Eric C. Ledbetter

In vivo confocal microscopy of equine fungal keratitis

OBJECTIVE To describe in vivo corneal confocal microscopy of horses with fungal keratitis and correlate findings with clinical, histopathological, and microbiological evaluations of clinical cases and an ex vivo experimental equine fungal keratitis model. ANIMALS STUDIED A total of 12 horses with naturally-acquired fungal keratitis and ex vivo equine corneas experimentally infected with clinical fungal isolates. PROCEDURES Horses with naturally-acquired fungal keratitis were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module. Confocal microscopy images of clinical isolates of Aspergillus fumigatus, Fusarium solani, and Candida albicans were obtained by examination of in vitro cultures and experimentally infected ex vivo equine corneas. RESULTS Non-specific in vivo corneal confocal microscopic findings in horses with fungal keratitis included leukocyte infiltrates, activated keratocytes, anterior stromal dendritic cell infiltrates, and vascularization. Linear, branching, hyper-reflective structures that were 2-6 μm in width and 200 to >400 μm in length were detected in all horses with filamentous fungal keratitis. Round to oval hyper-reflective structures that were 2-8 μm in diameter were detected in a horse with yeast fungal keratitis. The in vivo confocal microscopic appearance of the organisms was consistent with fungal morphologies observed during examination of in vitro cultures and infected ex vivo equine corneas. CONCLUSIONS In vivo corneal confocal microscopy is a rapid and non-invasive method of diagnosing fungal keratitis in the horse. This imaging technique is useful for both ulcerative and non-ulcerative fungal keratitis, and is particularly advantageous for confirming the presence of fungi in deep corneal stromal lesions.

Experimental reactivation of latent canine herpesvirus-1 and induction of recurrent ocular disease in adult dogs.

Latent canine herpesvirus-1 (CHV-1) infection is common in domestic dogs, but recrudescent CHV-1 diseases are poorly characterized. To determine if administration of an immunosuppressive dosage of prednisolone to adult dogs latently infected with CHV-1 results in recurrent ocular disease, adult beagles with and without experimentally induced CHV-1 latent infection were divided into groups: group 1 latently infected and administered prednisolone, group 2 latently infected and administered placebo, and group 3 not latently infected and administered prednisolone. Prednisolone (3.0 mg/kg/day) was administered to dogs in groups 1 and 3 for seven consecutive days beginning on study day 1. Samples for CHV-1 polymerase chain reaction and serum neutralization (SN) assays were collected, and physical, ophthalmologic, and in vivo ocular confocal microscopic examinations were performed at intervals for 42 days. Bilateral ocular disease (i.e., conjunctivitis or keratitis) was detected in 83% of group 1 dogs between study days 3 and 18. In vivo confocal microscopic abnormalities included conjunctival leukocyte infiltration and corneal leukocyte infiltration, abnormal epithelial cell morphology, and Langerhans cell infiltration. Ocular viral shedding was detected in 50% of group 1 dogs on study days 10 and 13. Fourfold elevations in CHV-1 SN titers were detected in 100% of group 1 dogs by study day 14. Dogs in control groups did not develop clinical ocular disease (P<0.05), CHV-1 titer elevations (P<0.005), or viral shedding. Administration of an immunosuppressive dosage of systemic prednisolone to adult dogs latently infected with CHV-1 may result in viral reactivation and ocular disease recrudescence.

In vivo confocal microscopy of the normal equine cornea and limbus.

OBJECTIVE To describe morphologic features, pachymetry and endothelial cell density of the normal equine cornea and limbus by in vivo confocal microscopy. ANIMALS STUDIED Ten horses without ocular disease. PROCEDURE The central and peripheral corneas were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module using a combination of automated and manual image acquisition modes. Thickness measurements of various corneal layers were performed and endothelial cell density determined. RESULTS Images of the constituent cellular and noncellular elements of the corneal epithelium, stroma, endothelium, and limbus were acquired in all horses. Corneal stromal nerves, the subepithelial nerve plexus, and the sub-basal nerve plexus were visualized. Cells with an appearance characteristic of Langerhans cells and corneal stromal dendritic cells were consistently detected in the corneal basal epithelium and anterior stroma, respectively. Median central total corneal thickness was 835 microm (range 725-920 microm) and median central corneal epithelial thickness was 131 microm (range 115-141 microm). Median central endothelial cell density was 3002 cells per mm(2) (range 2473-3581 cells per mm(2)). CONCLUSIONS In vivo corneal confocal microscopy provides a noninvasive method of assessing normal equine corneal structure at the cellular level and is a precise technique for corneal sublayer pachymetry and cell density measurements. A resident population of presumed Langerhans cells and corneal stromal dendritic cells was detected in the normal equine cornea. The described techniques can be applied to diagnostic evaluation of corneal alternations associated with disease and have broad clinical and research applications in the horse.

Experimental primary ocular canine herpesvirus-1 infection in adult dogs

OBJECTIVE-To characterize clinical ocular disease, viral shedding, and serologic response associated with primary canine herpesvirus-1 (CHV-1) ocular infection in naïve adult dogs. ANIMALS-12 specific pathogen-free adult Beagles. PROCEDURES-Dogs were topically inoculated in the right eye with CHV-1 (infection group; n = 8) or virus-free medium (control group; 4). Dogs were inoculated with or without corneal microtrephination and subconjunctivally administered corticosteroids. Conjunctiva, buffy coat, and serum samples for real-time PCR assay, virus isolation, and serum neutralization (SN) antibody titers were collected until postinfection day (PID) 224, and general physical and ophthalmologic examinations were performed. RESULTS-Dogs in the infection group developed bilateral, mild to moderate conjunctivitis that reached maximal intensity on PIDs 7 to 10. Ocular viral shedding was detected in all dogs in the infection group between PIDs 3 and 10. Infected dogs developed CHV-1 SN antibody titers, beginning at PID 7 and peaking on PID 21. All buffy coat PCR assay results were negative. Corneal microtrephination and subconjunctival corticosteroid administration did not significantly affect clinical disease or viral shedding. Following recovery from primary infection, dogs remained clinically normal, did not shed virus, and had slowly decreasing SN antibody titers. Dogs in the control group did not develop conjunctivitis, shed virus, or develop CHV-1 SN antibody titers. CONCLUSIONS AND CLINICAL RELEVANCE-Primary ocular infection of adult dogs with CHV-1 was associated with self-limiting conjunctivitis and ocular viral shedding, which was evident in the absence of clinically detectable keratitis or systemic disease. Features of this infection resembled herpes simplex virus primary ocular infection in humans.

Canine Reproductive, Respiratory, and Ocular Diseases due to Canine Herpesvirus

This review documents how clinical inquiry expands as our knowledge base about canine herpesvirus (CHV) increases. We must understand the various forms of CHV infection that may occur in the dog population. This has prompted the veterinary community to develop more sensitive diagnostic assays. CHV is more common than we considered a decade ago. Up to 70% of some high-risk dog populations have been infected with and are latent carriers of CHV. Recognition of the various forms of CHV-induced disease, availability of diagnostic assays with increased sensitivity, and the formation of reliable biosecurity measures will allow for better control steps.

Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals.

OBJECTIVE To determine the frequency of obligate anaerobic bacterial isolation from corneal samples of domestic animals with ulcerative keratitis and to characterize the historical, clinical, cytological, and microbiological features of culture-positive cases. ANIMALS STUDIED Three hundred and thirty domestic animals with ulcerative keratitis. PROCEDURES Anaerobic bacteriologic culture and Gram stain were performed on corneal samples from consecutive animals examined with suspect septic ulcerative keratitis. Additional corneal diagnostics included: aerobic bacteriologic culture for all species; fungal culture for ungulates; Mycoplasma culture and virus isolation or feline herpesvirus-1 (FHV-1) polymerase chain reaction (PCR) for cats. Historical, clinical, and cytological findings were correlated with microbiologic data. RESULTS Anaerobic bacteria were isolated from 13.0% of corneal samples (dogs: 14.0%; horses: 12.9%; cats: 7.9%; alpacas: 18.8%). The most frequent isolates were Clostridium, Peptostreptococcus, Actinomyces, Fusobacterium, and Bacteroides species. The majority of these infections were mixed anaerobic and aerobic bacteria, unless antimicrobial therapy had been administered prior to presentation. The clinical appearance of anaerobic bacterial culture-positive cases was highly variable. Ocular trauma, pre-existing corneal disease, previous corneal surgery, and chronic dermatological disease were significantly (P < or = 0.05) correlated with positive anaerobic cultures in one or more species. CONCLUSIONS The results of the present study demonstrate that obligate anaerobic bacteria are present within the intralesional flora of ulcerative keratitis in domestic animals. In most species evaluated, these bacteria were identified infrequently. Anaerobic bacterial infection of the cornea most frequently occurs in association with other ocular pathogens and previous corneal abnormalities.

Orbital abscess bacterial isolates and in vitro antimicrobial susceptibility patterns in dogs and cats.

OBJECTIVE To determine bacterial populations, in vitro antimicrobial susceptibility patterns, and sources of microorganisms for dogs and cats with orbital abscess. ANIMALS STUDIED In total, 34 dogs and 7 cats with orbital abscess participated in the study. PROCEDURE Medical records of dogs and cats with a clinical diagnosis of orbital abscess, confirmed by cytologic or histopathologic evaluation of orbital specimens, were reviewed from the years 1990 to 2007. Animal signalment, presumptive source of microorganisms and mechanism of orbital introduction, bacterial isolates, and aerobic bacterial in vitro antimicrobial susceptibility test results were recorded. Percentages of susceptible aerobic bacterial isolates were compared among antimicrobials. RESULTS Twenty dogs and five cats had positive culture results. The most frequent bacterial genera isolated from dogs were Staphylococcus, Escherichia, Bacteroides, Clostridium and Pasteurella. The most frequent bacterial genera isolated from cats were Pasteurella and Bacteroides. Aerobic bacterial isolates from dogs had the highest percentage of susceptibility to amikacin, ceftiofur, gentamicin, imipenem, ticarcillin and trimethoprim-sulfamethoxazole. Aerobic bacterial isolates from dogs had the lowest percentage of susceptibility to ampicillin, clindamycin, erythromycin and penicillin. Antimicrobial resistance was uncommon among feline aerobic bacterial isolates. The most commonly identified routes of orbital bacteria introduction were extension from adjacent anatomical structures, penetrating exogenous trauma, and foreign bodies. CONCLUSIONS Mixed aerobic and anaerobic bacterial infections of the orbit occur commonly in dogs and cats. On the basis of aerobic and anaerobic bacterial isolates and in vitro susceptibility testing of aerobic bacterial isolates, cephalosporins, extended-spectrum penicillins, potentiated-penicillins and carbapenems are recommended for initial antimicrobial therapy of orbital abscess in dogs and cats.

Brucella canis endophthalmitis in 3 dogs: clinical features, diagnosis, and treatment.

OBJECTIVE To describe historical, clinical and diagnostic features of dogs with Brucella canis endophthalmitis and the response to medical therapy. ANIMALS STUDIED Three dogs with naturally acquired B. canis endophthalmitis. PROCEDURE Dogs were treated symptomatically with topical ophthalmic anti-inflammatories and a novel antimicrobial protocol that included doxycycline, enrofloxacin, rifampin and streptomycin. RESULTS All dogs presented with chronic or recurrent uveitis in the absence of overt systemic disease. Clinical ophthalmologic abnormalities were unilateral in each dog and included mild-to-moderate anterior uveitis, iris hyperpigmentation, marked vitreal infiltrates, and multifocal chorioretinitis. Dogs were diagnosed with canine brucellosis serologically and by blood culture (n = 2 dogs) or polymerase chain reaction of aqueous humor and blood (n = 1 dog). Active ocular inflammation resolved in all dogs during treatment, with preservation of vision in 2 dogs. Following treatment, B. canis could not be cultured from blood samples and serological values declined with seronegativity achieved in all dogs after a median of 96 weeks (range: 36-112 weeks) of therapy. CONCLUSIONS Brucella canis infection should be included in the differential diagnosis for dogs with intraocular inflammation, regardless of previous history or neuter status. This is the first report of apparently successful medical therapy of canine brucellosis with ocular involvement.

Virologic survey of dogs with naturally acquired idiopathic conjunctivitis.

OBJECTIVE To determine the frequency of viral detection in conjunctival samples from client-owned domestic dogs with naturally acquired idiopathic conjunctivitis and to identify signalment, historical, and clinical findings positively associated with viral detection. DESIGN Case-control study. ANIMALS 30 dogs with naturally acquired idiopathic conjunctivitis and a control population of 30 dogs without ocular disease. PROCEDURES Complete physical and ophthalmic examinations were performed for each dog. Conjunctival swab specimens were analyzed by use of virus isolation and PCR assays for the following viruses: canine adenovirus-2 (CAV-2), canine distemper virus, canine herpesvirus-1 (CHV-1), canine parainfuenza virus, canine respiratory coronavirus, infuenza A virus, and West Nile virus. Signalment, clinical, and historical information was recorded and compared between study groups. RESULTS Viruses were detected by either virus isolation or PCR methods significantly more frequently in conjunctival samples from dogs with conjunctivitis (7/30 [23.3%]) than dogs without conjunctivitis (0/30 [0%]). Canine herpesvirus-1 was isolated from 2 conjunctival samples and detected by use of PCR assay in 5 conjunctival samples. Canine adenovirus-2 was isolated from 1 conjunctival sample and detected by use of PCR assay in 2 conjunctiva samples. Sexually intact dogs and frequent exposure to dogs outside the household were positively associated with viral detection in the conjunctivitis group. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that CHV-1 and CAV-2 are common etiologic agents of conjunctivitis in domestic dogs. Risk factors for viral conjunctivitis in dogs reflected increased exposure to other dogs and opportunities for contact with infectious secretions.

Pathogenic Phenotype and Genotype of Pseudomonas aeruginosa Isolates from Spontaneous Canine Ocular Infections

PURPOSE This study was designed to determine whether the ability to adversely affect corneal epithelial cell health is a factor common to Pseudomonas aeruginosa keratitis strains and to assess the prevalence of each pathogenic phenotype and genotype in a canine model of naturally-acquired P. aeruginosa ocular infection. METHODS P. aeruginosa ocular isolates were collected by sampling 100 dogs without disease (six isolates collected) and by sampling dogs with conjunctivitis (two isolates), endophthalmitis (one isolate), active keratitis (12 isolates), and resolved P. aeruginosa keratitis (four isolates). Phenotype was determined in vitro by quantifying corneal epithelial cell invasion by gentamicin survival assays, and cytotoxic activity by Trypan blue exclusion assays. Genotyping was performed for genes encoding the type III secreted effectors. RESULTS The ratio of invasive to cytotoxic strains with 95% confidence intervals (CI) was 0.83 (CI, 0.42-0.99) for conjunctival microflora isolates, 0.80 (CI, 0.54-0.94) for ocular infection isolates, and 1.0 (CI, 0.45-1.0) for strains isolated post-resolution of keratitis. Among ocular infection isolates, invasive and cytotoxic strains were significantly (P <or= 0.02) associated with older and younger dogs, respectively. Visible adverse effects on epithelial cells were significantly (P <or= 0.03) more frequent for keratitis strains (6/12) than other strains (1/13), but only three of these keratitis strains and the single non-keratitis strain possessed ExoU. CONCLUSIONS Invasive strains predominated in the dogs of this study. Only keratitis strains had visible adverse effects on epithelial cells without overt cytotoxicity, suggesting virulence strategies affecting live corneal epithelial cell health are selected for among keratitis strains.