. This insight is critical for identifying virulence factors, evaluating vaccine candidates, and uncovering mechanisms of disease or attenuation. In support of Objective 5, we have analyzed publicly available genomes of MmmSC to identify potential vaccine targets. Based on the analyses, we have designed numerous MmmSC proteins as vaccine candidates and some have already been extracted in preparation for vaccine trials. Although we have experienced delays in obtaining genetic material (DNA) samples from newly collected field isolates of MmmSC, we continue efforts in analyzing existing datasets to identify further virulence determinants. In collaboration with INRAE, the UConn team are also actively refining a technique called genome transplantation. Genome transplantation of Mycoplasma into yeast is a method that involves replacing a recipient (yeast) cell’s genome with a donor’s (MmmSC) genome. This method allows and modifies MmmSC genome safely and easily inside the yeast cells. We can then insert the modified genome back into the Mycoplasma cell (via a process called transplantation) to generate an attenuated Mycoplasma strain that can be used as a vaccine candidate. We are continuing to improve MmmSC genome transplantation methods by overcoming barriers that reduce transplant success.This includes addressing key biological barriers such as protecting chemical markers on the DNA (called methylation) and disabling defense mechanisms of the recipient cells (restriction modification systems) that would otherwise reject or destroy the incoming genetic material. Genome transplantation allows for the creation of cells with novel genetic information, potentially also leading to the identification of virulence factors and new vaccine targets.

Accomplishments
1. Mannheimia haemolytica adhesion mutant showed enhance ability to colonize in the upper respiratory tract. Bovine respiratory disease complex (BRDC) has a significant economic impact to the U.S. cattle industry and consequently, affecting higher meat prices for the U.S. consumers. Mannheimia haemolytica resides in the upper respiratory tract of cattle and is a major contributor to BRDC under stress conditions such as transport, crowding, or viral infections. Therefore, it is important to identify effective BRDC control measures. ARS scientists in Ames, Iowa, studied how well modified strains of Mannheimia haemolytica could colonize the upper respiratory tract of animals. Normal wildtype strain and two weakened versions (one missing a "stickiness" factor (adhesin mutant) and one missing a protective outer layer (capsular mutant)) bacteria were administered into the animals' noses and checked how well each strain was able to colonize and grow in the nose and throat areas. Adhesin mutant strain showed the highest colonization while capsular mutant strain failed to colonize. Our findings suggest that a protective outer layer (capsule) is needed for long-term colonization. Thus, the Mannheimia haemolytica adhesin mutant containing intact capsule may serve as a potential vaccine candidate.

2. Identification of virulence factors in bovine respiratory disease complex. Bovine respiratory disease complex (BRDC) causes severe economic losses of over one billion dollars to the U.S. beef and dairy cattle industries, resulting in increased meat prices for the U.S. consumers. Mannheimia haemolytica colonizes the upper respiratory tract of cattle and is a significant cause of BRDC under stress conditions such as transport, crowding, or viral infections. Identifying genes linked to virulence is essential for understanding how this bacterium becomes pathogenic and developing effective control measures. ARS scientists in Ames, Iowa, determined that the outer coating (capsule and sialic acid attached to outer membrane) of the bacterium was critical for virulence. They confirmed this by deleting the genes involved in this coating and showing that the mutants caused significantly less lung lesions and reduced bacterial loads in calves. Hence, Mannheimia haemolytica capsular or sialic acid mutants could serve as a potential vaccine candidate.

3. Improved vaccines for protection to Mycoplasma bovis infections in American bison. Mycoplasma bovis is a significant pathogen and causes severe pneumonia outbreaks with high fatality rates in bison leading to significant economic losses to the bison ranchers through mortality, decreased production, and treatment costs. Therefore, it is important to develop preventive measures such as a vaccine against Mycoplasma bovis to protect American bison from this deadly pathogen. ARS scientists in Ames, Iowa, formulated an improved vaccine that only required administration of two doses for the bison to have a robust antibody and cellular immune response to the vaccine antigens. Although the vaccine failed to provide full protection, vaccinated bison had lower bacteria counts in the lung and less lung damage compared to unvaccinated bison after intranasal Mycoplasma bovis infection. These data demonstrate this vaccine can provide partial protection against Mycoplasma bovis infection in bison which is a priority to assist the bison industry. Therefore, ARS researchers in Ams, Iowa, are currently refining protein subunit vaccines to improve their protective effectiveness.

4. Characterization of Mycoplasma bovis subunit vaccine induced transcriptomic response. The American bison is an icon of the U.S. revered by native Americans and currently a thriving livestock and tourist industry. Herds in the U.S. need to be protected against disease that could deplete the national inventory. Mycoplasma bovis is a pathogen affecting the American bison, producing devastating respiratory disease problems. It causes chronic respiratory disease with high mortality rates. ARS scientists in Ames, Iowa, demonstrated that two Mycoplasma bovis proteins induced immunity in bison, leading to reducing lung lesions and bacterial loads following an experimental Mycoplasma bovis. The objective of this study was to identify genes that were expressed differently in bison following vaccination with the two Mycoplasma bovis proteins, compared to non-vaccinated animals. Differentially expressed genes were identified in all tissues associated with the response of bison against the pathogen. Spleen-specific upregulation included genes involved in immune response. These genes demonstrate a broad, temporally regulated immune response which can be detected in the bison, supporting the efficacy of the two Mycoplasma bovis proteins as potential vaccines.

5. Identified vaccine targets and developed a modified-live attenuated vaccine candidate against Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC). MmmSC is the causative agent of contagious bovine pleuropneumonia (CBPP), a severe respiratory disease affecting cattle mostly in sub-Saharan African countries. CBPP has been eradicated in the U.S.; however, due to global livestock and animal product movement, as well as the potential for intentional introduction, CBPP still poses a threat to the U.S. livestock. Therefore, it is important to develop efficacious vaccine against CBPP. ARS scientists in Ames, Iowa, used genome sequences of MmmSC strains isolated from cattle and identified 86 potential novel vaccine targets. The researcher used two of the identified proteins (EFTu and HSP70) to develop a modified-live attenuated Mannheimia haemolytica vaccine strains. Calves were orally and intranasally vaccinated and a strong antibody response was observed three-to-four weeks post-vaccination. The observed immune response was comparable to previously developed vaccines suggesting the newly developed vaccine may provide similar protection to CBPP.

U.S. DEPARTMENT OF AGRICULTURE

Ruminant Diseases and Immunology Research: Ames, IA