Cecil P. Moore

In vitro susceptibility patterns of Aspergillus and Fusarium species isolated from equine ulcerative keratomycosis cases in the midwestern and southern United States with inclusion of the new antifungal agent voriconazole

OBJECTIVE To evaluate and compare the in vitro susceptibility of Aspergillus and Fusarium spp. isolated from horses with ulcerative keratomycosis, address regional differences in equine keratomycosis isolates, and provide susceptibility data to update prior studies. ANIMAL STUDIED Fourteen horses with ulcerative keratomycosis. PROCEDURES Banked fungal isolates from equine corneal ulcers (eight Aspergillus spp. and six Fusarium spp.) were identified at The University of Texas Health Science Center at San Antonio. In vitro minimum inhibitory concentration and susceptibility to natamycin, fluconazole, itraconazole, voriconazole, ketoconazole, and miconazole were determined for each isolate. RESULTS Fungi were significantly more susceptible to voriconazole than to natamycin, itraconazole, fluconazole, and ketoconazole, but miconazole susceptibility did not differ significantly from voriconazole. Aspergillus spp. were most susceptible to voriconazole, miconazole, and itraconazole, which were significantly better to fluconazole and ketoconazole. Fusarium spp. susceptibility was greatest to natamycin and voriconazole and lowest to itraconazole and ketoconazole. Fusarium spp. were significantly less susceptible to itraconazole and ketoconazole compared to natamycin. No significant differences in susceptibility were found when isolates from Florida were compared with isolates from other states. CONCLUSIONS AND CLINICAL RELEVANCE Based on in vitro evidence, voriconazole appears to be the most effective antifungal for initial treatment of equine keratomycosis in the midwestern and southern United States. Results are comparable with previous studies in that isolated fungi from equine keratomycosis cases showed consistently poor susceptibility to fluconazole. Organisms isolated in different geographic locations of the midwestern and southern United States appeared to have similar patterns of antifungal susceptibility.

Morphological evidence of M cells in healthy canine conjunctiva-associated lymphoid tissue

Abstract.Purpose: To characterize the follicle-associated epithelium (FAE) and organized lymphoid nodules from dog nictitating membranes to determine if canine conjunctiva-associated lymphoid tissue (CALT) contains M cells analogous to those described in other regions of mucosa-associated lymphoid tissue (MALT). Methods: Nictitan lymphoid follicles from 15 healthy dogs (30 eyes) were harvested immediately post-mortem. Twelve follicles from each nictitating membrane were isolated. Four follicles from each eye of 10 dogs were examined by light microscopy, transmission electron microscopy and scanning electron microscopy. Five of the 10 dogs were treated with a heat-killed staphylococcal topical suspension immediately prior to euthanasia. Nictitan follicles from five other dogs were processed for immunohistochemistry to characterize follicle lymphocyte populations. Results: The FAE overlying CALT demonstrated morphology characteristic of M cells, including attenuated apical cell surface with blunted microvilli and microfolds, invaginated basolateral membrane forming a cytoplasmic pocket containing lymphocytes and macrophages, and diminished distance between the apical and pocket membrane. Heat-killed bacteria were bound to the surface and transcytosed to the cytoplasmic pocket of CALT M cells. Immunohistochemistry of organized lymphoid tissue subtending the FAE demonstrated B-cell germinal centers with T-cell predominant apical caps. Conclusions: In canine CALT, the FAE overlying lymphoid follicles, as well as the distribution of T and B lymphocytes subtending this region, contain morphologic and functional features analogous to MALT described in other regions. Documentation of canine conjunctival M cells is of clinical relevance in the study of primary ocular diseases, as well as a potential means of vaccination or drug delivery.

The pharmacologic assessment of a novel lymphocyte function-associated antigen-1 antagonist (SAR 1118) for the treatment of keratoconjunctivitis sicca in dogs.

PURPOSE Keratoconjunctivitis sicca (KCS) is characterized by inflammation and decreased production of tears containing increased levels of cytokines. The release occurs in the setting of conjunctival and lacrimal gland inflammation, potentially mediated by the interaction between lymphocyte function-associated antigen (LFA)-1, a cell surface protein found on lymphocytes, and its cognate ligand intercellular adhesion molecule (ICAM)-1. SAR 1118 is a novel LFA-1 antagonist and may be an effective therapeutic agent for the treatment of KCS. The following studies were performed to assess the in vitro activity of SAR 1118 and to evaluate the clinical efficacy of topical SAR 1118 for the treatment of idiopathic canine KCS. METHOD Pharmacodynamics were assessed by measuring the ability of SAR 1118 to inhibit Jurkat T-cell binding with recombinant human ICAM-1 and to inhibit cytokine release from human peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin B. For the assessment of clinical efficacy, 10 dogs diagnosed with idiopathic KCS were treated with SAR 1118 1% topical ophthalmic solution three times daily for 12 weeks. Schirmers tear test (STT) was used to measure tear production. RESULTS SAR 1118 demonstrated concentration-dependent inhibition of Jurkat T-cell attachment, inhibition of lymphocyte activation, and release of inflammatory cytokines, particularly the Th1, Th2, and Th17 T-cell cytokines IFN-γ, IL-2, and IL-17F, respectively. Mean STT values increased from 3.4 mm during week 1 to 5.8 mm at week 12 (P < 0.025). No SAR 1118-related adverse events were observed. CONCLUSIONS SAR 1118 appears to be an effective anti-inflammatory treatment for KCS. Additional studies are warranted to establish the efficacy of SAR 1118 for the treatment of KCS in humans.

Surgery of the Adnexa

Review of the functional anatomy of ocular adnexal tissues is followed by presentation of surgical procedures aimed at correcting conditions of the eyelids, conjunctiva, and third eyelids of small animals. Procedures used effectively by the primary author are described in detail including instances where combination procedures may be indicated. Some newer, recently described techniques are also briefly discussed. Illustrations of applied anatomy and multiple surgical techniques are provided by illustrator and coauthor Dr. Gheorghe Constantinescu. References are given to encourage readers to further explore alternative techniques focusing on the surgical correction of adnexal diseases of dogs and cats.

Eyelid and nasolacrimal disease.

An understanding of normal structure and function of the equine eyelid is essential to make an accurate diagnosis and appropriately treat equine eyelid diseases. Entropion, eyelid trauma, neoplasia, and nasolacrimal disorders are reviewed. Methods of diagnosis and treatment are presented.

Detection of Leptospira Interrogans DNA and Antigen in Fixed Equine Eyes Affected with End-Stage Equine Recurrent Uveitis

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.

Cyclosporine has a direct effect on the differentiation of a mucin-secreting cell line

Cyclosporine is a potent immunosuppressant used in the treatment of ulcerative colitis and keratoconjunctivitis sicca. Neither the etiologies of these diseases nor the mechanism by which cyclosporine exerts its therapeutic effect is well understood. Since both diseases are linked by a common decrease in mucin‐filled goblet cells, this study tests a hypothesis that cyclosporine acts directly on goblet cells to promote their differentiation and production of secretory mucins. The HT29‐18N2 human colon adenocarcinoma cell line, which is capable of forming monolayers of well‐differentiated goblet cells, was used as a model system. Cyclosporine induced a dose‐dependent increase in intracellular mucin stores. A 2‐week exposure to 1 μM cyclosporine resulted in an average increase in mucin volume of 94%. This increase resulted from both a higher percentage of cells with mucin stores and an increased volume of mucin per cell. PSC‐833, a nonimmunosuppressive analog of cyclosporine, also increased mucin production. The intracellular accumulation of mucin was not a result of reduced secretion, since the time required for the release of pulse‐radiolabeled glycoproteins was similar for both control and cyclosporine‐treated monolayers. The effect of cyclosporine was not mediated by the drugs previously documented abilities to decrease cellular proliferation rates, inhibit calmodulin, antagonize prolactin receptor binding, or modulate prostaglandin production. J. Cell. Physiol. 184:400–408, 2000.

Long-term outcome of sudden acquired retinal degeneration syndrome in dogs.

OBJECTIVE To investigate long-term outcomes and owner-perceived quality of life associated with sudden acquired retinal degeneration syndrome (SARDS) in dogs. DESIGN Survey study. ANIMALS 100 dogs with SARDS examined at 5 academic veterinary institutions from 2005 to 2010. PROCEDURES The diagnosis was based on documented acute vision loss, normal results of ophthalmic examinations, and evaluation of extinguished bright-flash electroretinograms. Primary owners of affected dogs completed a questionnaire addressing outcome measures including vision, systemic signs, and perceived quality of life for their dogs. RESULTS Age at diagnosis was significantly correlated with positive outcome measures; dogs in which SARDS was diagnosed at a younger age were more likely to have alleged partial vision and higher owner-perceived quality of life. Polyphagia was the only associated systemic sign found to increase in severity over time. Medical treatment was attempted in 22% of dogs; visual improvement was not detected in any. Thirty-seven percent of respondents reported an improved relationship with their dog after diagnosis, and 95% indicated they would discourage euthanasia of dogs with SARDS. CONCLUSIONS AND CLINICAL RELEVANCE Blindness and concurrent systemic signs associated with SARDS appeared to persist indefinitely, but only polyphagia increased in severity over time. Most owners believed their pets had good quality of life and would discourage euthanasia of dogs with SARDS.

The effect of photodynamic therapy on squamous cell carcinoma in a murine model: Evaluation of time between intralesional injection to laser irradiation

Successful treatment of naturally occurring periocular squamous cell carcinoma (SCC) in horses with photodynamic therapy (PDT) has been performed by injecting residual tumor with verteporfin and applying laser irradiation immediately following injection. This study used a murine model to evaluate the influence of time between intralesional injection of verteporfin to laser irradiation on tumor growth inhibition with PDT. Mice were randomized into six groups (n=10/group). Each tumor was injected with either 0.1mg/cm(3) of verteporfin (Tx) or 5% dextrose in water (C). Tx and C groups 1, 2, and 3 were irradiated at 1, 30, and 180min after injection. Wilcoxon-rank sum test (P< or =0.05) was performed to determine the relative change in tumor volume (RCTV) between groups. Statistical significance was demonstrated between treatment groups. Although verteporfin-PDT treated mice in Tx1 and Tx2 demonstrated a lower RCTV compared to C1 and C2 mice, the differences were not statistically significant.

Cholesterylene, a newly recognized tissue lipid, found at high levels in the cornea.

In the course of measuring the concentration of cholesterol in an opacified dog cornea by gas-chromatography, relatively large amounts of an unidentified non-saponifiable lipid were recognized. When the unknown lipid was subjected to gas chromatographic-mass spectral analysis it displayed a major ion at m/z 368 M+. and was identified as cholesta-3,5-diene, cholesterylene, by computer match with mass spectral-registry data. Cholesterylene was then shown to be present in the corneas of normal dogs, cows and humans, accounting for 20-25% of the total steroid-sterol in dog corneas and 5-10% in cow and human. Cholesterylene, which can be considered as an extremely nonpolar dehydration product of cholesterol, has not previously been recognized in animal tissues. Although the source of corneal cholesterylene is unknown, preliminary results suggest non-enzymatic formation from cholesterol.