In vitro antifungal susceptibility of Fusarium species and Aspergillus fumigatus cultured from eleven horses with fungal keratitis.

PURPOSE: To examine the relationship between Minimum Inhibitory Concentration (MICs) and response to therapy of 6 Fusarium spp. and 5 Aspergillus fumigatus isolated from equine ulcerative keratitis cases. PROCEDURE: Fungi were identified by morphology and Internal Transcribed Spacer (ITS) polymerase chain reaction (PCR) with sequencing and evaluated at the University of Texas Fungal Testing Laboratory for susceptibility to three azole antifungals (miconazole, voriconazole, posaconazole), natamycin, and two echinocandins (anidulafungin, caspofungin). A Mann-Whitney rank sum test was used for the comparison of time to heal between infections of different fungal genera and in vitro susceptibility to the drug administered. RESULTS: Fusarium spp. were resistant to azole antifungals in 6/6 cases (100%). Fusarium spp. were susceptible to echinocandins and natamycin in all cases. A. fumigatus was resistant to anidulafungin in 1/5 cases (20%) and posaconazole in 1/5 cases (20%) The remainder of A. fumigatus isolates were susceptible to all antifungal agents tested. Fusarium isolates were treated with antifungals to which they were not susceptible; however, all cases of A. fumigatus were treated with antifungals to which they were susceptible. All Fusarium cases and A. fumigatus cases experienced clinical resolution, regardless of surgical intervention. There was no statistical correlation between fungal genus and time to heal (p < .082). CONCLUSIONS: The in vitro susceptibility indicated that all cases of Fusarium spp. were resistant to azole antifungal drugs which were used as treatment. Clinical outcomes, however, showed that all cases healed despite resistance to antifungals.

Therapeutic effects of equine amniotic membrane suspension on corneal re-epithelialization and haze in a modified lagomorph ex vivo wound healing model.

OBJECTIVE: To investigate the therapeutic effects of topical equine amniotic membrane (eAM) suspension following corneal wounding in a controlled experimental setting. PROCEDURES: Equine amniotic membrane was collected, gamma irradiated, homogenized for topical suspension preparation, and cryopreserved. Corneoscleral rims harvested from fresh rabbit globes were wounded via keratectomy and were maintained in an air-liquid interface ex vivo corneal culture model. Treatment groups included topical gamma irradiated eAM suspension (n = 20) and a control group (n = 20). Re-epithelialization of the wound was assessed with daily photographic evaluation of area of fluorescein uptake (mm2 ). Corneal wound haze after a 21-day period was assessed by photographic analysis of haze area (mm2 ) and pixel intensity (0-255). Histologic processing of corneal tissue was performed, and protein identification of eAM suspension using Liquid chromatography-mass spectrometry (LC-MS). RESULTS: The average day of complete corneal re-epithelialization in controls (5.5 ± 1.1) and topically treated (5.5 ± 0.6) corneas, and rates of reduction in area of fluorescein uptake over time did not significantly differ (p = .44). The corneal wound haze was significantly reduced in mean area by approximately 52% and intensity by 57% in corneas treated with topical eAM suspension (p < .05), compared to controls 21 days following wounding. Protein analysis identified numerous proteins, specifically decorin, dermatopontin, and lumican, which have previously been documented in eAM. CONCLUSIONS: Area and intensity of corneal wound haze were significantly reduced in corneas treated with gamma irradiated eAM suspension, which may be due to previously identified therapeutic proteins which promote corneal clarity.

Assessment of Topical Therapies for Improving the Optical Clarity Following Stromal Wounding in a Novel Ex Vivo Canine Cornea Model.

Purpose: To evaluate the effect of topical suberanilohydroxamic acid (SAHA) and 5-methyl-1-phenyl-2[1H]-pyridone (pirfenidone) on the degree of corneal haze in the stromal wounded ex vivo canine cornea. Methods: Twenty-four corneoscleral rims from normal dogs were uniformly wounded with an excimer laser and placed into culture medium with an air-liquid interface. The control group (n = 8) contained placebo-treated corneas. Treatment group 1 (n = 8) received SAHA topically every 6 hours. Treatment group 2 (n = 8) received pirfenidone topically every 6 hours. Each cornea was fluorescein stained and macrophotographed every 6 hours to assess epithelialization rate. All corneas were also macrophotographed weekly to assess optical clarity (haze). Images were analyzed for differences in pixel intensity between wounded (haze) and unwounded (nonhaze) regions, and haze surface area for each cornea was calculated. Results: The mean epithelialization time was 47.25 hours in the control group, 45.00 hours in the SAHA group, and 43.50 hours in the pirfenidone group, revealing no significant difference (P = 0.368). The median difference in pixel intensity between haze and nonhaze areas was 21.5 in the control group, 8.0 in the SAHA group, and 8.0 in the pirfenidone group, which is significant (P < 0.01). The median haze surface area was 12.96 mm2 in the control group, 5.70 mm2 in the SAHA group, and 5.92 mm2 in the pirfenidone group, which is significant (P < 0.01). Conclusions: Stromal-wounded ex vivo canine corneas exhibited greater optical clarity when treated with SAHA and pirfenidone than when placebo treated at 21 days. There was no significant difference in epithelialization rate between groups. Corneal contour was correlated with geographic haze distribution.

Schirmer tear test I in dogs: results comparing placement in the ventral vs. dorsal conjunctival fornix.

OBJECTIVE: To compare Schirmer tear test I (STT I) values obtained from placement of tear strips in the ventral and dorsal conjunctival fornices in dogs. PROCEDURE: Schirmer tear test I was performed on each eye of 16 clinically normal dogs (32 eyes) in a crossover study. Initial tear strip placement site was randomized for each eye. Alternative placement site measurements were obtained after 1 week. RESULTS: The mean (± standard deviation, SD) STT I for dorsal and ventral conjunctival fornices was 20.44 (±4.46) mm/min and 23.56 (±3.98) mm/min, respectively. STT I values obtained from the ventral conjunctival fornix were significantly greater than those obtained from the dorsal conjunctival fornix (P = 0.004). CONCLUSIONS: Schirmer tear test I values were significantly greater with tear strips placed in the ventral conjunctival fornix.

Development and Assessment of a Novel Canine Ex Vivo Corneal Model.

PURPOSE: To develop a novel ex vivo extended culture model of canine corneal epithelial cell wound healing. MATERIALS AND METHODS: Canine corneoscleral rims (CSR) were obtained and, after preparation for culture, were placed on a nutating scaffold and incubated in physiological conditions. In experiment 1, eight CSR in a serum-containing antimicrobial-fortified medium were monitored for epithelial integrity and bacterial infection up to 28 days in culture. CSR were assessed histologically at the end of the culture period end points 0, 7, 14, and 28 days with accompanying scanning electron microscopic (SEM) and transmission electron microscopic (TEM) evaluation. Samples for microbial culture were obtained at days 0, 3, 7, 14, and 28. In experiment 2, uniform 8-mm-diameter superficial corneal epithelial wounds were created and monitored for re-epithelialization in the same culture conditions or in a serum-free protein equivalent medium, with four CSR per group. Standardized digital images were obtained with cobalt filter at the time of fluorescein staining and media change every six hours. Image J imaging software was used to measure the area of fluorescein retention. Re-epithelialization rates were calculated and CSR then fixed for immunohistochemistry (IHC). RESULTS: All corneas survived to end points as described in experiment 1 with no evidence of contamination or compromised epithelial integrity. Histologically, a multilayered epithelium was maintained and corneal edema was not appreciated until day 14. SEM examination revealed epithelial cell layer confluence and migrating epithelial cells of normal cellular morphology with normal cell-cell interactions on TEM. In experiment 2, all eight corneas healed with a healing rate of 0.702 ± 0.130 mm2/h (1.25 mm/day epithelial cell migration rate) and were positive in IHC evaluation for markers of corneal fibrosis. CONCLUSION: This ex vivo canine corneal wound healing model is an appropriate and clinically relevant tool for assessment and modulation of epithelial wound healing.

Effect of topical ophthalmic latanoprost 0.005% solution alone and in combination with diclofenac 0.1% solution in healthy horses: a pilot study.

OBJECTIVE: To evaluate the effect of topical ophthalmic 0.005% latanoprost alone and in combination with 0.1% diclofenac on healthy horses. ANIMALS STUDIED: Twelve healthy adult horses. PROCEDURES: A randomized, masked crossover design was used with horses divided into three groups for once daily treatment in one randomly selected eye. For arm 1 of the study, Group D (n = 3) received 0.1% diclofenac, Group L (n = 3) received 0.005% latanoprost, and Group DL (n = 6) received 0.005% latanoprost and 0.2 ml of 0.1% diclofenac. For arm 2 of the study, horses from Group D and L were placed into Group DL and horses from Group DL were placed into either Group D or L. Evaluations of intraocular pressure (IOP), vertical pupil diameter, aqueous flare, conjunctival hyperemia, epiphora, blepharospasm, and blepharoedema were performed 4 times daily on days 1 and 2 (baseline), days 3 to 7 (arm 1 treatment), days 8 to 11 (washout), days 12 to 16 (arm 2 treatment), and days 17 and 18 (return to baseline). RESULTS: During the treatment period, significant reduction in IOP and vertical pupil diameter occurred in treated eyes of Groups L and DL, but not Group D. These variables did not differ significantly between Groups L and DL. Blepharospasm, blepharoedema, epiphora, and conjunctival hyperemia scores were significantly higher in Group L than in Groups D and DL. CONCLUSIONS: Latanoprost reduced IOP in healthy horses and signs of drug-induced discomfort were mitigated by concurrent use of diclofenac.

Effect of auriculopalpebral nerve block on Schirmer tear test I values in normal horses.

OBJECTIVE: To compare Schirmer tear test I (STTI) values collected in normal horses with and without an auriculopalpebral nerve block. PROCEDURE: Schirmer tear test I values were measured in 20 clinically normal horses (38 eyes) with a median age of 12 years. The order of eyes tested was randomized. Within 24-48 h, at the same time of day, tear measurements were collected again after administration of an auriculopalpebral nerve block. Each block was performed a minimum of 5 min prior to each STT I. A repeated-measures model was used to analyze differences between STT I values in eyes with and without nerve blocks incorporating within horse correlation between eyes. The mixed-model included fixed factors of treatment and eye and a random intercept for each horse. An unstructured covariance structure was used. RESULTS: On average, STT I values measured in eyes after auriculopalpebral nerve blocks were 0.55 mm/min greater than those without nerve blocks. This difference was not statistically significant (P = 0.5268). CONCLUSIONS: There was no effect of auriculopalpebral nerve block on STT I values in normal horses.

Hypokalemia and suspected renal tubular acidosis associated with topical carbonic anhydrase inhibitor therapy in a cat.

OBJECTIVE: To describe the occurrence of hypokalemia, metabolic acidosis, and suspected renal tubular acidosis associated with the administration of topical ophthalmic carbonic anhydrase inhibitor (CAI) in a cat. CASE SUMMARY: A 2-year-old, 5.3 kg, male, castrated, domestic short-haired cat developed hyporexia 6 weeks after starting topical ophthalmic dorzolamide 2% therapy for treatment of ocular hypertension. Two weeks later, the cat was evaluated for severe weakness, cervical ventroflexion, and anorexia. Plasma electrolyte and acid-base measurement revealed hypokalemia (K+ = 2.9 mmol/L; reference interval 3.8-5.4 mmol/L) and metabolic acidosis (plasma HCO3- = 9.8 mmol/L; reference interval 15-23 mmol/L) in the presence of a urine pH of 7.5 (reference interval 6.5-7.5). The pH abnormalities were consistent with a renal tubular acidosis. Clinical and biochemical abnormalities resolved with short-term supportive care, potassium supplementation, and discontinuation of dorzolamide therapy. NEW OR UNIQUE INFORMATION PROVIDED: This is the first report of hypokalemia and metabolic acidosis associated with topical CAI therapy in a cat.

Comparison of two- and three-times-daily topical ophthalmic application of 0.005% latanoprost solution in clinically normal dogs.

OBJECTIVE: To determine whether 2- or 3-times-daily application of topical ophthalmic 0.005% latanoprost solution is more effective at lowering intraocular pressure (IOP) in clinically normal dogs. ANIMALS: 9 clinically normal dogs. PROCEDURES: For each dog, I drop of latanoprost 0.005% solution was applied to 1 eye every 8 or 12 hours each day for 5 days; the contralateral eye received topical ophthalmic treatment with 1 drop of saline (0.9% NaCl) solution at the times of latanoprost application. Ocular examinations of both eyes were performed every 6 hours starting 48 hours prior to and ending 42 hours after the treatment period. Following a 5-week washout interval, the procedures were repeated but the previously latanoprost-treated eye of each dog received latanoprost application at the alternate frequency. RESULTS: Mean ± SD IOP reduction in the latanoprost-treated eyes was 31 ± 6.9% with 2-times-daily application and 33 ± 8.2% with 3-times-daily application. A 2-way repeated-measures ANOVA revealed significant differences in IOP with contributions by treatment (2 or 3 times daily), time of day (diurnal variation), and individual dog. The maximum mean daily IOP reduction in latanoprost-treated eyes was detected on day 3 of latanoprost treatment in each group. Eyes treated 3 times daily had significantly smaller pupil diameter and greater conjunctival hyperemia than eyes treated 2 times daily. CONCLUSIONS AND CLINICAL RELEVANCE: The clinical importance of the ocular hypotensive effects of 3-times-daily topical ophthalmic application of 0.005% latanoprost solution in dogs with glaucoma warrants investigation.

Electroretinogram evaluation of equine eyes with extensive 'bullet-hole' fundic lesions.

OBJECTIVE: To evaluate the impact of extensive bullet-hole nontapetal fundic lesions in horses on retinal function as measured by full-field electroretinography (ERG). MATERIALS AND METHODS: Full-field ERG was performed on two horses with numerous bullet-hole lesions in the nontapetal fundus of both eyes. The ERG was first recorded from the eye with the more extensive lesions in response to a low-intensity light stimulus (0.03 cd s/m(2) ) that was given at times (T) T = 5, 10, 15, 20 min of dark adaptation. Consecutively, combined rod-cone response was evaluated bilaterally in response to high-intensity light stimulus (3 cd s/m(2) ), followed by cone function evaluation by flicker stimulus (3 cd s/m(2) at 30 Hz). Off-line analysis of the ERG recordings was then performed. RESULTS: Despite extensive bullet-hole lesions in the nontapetal fundus bilaterally in both horses, retinal function as measured by ERG did not show any observable deficits. The b-wave amplitude of the full-field ERG increased continuously from 5 to 20 min of dark adaptation peaking at 446 µv and 377 µv for horse number 1 and 2, respectively. The b-wave amplitudes of the combined rod-cone response were OS- 459 µv and OD- 392 µv for horse number 1 and OS- 491 µv and OD- 608 µv for horse number 2. The amplitude of the flicker ERG for horse number 1 was OS- 86 µv and OD- 110 µv and for horse number 2, OS- 80 µv and OD- 74 µv. CONCLUSIONS: Extensive bullet-hole chorioretinal lesions do not appear to compromise outer retinal function in these horses.

Episodic blindness and ataxia in a horse with cholesterinic granulomas.

An 11-year-old Oldenburg mare presented following three episodes of acute, transient blindness, ataxia, and disorientation within the preceding 7 months. Clinical improvement, including return of vision, occurred within 1 week of initiating corticosteroid therapy for each of the three episodes. However, mild right-sided miosis was a consistent finding on ophthalmic examinations. Routine clinicopathologic testing revealed no significant abnormalities, and testing of cerebral spinal fluid for selected infectious diseases was unrewarding. Computed tomography of the brain demonstrated a hyperattenuating mass with peripheral mineralization in the rostroventral aspect of each lateral ventricle. The mare was euthanized due to a guarded to poor prognosis. On histopathology, the masses consisted of clusters of cholesterol clefts admixed with leukocytes, mineral deposits, and connective tissue. Cholesterinic granulomas of the lateral ventricles and hydrocephaly were diagnosed. Cholesterinic granulomas should be considered a differential diagnosis in horses presenting for intermittent blindness.

Replacing the enzyme alpha-L-iduronidase at birth ameliorates symptoms in the brain and periphery of dogs with mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by loss of activity of α-l-iduronidase and attendant accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate. Current treatments are suboptimal and do not address residual disease including corneal clouding, skeletal deformities, valvular heart disease, and cognitive impairment. We treated neonatal dogs with MPS I with intravenous recombinant α-l-iduronidase replacement therapy at the conventional 0.58 mg/kg or a higher 1.57 mg/kg weekly dose for 56 to 81 weeks. In contrast to previous results in animals and patients treated at a later age, the dogs failed to mount an antibody response to enzyme therapy, consistent with the induction of immune tolerance in neonates. The higher dose of enzyme led to complete normalization of lysosomal storage in the liver, spleen, lung, kidney, synovium, and myocardium, as well as in the hard-to-treat mitral valve. Cardiac biochemistry and function were restored, and there were improvements in skeletal disease as shown by clinical and radiographic assessments. Glycosaminoglycan levels in the brain were normalized after intravenous enzyme therapy, in the presence or absence of intrathecal administration of recombinant α-l-iduronidase. Histopathological evidence of glycosaminoglycan storage in the brain was ameliorated with the higher-dose intravenous therapy and was further improved by combining intravenous and intrathecal therapy. These findings argue that neonatal testing and early treatment of patients with MPS I may more effectively treat this disease.

Ahmed valve implantation to control intractable glaucoma after phacoemulsification and intraocular lens implantation in a dog.

An 11-year-old spayed female Miniature Poodle presented with bilateral senile cataracts. Treatment was cataract removal by phacoemulsification and intraocular lens implantation. Five hours after surgery the operated right eye was partially closed and painful. The right pupil was fully dilated, there was generalized corneal edema, and intraocular pressure (IOP) was increased. After 12 h of medication IOP was controlled. Ten weeks after surgery the owners reported cloudiness of right eye, and the dog was again evaluated. The IOP was again increased and an Ahmed valve was implanted. This case describes describe the progression of this case and benefits of controlling postcataract surgery glaucoma by the implantation of an Ahmed valve.