In vivo confocal microscopy for the detection of canine fungal keratitis and monitoring of therapeutic response.

OBJECTIVE: To describe in vivo corneal confocal microscopy of dogs during the clinical course of fungal keratitis and correlate findings with clinical evaluations and an ex vivo experimental canine fungal keratitis model. ANIMALS STUDIED: Seven dogs with naturally acquired fungal keratitis and ex vivo canine corneas experimentally infected with clinical fungal isolates. PROCEDURES: Dogs with naturally acquired fungal keratitis were examined by in vivo laser scanning confocal microscopy. Initial confocal microscopic examinations were performed to assist in establishing the diagnosis of fungal keratitis. Serial confocal microscopic examinations were performed to guide antifungal chemotherapy. Confocal microscopy images of canine corneal fungal isolates were obtained by examination of experimentally infected ex vivo canine corneas to corroborate in vivo findings. RESULTS: Fungi cultured and detected by PCR from canine corneal samples included Candida albicans, Fusarium incarnatum-equiseti, Malassezia pachydermatis, and a Rhodotorula sp. Linear, branching, interlocking, hyperreflective structures were detected by confocal microscopy in dogs with filamentous fungal keratitis and round to oval hyperreflective structures were detected in dogs with yeast fungal keratitis. Antifungal chemotherapy was associated with a progressive reduction in the distribution and density of corneal fungal elements, alterations to fungal morphology, decreased leukocyte numbers, restoration of epithelial layers, and an increased number of visible keratocyte nuclei. No dogs had a recurrence of fungal keratitis following medication discontinuation. Confocal microscopic fungal morphologies were similar between in vivo and ex vivo examinations. CONCLUSIONS: In vivo corneal confocal microscopy is a rapid method of diagnosing fungal keratitis in dogs and provides a noninvasive mechanism for monitoring therapeutic response.

Malassezia pachydermatis keratomycosis in a dog.

A 13-year-old female Lhasa Apso was presented for blepharospasm and conjunctival hyperemia of the right eye. Ophthalmic examination revealed an anterior stromal ulcer associated with a raised yellow corneal plaque. In vivo confocal microscopy and cytology of the cornea identified neutrophilic inflammation and yeast cells. Malassezia pachydermatis was isolated from a corneal scraping. Treatment with topical voriconazole ophthalmic solution resolved the keratitis.

Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit.

Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.

Serologic responses of dogs given a commercial vaccine against Leptospira interrogans serovar pomona and Leptospira kirschneri serovar grippotyphosa.

OBJECTIVE: To evaluate serum titers obtained by use of the microscopic agglutination test (ie, MAT titers) to Leptospira interrogans serovar pomona and autumnalis and Leptospira kirschneri serovar grippotyphosa in dogs given a commercial vaccine against serovars pomona and grippotyphosa. ANIMALS: Forty 12-week-old puppies and 20 mature Beagles. PROCEDURE: Puppies received a commercial vaccine against serovars pomona and grippotyphosa at 12 weeks of age, then received a booster vaccine and 3 weeks later; mature dogs received the vaccine once. Serum MAT titers to serovars pomona, autumnalis, and grippotyphosa were measured before vaccination and at 2, 4, 6, 10, and 16 weeks after the first or only vaccination. RESULTS: Of the 40 puppies vaccinated, 40, 0, and 40 developed MAT titers of > 100 after vaccination to serovars pomona, grippotyphosa, and autumnalis, respectively. Microscopic agglutination test titers to serovar autumnalis were higher than MAT titers to serovars pomona and grippotyphosa and persisted in some dogs for 16 weeks (6 weeks longer than for titers to serovar pomona). Of the 20 mature dogs, 13, 5, and 20 developed MAT titers of > 100 at 2 weeks to serovars pomona, grippotyphosa, and autumnalis, respectively. Titers to serovar pomona were higher and persisted in some dogs beyond 16 weeks after vaccination, compared with titers to serovars pomona and grippotyphosa, which persisted for 10 and 6 weeks, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Subunit vaccines against serovars pomona and grippotyphosa induce MAT titers not only to homologous antigens but also to serovar autumnalis, which could lead to a misdiagnosis of leptospirosis caused by serovar autumnalis.