Performing Department
Veterinary Clinical Sciences
Non Technical Summary
Equine infectious keratitis is frequently reported in horses. Aggressive medical and/or surgical therapy is required to control the infection, inflammation, and pain. Treatment is typically prolonged and expensive. Infectious keratitis causes marked discomfort, and the resultant pain can be difficult to control. Significant vision loss or loss of the eye is not uncommon. Detection of the underlying etiologic agent is often difficult due to the limited sample size available for culture and the propensity for mycotic organisms to reside deep within the corneal stroma, which often precludes collection of a suitable diagnostic sample. Furthermore, mycotic cultures may take several weeks before fungal growth ensues during which the corneal infection will be controlled or the progress to corneal perforation, all without the benefit of a definitive diagnosis. While pharmacologic advances have bolstered the armamentarium available to address the underlying etiologic agents, infectious keratitis remains a therapeutic problem. Achieving a therapeutic concentration of any drug within the cornea is a formidable task due to the short contact time with the ocular surface and the difficulty in applying the drugs. The corneal epithelial toxicity of most pharmacologic agents further complicates the treatment regimen. Additionally, the reflex anterior uveitis associated with infectious keratitis can be very severe and result in significant intra-ocular scarring. The prolonged use of non-steroidal anti-inflammatory agents (NSAIDS) to control the reflex uveitis can result in gastro-intestinal and renal complications. Improved diagnostic modalities, less epithelio-toxic therapies, and enhanced treatments to resolve reflex uveitis would greatly facilitate management of equine infectious keratitis. The other principal equine ocular challenge is equine recurrent uveitis (ERU). Equine recurrent uveitis was first described in the 4th century AD and remains the most common cause of blindness in horses. The economic impact is significant especially when one considers training disruptions, decreased performance, and the withdrawal from competition in addition to the treatment costs themselves. The prevalence of equine recurrent uveitis in the United States is approximately 8%. Successful management of ERU focuses on halting inflammation. However, horses afflicted with the insidious form of ERU frequently do not demonstrate overt clinical signs of ocular discomfort. Therefore owners often fail to recognize the presence of intraocular inflammation. In addition, detection of a low-grade inflammatory response can be difficult, particularly for general practitioners in a field setting. Consequently, despite advances in therapy and diagnosis, 1 to 2% of horses continue to suffer clinical disease severe enough to threaten vision. The sequelae of the persistent inflammation include marked visual compromise and chronic discomfort. Unfortunately the underlying etiologic agent responsible for ERU has yet to be elucidated. Therefore, we have no means of preventing ERU. All current therapies are directed at preventing further bouts of uveitis.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall objectives of the research are as follows: Objective #1: Refine the therapeutic modalities used to treat glaucoma, a sequelae of infectious keratitis and equine recurrent uveitis (ERU). Hypothesis: Placement of an aqueous shunt will not result in serious complications including endophthalmitis, persistent inflammation, anterior chamber collapse, marked hypotony, hyphema, corneal edema, corneal ulceration, cataract formation, retinal detachment, anterior chamber tube displacement, slippage of the explant plate, scleral thinning, or scleral perforation. Expected outcomes: The aqueous shunts will be placed successfully and remain in situ for one month without causing serious ocular complications. Objective #2: Determine the value of advanced molecular technologies to detect infectious pathogens associated with infectious keratitis and ERU. Hypothesis: Universal bacterial and fungal primer PCR will have a high level of agreement with routine bacterial and fungal culture. Expected outcomes: The universal bacterial and fungal primer PCR will have a high sensitivity, high specificity, high positive predictive value, and high negative predictive value when compared to routine bacterial and fungal culture. Objective #3: Determine the corneal toxicity of tpical ocular therapeutic agents using cell culture models. Hypothesis: Prednisolone acetate will delay healing in the EpiOcularTM and ex vivo models, but diprofloxacin will not delay healing when compared to balanced salt solution. Expected outcomes: The EpiOcularTM and ex vivo models will demonstrate epithelial migration as expected during healing of in vivo corneal defects. The models will demonstrate delayed healing when exposed to prednisolone acetate.
Project Methods
Objective #1: Aqueous shunts will be placed in normal eyes of horses under general anesthesia. Routine anti-inflammatory and anti-bacterial therapy will be used post-operatively. Complete ocular examinations will be performed PI daily for the first week and then every 3rd day until the end of the study. Four weeks after implantation of the aqueous shunt, each horse will be euthanized and the globes collected for histologic evaluation. The following features will be graded histologically: degree of inflammation, degree of fibrosis surrounding the explant plate, corneal endothelial cell damage, scleral thinning, retinal degeneration, and cataract.Statistical analysis: Descriptive data will be presented for each of the criteria recorded during the clinical and histologic examinations. The cumulative 95% binomial confidence interval will be reported for serious complications. Objective #2: Horses presenting for suspected infectious keratitis will be enrolled. A swab will be used to collect samples for routine cytologic evaluation. Three culture swabs will be used to collect samples. Two will be sent to the Indiana Animal Disease Diagnostic Laboratory for routine fungal culture and bacterial culture and sensitivity. A third swab will be used for the PCR assay. DNA will be routinely extracted from the culture swab and used in the PCR assay. The primers target the bacterial and fungal internal transcribed spacer region (ITS). Negative and positive controls will be included. Statistical analysis: The sensitivity, specificity, positive predictive value, and negative predictive value of the PCR assay as compared to routine culture will be reported. Objective #3: At t=0 all samples will be wounded. The EpiOcularTM and rabbit corneas will be incubated at an air-liquid interface. Four times daily, either balanced salt solution, prednisolone ophthalmic or ciprofloxacin ophthalmic solution will applied to the apical surface of the construct and then progressively diluted. The rate of wound closure will be characterized. Histology: Samples from each group will be collected, routinely processed, and stained. Sections will be blindly evaluated for cell layer thickness, degree of cell degeneration and necrosis/apoptosis, and degree of epithelial detachment of the basement membrane. Immunohistochemistry: Sections will be incubated with mouse monoclonal anti-Ki67 primary or rabbit polyclonal anti-caspase 3 antibodies and then blindly evaluated for expression of each immunohistochemical stain. Cells with nuclear labeling will be counted and the staining index will be expressed as a percentage. In Situ Hybridization: The TUNEL assay will be performed. The number of TUNEL-positive nuclei will be counted and the staining index expressed as a percentage. Data Analysis: The rate of wound closure will be analyzed using a one-way ANOVA. The immunohistochemical and in situ hybridization data will undergo an arc sine square root transformation and then be analyzed using a one-way ANOVA. The histomorphologic alterations will be analyzed using the Kruskal-Wallis test and the Friedman test. For all significance will be set at p<0.05.