1a. Objectives (from AD-416):
1) Define the virulence determinants and mechanisms involved with the primary bacteria associated with bovine respiratory disease. Subobjectives: (1a) Determine the role of selected molecules at the host-bacterial interface in mucosal colonization and disease; (1b) Comparative genomic analysis of Pasteurella multocida and Mannheimia haemolytica. 2) Design disease models and experiments to evaluate the interaction, pathology, temporal pathogenesis, and immune response induced by bacteria and viruses associated with bovine respiratory disease. Subobjectives: (2a) Development of BRSV/bacterial and BVDV/bacterial co-infection models to be used in studies on pathogenesis and immune responses induced by bacteria and viruses associated with bovine respiratory disease (BRD); (2b) Determine the interaction of Mannheimia haemolytica in commensal and pathogen status from virus-infected calves. 3) Develop intervention strategies to control bovine respiratory disease by developing novel vaccines and novel immunomodulators. Subobjectives: (3a) Evaluate the immune response and efficacy against pulmonary challenge using modified-live P. multocida deficient in LPS sialylation and/or capsule expression; (3b) Evaluate the effects of vitamin D status on growth and immune response capacity of the calf and its response to experimental challenge with pathogens in causing BRD.
1b. Approach (from AD-416):
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 focus initially on 2 relevant molecular factors for this endeavor: LPS which, when sialylated, was shown to be pivotal to disease progression in a model of P. multocida disease; and filamentous hemagglutinin which has been implicated as an important mediator of mucosal adhesion and systemic invasion. We propose to establish a reference genome sequence for bovine P. multocida for use in transcriptomic analysis and to compare the reference genome to other isolates. We further propose to compare bovine lung isolates of M. haemolytica serotypes 1, 2, and 6 in order to determine potential molecular mechanisms of serotype specificity in disease pathogenesis. While much knowledge has been gained regarding the individual pathogens involved in BRD, less is known concerning co-infections involving viral and bacterial respiratory pathogens. Given the expertise of our research team, and specific etiologic agent prevalance in the field, we will focus on BVDV and BRSV as the viral pathogens. For bacterial co-challenge, P. multocida and M. bovis were selected because both are commonly considered highly opportunistic compared to M. haemolytica and H. somni which are much more capable of primary lung infection. We plan to develop reproducible models of viral predisposition to bacterial disease and to characterize the host and infectious agents’ response using a comprehensive transcriptomic approach. Sialylation of LPS has been found to be a critical feature in disease pathogenesis wherein the organism is greatly attenuated by inactivation of the system. There is evidence that sialylation may confer immunologic stealth to the organism, whereby the organism is not recognized as foreign to the host. The sialylated LPS enables the pathogen to evade recognition by the host’s immune system. We propose to evaluate and compare the efficacy of acapsular, asialic, and combined acapsule/asialic mutants as vaccines in calves. Mechanisms predisposing the calf to BRD have not been completely elucidated; however, in addition to roles for viral/bacterial interactions and immune modulation, physiological stresses related to management practices may alter immune compentency. Vitamin D, a fat-soluble hormone critical for assuring calcium homeostasis modulates both innate and adaptive immune responses in other species. We expect Vitamin D status will influence the functional capacity of the immune system of the calf pre-challenge and reduce the severity and duration of respiratory disease following experimental infection with BRSV alone or in combination with bacterial pathogens.
3. Progress Report:
Supporting objective 2, two co-infection challenge experiments were conducted with 8 calves in each experiment. Two calves randomly assigned to each group: controls, challenged with BRSV, challenged with Mannheimia haemolytica, or challenged with BRSV and then with M. haemolytica. Animals were euthanized and tonsil samples collected. RNA was used to generate cDNA for sequencing. Approximately 150 million sequences generated from each experiment. Sequences are being used to establish genes differentially expressed among the infected and co-infected groups compared to the control group. Analysis of the data is in progress. For objective 2, a study to evaluate the effects of vitamin D status on BRSV infection at day 14 post-infection, it was noted upon histological evaluation of lung tissue that a prominent feature observed is bronchiolitis obliterans. These fibrous polyps result in permanent decreases in airflow and ventilation. Thus, we have determined that it may be advantageous to provide a secondary bacterial challenge at a later time point than we had attempted in previous viral-bacterial co-infections. Supporting objective 2, a study evaluating effects of vitamin D status on immune response was completed. Results suggest vitamin D status has a moderate influence on adaptive immune response of neonatal calves. Data from a second study is being used to evaluate the effects of vitamin D status on CD14 cell functional capacity. These data include cytokine and chemokine expression by monocytes in response to toll-like receptor agonists. Cells were collected at several time points during the first two months of life from vitamin D deficient and replete calves. Supporting objective 1, twelve M. haemolytica and 7 Pasteurella multocida genomes were sequenced. Reference genomes of 4 isolates were closed, the remaining genomes are draft genomes. At this time 4 genomes have been annotated and uploaded to the National Center for Biotechnology Information (NCBI) database. It is anticipated at least eight more genomes will be annotated, uploaded, and available to the public this year. Among the early findings of this project, it is evident integrative-conjugative elements are relatively common in both M. haemolytica and P. multocida. Experimental evidence was obtained separately that showed at least some of these elements can transfer, carrying antimicrobial-resistance traits with them, within and across species. A highly virulent multiple-resistant strain of M. haemolytica Texas feedlots was found to contain an 88 kb integrative-conjugative element nearly identical to that identified in a P. multocida recovered from Nebraska cattle. Supporting objective 3, P. multocida asialic, acapsular, and acapsular/asialic double mutants were utilized in both mucosal and systemic avian models of infection. It was determined the acapsular mutants were modestly attenuated compared to virulent parent strains, whereas asialic mutants were nearly non-virulent with respect to acute disease.
Abrahante, J.E., Johnson, T.J., Hunter, S.S., Maheswaran, S.K., Hauglund, M.J., Bayles, D.O., Tatum, F.M., Briggs, R.E. 2013. Draft genome sequences of two virulent serotypes of avian Pasteurella multocida. Genome Announcements. 1(1):e00058. DOI:10.1128/genomeA.00058-12.