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. In contrast to human pinkeye, the disease in cattle is characterized by a painful corneal ulcer that may lead to permanently reduced vision and even blindness due to eyeball rupture in severe cases. In less severe cases ocular scarring occurs that impairs vision and leads to lost marketability of affected animals. Because cattle that develop IBK often require antibiotic treatment, the disease can be especially costly to organic and natural beef producers. For these reasons identifying better ways to prevent IBK is important for improving overall cattle health and welfare, and conducting research to discover better ways to prevent pinkeye is extremely relevant to the US cattle industry.
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 evaluate the ability of an intranasal vaccine to prevent infectious bovine keratoconjunctivitis (IBK; 'pinkeye') and reduce morbidity associated with this disease. The vaccine to be tested will be composed of antigens derived from Moraxella bovis and Moraxella bovoculi plus uninfected bacterial growth medium and adjuvanted with polyacrylic acid (Carbigen) and dimethyldioctadecylammonium bromide (Emulsigen-D). To achieve this goal the following specific aims will be undertaken: #1) vaccinate healthy beef calves at the Sierra Field Station (SFS) in early April with either: A) Moraxella spp antigens plus concentrated bacterial growth medium adjuvanted with Carbigen and Emulsigen-D; or B) adjuvant alone; #2) examine calves once weekly for 16 weeks for the development of IBK; #3) photograph and measure corneal ulcer sizes and ulcer scores in eyes of IBK-affected calves and record antibiotic and non-steroidal anti-inflammatory drug (NSAID) treatments in study calves; #4) determine if cattle vaccinated with the proposed vaccine have less severe IBK as determined by measurement of cumulative corneal ulcer surface areas, corneal ulcer scores, and requirements for antibiotic and NSAID treatments.
Project Methods
To prepare the Moraxella bovis and Moraxella bovoculi antigens, cultures of both organisms will be streaked onto cow blood agar plates to create lawns of bacteria and incubated at 35C. Following incubation the bacteria will be scraped from blood agar plates and resuspended in bacterial growth medium containing 1.5 mM CaCl2 (calcium chloride). This suspension will be used to inoculate flasks of growth medium containing 1.5 mM CaCl2. Flasks will be incubated at 35C with shaking at 200 rpm for 5 hours. Following incubation growth medium will becentrifuged, and supernatant will be sterile filtered. The filtrate will then be concentrated approximately 60 fold. The total protein in the finalproducts will be quantitated. An uninoculated growth medium antigen will be created following the same procedure, but without bacterial inoculation of the growth medium. The final vaccine antigens will be frozen at -20C until preparation of the vaccine.To prepare the final vaccine, the Moraxella antigens and growth medium antigens will be thawed and dialyzed against deionized water. The amount of antigen in the final vaccine will be calculated to deliver 500 ug of each antigen in a 2 cc dose. Adjuvanting with Carbigen + Emulsigen D will be performed according to adjuvant manufacturer's instructions. The final vaccines will be stored at 4C until use. For the control vaccine, sterile water will be adjuvanted with Carbigen + Emulsigen D per manufacturer's instructions.Enrollment/vaccination: Calves will be vaccinated at the SFS study site during early April. Prior to enrollment calves will be restrained in a hydraulic squeeze chute and both eyes will be examined grossly for evidence of preexisting corneal pathology. Only calves with 2 normal corneas (i.e., no presence of corneal ulcer/opacities/scars suggestive of previous pinkeye) will be enrolled. Calves will be allocated to either the control or vaccine groups by use of a blocked randomization scheme to maintain an approximate balance between the treatment groups. The individual administering the vaccines as well as the investigator performing all weekly ocular examinations will be blinded as to the contents of the vaccines that are being administered. On the day of enrollment (day 0) and on day 21 (booster vaccination) calves will be administered either the experimental vaccine or the control vaccine. The total vaccine dose will be 2 ml administered into one nostril through a flexible plastic tube.Weekly examinations/sample and data collection: Both eyes of each enrolled animal will be examined once weekly for 16 weeks following vaccination. Cattle eyes with grossly visible opacities will be stained with fluorescein to identify if an ulcer is present. Eyes with ulcers that are associated with mechanical trauma or the presence of a foreign body such as a plant awn will not be considered to have pinkeye unless an ulcer is still present in the affected eye during the next weekly examination. Each eye of each calf with a pinkeye-associated ulcer will be assigned a corneal ulcer score (CUS) based on the widest diameter of the ulcer (0 = no ulcer; 1 = diameter ≤ 5 mm; 2 = diameter > 5 mm; and 3 = perforated corneal ulcer). Eyes with IBK-associated ulcers will also be digitally photographed with a ruler positioned next to the eye for subsequent measurement of the corneal ulcer surface area. Cattle that develop a corneal ulcer with widest diameter greater than 5 mm (CUS=2) will be administered a subcutaneous (SC) dose of oxytetracycline (20 mg/kg). At the next weekly visit, calves showing further progression of corneal ulceration will be treated with florfenicol (40 mg/kg SC). Also, cattle that are affected with pinkeye and displaying evidence of severe ocular pain and visible discomfort will be administered flunixin meglumine (1 mg/kgIV).Ulcer area measurement: During the course of the summer field work, ulcer areas will be determined in the laboratory by tracing the ulcerated areas on a computer by use of publicly available image analysis software (ImageJ). Differences in magnification between photographs will be accounted for by standardizing the ruler scale from the ruler that was held next to the eye when it was photographed.The mean of 3 tracings of each ulcer will be used to calculate the corneal ulcer surface area. The limit of detection will be considered to be 0.008 cm2; this corresponds to the area of a 1-mm-diameter circle. Areas of fluorescein uptake that are <0.008 cm2 will not be considered to be ulcers in our data analysis.Means by which results will be analyzed, assessed, or interpreted: The primary outcome of interest to be evaluated will be cumulative (by week) corneal ulcer surface area in each group. Secondary variables of interest will be the number of antibiotic and NSAID treatments, cumulative corneal ulcer sizes by week, and corneal ulcer scores. The differences between groups in the cumulative corneal ulcer surface areas will be evaluated with the Mann-Whitney test and differences in the numbers of IBK-affected calves, and calves treated with oxytetracycline, florfenicol, and NSAIDs will be evaluated with the Fisher's exact test. A value of P < 0.05 will be considered significant.