2012 Annual Report
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.
Isogenic mutants were constructed of Pasteurella multocida. (P. multocida) Each has specific chromosomal DNA deletions, without insertion of foreign DNA, which impact the function of a single gene product or metabolic process without probable impact on the function of adjacent regions of the chromosome. Mutants were sequenced to demonstrate the presence of the desired deletion and tested for absence of the associated phenotypic trait and will be used in studies of disease pathogenesis in pasteurellosis of cattle and potentially in vaccines.
Chromosomal DNA sequence was obtained from reference P. multocida strains P1062, TT94, X73, and P1059 by 454 pyrosequencing. These strains are representative isolates originally obtained from cattle and poultry that are virulent in the respective species. Assembly of obtained sequence resulted in 23-42 contiguous DNA segments for the respective strains. Analysis of genomic sequence showed that bovine isolates are closely related. Avian isolates are more diverse, but more closely related to one another than to bovine isolates. The reference strain (Pm70, an avian isolate) is more closely related to bovine than avian isolates, potentially related to relative lack of Pm70 virulence in birds. Considerable similarity was found among all P. multocida isolates, indicating that mechanisms whereby these bacteria survive and cause disease in their respective hosts is largely shared across host species. Further evaluation of these and other genomes will yield valuable information regarding host specificity of disease and facilitate identification of specific gene targets for disease intervention strategies. A study evaluated varying vitamin D (vitD) levels on the response to bovine respiratory syncytial virus (BRSV) infection. Calves were fed milk replacer diets differing in levels of vitD to establish two treatment groups, either sufficient or deficient vitD. Animals were experimentally infected with BRSV. No differences in susceptibility to BRSV were found between the two groups. Identifying a role of bovine yd T cells in immunity from BRD, as well as how this role can be modulated by vitD status in the calf. Subsets of bovine yd T cells were purified from blood of BRSV-infected, neonatal calves with normal or deficient levels of vitD. Purified cells were re-stimulated in vitro with BRSV +/- vitD, then analyzed for RNA and protein production. While vitD status does not appear to have a significant effect on yd T cell function in vivo or in vitro, the cells respond to stimulation with BRSV with chemokine and inflammatory cytokine production. Further, yd T cell subsets appear to have unique roles in immunity to bovine viral infection, as their cytokine profiles differed significantly to in vitro re-stimulation. Future studies will aim to further elucidate the function of each T cell subset, as well as clarify a role for yd T cells in immunity from BRD. An experiment was conducted to develop BRSV-bacterial co-infection in calves. Calves were challenged with BRSV followed by M. haemolytica. At necropsy, tissues were collected for histological evaluation, and for real-time PCR and ongoing transcriptomics analyses.
Vitamin D (vit D) modulates the immune response to bovine respiratory syncytial virus (BRSV). ARS scientists in Ames,Iowa conducted research to determine whether calves with low circulating levels of vit D would be more susceptible to RSV infection than calves with high levels of vit D. Calves were fed milk replacer diets with different levels of vit D for a 10 wk period to establish two treatment groups, one with high and one with low circulating vit D. Animals were experimentally infected with BRSV. The results provide the first evidence that BRSV infection in the lung activates the vit D pathway within the cells that produce it. The data indicated that while increasing vitD levels may alter the immune response to BRSV infection, there was no significant impact on the disease. Thus, producers may be able to reduce the amount of vit D added to milk replacers without significantly increasing susceptibiliy to infection.
Mycoplasma bovis isolates from bison. US and Canadian colleagues provided clinical samples diseased bison, from which were derived and expanded seed stocks of Mycoplasma bovis isolates. ARS researchers at Ames, Iowa established and are responsible for ongoing maintenance and curation of authenticated seed stock cultures. This will impact workd on M. bovis by providing a way to facilitate the sharing of isolates among researchers.
Cultured Mycoplasma bovis from lung samples of bison. At the request of colleagues with the Animal, Plant Health Inspection Service, researchers at ARS in Ames, Iowa cultured samples from lung lesions of bison that had been submitted under the Mycobacterium Bison Slaughter Surveillance Program for which APHIS was unable to establish an etiology. ARS researchers isolated and definitively identified Mycoplasma bovis. The results impact the bison Mycobacterium surveillance program in that it shows other pathogens (e.g., mycoplasma) may cause similar leasions and should be evaluated as well.
New modified-live vaccine strains of Pasteurella multocida (P. Multocida). Pneumonic pasteurellosis is an ongoing cause of multi-million dollar losses to the beef and dairy cattle industries. Two new vaccine strains were constructed by ARS researchers in Ames, Iowa which may be delivered to calves by conventional injection or by a intranasal route. Effective vaccines to control P. multocida in susceptible cattle populations will save livestock producers millions of dollars annually while reducing therapeutic antibiotic usage and increasing the quality of beef products.
Briggs, R.E., Tabatabai, L.B., Tatum, F.M. 2012. Mucosal and parenteral vaccination against pneumonic pasteurellosis in cattle with a modified-live in-frame lktA deletion mutant of Mannheimia haemolytica. Microbial Pathogenesis. 52(5):302-309.
Brockmeier, S.L., Loving, C.L., Nelson, E.A., Miller, L.C., Nicholson, T.L., Register, K.B., Grubman, M.J., Brough, D.E., Kehrli, Jr., M.E. 2012. The presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus. Clinical and Vaccine Immunology. 19(4):508-514.
Casas, E., Garcia, M.D., Wells, J., Smith, T.P.L. 2011. Association of single nucleotide polymorphisms in the ANKRA2 and CD180 genes with bovine respiratory disease and presence of Mycobacterium avium subsp. paratuberculosis. Animal Genetics. 42:571-577.
Mullins, M.A., Register, K.B., Bayles, D.O., Butler, J.E. 2011. Haemophilus parasuis exhibits IgA protease activity but lacks homologs of the IgA protease genes of Haemophilus influenzae. Veterinary Microbiology. 153(3-4):407-412.
Mullins, M.A., Register, K.B., Bayles, D.O., Dyer, D.W., Kuehn, J.S., Phillips, G.J. 2011. Genome sequence of Haemophilus parasuis strain 29755. Standards in Genomic Sciences. 5(1):61-68.
Nicholson, T.L., Brockmeier, S.L., Loving, C.L., Register, K.B., Kehrli, Jr., M.E., Stibitz, S.E., Shore, S.M. 2012. Phenotypic modulation of the virulent Bvg phase is not required for pathogenesis and transmission of Bordetella bronchiseptica in swine. Infection and Immunity. 80(3):1025-1036.
Nonnecke, B.J., Waters, W.R., Goff, J.P., Foote, M.R. 2012. Adaptive immunity in the colostrum-deprived calf: Response to early vaccination with Mycobacterium bovis strain bacille Calmette Guerin and ovalbumin. Journal of Dairy Science. 95(1):221-239.
Register, K.B., Sukumar, N., Palavecino, E.L., Rubin, B.K., Deora, R. 2012. Bordetella bronchiseptica in a paediatric cystic fibrosis patient: possible transmission from a household cat. Zoonoses and Public Health. 59(4):246-250.
Sacco, R.E., Nonnecke, B.J., Palmer, M.V., Waters, W.R., Lippolis, J.D., Reinhardt, T.A. 2012. Differential expression of cytokines in response to respiratory syncytial virus infection of calves with high or low circulating 25-hydroxyvitamin D3. PLoS One. 7(3):e33074. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0033074.
Sharma, V.K., Sacco, R.E., Kunkle, R.A., Bearson, S.M., Palmquist, D.E. 2012. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle. Infection and Immunity. 80(4):1333-1342.
Waters, W.R., Thacker, T.C., Nonnecke, B.J., Palmer, M.V., Schiller, I., Oesch, B., Vordermeier, H.M., Silva, E., Estes, D.M. 2012. Evaluation of gamma interferon (IFN-gamma)-induced protein 10 responses for detection of cattle infected with Mycobacterium bovis: comparisons to IFN-gamma responses. Clinical and Vaccine Immunology. 19(3):346-351.