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  • Author or Editor: Lynne S. Sandmeyer x
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Abstract

Objective—To isolate and characterize pure cultures of feline corneal epithelial cells and to assess the extent and nature of feline herpesvirus (FHV)-1 infection in these cells.

Sample Population—Healthy eyes from 23 recently euthanatized cats.

Procedure—Stroma and epithelium of the rostral portion of the cornea were surgically isolated, and epithelial cells were detached from the stroma by enzymatic incubation. Epithelial cells were cultured in hormone-supplemented media. Cells were passaged, and cytokeratin expression was assessed. Cells were then infected with FHV-1, and cytopathic effects were determined.

Results—Cell cultures were readily established from samples obtained from each eye and could be maintained through 6 passages. Cultured cells expressed cytokeratins 3 and 12 but not other cytokeratins. Infection with FHV-1 was rapid and caused widespread cytopathic effects.

Conclusions and Clinical Relevance—Feline corneal cells cultured in vitro during multiple passages maintain consistent morphologic characteristics and intermediate filament expression. They are susceptible to infection with FHV-1 and may provide a useful in vitro model for investigation of ocular drugs. (Am J Vet Res 2005;66:205–209)

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in American Journal of Veterinary Research

Abstract

Objective— To assess the effect of cidofovir on viability of feline corneal epithelial (FCE) cells, replication of feline herpesvirus (FHV)-1, and virus-induced cytopathic changes.

Sample Population—Healthy eyes from 14 recently euthanatized cats.

Procedure—Cidofovir at concentrations ranging from 0.05 to 0.000005 mg/mL was added to primary cultures of FCE cells, and cytopathic changes and effects on cell proliferation and cell viability were determined during the subsequent 48 hours. Efficacy of cidofovir (0.02 and 0.05 mg/mL) to prevent in vitro infection of FCE cells with FHV-1 was determined during 72 hours of culture by assessing viral cytopathic effects and viral titers.

Results—Cidofovir at concentrations of 0.05, 0.005, and 0.0005 mg/mL significantly reduced mean viable cell counts, and cidofovir at a concentration of 0.05 mg/mL significantly reduced the percentage viability of cultured FCE cells. Minimal cytopathic changes were observed at concentrations of 0.02 and 0.05 mg of cidofovir/mL. Cidofovir at concentrations of 0.05 and 0.02 mg/mL abrogated the cytopathic effects attributable to FHV-1 infection and reduced viral titers from ≥ 1014 TCID50/mL to ≤ 103.5 TCID50/mL.

Conclusions and Clinical Relevance—Cidofovir in vitro was highly efficacious against FHV-1 infection of a primary culture of FCE cells but had cytostatic effects on cultured cells. (Am J Vet Res 2005;66:217–222)

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in American Journal of Veterinary Research

Abstract

Objective—To assess the effect of interferon (IFN)-α on viability of feline corneal epithelial cells, replication of feline herpesvirus (FHV)-1, and virus-induced cytopathic changes.

Sample Population—Healthy eyes from 10 recently euthanatized cats.

Procedure—4 replicate primary cultures of feline corneal epithelial cells were grown after the addition of 102 to 106 IU of IFN-α/mL. Cultures were examined every 24 hours for evidence of cytotoxic changes. Viable cell counts and percentage of viable cells were determined 48 hours after initiation of culture. In a separate experiment, cultures of corneal cells were inoculated with FHV-1 and cultured for 72 hours with or without 105 IU of IFN-α/mL. The FHV-1–infected cultures were evaluated for viral-induced cytopathic effects, and viral titers were determined in samples of culture supernatant.

Results—Interferon-α did not have cytotoxic effects on corneal epithelial cells at concentrations ranging from 102 to 106 IU of IFN-α/mL. Interferon-α at a concentration of 105 IU/mL significantly reduced the cytopathic changes and FHV-1 titers.

Conclusions and Clinical Relevance—Lack of in vitro cytotoxic effects and efficacy against FHV-1 infection in primary cultures of feline corneal cells suggests that the in vivo therapeutic effect of IFN-α should be assessed in controlled clinical trials. (Am J Vet Res 2005;66:210–216)

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in American Journal of Veterinary Research

Abstract

Objective—To compare relative amounts of WBC mitochondrial DNA (mtDNA; assessed via real-time PCR assay) and morphology of lymphocyte mitochondria (assessed via transmission electron microscopy [TEM]) in blood samples collected from English Springer Spaniels with and without retinal dysplasia.

Animals—7 and 5 client-owned English Springer Spaniels (1 to 11 years old) with and without retinal dysplasia, respectively.

Procedures—Blood samples were obtained from affected and unaffected dogs via venipuncture. Genomic DNA was extracted from WBCs of the 7 affected and 5 unaffected dogs, and relative quantification of the cytochrome c oxidase subunit 1 gene (COX1) was determined via analysis of real-time PCR results. White blood cells from 3 affected and 4 unaffected dogs were embedded in epoxide resin for TEM; cross sections were examined for lymphocytes, which were measured. The mitochondria within lymphocytes were quantified, and the mitochondrial surface area per lymphocyte cross section was calculated. A masked technique was used to compare mitochondrial morphology between the 2 groups.

Results—Compared with the smallest measured quantity of mtDNA among unaffected dogs, mtDNA amounts varied among unaffected (1.08- to 4.76-fold differences) and affected dogs (1- to 2.68-fold differences). Analysis of lymphocyte measurements and mitochondrial surface area, morphology, and quantity revealed no significant differences between affected and unaffected dogs.

Conclusions and Clinical Relevance—No significant differences were detected in relative amounts of WBC mtDNA or the size, number, or morphology of lymphocyte mitochondria in English Springer Spaniels affected with retinal dysplasia, compared with results for unaffected control dogs.

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in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate changes in conjunctival bacteria and antimicrobial susceptibility of bacteria after cataract surgery in dogs.

ANIMALS 16 client-owned dogs.

PROCEDURES Samples for aerobic and anaerobic culture were obtained from the conjunctival fornices of both eyes of dogs 24 hours before (week 0) and 1, 3, and 6 weeks after cataract surgery. Topical administration of ofloxacin (every 6 hours) was initiated 12 hours before surgery and continued for 3 weeks. In vitro antimicrobial susceptibility was determined by Kirby-Bauer disk diffusion and a commercially available test for ofloxacin.

RESULTS Frequency of positive culture results was significantly higher at week 6 than at weeks 0 and 1. Bacterial load was more likely to be moderate or high at weeks 3 and 6 than at weeks 0 and 1. The most frequently cultured organism was Staphylococcus pseudintermedius (21/78 [26.9%]), followed by coagulase-negative Staphylococcus spp (19/78 [24.4%]). Staphylococcus pseudintermedius was the organism most frequently cultured at weeks 0 (5/12), 1 (4/12), and 6 (8/19), whereas frequency of this organism was lowest at week 3 (1/20). In contrast, coagulase-negative Staphylococcus spp were the most frequently cultured organisms at week 3 (10/20). There was a significant increase in the proportion of organisms resistant to ofloxacin at week 3, compared with the proportion at week 0.

CONCLUSIONS AND CLINICAL RELEVANCE The number of bacterial organisms increased and the population of conjunctival bacteria was altered and had a higher proportion resistant to ofloxacin during the 6 weeks after cataract surgery for dogs treated by use of this protocol.

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in American Journal of Veterinary Research