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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Ruminant Diseases and Immunology Research » Research » Research Project #432020

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

Location: Ruminant Diseases and Immunology Research

2021 Annual Report

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.

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
This is the final report for the project 5030-32000-116-00D terminating September 30, 2021. The goal of this project is to reduce the incidence of bovine respiratory disease complex (BRDC), the leading cause of morbidity and mortality in U.S. feedlots and in weaned dairy heifers. Important bacterial respiratory pathogens in BRDC, include Mannheimia haemolytica (M. haemolytica), Pasteurella multocida (P. multocida), Histophilus somni (H. somni), and Mycoplasma bovis (M. bovis). Viral pathogens that may predispose to secondary bacterial infection in BRDC include bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3, bovine herpes virus 1, and bovine viral diarrhea virus. Understanding the roles of these pathogens and their interactions are key to developing strategies to reduce BRDC. Substantial progress has been made on the objectives of this research project over the five-year lifetime. Detailed below is the progress report over the life of the project, highlighting past progress on the objectives and including progress made this FY. Supporting Objective 1, we conducted a calf trial designed to document colonization and shedding of M. haemolytica mutants in conjunction with bovine herpesvirus-1 (BHV-1). The BHV-1 infection was found to enhance shedding of both serotypes 1 and 6 M. haemolytica, as well as a Mannheimia varigena ( M. varigena) which was part of the normal flora of some of the experimental calves upon arrival at the National Animal Disease Center. Genome sequence of the M. varigena isolate confirmed its identity and showed an intact leukotoxin gene consistent with its hemolytic phenotype. The data suggest M. varigena may be an emerging pathogen in cattle. We identified and solved a problem with a diagnostic laboratory test that led to false-negative results for Mycoplasma bovis, an important respiratory pathogen. Among the key tests employed to identify isolates as M. bovis is a widely used polymerase chain reaction (PCR) assay developed and reported by clinicians at the Iowa State University Veterinary Diagnostic Laboratory. The intended target of the PCR is a DNA repair gene, known to be highly conserved among isolates. Upon investigating the reason for negative results with some isolates confirmed to be M. bovis on the basis of 16S RNA gene sequence, we discovered that the PCR primers were mistakenly selected from a gene immediately adjacent to the DNA repair gene, predicted to encode a lipoprotein. Further analysis of over 200 isolates revealed the presence of nucleotide changes in one of the primer-binding regions and a gene insertion within the amplified region that leads to a false negative result. These recently published findings are of importance to clinicians using this diagnostic PCR, who should be aware that the amplified fragment lies within a gene that appears to be less well-conserved than the intended target and that occasional false negative results may be obtained. Genomes have been sequenced, assembled, and annotated for 40 bison isolates, 14 cattle isolates and two deer isolates of Mycoplasma bovis, and for two 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 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. It is a critically important resource that greatly benefits ARS research priorities and objectives as well as global animal health. Supporting Objective 2, RNA was extracted from animals challenged with bovine viral diarrhea virus (BVDV) and Mycoplasma bovis. The objective was to establish a relationship between expression of non-coding RNAs and messenger RNA. Animals were allocated in a control group, a group challenged with Mycoplasma bovis, and a group first challenged with BVDV, and then with Mycoplasma bovis. Blood, liver, spleen, thymus, and retropharyngeal, mesenteric, and tracheobronchial lymph nodes were collected. RNA was extracted and sequenced from each tissue and animal. Messenger and non-coding RNA sequence was obtained. The bioinformatic analysis of non-coding and messenger RNA is in progress. The goal was to determine if non-coding RNAs can be used as an alternative to antibiotics to prevent or minimize the effects of pathogens producing bovine respiratory diseases. Three modified live Pasteurella multocida strains were produced by mutating one of three genes encoding potential virulence factors (FhaB2, HyaE, and NanP). Calves were inoculated intranasally as follows: control (sham inoculation), wild-type P. multocida; FhaB2, HyaE, or NanP modified. There were differences in the colonization level of the mutants compared to wild-type parent strain. For example, the FhaB2 mutant colonized at a higher level than did its parent strain. Conversely, the acapsular HyaE mutant colonized to a lesser degree than did the parent strain. Serum, white blood cells, retropharyngeal lymph node, tracheobronchial lymph node, palatine tonsil, and liver were collected from all animals at necropsy. Total RNA was extracted from all samples obtained, and messenger RNA (mRNA) and small RNA libraries were separately sequenced. Expression of mRNA, and non-coding RNAs (microRNAs and long non-coding RNAs (ncRNAs)) were analyzed for each tissue and treatment group. Associations between mRNA and ncRNAs were established by mRNA target identification of the differentially expressed microRNAs and ncRNAs. For example, comparing RNAs isolated from post-challenged blood samples from control and wild-type treatment groups, three differentially expressed microRNAs regulated 30 coding regions of the differentially expressed genes (DEGs); while six DEGs were targeted by eight differentially expressed ncRNAs. These results will be used by scientists interested in how bacterial virulence factors differentially modulate host genes involved in colonization and how these processes are regulated in different tissues and cells. We have successfully cloned, expressed and purified surface proteins containing lipids from Pasteurella multocida and M. bovis and purified membrane molecules from Mannheimia haemolytica and P. 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. Supporting 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 M. bovis vaccine payload was highly significant protection evident against virulent M. 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 M. bovis vaccine product are patent-applied and transferred to industry under a material transfer agreement. Our proprietary M. haemolytica mucosal vaccine delivery platform was modified to deliver a Histophilus somni vaccine payload. Sequencing RNA of several tissues from animals challenged with a vaccine platform based on Mannheimia haemolytica, expressing Mycoplasma bovis antigens was initiated. Animals were assigned to a control group, a group vaccinated with an inactive form of M. haemolytica, or a group vaccinated with M. haemolytica expressing M. bovis antigens. After euthanasia, blood, liver, spleen, palatine tonsil, and retropharyngeal and tracheobronchial lymph nodes were collected. RNA was extracted and sequenced from each tissue and animal, and RNA was sent for sequencing. The goal is to understand how this vaccine is protecting cattle against pathogens producing respiratory disease. We previously evaluated an antigen delivery platform using Mannheimia haemolytica for colonization and immune responses in calves. M. haemolytica vaccine isolates efficiently colonized the upper respiratory tract of calves and antibodies against the vaccine delivery platform were readily detected in serum samples. Antigenic proteins of three BVDV strains were expressed in M. haemolytica vaccine platform as a fusion protein. Despite codon-optimization of BVDV antigens, BVDV antigen expression levels were low. A vaccine efficacy study in calves was conducted and M. haemolytica colonization in the upper respiratory tract was confirmed. Although animals responded to the vaccine platform, poor immune responses were observed for BVDV antigens possibly due to the lack of glycosylation and lower expression of BVDV antigens.

1. Identification of a distinct strain of Mycoplasma bovis associated with fatal pneumonia in free-ranging pronghorn antelope. Mycoplasma bovis (M. bovis) is one of several bacterial pathogens associated with pneumonia in cattle. Its role in pneumonia of free-ranging antelope has not been established. Over a 3-month period in early 2019, ˜60 free-ranging pronghorn antelope with signs of respiratory disease died in northeast Wyoming. A consistent finding in submitted carcasses was severe pneumonia. M. bovis was detected by two distinct diagnostic tests. ARS researchers in Ames, Iowa, collaborating with investigators from University of Wyoming and Washington State University utilized the DNA sequence of M. bovis isolates from four animals, which revealed that all have a deletion in one of the target genes, alcohol dehydrogenase-1. The results of this study indicate that a distinct strain of M. bovis was associated with fatal pneumonia in free-ranging pronghorn. The findings will be used by wildlife officials, veterinarians and veterinary diagnosticians, and scientists with the ultimate goal to determine causes of such die-offs among wild ungulates and to work to develop preventive measures against the causative organisms to reduce these die-offs.

2. Completed genome sequencing of 16 Mycoplasma bovis isolates from bison and cattle which will aid our understanding of host specificity and disease development. Mycoplasma bovis was first recognized as a pathogen in the 1960s, when it caused an outbreak of mastitis in an American dairy herd and it has since spread to nearly all countries. In the early 2000s, M. bovis began to appear as a primary disease agent in North American bison (bison) and subsequently became one of the most serious and costly infectious disease threats faced by these animals. One hypothesis is that the relatively recent appearance of M. bovis as a pathogen in bison is due to the emergence of unique, newly evolved strains with an expanded host range or increased ability to cause disease. ARS researchers in Ames, Iowa, collaborating with investigators from University of Calgary and University of Saskatchewan have reported the complete genome sequences of 12 M. bovis isolates isolated from Canadian bison and four isolated from Canadian cattle. These findings will be used by veterinarians, epidemiologists and other scientists to better understand the relationship between cattle and bison isolates and will aid in our understanding of disease development and host specificity.

3. Killing of intracellular bacterial pathogen by host white blood cell-derived small antimicrobial proteins (NK-lysins). Previously, it was demonstrated that white blood cell-derived small proteins can efficiently kill bacteria associated with Bovine Respiratory Disease Complex (BRDC). This year, ARS researchers in Ames, Iowa, assessed whether these small antimicrobial proteins can kill the intracellular bacterial pathogen, Mycobacterium avium subspecies paratuberculosis (MAP), which is the causative agent of Johne's disease or chronic enteritis in cattle and other ruminants. Two of the four small antimicrobial proteins were able to kill MAP very efficiently and the killing of MAP was mediated by membrane pore formation. Furthermore, artificial construction of one small antimicrobial protein fused with a cell-penetrating peptide was able to kill intracellular MAP within the infected white blood cells. These findings will be used by veterinarians and scientists seeking potential new therapies against MAP.

4. Development of a small antimicrobial protein-based biosensor to detect bacteria and bacterial products. It was previously demonstrated that small 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. In collaboration with researchers at the Iowa State University, ARS researchers in Ames, Iowa, have developed a label-free detection method for bacterial products using a small antimicrobial protein-coated membrane (biosensor). The small antimicrobial protein biosensor was able to quickly bind nanogram amounts of bacterial products (lipopolysaccharides) in the samples. Therefore, this novel detection system should assist in the rapid detection of bacterial products (or bacteria) from blood samples. These findings will be used by veterinarians, diagnosticians, and scientists seeking to detect bacteria or their products at lower levels than currently available methods.

5. Development of a single novel vaccine for two specific bovine respiratory disease pathogens. There is no commerically available vaccine against Mycoplsma bovis. A novel vaccine against two bovine disease pathogens Mannheimia haemolytica and Mycoplasma bovis was developed and tested by ARS scientists in Ames, Iowa. In calf vaccination/challenge studies it was demonstrated that a single intranasal dose of the vaccine confers protection against respiratory disease, and the associated symptoms of disease, following challenge with virulent Mannheimia as well as following challenge with virulent Mycoplasma. The protection was associated with major reductions in the levels of disease-causing organisms in lung tissue following challenge. The vaccine also conferred significant protection against M. bovis-induced polyarthritis and middle ear infection, major economically important signs of disease. The vaccine is being further developed as a potential commercial product. These findings will be used by veterinarians, scientists and vaccine manufacturers seeking to reduce bovine respiratory diseases.

Review Publications
Malmberg, J., O'Toole, D., Creekmore, T., Peckham, E., Killion, H., Vance, M., Ashley, R., Johnson, M., Anderson, C., Vasquez, M., Sandidge, D., Mildenberger, J., Hull, N., Bradway, D., Cornish, T., Register, K.B., Sondgeroth, K.S. 2020. Mycoplasma bovis infections in free-ranging pronghorn, Wyoming, USA. Emerging Infectious Diseases. 26(12):2807-2814.
Dassanayake, R.P., Wherry, T.L., Falkenberg, S.M., Reinhardt, T.A., Casas, E., Stabel, J.R. 2021. Bovine NK-lysin-derived peptides are bactericidal against Mycobacterium avium subspecies paratuberculosis. Veterinary Research. 52. Article 11.
Kumar, R., Register, K.B., Christopher-Hennings, J., Moroni, P., Gioia, Gloria, Garcia-Fernandez, N., Nelson, J., Jelinski, M., Lysnyansky, I., Bayles, D.O., Alt, D.P., Scaria, J. 2020. Population genomic analysis of Mycoplasma bovis elucidates geographical variations and genes associated with host types. Microorganisms. 8(10). Article 1561.
Register, K.B., Parker, M., Patyk, K.A., Sweeney, S.J., Boatwright Jr, W.D., Jones, L.C., Woodbury, M., Hunter, D., Treanor, J., Kohr, M., Hamilton, R., Shury, T., Nol, P. 2021. Serological evidence for historical and present-day exposure of North American bison to Mycoplasma bovis. BMC Veterinary Research. 17. Article 18.
Kaplan, B.S., Falkenberg, S.M., Dassanayake, R.P., Neill, J.D., Velayudhan, B., Li, F., Vincent, A.L. 2020. Virus strain influenced the interspecies transmission of influenza D virus between calves and pigs. Transboundary and Emerging Diseases.
Mosena, A.C., Falkenberg, S.M., Ma, H., Casas, E., Dassanayake, R.P., Watz, P., Canal, C., Neill, J.D. 2020. Multivariate analysis as a method to evaluate antigenic relationships between BVDV vaccine and field strains. Vaccine. 38(36):5764-5772.
Paredes-Sanchez, F.A., Sifuentes-Rincon, A.M., Casas, E., Arellano-Vera, W., Parra-Bracamonte, G., Riley, D.G., Welsh Jr., T.H., Randel, R.D. 2020. Novel genes involved in the genetic architecture of temperament in Brahman cattle. PLoS ONE. 15(8). Article e0237825.
Putz, E.J., Palmer, M.V., Ma, H., Casas, E., Reinhardt, T.A., Lippolis, J.D. 2020. Characterization of a persistent, treatment-resistant, Staphylococcus aureus infection causing chronic mastitis in a Holstein dairy cow. BioMed Central (BMC) Veterinary Research. 16. Article 336.
Shepherd, B.S., Ma, H., Han, Y., Palti, Y., Gao, G., Liu, S., Wiens, G.D. 2020. Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss). Developmental and Comparative Immunology. 116 (103961).
Falkenberg, S.M., Dassanayake, R.P., Terhaar, B., Ridpath, J., Neill, J.D., Roth, J. 2021. Evaluation of antigenic comparisons among BVDV isolates as it relates to humoral and cell mediated immunological measures. Frontiers in Veterinary Science. 8. Article 685114.