Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Medicine & Epidemology
Non Technical Summary
Infectious bovine keratoconjunctivitis (IBK; pinkeye) is the most common eye disease of US cattle. Cattle producers throughout California are adversely affected by IBK through reduced weight gains and high treatment costs. IBK causes animal pain and suffering; therefore IBK prevention is important for improving cattle health and welfare. Today's commercially available IBK vaccines have variable efficacy, most likely because of antigenic diversity amongst Moraxella spp associated with IBK.Characterized by a painful corneal ulcer, IBK can cause permanent visual impairment and even blindness due to eyeball rupture in severe cases. While a causal association between IBK and Moraxella bovis was established many years ago, our discovery of Moraxella bovoculi suggested that the disease may be multi-factorial. While recent studies implicate M. bovoculi in the majority of IBK cases, one challenge study that tested a lab-adapted strain of M. bovoculi could not demonstrate a causal association. Nevertheless, a commercially-available M. bovoculi bacterin to prevent IBK (Addison Biological Laboratory, Inc) was just released during spring 2017 and suggests that an effective challenge model may have been established within industry and therefore that M. bovoculi may play an important role in IBK pathogenesis. The pathogenicity of M. bovis requires expression of pilin and a cytotoxin (hemolysin). Pilus-based vaccines can reduce incidence and severity of IBK, however, the existence of multiple pilus serogroups and a pilin gene inversion mechanism leads to antigenic variability that allows M. bovis the ability to evade a host's immune response. In contrast, the M. bovis cytotoxin is highly conserved and cattle with IBK develop anticytotoxin antibodies that cross-neutralize cytotoxins from different strains of M. bovis. We have identified and characterized a related cytotoxin in M. bovoculi that also appears to be well conserved. Although our earlier studies with partially purified native M. bovis cytotoxin vaccines derived from bacterial culture supernatants showed efficacy against IBK, purification methods were labor-intensive and made scale-up for commercialization difficult. These obstacles led us to investigate recombinant cytotoxin vaccines against IBK. Over the past 10-15 years we developed and tested efficacy of parenterally-administered recombinant M. bovis and M. bovoculi cytotoxin subunit vaccines against naturally occurring IBK. Collectively our results suggest that additional vaccine improvements are necessary to improve efficacy of recombinant Moraxella spp cytotoxin-subunit vaccines. Fortunately, methods to partially purify native M. bovis and M. bovoculi cytotoxin from culture supernatants have improved during the past decade and feasibility of using culture supernatant enriched in native cytotoxin as a source for vaccine antigen is now better than it was previously. Use of a culture supernatant could also have advantages over a recombinant cytotoxin subunit vaccine as this material could also contain additional protective Moraxella spp. antigens. We also recently began investigating mucosal vaccines against IBK. Those projects arose out of observations that corneal ulcers caused by M. bovis infection were more shallow in immunosuppressed Holstein calves versus normal calves, possibly because neutrophils, attracted to the eye by complement factors released during IgG binding to antigen and complement fixation, release degradative enzymes into the corneal stroma and exacerbate corneal injury. Parenteral vaccination that stimulates a robust IgG response may therefore lead to high concentrations of ocular antigen-specific IgG since most IgG in bovine tears derives from plasma. Immunoglobulin A (IgA), the most abundant antibody type on mucosal surfaces such as the eye, does not fix complement. We have now developed and tested an intranasal recombinant cytotoxin subunit vaccine in cattle. Positive results that showed augmented ocular mucosal immunity in vaccinates led us to test this vaccine at the Sierra Field Station during summer 2016 (manuscript in preparation). Preliminary results suggest that ulcer sizes in vaccinates were less than in controls; also, control animals required more frequent antibiotic antibiotic and NSAID treatments versus vaccinates. Nevertheless, the group that received the recombinant cytotoxin subunit vaccine still experienced IBK and we believe that additional improvements are necessary. For this project we propose to use a native M. bovis and M. bovoculi cytotoxin derived from culture supernatants as antigen with the mucoadhesive polymer, polyacrylic acid, that we have previously tested.Selected References:Henson JB, Grumbles LC. Infectious Bovine Keratoconjunctivitis. I. Etiology. Am J Vet Res 1960;21:761-766.Angelos JA, Spinks PQ, Ball LM, et al. Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis. Int J Syst Evol Microbiol 2007;57:789-795.Gould S, Dewell R, Tofflemire K, et al. Randomized blinded challenge study to assess association between Moraxella bovoculi and Infectious Bovine Keratoconjunctivitis in dairy calves. Vet Microbiol 2013;164:108-115.Kagonyera GM, George LW, Munn R. Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells. Am J Vet Res 1989;50:10-17.Lehr C, Jayappa HG, Goodnow RA. Serologic and protective characterization of Moraxella bovis pili. Cornell Vet 1985;75:484-492.Lepper AW. Vaccination against infectious bovine keratoconjunctivitis: protective efficacy and antibody response induced by pili of homologous and heterologous strains of Moraxella bovis. Aust Vet J 1988;65:310-316.Angelos JA, Ball LM. Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis. Vet Microbiol 2007;124:382-386.Ostle AG, Rosenbusch RF. Immunogenicity of Moraxella bovis hemolysin. Am J Vet Res 1985;46:1011-1014.Angelos JA, Ball LM, Hess JF. Identification and characterization of complete RTX operons in Moraxella bovoculi and Moraxella ovis. Vet Microbiol 2007;125:73-79.George LW, Borrowman AJ, Angelos JA. Effectiveness of a cytolysin-enriched vaccine for protection of cattle against infectious bovine keratoconjunctivitis. Am J Vet Res 2005;66:136-142.Angelos JA, Bonifacio RG, Ball LM, et al. Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine. Vet Microbiol 2007;125:274-283.Angelos JA, Hess JF, George LW. Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine. Vaccine 2004;23:537-545.Angelos JA, Lane VM, Ball LM, et al. Recombinant Moraxella bovoculi cytotoxin-ISCOM matrix adjuvanted vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis. Vet Res Commun 2010;34:229-239.Angelos JA, Chigerwe M, Edman JM, et al. Systemic and ocular immune responses in cattle following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid. Am J Vet Res 2016;77:1411-1418.Angelos JA, Edman JM, Chigerwe M. Ocular immune responses in steers following intranasal vaccination with recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid. Clin Vaccine Immunol 2014;21:181-187.Kagonyera GM, George LW, Munn R. Light and electron microscopic changes in corneas of healthy and immunomodulated calves infected with Moraxella bovis. Am J Vet Res 1988;49:386-395.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The overarching goal of this project will be to determine if an intranasal native M. bovis/M. bovoculi cytotoxin vaccine adjuvanted with polyacrylic acid will prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye). To achieve this goal the following specific aims will be undertaken: #1: conduct a randomized controlled field trial to determine if the cumulative proportion of corneal ulcers due to IBK in calves vaccinated with either: a) M. bovis cytotoxin/M. bovoculi cytotoxin adjuvanted with polyacrylic acid (adjuvant); or b) adjuvant alone; #2: determine if calves vaccinated with this cytotoxin vaccine experience less severe IBK as determined by corneal ulcer surface areas and ulcer healing times; #3: determine if calves vaccinated with the cytotoxin vaccine have reduced requirements for antibiotic and non-steroidal anti-inflammatory drug treatments versus control calves; and #4: collect serum and tears from a cohort of vaccinates in each group for subsequent quantitation of M. bovis/M. bovoculi cytotoxin neutralizing antibody responses and tear/serum antigen specific IgA/IgG responses following vaccination.
Project Methods
Design - Frozen pathogenic isolates of Moraxella bovis Tifton 1 and Moraxella bovoculi 237 to generate native cytotoxin antigen are available in Dr. Angelos' laboratory. These isolates will be propagated in heart infusion broth. Native cytotoxin antigen retained in the diafiltered retentate (DR) of culture supernatants will be prepared according to published methods. Native cytotoxin retained in DR will be combined 1:1 (based on protein content) and adjuvanted with polyacrylic acid (Carbigen™; MVP Technologies (Phibro Animal Health)) according to manufacturer instructions. The final vaccine will be formulated to deliver 500 µg protein in a 2 cc total volume. Calves at the Sierra Field Station will be vaccinated intranasally starting in early March/April with either: A) native M. bovis and M. bovoculi cytotoxins adjuvanted with polyacrylic acid; or B) adjuvant alone. Based on a power of 80%, α=0.05, and historic proportions of calves developing IBK between vaccinates and controls, the minimum number of calves required in each of the two study groups is calculated to be 76. Calves will be randomly assigned to receive the intranasal cytotoxin vaccine or control vaccine (2 cc total volume delivered in one nostril) on day 0 and day 21. Pre- and once weekly (for 16 weeks) post-enrollment examinations including ulcer scoring and ulcer size determinations will be performed to identify animals that develop corneal ulcerations associated with IBK; calves with ulcer scores greater than 2 will be treated with oxytetracycline (20 mg/kg SC) and calves exhibiting a high degree of ocular pain/discomfort will receive intravenous flunixin meglumine (a non-steroidal anti-inflammatory drug; NSAID). The investigators will remain blinded as to what vaccine each calf receives at enrollment and throughout all post-enrollment examinations. Tears and whole blood for serum will also be collected from a randomly selected cohort of 25 calves in each group on day 0, day 42 (3 weeks post booster), and day 112 (final study day). The sera and tears will be subsequently assayed for M. bovis and M. bovoculi cytotoxin neutralizing antibody responses and cytotoxin-specific serum/tear IgG/IgA concentrations. Anticipated results - We predict that the proportion of calves with IBK, ulcer healing times, and requirements for antibiotic and NSAID treatments will be lowest in calves receiving the cytotoxin vaccine. Data analysis - The primary outcome of interest to be evaluated will be the cumulative proportion of calves that develop IBK over the course of the 16 week trial. Secondary variables of interest will be the cumulative corneal ulcer surface areas, median time for a first ulcer to heal, and the proportions of calves requiring antibiotics/non-steroidal anti-inflammatory drug treatments. Data will be analyzed with the Pearson Chi Square test. Risk ratios with 95% confidence intervals for developing IBK will be calculated for the control and vaccine groups at weeks 8 and 16; risk ratios will be considered significant if the confidence intervals do not include a value of 1. The Mann-Whitney test will be used to evaluate differences between groups with respect to median time for a first ulcer to heal and corneal ulcer surface areas. A value of P < 0.05 will be considered significant.