Project : USDA ARS

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Research Project: Identification of Disease Mechanisms and Control Strategies for Bacterial Respiratory Pathogens in Ruminants

Location: Ruminant Diseases and Immunology Research

2020 Annual Report

Objectives
Objective 1. Define the virulence determinants and mechanisms involved with the principal bacteria associated with bovine respiratory disease, including identifying microbial mechanisms used by commensal bacteria to become pathogens, and identifying the mechanisms of bacterial colonization in target hosts. 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 respiratory infections, including developing animal disease models to study respiratory disease complex, identifying the host factors that drive the early innate immune response to bacterial respiratory infections, and characterizing functional genomics of the host associated with respiratory infection. 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 reduce antibiotic use, including developing vaccines that will induce early mucosal immunity in young animals, and developing and evaluating immune-modulators to prevent and/or treat respiratory disease. Subobjective 3.1. Develop vaccines that induce early mucosal immunity in young animals. Subobjective 3.2. Develop and evaluate immune modulators to prevent and/or treat respiratory disease. Objective 4: Identify bacterial genes and proteins important for protective immunity against contagious bacterial pleuropneumonia (CBPP) for incorporation into existing and developing vaccine platforms for the development of a DIVA vaccine that can be used to protect the U.S. bovine herd from an incursion of CBPP. Objective 5: Establish a reliable infection and challenge model for CBPP when ability to work with select agent is available. Objective 6: Utilize comparative genomics, proteomics, transcriptomics, and systems biology approaches to identify molecular determinants of pathogenesis for CBPP and other diseases associated with Mycoplasma mycoides cluster agents. Objective 7: Utilize comparative immunologic approaches to elucidate host-mycoplasma immune responses in order to improve the understanding of host-species susceptibility and resistance differences, disease pathogenesis, and tissue tropisms.

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 will utilize a coordinated and multipronged approach to characterize molecular mechanisms promoting respiratory bacterial pathogen colonization, adherence, and persistence in cattle. We plan to use experimental ruminant models and specific mutants to describe molecular mechanisms enabling bacteria to colonize, adhere and grow in the respiratory tract, and to examine the influences of primary viral infection on secondary bacterial infections. While much knowledge has been gained regarding the individual pathogens involved in BRDC, less is known concerning co-infections involving bacterial and viral respiratory pathogens. Given the expertise of our research team, and specific etiologic agent prevalence in the field, we will focus on BVDV and BRSV as the viral pathogens, and Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis as the bacterial agents. We plan to continue the development of reproducible models of viral predisposition to bacterial disease and to characterize the host and infectious agents’ response using a comprehensive approach. Bacterial genes or gene products identified in these studies will be used, based on their importance in colonization, for developing and testing novel vaccines. Furthermore, we will examine the potential of immunomodulators to enhance the host response to infection with respiratory pathogens. The overriding goal of this plan is to develop preventative measures that aid in the reduction or elimination of BRDC in beef and dairy cattle. Reductions in BRDC will be of substantial economic benefit to cattle producers. However, as specific bacterial pathogens involved in BRDC are significant causes of morbidity and mortality in wild ruminant populations, there are aspects of this research plan that include those species. For example, bighorn sheep suffer severe die-offs as a result of respiratory disease and it is considered the major factor impacting the long-term sustainability of bighorn sheep populations. Moreover, M. bovis has additionally emerged in North American bison, causing substantial economic losses to producers and threatening the stability of heritage herds. Therefore, strategies to reduce respiratory disease in wild ruminant populations will be of substantial value to the public interest in sustaining wildlife populations, as well as reduce economic losses to bison producers.

Progress Report
In support of Objective 1, genomes have been sequenced, assembled and annotated for 40 bison isolates, 14 cattle isolates and 2 deer isolates of Mycoplasma bovis, and for 2 isolates of M. bovirhinis. Assemblies for 16 of the M. bovis isolates have been made public via submissions to GenBank and a related paper has been published in a peer-reviewed journal. Core and accessory genomes for bison and cattle isolates have been defined and additional comparative analyses are underway. Related collaborative, genomics-based studies with the University of Saskatchewan, South Dakota State University, Kimron Veterinary Institute (Israel) and the government of New Zealand are in progress. Genomics data were critical in completing a multinational effort, organized and led by ARS, to develop and optimize a genetic reference typing method for M. bovis. The newly developed method was installed as the reference scheme on the PubMLST.org web site. PubMLST is an open-access, curated, web-based database that integrates population sequence data with provenance and phenotype information for a wide variety of bacterial pathogens. The M. bovis PubMLST database, which is curated by ARS, is readily accessible to researchers and clinicians around the world. As of June 2020, it has more than 100 users who have thus far contributed over 1200 isolates. It is a critically important resource that greatly benefits ARS research priorities and objectives as well as global animal health. In support of Objective 1, ELISA testing, identification of Mycoplasma from clinical samples and related data analyses are complete for 3022 sera and 488 nasal or tonsil swabs obtained from healthy bison in the U.S. and Canada, collected between 1984 and 2019. These samples represent 32 different farms, herds or locations, some public and some private, including Yellowstone and other federally managed herds in the United States and several federally or provincially managed herds in Canada. Overall, 7.29 percent of sera have detectable antibodies to M. bovis. The proportion of bison testing positively during the years 1984-1999, before M. bovis began causing disease in bison, is essentially identical to the percentage of bison testing positive from 2000-2019 (7.32 percent and 7.26 percent, respectively). This information suggests M. bovis has been present in healthy bison since at least the 1980’s, long before it emerged and was recognized as a disease problem. Viable M. bovis was recovered from the nasal cavity or tonsil of 1.2 percent of bison sampled, demonstrating that a healthy carrier state exists that could serve as a reservoir for disease outbreaks. One of four additional Mycoplasma species was found in 31.4 percent of bison. Whether these species can trigger cross-reactive antibody responses or contribute to mycoplasmosis in bison requires further investigation. In support of Objective 2, gene expression profiles of white blood isolated from calves challenged with Pasteurella multocida (a bovine respiratory pathogen) challenge in calves previously exposed to related P. multocida strains. At the end of the study, serum, white blood cells, lymph nodes, tonsil, and liver were collected from all animals. Total RNA was extracted from tissues of all individuals in the experiment. Different RNAs were separately sequenced. Quality control was applied to the sequences and mapped to the bovine genome. The expression profiles of the RNAs were generated and statistical analysis is pending. In support of Objective 2, ARS researchers at Ames, Iowa, have successfully cloned, expressed and purified surface proteins containing lipids from Pasteurella multocida and Mycoplasma bovis and purified membrane molecules from Mannheimia haemolytica and Pasteurella multocida. Gene expression profiles of pro- and anti-inflammatory molecules were examined from bovine peripheral blood mononuclear leukocytes. These studies will provide important information on bacterial molecules that stimulate pro- and/or anti-inflammatory cytokine expression in bovine leukocytes. In support of Objective 2, the biofilm formation properties of BRDC pathogen Mannheimia haemolytica and mastitis pathogen Staphylococcus aureus were evaluated. A biofilm is an assemblage of bacterial cells, in which cells stick to each other and to surfaces. They are important, as biofilms are thought to play a role in chronic infections. Robust biofilm formation was observed for both bacteria under in vitro assay conditions. Furthermore, complex three-dimensional structure of biofilms produced by both bacteria were clearly visible under confocal and electron microscopic examination. Extracellular polymeric substances which connect bacterial microcolonies were visible on the top, in the middle and in the bottom of biofilms. Both bacteria produced more biofilms in complex culture medium as compared to defined medium. However, regardless of the type of medium used, Staphylococcus aureus produced higher amounts of biofilms as compared to Mannheimia haemolytica. ARS researchers were able to identify a superior fixative solution to preserve complex architecture of biofilms which could be used with the study of other bacterial biofilms. In support of Objective 3, ARS researchers at Ames, Iowa, used a goat pneumonia model to assess whether small antimicrobial proteins are effective against Mannheimia haemolytica infection. However, in vivo effectiveness of the small proteins could not be determined due to the presence of preexisting pneumonic lung lesions in control, uninfected animals. Therefore, another study will be performed in a colostrum-deprived calf pneumonia model this next fiscal year. In support of Objective 3, a modified-live Mannheimia haemolytica mucosal vaccine delivery platform was evaluated in calves. With or without a Mycoplasma bovis vaccine payload, the platform vaccine induced highly significant protection against virulent Mannheimia lung challenge associated with reduced clinical signs, lung lesions, lung bacterial loading, and mortality. Only with the Mycoplasma bovis vaccine payload was highly significant protection evident against virulent Mycoplasma bovis challenge. Protection against both agents was associated with significant systemic and local (mucosal) specific IgG1, IgG2, IgM, and IgA antibodies. The platform vaccine technology and Mycoplasma bovis vaccine product are patent-applied and transferred to industry under a material transfer agreement. Our proprietary M. haemoytica mucosal vaccine delivery platform was modified to a deliver Histophilus somni vaccine payload. It is planned to evaluate protective efficacy of the new vaccine product in FY21.

Accomplishments
1. A reference typing method and related public database for Mycoplasma bovis. M. bovis-related disease in cattle and bison is poorly controlled. Reducing the number of outbreaks and minimizing the impact of those that occur requires an objective, standardized and discriminatory method to categorize isolates that will enable an understanding of how this bacterium spreads and whether particular families of isolates have an enhanced ability to cause disease. Two different genetic typing methods have recently been developed for M. bovis, but the underlying data fail to make clear whether one is superior to the other. To resolve this issue, a subcommittee of the International Organization for Mycoplasmology requested ARS researchers in Ames, Iowa, to organize and lead a multinational effort to compare the two methods and identify a single approach for use as a universal typing scheme. Using bioinformatics tools to analyze genome sequences from over 450 isolates obtained from every major geographic region of the world we defined a single, highly informative method now employed as the reference typing scheme for M. bovis. The scheme and a related open-access, curated database are freely available online at pubmlst.org/mbovis. The database integrates genetic typing data with isolate-specific information, such as geographic and anatomic origin, year of origin, clinical presentation of the animal of origin, etc. This comprehensive resource currently includes over 1200 isolates and has been accessed by more than 100 animal health researchers and clinicians around the world. Information from the database was a critical part of several recent studies that defined local, regional and global transmission patterns of M. bovis. Such insights into the population structure and epidemiology of M. bovis will support the development of rational, data-driven management and treatment practices that will positively impact livestock farmers and consumers of related products.

2. Monoclonal antibodies for specific cytokines recognize conserved molecules in cattle, sheep and goats. There is a need for the development of improved methods for the study of the immune systems of veterinary species to further enhance our understanding of comparative animal immunology. ARS researchers in Ames, Iowa, have been collaborating with investigators at Washington State University Monoclonal Antibody Center in Pullman, Washington, on a project to develop experimental methods for the study of white blood cells in veterinary species. This work resulted in the development of new reagents for immunological studies which allow researchers to define cattle, sheep and goat white blood cell subsets. To be able to use these tools that are recently developed would be beneficial to examine changes in white blood cells of these animals as part of the current effort to monitor the health of ruminants and for future studies of their immune response to vaccination. These findings will be used by veterinarians, scientists and vaccine manufacturers in reducing the negative effects of bovine respiratory disease.

3. Identification of superior fixative solution to preserve the complex architecture of bacterial biofilms. A biofilm is an assemblage of bacterial cells, in which cells stick to each other and to surfaces. They may play a role in chronic infections by helping to maintain colonization. Although specific bacteria are known to produce complex biofilms, if a reliable fixative, to preserve biological materials, is not used, biofilms can be destroyed. ARS researchers at Ames, Iowa, have compared several fixative solutions to identify a superior fixative to preserve complex architecture of Mannheimia haemolytica and Staphylococcus aureus biofilms produced under in vitro assay conditions. Both bacteria produced complex biofilms as assessed by biomass evaluation, confocal laser scanning microscopy, and scanning electron microscopy. Among the fixatives tested, only Methacarn solution was able to preserve both bacterial cell morphology and also extracellular polymeric substances in the biofilm as assessed by scanning electron microscopy. Identification of a superior fixative solution to preserve both Mannheimia haemolytica and Staphylococcus aureus biofilms will now help ARS researchers to evaluate whether both bacteria are able to produce biofilms in the upper respiratory tract and mammary glands in vivo. These findings will be used by scientists with the ultimate goal to reduce the negative effects of bovine respiratory disease.

U.S. DEPARTMENT OF AGRICULTURE

Ruminant Diseases and Immunology Research: Ames, IA