Abstracts: 36th Annual Meeting of the American College of Veterinary Ophthalmologists, Nashville, TN, USA, October 12 15, 2005

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1 Veterinary Ophthalmology (2005) 8, 6, Blackwell Publishing, Ltd. Abstracts: 36th Annual Meeting of the American College of Veterinary Ophthalmologists, Nashville, TN, USA, October 12 15, 2005 NO.: 01 Effects of latanoprost on aqueous humor flow rate in normal dogs D. A. Ward Department of Small Animal Clinical Sciences, University of Tennessee College of Veterinary Medicine, Knoxville TN, USA Purpose: To evaluate the effects of topical application of latanoprost 0.005% (Xalatan ; Pfizer Inc., New York NY USA) on aqueous humor flow rate and intraocular pressure in normal dogs. Methods: Twelve healthy adult dogs were used in this study. All dogs were free of ocular abnormalities based on slit lamp examination, indirect ophthalmoscopy, applanation tonometry, and direct gonioscopy. Baseline aqueous humor flow rate (AHFR) was measured in both eyes of each dog using anterior chamber fluorophotometry. On the same day that baseline flow rates were measured, one drop of 0.005% latanoprost was applied to both eyes of each dog, and flow rate measurements were repeated using an interventional fluorophotometry protocol. Intraocular pressure (IOP) measurements were repeated 3 h after latanoprost administration. IOP and AHFR data for both eyes were averaged for each dog, and the means of the 2 eyes were used in data analyzes. Pre-latanoprost AHFRs and IOPs were compared to postlatanoprost values using Wilcoxon signed rank test and paired t-test, respectively. Results: Mean (SD) pre- and postlatanoprost IOPs were (2.49) and (2.99) mmhg, respectively (P = 0.048). Intraocular pressures declined in 8/11 dogs (IOP data was lost in 1 dog), with an average decline of 12%. Median (25th, 75th percentiles) pre- and postlatanoprost AHFRs were 5.60 (4.36, 7.83) µl/min and 0.36 ( 0.39, 1.00) µl/min, respectively (P < 0.001). Flow rates declined dramatically in 12/12 dogs, with an average decline of 93%. Conclusions: The reduction in aqueous humor flow rate following latanoprost administration indicates a reduction in aqueous humor production. The extent of aqueous suppression was rapid and dramatic, corroborating clinical observations following topical latanoprost administration in dogs. Supported by the UTCVM Department of Small Animal Clinical Sciences. Commercial NO.: 02 Canine conjunctival vascular tumors of endothelial origin: a retrospective evaluation of 108 cases C. G. Pirie,* C. B. Thomas,* A. M. Knollinger* and R. R. Dubielzig *Eye Care for Animals, Salt Lake City, UT; and Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin Purpose: To evaluate the epidemiology and potential risk factors of canine conjunctival vascular tumors of endothelial origin. Methods: A case series of 108 cases (70 hemangiomas, 38 hemangiosarcomas) from a collection of 8300 canine submissions between 1989 and 2004 were evaluated using routine H&E. Signalment, location, pigmentation, size, duration, margins, adjuvant therapy, and geographic location, comparing ultraviolet exposure levels by state, were recorded. Follow up information was available for 49 cases. Cases were matched with two unaffected controls and compared using logistic regression analysis. Results: Average age was 8.6 years, with no sex predilection. The Basset hound, Boxer, Dalmatian, English Setter and Springer spaniel were over-represented. Breed class was statistically significant (P < ) with the Hound, Working, and Herding groups at increased risk, as compared to mixed breeds. Evaluation of UV exposure demonstrated a linear trend, compared to controls, with a significant correlation between hemangiosarcomas and increased exposure (P < ). Primary sites of involvement occurred within the nonpigmented leading edge of the third eyelid (41 of 108) and lateral bulbar conjunctiva (33 of 108). None of the tumors had evidence of metastasis, however, recurrence was reported and more likely with hemangiosarcomas (11 of 20) than hemangioma (3 of 29). Conclusion: These are benign tumors of endothelial origin with their predilection sites, involvement of the nonpigmented epithelium, and development within breed classes of likely increased outdoor activity strongly supportive of ultraviolet light as a significant risk factor. Surgical excision may be curative, however, recurrence is possible and more likely with hemangiosarcomas. Commercial interest: None. NO.: 03 Feline primary conjunctival tumors of endothelial origin: evaluation of 8 cases C. G. Pirie and R. R. Dubielzig Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, Madison WI USA Purpose: The purpose of this retrospective study was to evaluate feline primary conjunctival vascular tumors of endothelial origin. Methods: A case series of 8 cases (6 hemangiomas, 2 hemangiosarcomas) from a collection of 3460 feline submissions between 1993 and 2004 were evaluated using routine H&E. Signalment, outdoor activity, location, epithelial pigmentation, size, duration, margins, adjuvant therapy, and geographic location, comparing ultraviolet exposure levels by state, were recorded. Follow up information was available for 5 cases. Results: The average age was 10.6 years, with neutered males over-represented (6 of 8). The domestic shorthair cat was most commonly affected (5 of 8) and where available, 3 of 4 cases were from indoor/outdoor cats. The nictitans membrane was the most common anatomical site (3 of 8), with the left conjunctival surface most commonly involved (6 of 8). Average size and duration was 7.5 mm and 4.4 months, respectively. Seven of 8 cases were devoid of epithelial pigmentation in nonaffected areas and 6 of 8 cases were from states of high annual ultraviolet light exposure. Only cases of hemangiosarcoma recurred, however, following surgical revision, no further recurrence was reported. Conclusion: Feline conjunctival hemangioma and hemangiosarcoma are benign tumors of endothelial origin. No cases evaluated had evidence of metastatic disease at the time of excision. Surgical excision alone may be curative, however, recurrence is possible. These tumors demonstrate similar predilection sites and involvement of nonpigmented epithelium, as is true of canine involvement, which may relate to risk factors as well. Commercial NO.: 04 Septic implantation syndrome: analysis of 29 canine and 11 feline cases R. R. Dubielzig and C. G. Pirie Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW), School of Veterinary Medicine, University of Madison-Wisconsin, Madison WI USA Purpose: To evaluate clinical data and histological features of ocular submissions following penetrating ocular injuries, often due to a cat scratch. Methods: Twenty-nine of 9300 canine and 11 of 3400 feline submissions to COPLOW were selected and stained with H&E and a gram stain. Results: Histories of a cat scratch were obtained in 10 of 29 canine and 2 of 9 feline cases, respectively. No breed predilection was noted, however, small breed and adolescent large breed dogs were over represented. The Domestic shorthaired was most commonly affected. The average age was 7.4 years (3.5 months to 15 years) and 5.3 years (1 14 years) for canine and feline submissions, respectively, with no sex predilection. There was no globe predilection and the majority of submissions reported a chronic, poorly response, low-grade uveitis, with cataract development, in addition to, corneal scar, anterior or posterior synechiae, and secondary glaucoma. Duration was on average 2 months and 1.5 months, respectively. Histologically, features of chronicity including lymphogranulomatous endophthalmitis, preiridal fibrovascular membrane, and a collagen-rich, cell-poor membrane lining the anterior lens capsule, associated with anterior lens capsule rupture, cataractous changes, and intralenticular pyogranulomatous inflammation was noted. A full thickness corneal scar and intralenticular bacteria were often noted within the plane of section, with gram-positive cocci being the most common organism identified. Conclusion: Septic implantation demonstrates a clinical course and histological features of chronicity, with a pathogenesis believed to relate primarily to sepsis, rather than release of lens proteins (phacoclastic). Commercial NO.: 05 Glaucoma drainage implant surgery has a higher survival rate as a primary procedure in a rabbit model D. W. Esson,* M. B. Sherwood, E. M. Sampson, G. S. Schultz and D. A. Samuelson *Eye Care for Animals, Tustin, CA; Ophthalmology, Institute for Wound Healing, College of Veterinary Medicine, University of Florida, Gainesville FL USA Purpose: The purpose of this study was to investigate the effect of prior trabeculectomy on the survival of drainage implant surgery in a rabbit model. Methods: The study was divided into 3 parts; In part one, 12 rabbits underwent a trabeculectomy procedure and the blebs were allowed to fail. A second procedure was performed consisting of the insertion of a drainage cannula without a plate, to allow aqueous to shunt to the subconjunctival space in the vicinity of the previous, failed bleb. The length of time to bleb failure was monitored. Twelve further, un-operated rabbits underwent similar cannula insertion surgery and served as a control. In part two, pediatric Molteno drainage implants were placed in an additional 12 rabbits. Half of these rabbits underwent prior failed sclerostomy surgery, before Molteno implant insertion, and the remaining half had not undergone previous surgery. The bleb capsule surrounding the plate was examined histologically. In part three, conjunctival, Tenons capsule and scleral tissues from another 8 rabbits were examined on the fifth day following either cannula tube or pediatric Molteno implant insertion (performed either as primary procedures or following prior trabeculectomy surgery n = 2/group) and levels of Transforming Growth Factor Beta 2 (TGFβ 2 ) were measured by ELISA. Results: Bleb survival was significantly longer for drainage cannula tubes in eyes that had not undergone prior failed trabeculectomy surgery (18 days vs. 13 days P < ). Histological analysis of the capsules surrounding the Molteno implants showed that these were thicker and associated with greater number of inflammatory cells in those which had undergone prior failed surgery. Examination by ELISA showed significantly higher levels of TGFβ 2 in the tissues of rabbits undergoing either cannula or Molteno implant that had undergone prior (failed) sclerostomy surgery. Conclusions: This study supports the idea that drainage implant surgery may be more successful if performed as a primary procedure in cases of medically intractable glaucoma. Supported by Departmental Unrestricted RPB Grant. Commercial NO.: 06 Expression of transforming growth factor-β 2 following sclerostomy and ex-press glaucoma drainage implant beneath a scleral flap in a rabbit model D. W. Esson,* M. B. Sherwood, E. M. Sampson and G. S. Schultz *Eye Care for Animals, Tustin, CA; Ophthalmology, University of Florida; Institute for Wound Healing, University of Florida, Gainesville FL USA Purpose: Sclerostomy surgery may be successfully employed for the temporary relief of intraocular pressure spikes such as those occurring following uncontrolled uveitic glaucoma or cataract phacoemulsification. Transforming Growth Factor Beta 2 (TGFβ 2 ) has been shown to be locally induced following glaucoma surgery and to stimulate fibrosis and subsequent bleb failure. Clinically, eyes undergoing Ex-Press shunt implantation have anecdotally been noted to be less inflamed in the early postoperative period. This study was designed to determine whether placement of an Ex-Press glaucoma drainage shunt underneath a scleral flap excites less TGFβ 2 formation in the bleb tissues postoperatively compared to sclerostomy filtration surgery. Methods: One eye of 20 NZW rabbits was randomly selected to receive either sclerostomy filtration surgery or implantation of an Ex-Press drainage shunt beneath a partial thickness scleral flap. Conjunctival, tenon s capsule and scleral tissues were harvested from the bleb site 5 days after the surgery and levels of TGFβ 2 measured by ELISA. Tissues 180 from the bleb were also harvested to serve as controls. Results: Significantly higher levels of TGFβ 2 were detected in the bleb tissues of the eyes that underwent sclerostomy filtration procedure (mean of 150 pg/mg tissue) than those which received the Ex-Press shunt under a scleral flap (mean 70 pg/mg tissue). Conclusions: Ex-Press drainage shunts are easy to place and may 2005 American College of Veterinary Ophthalmologists

2 438 S induce a less aggressive postoperative inflammatory and subsequent fibrous response. Supported by Departmental Unrestricted RPB Grant. Commercial NO.: 07 Evaluation of the effect of laser cyclophotocoagulation on the success of glaucoma drainage implant surgery in a rabbit model D. W. Esson,* G. Gum, J. Brinkis,* R. H. Scagliotti,* M. B. Sherwood, E. M. Sampson and G. S. Schultz *Eye Care for Animals, Tustin, CA; Biological Test Center, Irvine, CA; Ophthalmology and Institute for Wound Healing, University of Florida, Gainesville FL USA Purpose: Cyclo-destructive surgery and drainage implant surgery are commonly performed in glaucomatous veterinary patients and may both be required in order to achieve optimal intraocular pressure (IOP) control. No guidelines exist regarding the sequencing of these procedures. The aim of this study was to investigate the effect of cyclo-destructive surgery on the survival of drainage implant surgery in a rabbit model. Methods: Ten rabbits were used in each of four parts of the study (n = 40). In part one, glaucoma filtration surgery (GFS) to allow aqueous to shunt to a subconjunctival bleb was performed (n = 10). In part two of the study, rabbits underwent laser cyclophotocoagulation 48 h prior to similar GFS (n = 10). In part three rabbits underwent laser cyclophotocoagulation 48 h following similar GFS (n = 10) and in part four of the study, rabbits underwent combined laser cyclophotocoagulation and GFS (n = 10). Two rabbits from each group were sacrificed 5 days postoperatively so that levels of Connective Tissue Growth Factor (CTGF) and Transforming Growth Factor Beta 2 (TGFβ 2 ) could be assessed in conjunctival, tenons and scleral tissues. An additional rabbit was sacrificed on each of days 5 and 14 postoperatively from each of the four groups, for histological analysis. Clinical bleb survival was monitored in the remaining animals. Results: Rabbits undergoing cannula filtration only, exhibited bleb failure by day 16 postoperatively, rabbits undergoing staggered cyclophotocoagulation and GFS surgeries exhibited similar bleb failure by an average of 25 days postoperatively and rabbits undergoing combined cyclophotocoagulation and GFS surgeries exhibited bleb failure by an average of 17 days postoperatively. Levels of CTGF and TGFβ 2 were lowest in rabbits undergoing cannula filtration only (323.6 pg/ml & 23.7 ng/ml, respectively), higher in rabbits undergoing staggered cyclophotocoagulation and GFS (422.3/686.8 pg/ml & 44.3/45.4 ng/ml, respectively) and highest in rabbits undergoing combined cyclophotocoagulation and GFS surgeries (303.9 pg/ml & 59.8 ng/ml, respectively). Histological analysis of bleb tissues revealed that animals undergoing combined cyclophotocoagulation and GFS surgeries exhibited a more intense inflammatory reaction 5 days postoperatively, characterized by greater numbers of heterophils and organizing fibroblasts. Conclusions: The results of this study suggest that while combined cyclo-destructive surgery and drainage implant surgery may provide increased long-term IOP control, these procedures may be more effective if performed separately. Supported by ACVO Resident Grant. Commercial NO.: 08 Development of gene microarray chips for canine and rabbit ocular tissues D. W. Esson,* K. N. Gelatt, E. Mackay, M. Popp, L. Liu, G. Wistow, J. M. Hill, G. S. Schultz** and M. B. Sherwood *Eye Care for Animals, Tustin, CA; College of Veterinary Medicine, University of Florida; UFSCC/ICBR Microarray Core, University of Florida; Molecular Structure & Functional Genomics, National Eye Institute, Bethesda, MD; Department of Ophthalmology, Louisiana State University; and **Institute for Wound Healing and Ophthalmology, University of Florida, Gainesville FL USA Purpose: Microarray technology allows broad analysis of changes in gene expression across biological processes, using probes which simultaneously represent thousands of genes. In spite of the importance of the rabbit and the dog in research and veterinary medicine, microarray gene chips have not been available for these species. This project was designed to develop and test microarrays for rabbit and dog ocular tissues. Methods: Normal ophthalmic tissues were harvested from NZW rabbit and beagle dog eyes and processed through the NEI bank. Five cdna libraries (rabbit lens library failed) were constructed and almost 4000 clones from each library sequenced. Sequences were assembled and filtered by UF/ICBR to derive 2 sets of nonredundant sequences for dog and rabbit, respectively, which were annotated with sequence orientation, gene identity and GeneOntology terms. Custom arrays were manufactured by Agilent Technologies. Dog chips were manufactured as two individually hybridizable 11K arrays per slide, covering ophthalmic genes. Rabbit chips were manufactured as 2 sets of 8 individually hybridizable 1.5K arrays per slide, covering 1578 cornea-specific genes and 1578 genes expressed in the rest of the eye. Results: Initial array testing indicated that over 99% of probe genes were present above background levels. Tissues from rabbits undergoing glaucoma filtering surgery indicated changes in the expression of genes known to be involved in wound healing, including TGFβ 2, MMP2, MMP9, collagen, vimentin and fibronectin. Tissues from rabbits undergoing latent Herpesviral keratitis re-activation indicated changes in the expression of a number of defense related genes, some whose expression (MHC class II histocompatibility antigen, cytochrome c oxidase subunit II) have been altered in previous studies. Conclusions: Initial results demonstrate these new microarray chips may provide a valuable tool for ocular research. Supported by University of Florida Research Opportunity Fund Grant. Commercial NO.: 09 A sequential multiple treatment approach to influence wound healing following glaucoma filtration surgery in a rabbit model D. W. Esson,* M. B. Sherwood, S. S. Tuli, E. M. Sampson, G. S. Schultz and D. A. Samuelson *Eye Care for Animals, Tustin, CA; Ophthalmology, Institute for Wound Healing, and College of Veterinary Medicine, University of Florida, Gainesville FL USA Purpose: The purpose of this study was to evaluate the concept of targeting specific mediators of the wound healing process following glaucoma filtration surgery (GFS) at multiple points across the course of bleb failure, in order to prolong bleb survival and minimize the unwanted side-effects of the antimetabolites which are currently in use. Methods: In part one of the experiment, cannula GFS was performed on 32 NZW rabbits. In each of four eyes, filtering blebs were treated with Balanced Salt Solution (BSS), mitomycin-c 0.4 mg/ml (MMC), Transforming Growth Factor Beta 2 (TGFβ 2 ) antibody, TGFβ 2 antisense, Connective Tissue Growth Factor (CTGF) antibody, CTGF antisense, Mannose-6-Phosphate (M-6-P) or the broad spectrum MMP inhibitor Ilomastat (GM6001). Bleb survival was assessed by grading bleb height and width and IOP was followed. In part two, the same GFS surgery was performed on18 NZW rabbits. Six of the filtering blebs were treated with either BSS only, MMC (0.4 mg/ml) only or a sequential protocol consisting of M-6-P (200 mm) at the time of surgery, subconjunctival injections of CTGF antibody at days 2 & 4 and Ilomastat at days 7, 12 and 20 postoperatively. Bleb survival was again assessed by grading bleb height, width and IOP. Blebs were also examined histologically, 14 days postoperatively. Results: All the individual therapies resulted some improvement in bleb survival compared to the BSS control. Blebs treated with the sequential, multiple treatment protocol survived an average of 31 days (regression slope P < compared to control), those receiving BSS an average of 19 days and those treated with MMC (0.4 mg/ml) an average of 39 days. Histology of the conjunctival tissues showed a marked reduction in epithelial thinning and goblet cell loss in the multiple-treatment group, when compared with the MMC blebs. Conclusions: A sequential multiple therapy approach in a rabbit model of GFS, prolonged bleb survival and decreased the unwanted side-effects of the currently used, nonspecific anti metabolites. Supported by Departmental Unrestricted RPB Grant. Commercial NO.: 10 Brainstem auditory evoked responses and ophthalmic findings in llamas and alpacas in eastern Canada A. A. Webb,* C. L. Cullen and L. A. Lamont *Department of Biomedical Sciences and Department of Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Canada Purpose: To determine if coat or iris color or both are related to congenital deafness in llamas and alpacas in eastern Canada, and to describe the ophthalmic findings in this group of animals. Methods: Seventeen llamas (females) and 23 alpacas (17 females; 4 males; 2 geldings) were examined. Age, and coat and iris colors were recorded. Hearing was evaluated by using brainstem auditory evoked response (BAER) testing. BAERs were recorded using a portable BAER evaluation system (BAERCOM, UFI). Two to three thousand monaural clicks were delivered at 80 db at a rate of approximately 40/s via an earphone that was inserted deep within the external ear canal. Positive hearing ability was determined by identifying peaks I and V on the BAER recording. Latencies were determined manually for peaks I and V of the BAER. Ophthalmic examination included neuro-ophthalmic examination, applanation tonometry, and slit-lamp biomicroscopic and indirect ophthalmologic examinations. Results: Mean age (± SD) of llamas and alpacas was 9.6 (± 5.1) and 3.8 (± 3.6) years, respectively. The llamas were significantly older than the alpacas (P < 0.001). Latencies for peaks I and V for llamas were (mean ± SD) 1.8 ± 0.2 ms and 4.9 ± 0.3 ms, respectively. Latencies for peaks I and V for alpacas were (mean ± SD) 1.9 ± 0.2 ms and 4.9 ± 0.2 ms, respectively. Regardless of the coat or iris color, all animals examined by BAER testing were found to be hearing bilaterally. Ophthalmic examination revealed overall mean (± SD) IOPs for llamas (33 eyes) and alpacas (46 eyes) were 16 (± 5) mmhg and 19 (± 4) mmhg, respectively. Llamas had significantly lower mean IOP compared to alpacas (P < 0.001). There were no differences between IOPs for different genders of alpacas (P > 0.05) nor were there differences in IOP measurements between eyes within each species (P > 0.05). Main ocular findings included iris-to-iris persistent pupillary membranes (PPMs) (14/17 llamas; 5/23 alpacas); p.p.m. pigment spots (anterior lens capsule) (1/17 llamas; 1/23 alpacas); incipient or immature capsular and/or cortical cataracts (9/17 llamas; 4/23 alpacas); superficial corneal scarring (1/17 llamas; 1/23 alpacas); anterior stromal corneal dystrophy (2/23 alpacas); conjunctival or palpebral mass (2/23 alpacas); unilateral enucleation (1/17 llamas); and no abnormal ocular findings (1/17 llamas; 10/23 alpacas). Conclusions: No animals were deaf. The most common ocular abnormalities noted were iris-to-iris PPMs and cataracts. Llamas and alpacas should be screened prior to breeding for potentially inherited ocular conditions. Supported by a grant from the Atlantic Veterinary College Research Fund, UPEI. Commercial NO.: 11 Schirmer tear test values and tear film break-up times in cats with conjunctivitis C. L. Cullen* and C. C. Lim *Department of Companion Animals and Veterinary Teaching Hospital, Atlantic Veterinary College, University of Prince Edward Island, Canada Purpose: (1) To document tear film break-up time (TFBUT) in a group of cats with conjunctivitis; (2) to determine if TFBUTs from cats with conjunctivitis vary significantly from previously established normal values for TFBUT in young cats without ocular disease; (3) to determine if a correlation exists between Schirmer tear test (STT) values and TFBUTs in cats with conjunctivitis; (4) to determine if the TFBUTs in cats with conjunctivitis are influenced by the detection of DNA from feline herpesvirus-type 1 (FHV-1), Chlamydophila felis, Mycoplasma spp., and feline calicivirus. Methods: Fourteen cats between the ages of 0.8 years to 12 years with active, untreated conjunctivitis and without active keratitis or other ocular or systemic abnormalities were included in this study. Complete ophthalmic examinations, including, STT and TFBUT, were performed on all cats. Polymerase chain reaction (PCR) screening for FHV- 1, Chlamydophila felis, Mycoplasma spp., and feline calicivirus was performed on conjunctival swabs from affected eyes and blood samples from all cats. Results: Mean (± SD) STT values for both eyes combined was 14.9 (± 8.5) mm/minute. Mean (± SD) TFBUT for cats in this study was 8.9 (± 4.8) seconds in the right eye (OD) and 8.1 (± 4.6) seconds in the left eye (OS). No correlation existed between mean TFBUTs and mean STT values OD or OS. Conjunctival swabs from 7 cats (n = 9 eyes) tested positive via PCR for one of the above infectious agents. Blood samples from 9 cats tested positive for FHV-1. Mean TFBUTs for cats from which the DNA from FHV-1 was isolated from the blood were significantly lower than mean TFBUTs for cats from which no such DNA was isolated from the blood. Conclusions: In this study, the mean TFBUT in cats with conjunctivitis was significantly lower than previously established values for clinically healthy cats. This supports the theory that qualitative tear film deficiency, and thus tear film instability, may play a role in the pathogenesis of feline conjunctivitis. Qualitative tear film deficiency may predispose to the development of conjunctivitis or may occur secondarily to this condition. Supported by a grant from the Companion Animal Trust Fund, Atlantic Veterinary College, University of Prince Edward Island. Commercial

3 S 439 NO.: 12 Quantification of FHV-1 DNA from the conjunctiva of cats with and without conjunctivitis H. C. Low, C. C. Powell and M. R. Lappin Department of Clinical Sciences, Colorado State University, Fort Collins CO USA Purpose: The purpose of this study was to determine if FHV-1 DNA copy numbers, measured by fluorogenic (real-time) polymerase chain reaction assay (RT-PCR), correlate to the presence or absence of conjunctivitis. Methods: Cats sampled to date (n = 67) included those with active conjunctivitis (20), those with a history of conjunctivitis which was resolved for at least 3 months (18), and those that have never had conjunctivitis (29). Cotton tipped applicators were rolled in the ventral conjunctival cul-de-sac and placed in 1.0 ml sterile 0.01 M PBS. Samples were kept 2 3 h at room temperature then stored at 70 C until analyzed. DNA was extracted using a commercial kit (QIAamp DNA minikit, Qiagen, Inc, Valencia, CA) and assayed for FHV-1 DNA using adaptations of a previously published protocol. Results: By use of RT- PCR, FHV-1 DNA was amplified from 4/20 cats with conjunctivitis (20%, mean FHV-1 copy/cell equivalent), 1/18 cats with previous conjunctivitis (5%, FHV-1 copy/cell equivalent), and 1/29 cats that never had conjunctivitis (3.4%, FHV-1 copy/ cell equivalent). Conclusions: FHV-1 prevalence rates were similar to those previously reported but more cats are needed to determine if results of this RT-PCR correlate to presence or absence of disease. Additional cats are currently being tested. Supported by the American College of Veterinary Ophthalmologists Vision for Animals grant, # Commercial interests: None. NO.: 13 Ophthalmic inflammation and increased metalloproteinase activity in ocular tissues arise in parallel with the development of laminitis in horses M. K. Zarfoss,* P. J. Johnson and E. A. Giuliano *University of Wisconsin-Madison; and University of Missouri-Columbia, Columbia MO USA Purpose: Demonstration of uveitis and increased matrix metalloproteinase (MMP) activity in ocular tissues from horses with alimentary laminitis suggests that laminitis is a component of a systemic inflammatory process with ophthalmic consequences. Methods: Four healthy adult horses with normal ophthalmic examination results (indirect fundic examination and slit-lamp biomicroscopy) were subjected to a laminitis-inducing dose of starch via nasogastric tube. Pre-treatment aqueous humor was collected via paracentesis under general anesthesia. Ophthalmic examination and tear collections were performed once daily, both pre- and posttreatment. Analysis of tears and aqueous humor for MMP activity was performed using gelatin zymography on samples with equivalent protein content (Bradford protein assay). Results: Ocular inflammatory changes, including the development of aqueous flare (n = 4) and keratic precipitates (n = 1), were identified in all horses. Clinical laminitis occurred in 3 of 4 horses. Metalloproteinase-9 activity was increased post-treatment in aqueous humor from 2 of 4 horses and in tears from 1 of 4 horses. Conclusions: Ophthalmic inflammation and increased MMP-9 activity occurred with the development of laminitis in horses subjected to carbohydrate overload. Laminitic horses may benefit from complete ophthalmic examination and topical anti-inflammatory medications. The carbohydrate overload model represents a potential model of equine uveitis. Supported by a Phi Zeta Research Grant. Commercial NO.: 14 Aqueous humor myocilin in dogs with glaucoma and cataract K. N. Gelatt,* E. O. MacKay,* M. E. Kallberg,* D. A. Samuelson,* K. P. Barrie,* F. J. Ollivier,* C. E. Plummer,* K. Tenorio,* K. Kligman,* B. Cartlidge,* W. Miller, J. S. Sapienza, S. E. Andrew and H. Denis *University of Florida, Gainesville FL; Advanced Animal Eye Care, Memphis TN; Long Island Veterinary Specialists, Plainview, NY; Georgia Veterinary Specialists, Atlanta, GA and Animal Eye Associates, Maitland FL USA Introduction: Myocilin has been linked to the increase in intraocular pressure in juvenile- and adult-onset POAG in man, and more recently to POAG in the Beagle. Three major posttranslation complexes of myocilin have been described in POAG in man. The exact role of myocilin in the genesis of ocular hypertension is still unknown. Purpose: The goal of this study is to compare the aqueous humor levels of myocilin between primary glaucoma, secondary glaucoma, primary cataracts and diabetic cataracts of different breeds of dogs. Methods: Aqueous humor samples ( ml) were obtained from the anterior chambers of over 300 dogs of various breeds. Each dog s aqueous humor samples was diagnosed as either primary glaucoma, secondary glaucoma, cataracts, or cataracts due to diabetes. Two ml aqueous samples from different dogs were loaded and run on 12% Bis-Tri gels (Invitrogen, Carlsbad CA). The separated proteins were then stained using the microwave Simply Blue Safestain protocol described in the Invitrogen 12% Bis-Tris gel manual, and imaged using a Bio-Rad Scanner and Quantity One software for analysis. Western blot analysis was performed by running the samples on a 12% Bis-Tris Gel. The separated proteins were then transferred onto nitrocellulose membranes and the blot incubated with an antimyocilin rabbit polyclonal antibody and stained (Supra Signal West Pico Chemiluminescent, Pierce, Rockford IL) for scanning. Results: Myocilin levels were detected in all dogs. The overall levels of myocilin were highest in the primary and secondary glaucoma dogs; there was no significant difference between the two groups. Low levels occurred in the primary cataract and the diabetic cataract eyes. In the glaucoma eyes the lightest band of myocilin (Myocilin Complex A) tended to show the greatest change, with great diffusion and an increase in estimated amount relative to the other two myocilin bands (Myocilin Complex B and C). Conclusions: Aqueous myocilin levels were significantly increased in glaucomatous eyes of many breeds of dogs with three discreet bands observed, similar to that reported in POAG of the Beagle. Aqueous myocilin levels in cataractous canine eyes were similar to those reported in normal Beagles. Increased levels of myocilin in the aqueous humor occurs in many breeds of dogs with primary glaucoma. Supported by the Jaqua Foundation and Gwathmey-Adams Laboratory for Vision Science. Commercial NO.: 15 Retinopathy of Coton de Tulear dogs B. H. Grahn, L. S. Sandmeyer and C. B. Breaux Department of Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Purpose: To document the clinical manifestations, development, and progression of the retinopathy of Coton de Tulear dogs. Methods: Ten Coton de Tulear dogs were examined with biomicroscopy, indirect ophthalmoscopy, photopic and scotopic electroretinography, fluorescein and indocyanine green angiography, and fundic photography for two years. Results: The retinopathy of Coton de Tulear dogs manifests between 3 and 4 months of age. Photopic and scotopic electroretinography failed to identify significant retinal impairment. Leakage of fluorescein or indocyanine green was not detected during repeated angiograms. Serial fundic photography confirmed minimal progression of lesions beyond one year of age. Conclusions: Retinopathy of Coton de Tulear dogs manifests early in life as nonprogressive multifocal bullous retinal detachments. The blood ocular barrier remains intact. Photopic and scotopic electroretinography were not significantly different from age-matched dogs. Supported by CAHF grant Commercial NO.: 16 Incidence of congenital stationary night blindness in a population of Appaloosa horses and clinical and electroretinographic findings in affected horses L. S. Sandmeyer,* B. H. Grahn,* C. B. Breaux* and S. Archer *Department of Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan; and Moose Jaw, Saskatchewan, Canada Purpose: To investigate a population of Appaloosa horses for incidence of congenital stationary night blindness (CSNB), and further characterize the clinical and electroretinographic findings in affected animals. Methods: Twenty Appaloosas underwent examination including neuroophthalmic examination, slit-lamp biomicroscopy, indirect ophthalmoscopy and streak retinoscopy. Photopic and scotopic flash electroretinography (ERG), rod and cone flickers, and oscillatory potentials were recorded. Results: Five Appaloosa horses were determined to have CSNB. Of the affected horses, two were related. Three horses had a history of behavioral changes in the dark. Dorsolateral strabismus was noted in one horse. Streak retinoscopy showed no difference in refractive errors between affected and nonaffected horses. Affected horses had negative scotopic ERGs with absence of a b-wave. Photopic a and b-wave implicit times were increased, b-wave amplitudes were decreased, and a- wave amplitudes were increased in affected compared to nonaffected horses. Cone flickers were not significantly different from nonaffected horses. Rod flicker waveforms appeared reversed in polarity and were reduced in amplitude in affected horses. Oscillatory potentials were reduced in amplitude in all affected horses. Conclusions: CSNB may be more prevalent in the Appaloosa population than is recognized. Myopia was not a significant feature of CSNB. ERG findings confirm abnormalities in the outer plexiform layer or inner nuclear layer. Supported by Equine Health Research Fund, Western College of Veterinary Medicine. Commercial NO.: 17 Retrospective immunohistochemical and clinical investigation of episcleritis in the dog C. B. Breaux, B. H. Grahn and L. S. Sandmeyer Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada Purpose: The purposes of this study were: (1) to use immunohistochemistry to characterize the inflammatory cell types present in dogs with episcleritis, and (2) to determine if the cell types present were predictive of clinical outcome. Methods: Twenty-seven cases of canine episcleritis confirmed by episcleral biopsy were selected. Biopsies were immunohistochemically stained with CD3, HM47, and CD18 to identify T cells, B cells and histiocytes, respectively. They were also stained with hematoxylin & eosin and mason s trichrome. Slides were digitally photographed and positive cells were counted. Signalment, duration of illness, affected eye(s), treatment, and clinical outcome were reviewed retrospectively. Results: There were 6 cases of nodular granulomatous episclerokeratitis (NGE) and 21 cases of episcleritis (9 bilateral, 12 unilateral). The age range was 1 12 y for NGE (avg 5 y) and the age range for episcleritis was 1 15 (avg 5.5 y). The Cocker Spaniel was overrepresented in the episcleritis group and the Collie was overrepresented in the NGE group. Medical therapy for > 1 years was commonly required to maintain remission in all groups. The ratio of T cells to B cells was 1 : 1.8 for NGE cases, 1 : 1.9 for unilateral cases, and 1 : 1.2 for bilateral cases. The ratio of histiocytes to lymphocytes was 1 : 4.8 in NGE cases, 1 : 4.3 in unilateral cases, and 1 : 10 in bilateral cases. Conclusions: The variation in lymphocyte and histiocyte ratios between the NGE, unilateral and bilateral groups may represent differences in the pathogenesis of canine episcleritis. Supported by a Companion Animal Health Fund grant ( ). Commercial NO.: 18 Tracing ocular characteristics of vertebrates by phylogeny using a computer program to assess a likely evolutionary history M. K. Zarfoss,* N. A. Dillon,* J. L. Skulan and R. R. Dubielzig* *School of Veterinary Medicine, and Geology and Geophysics, University of Wisconsin-Madison, Madison, WI USA Purpose: As a preliminary trial-run, using morphologic criteria of normal ungulate eyes we will assess the likely evolutionary pathway making use of modern technologies to calculate the most efficient evolution. Methods: Using a collection with more than 900 examples of normal vertebrate eyes from a wide range of species in all major and most minor groupings of vertebrates, we will sample the ungulates (perissodactylous and artiodactylic), recording the presence or absence of morphologic features from a long list of features, chosen for the purpose. This data has been recorded and analyzed using the phylogeny programs PAUP which calculates the evolutionary path which fits the data in the simplest pathway and the phylogeny graphics program MacClade which allows the user to visually analyze and manipulate a minimum length phylogenic tree. Results: The results are in the form of a phylogenic tree. The findings in this preliminary study match well with accepted evolutionary schemes. Conclusions: The

4 440 S Ungulates with odd numbers of toes (Order Perissodactyla) and the ungulates with even numbers of toes (Order Artiodactyla) share many features within each groups but differ in many ways between the two groups other. This and the differences noted within each group, allow us to predict a phylogeny. NO.: 19 Functional assessment of the efficacy of COP-1 treatment for laser-induced retinal damage in the rat G. Ben Shlomo,* M. Belokopytov, M. Belkin, M. Rosner, Y. Epstein and R. Ofri* *Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Rehovot, Israel; and Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel Aviv University, Tel Hashomer, Tel. Aviv, Israel Purpose: To assess the neuroprotective effect of Glatiramer acetate (Copolymer-1, Cop-1, trade name Copaxone) treatment on outer retinal function following laser induced retinal damage in the rat. Methods: Standard argon laser lesions were created in the right eye of 16 adult, DA pigmented rats. The number of lesions was enough to cover half of the visible retina. The laser settings were found in previous studies to result in lesions of the uniform size and configuration, involving mainly the outer retinal layers. The left eye served as an untreated control. Group 1 (n = 8) was treated with 200 µg Cop-1 (0.2 ml in volume emulsified with complete Freund s adjuvant) subcutaneously, seven days prior to photocoagulation. Group 2 (n = 8) was treated with saline (at the same volume) served as a control. Flash electroreinography (ERG) was performed to assess outer retinal function 3, 21 and 60 days post lasering. Ten stimuli (0.1 Hz) were recorded and averaged from each eye, and saved for further analysis. Results: Three days after laser treatment we noticed a significant decrease of ERG responses in the lasered eyes compared to the contra lateral intact eyes, though at this time there were no significant differences in ERG amplitudes between the treated an untreated animals (P > 0.05). However, 21 days post lasering we noticed a significant recovery of the ERG responses in the eyes of the treated group compared to the control group, as reflected by the ratio between lasered and intact eyes (99.7 ± 7% and 83.5 ± 8%, respectively, P < 0.05). This trend continued after 60 days, with a lasered eye/intact eye response ratio of 99.5 ± 10% in the treated animals, and 85.8 ± 6% in the untreated animals (P < 0.05). Conclusions: In this work we demonstrated that treatment by Cop-1 offers functional protection from argon laser retinal damage. Though outer retinal function decreased in the immediate post laser period, it recovered days afterwards in the treated animals. The untreated group showed a significant (P < 0.05) reduction of outer retinal function in the injured eyes during this period. Commercial NO.: 20 In vitro fungistatic and fungicidal activity of silver sulfadiazine and natamycin on pathogenic fungi isolated from equine eyes C. M. Betbeze,* C. C. Wu, J. Stiles,* S. G. Krohne* and M. J. Finn* *Department of Veterinary Clinical Sciences and Department of Veterinary Pathobiology, Purdue University School of Veterinary Medicine, West Lafayette, IN USA Purpose: To evaluate the in vitro fungistatic and fungicidal activity of silver sulfadiazine (SSD) against filamentous fungi isolated from equine eyes and compare it to natamycin (NAT). Methods: 17 filamentous fungal isolates were cultured from equine eyes, including 6 Fusarium spp., 7 Aspergillus spp., and 1 each of Curvularia spp., Scopulariopsis spp., Penicillium spp., and Chrysosporium spp. Fungal cultures were grown, saved, and isolated using standard microbiological methods. A broth macrodilution method was used to determine the minimum inhibitory concentration (MIC) of SSD and NAT against these isolates. Minimum fungicidal concentrations (MFC) were also determined. Results: For SSD, the MIC distribution for all fungal isolates (n = 17) was = 1 µg/ml to > 64 µg/ml. The MIC 50 and MIC 90 were 4 µg/ml and 32 µg/ml, respectively. The MFC distribution for all fungal isolates was = 1 µg/ml to > 64 µg/ml. The MFC 50 and MFC 90 were 8 µg/ml and > 64 µg/ml, respectively. For NAT, the MIC distribution for all fungal isolates was 256 µg/ml to > 1000 µg/ml. The MIC 50 and MIC 90 were 512 µg/ml and > 1000 µg/ml, respectively. The MFC distribution for all fungal isolates was 512 µg/ml to > 1000 µg/ml. The MFC 50 and MFC 90 for all fungal isolates was > 1000 µg/ml. Conclusions: Based on the in vitro MIC and MFC results, silver sulfadiazine is fungistatic and fungicidal against the evaluated fungal isolates from equine eyes. Natamycin is fungistatic and fungicidal against some of the evaluated fungal isolates at the drug concentrations evaluated. Silver sulfadiazine may be a good therapeutic choice for equine keratomycosis. Supported by a grant from the ACVO Vision for Animals Foundation. Commercial NO.: 21 Corneal ulceration associated with naturally occurring canine herpesvirus-1 infection in two adult dogs E. C. Ledbetter,* R. C. Riis,* T. J. Kern,* N. J. Haley and S. J. Schatzberg* *College of Veterinary Medicine, Cornell University, Ithaca, NY USA and College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft Collins CO USA Purpose: To describe two canine cases of corneal ulceration associated with naturally occurring canine herpesvirus-1 (CHV-1) corneal infection. Methods: A virus was isolated on presentation, and repeatedly during rechecks, from corneal swabs of two dogs presented with multiple, dendritic, superficial corneal ulcers. Oropharyngeal and genital virus isolations were negative. One dog was affected unilaterally and one dog bilaterally. CHV-1 immunofluorescent antibody staining of the cell cultures was positive. The virus, when viewed with transmission electron microscopy, appeared to be a herpesvirus. Three separate PCR reactions (two CHV-1 specific and one pan-herpesvirus) were performed. All resultant PCR products were cloned and sequenced bidirectionally. Positive controls consisted of DNA extracted from CHV-1 grown in culture. Negative controls consisted of conjunctival swabs from 50 dogs without extraocular disease for PCR and corneal swabs from 25 dogs with corneal ulcers for virus isolation. Results: Both viral isolates tested positive by all three PCR primer pairs. Cloning products had a 100% sequence identity with CHV-1. All negative control PCR assays and virus isolations were negative. Topical antiviral treatment resulted in ulcer healing and virus isolations of corneal swabs were repeatedly negative following ulcer resolution. Conclusions: These dogs are the first documented cases of naturally occurring CHV-1 associated corneal ulcers. Immunosuppression may be important in the pathogenesis of CHV-1 corneal infection. The virus isolated was positively identified as CHV-1 and displayed a similar morphology, antigenicity, and genotype to CHV-1 isolates from puppies with fatal, systemic infections; however, a virus strain with specific corneal virulence cannot be ruled-out. Commercial NO.: 22 Estrogen localization and synthesis by cataractous LEC C. M. H. Colitz, H. L. Chandler, P. Lu, Y. Sugimoto, C. A. Barden, A. G. J. Metzler, D. A. Wilkie, I. D. Bras, V. J. Kuonen, T. E. Robbin and E. B. Belknap The Ohio State University, College of Veterinary Medicine, Columbus OH USA Purpose: Our laboratory has identified overexpression of estrogen receptor alpha (ERα) in cataractous LEC. ERα can function in both an estrogen-dependent and -independent manner. Furthermore, sulfatase and/or aromatase enzyme activity are necessary for the synthesis of active estrogen (17-β estradiol). We hypothesized that in order for ERα to function in an estrogen-dependent manner in cataractous LEC, estrogen would have to be not only present in the LEC or aqueous humor, but there had to be local production of 17-β-estradiol within the anterior segment of the eye. Methods: Samples included normal whole canine lenses, anterior capsulotomy samples from dogs with naturally occurring cataracts, and aqueous humor collected from both populations prior to dissection or surgery, respectively. Immunohistochemical staining was performed using antibodies against 17-β-estradiol and sulfatase. In addition, quantitative RT-PCR was used to measure aromatase and sulfatase transcription. Results: Immunohistochemistry found that cataractous LEC had more intense immunostaining for 17-β estradiol and sulfatase in the cytoplasm and nucleus than normal LEC, which had primarily cytoplasmic immunolocalization of both proteins. RT-PCR found significantly up-regulated aromatase transcription in cataractous LEC and only negligible levels in normal LEC. Conclusions: The synthesis of 17-β estradiol requires the activity of at least one of 2 enzymes, sulfatase and aromatase. This is the first evidence of 17-β estradiol production by cataractous LEC supported by the positive immunostaining for 17-β estradiol and sulfatase, and up-regulated transcription of aromatase by RT-PCR. These findings, combined with overexpression of ERα in cataractous LEC, suggest that LEC may become estrogen-responsive during or due to cataractogenesis. Perhaps the presence of estrogen in normal LEC, without excessive local production or ERα overexpression, is a protective mechanism against cataractogenesis, but the up-regulation of ERα and increased production of estrogen result in cataractogenesis. Commercial NO.: 23 Markers of epithelial-mesenchymal transition during canine cataractogenesis H. L. Chandler, C. A. Barden, D. F. Kusewitt and C. M. H. Colitz The Ohio State University, College of Department of Veterinary Biosciences, Columbus, OH USA Purpose: During cataractogenesis, lens epithelial cells (LEC) on the anterior surface of the lens proliferate, transform into myofibroblast-like cells, and migrate posteriorly. These events resemble epithelial-mesenchymal transition (EMT), the transformation of anchored epithelial cells into migrating fibroblastic cells, occurring during development. EMT can be initiated by growth factors, such as epidermal growth factor (EGF), that stimulate expression of the transcription factors Slug and Snail. These transcription factors then coordinate the many downstream events required for EMT. We hypothesize that enhanced expression of Slug or Snail, due to EGF stimulation, controls the EMT-like changes in LEC during canine cataractogenesis. As well, expression of other markers of EMT such as lumican, α-smooth muscle actin (αsma), and COX-2 can be used to determine the temporal changes associated with catatactogenesis. Methods: LEC were wounded in vitro and treated with EGF. Wound closure was monitored and real time RT-PCR was performed to measure Slug and Snail expression. Real-time RT-PCR was also used to examine the expression of lumican, αsma, and COX-2. Results: EGF-treated LEC migrated faster to heal defects than untreated LEC. Following EGF treatment, Slug expression increased after 8 h and remained elevated until 12 h. Snail expression increased following 4 h of EGF treatment and did not decrease until after 12 h. During LEC migration, there was up-regulation of lumican, αsma, and COX-2. Conclusions: Cultured LEC treated with EGF express more Slug and Snail mrna than normal LEC, suggesting enhanced stimulation of cell migration. Temporal changes in Snail, Slug, lumican, αsma, and COX-2 expression, can be used to understand the molecular mechanisms occurring during cataract formation. Future aims of this project are to treat whole lenses with EGF in vitro and measure Slug and Snail expression during early stages of cataract formation. Commercial NO.: 24 COX-2 expression in canine lens epithelial cells C. Elligott, H. L. Chandler, P. Lu, C. A. Barden and C. M. H. Colitz The Ohio State University College of Veterinary Medicine, Columbus OH USA Purpose: Epithelial cell migration is accompanied by increased expression of cyclooxygenase- 2 (COX-2). The purpose of this study was to evaluate normal and cataractous canine lens epithelial cells (LEC) to determine if COX-2 expression is associated with cataract formation, and if inhibition of the COX-2 pathway could prevent cataractous changes. Methods: Sample population included normal canine lens capsules (n = 14) without evidence of cataract and anterior capsulotomy samples from 31 canine cataract patients. Samples underwent immunohistochemical staining using anti-cox2 antibody. Quantitative RT-PCR (qrtpcr) was used to evaluate COX-2 mrna expression in normal and cataractous LEC. The LEC wound healing model was used to evaluate whether inhibition of COX-2 would impede LEC migration, a characteristic of cataractous LEC. Briefly, a 1 mm wound was created in confluent cultured LEC followed by treatment with rofecoxib, celecoxib, or unsupplemented media (control). LEC migration was monitored and Western immunoblotting was used to confirm decreased COX-2 expression during treatment. Results: Cataractous LEC stained more intensely for COX-2 than normal LEC. qrtpcr demonstrated significantly increased COX-2 mrna expression in canine cataracts compared to normal LEC. When wounded LEC were treated with either rofecoxib or celecoxib, there was decreased LEC migration compared to controls, and Western immunoblotting confirmed inhibition of COX-2. Conclusions: COX-2 protein and mrna expression is increased in cataractous LEC, wherein LEC are undergoing epithelial mesenchymal transformation and posterior migration. COX-2 inhibitors prevented migration of cultured LEC. Utilization of COX-2 inhibitors following cataract surgery may