A case report: Null-cell cardiac lymphoma in an English bulldog.

This case report describes a novel example of an extranodal null-type lymphoma in the myocardium of a middle-aged English bulldog who presented with signs of right heart failure. An echocardiogram found, in addition to the pericardial effusion, thickened right and left ventricular free walls and the interventricular septum. The right ventricular free wall myocardium had multinodular lesions, suspicious for infiltrative disease. The owner elected humane euthanasia, and permission for necropsy was obtained. Multifocal left and right ventricular nodules and an incidental aortic root mass were detected, the latter of which was later confirmed as a chemodectoma. Microscopically, the myocardial nodules were sheets of round cells consistent with a high-grade lymphoma. Neoplastic cells were not immunoreactive to CD3 (T-cell) or CD20 and CD79a (B-cell), Mum-1 (plasma cell), CD117 (mast cell), or CD18 (histiocyte). These findings are consistent with a high-grade, null-cell-type lymphoma.

CLINICAL AND HISTOPATHOLOGIC OCULAR FINDINGS IN AQUARIUM-HOUSED COWNOSE RAYS (RHINOPTERA BONASUS).

Cownose rays (Rhinoptera bonasus) are susceptible to ocular disease with their prominent globes, but despite being popular animals housed in aquaria, there is little published information about their normal ocular anatomy and common pathologic ocular findings. A total of 63 live cownose rays (CNR) from three unrelated, separately housed groups had ocular examinations, and 5 adult rays were selected for ocular ultrasound. All examinations were performed out of the water, and most without anesthesia. Clinical findings were described, categorized, and scored by severity. Sixty-two of 63 rays (123 eyes) had clinical abnormalities, including 110 eyes with corneal pathology (mild = 76, moderate/severe = 34) and 74 eyes with intraocular pathology (mild = 44, moderate/severe = 30). Grey-to-white corneal opacities were the most common pathology (n = 58 rays/100 eyes) followed by cataracts (n = 41 rays/58 eyes), then persistent (or dysplastic) pupillary membranes (n = 14 rays/15 eyes). Most pathologic findings appeared inactive, but one aquarium had several CNR with active ocular pathology. There was a significant association between the diagnosis of moderate/severe corneal and intraocular pathology with age (P = 0.008 and P = 0.014, respectively) and weight (P = 0.001 and P = 0.039, respectively), as well as moderate/severe corneal pathology and group sampled (P = 0.03). There were no other significant variables identified. Additionally, histopathology of 14 eyes (11 rays) from two different facilities were examined, with keratitis (n = 8) and uveitis (n = 2) as the most common lesions. This study shows a high prevalence of pathologic ocular findings in cownose ray eyes with heavier adults more likely to be affected than lighter juveniles. Comprehensive ocular evaluation is important in this species and serial ocular exams and future studies should be pursued to monitor ocular disease progression and better understand possible etiologies.

Retinoschisis: a retrospective study of an uncommon retinal change in cats and dogs.

Retinoschisis is a poorly documented form of retinal degeneration characterized by cyst-like splitting that occurs between the inner nuclear and outer plexiform layers. The pathogenesis of retinoschisis is incompletely understood, but congenital, acquired and secondary aetiologies (glaucoma, inflammation, neoplasia) are described in humans. This retrospective study investigated the prevalence and associated histological and clinical features of retinoschisis in cats and dogs submitted for biopsy over a 10-year period. Of 140 samples with documented 'retinal vacuolation', four out of 120 (3%) canine samples and one out of 20 (5%) feline samples had changes consistent with retinoschisis. In most cases (80%), there was concurrent retinal detachment. In cases with available histories, increased intraocular pressure, proptosis and retinal detachment were reported clinical findings. In cats and dogs, retinoschisis is a retinal change that is generally secondary to other ocular lesions.

Prevalence of Cardiac Lesions in Cases of Bovine Blackleg in Tennessee (USA), 2004-2018.

Blackleg is a common cause of death in cattle, mostly caused by the bacterium Clostridium chauvoei. Cardiac lesions were traditionally considered uncommon in cases of blackleg in cattle until a 2018 study reported otherwise. This study was aimed at determining the prevalence of cardiac disease among cattle that died of blackleg in Tennessee, USA. The outcome of this study would reinforce the importance of assessing cardiac lesions in suspected cases of blackleg in cattle. The University of Tennessee Veterinary Medical Center database searched for cattle with a confirmed diagnosis of blackleg necropsied between 2004 and 2018. Of the 120 necropsy reports, 37 had a diagnosis of blackleg. Histology slides of skeletal muscle (26/37) and the heart (26/37) were reviewed to confirm the presence of supportive lesions. Of the 37 cases of blackleg identified, 26 animals (70.3%) had cardiac lesions, including 4 (10.8%) that had only cardiac involvement without skeletal muscle lesions. Specifically, (5.4%; 2/37) had only necrotizing myocarditis; (13.5%; 5/37) had only fibrinous or fibrinosuppurative pericarditis, epicarditis, or endocarditis; (51.4%; 19/26) had a combination of myocarditis and pericarditis, epicarditis, or endocarditis; and (29.7%; 11/37) had no lesions. Furthermore, of the 26 cases with cardiac lesions, 24 cases had gross lesions, while 2 cases were identified only by microscopic examination. This indicates that gross examination alone is insufficient to identify cardiac involvement in blackleg cases in cattle. Contrary to traditional perceptions, cardiac lesions in cases of bovine blackleg could be as high as 70% and are most often associated with skeletal muscle lesions. The prevalence of cardiac lesions in cases of blackleg in cattle may be higher when the heart is examined microscopically than if it is only evaluated grossly. Pathologists should specifically evaluate the heart for lesions in suspected cases of blackleg in cattle and utilize microscopic examination when gross lesions are absent.

Qualitative investigation of µ- and κ-opioid receptor distribution in the brains of budgerigars (Melopsittacus undulatus).

OBJECTIVE: To perform a qualitative analysis of the distribution of µ- and κ-opioid receptor mRNA in the forebrain and midbrain of budgerigars (Melopsittacus undulatus). SAMPLE: 8 brains of male budgerigars. PROCEDURES: Custom-made RNA hybridization probes (RNAscope; Advanced Cell Diagnostics Inc) were used for fluorescent in situ hybridization (FISH) assays performed on selected fresh frozen prepared sections of brain tissue to identify µ- and κ-opioid receptor mRNA. RESULTS: There was κ-opioid receptor mRNA present in the nucleus dorsomedialis posterior thalami, lateral striatum, mesopallium, tractus corticohabenularis et corticoseptalis, griseum et fibrosum, stratum griseum centrale, medial striatum, and area parahippocampalis. There was µ-opioid receptor mRNA present in the stratum griseum centrale, stratum opticum, dorsomedialis posterior thalami, area parahippocampalis, medial striatum, and nidopallium intermedium. CLINICAL RELEVANCE: Consistent with previous studies in pigeons and domestic chicks, κ-opioid receptors were more abundant than µ-opioid receptors in the samples of the present study. The results of this study may also help explain the hyperexcitability or lack of response that can occur with administration of pure µ-opioid receptor agonists, but not κ-opioid receptor agonists. This study was not quantitative, so further research should endeavor to compare the various regions of the brain using FISH technology.

Unilateral mixed adenoma of the third eyelid lacrimal gland in a boxer dog.

A female boxer dog was presented with a mass expanding the left third eyelid. Histopathology revealed a mass composed of pleomorphic cells arranged in tubuloacinar structures, a fibrovascular myxoid stroma, and intermixed trabeculae of woven bone. The mass was diagnosed as a mixed adenoma of the third eyelid lacrimal gland.

Margin of safety of extended-duration transdermal buprenorphine solution following multiple-dose administrations to cats.

Transdermal buprenorphine solution (TBS) is approved for the control of postoperative pain in cats where a single preoperative dose provides 4 days of analgesia. It is administered as a unit dose of 8 mg to cats weighing 1.2-3 kg and 20 mg to cats weighing to >3-7.5 kg, which is equivalent to a dosage on a bodyweight basis of 2.7-6.7 mg/kg. In this safety study, the 1X dose was defined as 6.7 mg/kg. Thirty-two cats (16 males and 16 females) were randomly allocated to placebo, 1, 2, and 3X TBS administered topically to the dorsal cervical skin every 4 days for 3 doses. Clinical observations, behavioral scores, mydriasis score (yes/no), and physiological variables were assessed or measured prior to each dose administration (0 h) and at 1, 2, 4, 8, 12, 24, 36, 48, and 72 h following each treatment and prior to euthanasia on Day 12 or 13. Blood samples for clinical pathology were collected on Days - 1, 4, 8, and prior to euthanasia. There was little evidence of respiratory, cardiovascular, or gastrointestinal effects. Respiratory rates were above the reference range in all groups and lower by 10 breaths/min in the 3X group during the third dosing interval compared to placebo. There were no differences in heart rates. Constipation was transiently observed in approximately equal numbers in placebo- and TBS-treated cats. Behavioral scores showed sedation or euphoria was transient in the first dosing interval but became more prolonged with each dosing interval. Mydriasis was prolonged in the first dosing interval and diminished by the third dosing interval consistent with accommodation. Mean body temperatures in TBS-treated cats were up to 0.6°C (1.8°F) greater than placebo-treated cats. There were no clinically relevant changes to serum chemistry, hematology, or urinalysis outcomes nor gross or microscopic observations attributable to TBS. These data demonstrate that TBS is safe and well-tolerated when administered to 16-week-old cats at multiples of the approved dose and duration and supports clinical safety in the event of delayed buprenorphine metabolism, medication errors, or alterations in the dosing regimen.

Pulmonary silicosis in 2 rock hyraxes, and literature review.

Two rock hyraxes (Procavia capensis), from the Chattanooga Zoo, were submitted separately for autopsy at the University of Tennessee Veterinary Medical Center. The first was a 4-y-old intact female that died without premonitory signs and the second was a 10-y-old intact male that was euthanized because of severe renal disease. Microscopically, the lungs of both hyraxes had multifocal-to-coalescing, <1-mm diameter aggregates of epithelioid macrophages separated by streams of fibrous tissue. Macrophages contained intracytoplasmic, clear, acicular, birefringent crystals. Transmission electron microscopy and energy-dispersive x-ray spectroscopy findings on the lung samples were consistent with silica crystal deposition. The hyraxes had been housed together on commercially sourced play sand composed of 99-99.5% quartz, a crystalline silica polymorph. The microscopic findings, transmission electron microscopy, and energy-dispersive x-ray spectroscopy of the intrahistiocytic crystals, in addition to the history of exposure to crystalline silica, were consistent with pulmonary silicosis. Pulmonary silicosis has not been reported previously in rock hyraxes, to our knowledge.

Inhibiting Glucose Metabolism Results in Herpes Simplex Encephalitis.

This report evaluates how HSV enters the brain to cause herpes simplex encephalitis following infection at a peripheral site. We demonstrate that encephalitis regularly occurred when BALB/c mice were infected with HSV and treated daily with 2-deoxy-d-glucose (2DG), which inhibits glucose use via the glycolysis pathway. The outcome of infection in the trigeminal ganglion (TG), the site to which the virus spreads, replicates, and establishes latency, showed marked differences in viral and cellular events between treated and untreated animals. In control-untreated mice, the replicating virus was present only during early time points, whereas in 2DG recipients, replicating virus remained for the 9-d observation period. This outcome correlated with significantly reduced numbers of innate inflammatory cells as well as T cells in 2DG-treated animals. Moreover, T cells in the TG of treated animals were less activated and contained a smaller fraction of expressed IFN-γ production compared with untreated controls. The breakdown of latency was accelerated when cultures of TG cells taken from mice with established HSV latency were cultured in the presence of 2DG. Taken together, the results of both in vivo and in vitro investigations demonstrate that the overall effects of 2DG therapy impaired the protective effects of one or more inflammatory cell types in the TG that normally function to control productive infection and prevent spread of virus to the brain.

Correlation of Mock Board Examination Scores During Anatomic Pathology Residency Training with Performance on the Certifying Examination.

Mock board exams are common in residency programs across many disciplines. However, the value of mock board results in predicting success on the actual certifying examination is largely anecdotal and undocumented. The University of Tennessee anatomic pathology residency program has a long history of giving mock board exams twice a year during the course of the 3-year diagnostic training program. The mock exams give residents a sense of the types of questions that may appear on the actual certifying examination. The resulting scores serve to help identify improvement areas to focus additional study. In addition, by providing residents the mental and physical experiences designed to mimic the test day, we hope to better prepare these trainees for optimal performance on the certifying examination. This study correlated mock board results of 16 anatomic pathology residents, from July 2006 through January 2016, with their subsequent performance on the certifying exam. The results of these biannual exams were significantly correlated (p < .001) with results for the American College of Veterinary Pathologists Certifying Examination.

Suspected Iatrogenic Seeding of Oral Melanoma Secondary to Endotracheal Intubation in a Dog.

A 10 yr old castrated male pug was presented with a 3 day history of intermittent dyspnea, cough, inappetence, and inability to breathe while sleeping. He had previously received hypofractionated radiation therapy for an amelanotic oral malignant melanoma (OMM) 7 mo prior to presentation. At presentation, the dog was gasping and dyspneic. Oral examination identified the OMM on the right hard palate. Thoracic radiographs revealed an angular soft-tissue opacity within the trachea just distal to the thoracic inlet. No evidence of pulmonary metastatic disease was seen. Tracheoscopy identified a pedunculated, nonpigmented mass within the lumen of the distal trachea near the carina. Treatment options were presented to the owners and included tracheal stenting or tracheal resection and anastomosis. Because of the poor prognosis, the owners elected humane euthanasia. Postmortem examination confirmed the presence of melanoma in the distal trachea; no other sites of OMM metastasis were identified. The cause of OMM development in the distal trachea in this case is suspected to have resulted from mechanical tumor cell seeding during endotracheal tube placement for general anesthesia 7 mo prior to presentation. Despite the reported rarity of mechanical tumor seeding, this potential complication warrants consideration in dogs with OMM.

Effects of low-level laser therapy on the healing of surgically closed incisions and surgically created open wounds in dogs.

OBJECTIVE: To determine the effects of low-level laser therapy (LLLT) on primarily closed incisions and full thickness open wounds in dogs. STUDY DESIGN: Prospective, masked, placebo-controlled design. ANIMAL POPULATION: Healthy intact female dogs (n = 10). METHODS: Dogs underwent bilateral flank ovariectomy procedures, and open wounds were created bilaterally with a punch biopsy. Each side of the dog (incision and open wound) was randomly assigned to the treatment (TX) group or the control (CN) group. The TX group received LLLT once daily for 5 days with a 980-nm laser and a total energy density of 5 J/cm2 . The CN group received a sham treatment (laser turned off) for an identical amount of time each day. The wounds were assessed visually; measured; photographed at postoperative days 3, 7, 11, and 14; and biopsied on postoperative days 7 and 14. A 2-way repeated measures multivariate analysis of variance was used to analyze differences between groups. RESULTS: There was no difference between groups for subjective assessment of healing time and wound measurements (P = .7). There was no difference in histopathologic assessment except that the CN group had more necrosis and perivascular lymphocytes and macrophages at day 7 (P = .03). The TX group had more perivascular lymphocytes and macrophages at day 14 (P = .01). CONCLUSION: LLLT did not appear to influence the healing of surgically created incisions and small wounds with the methodology reported here. CLINICAL SIGNIFICANCE: Results of this study do not support recommending LLLT to stimulate healing of uncomplicated, small wounds and incisions.

Immunohistochemical expression of insulin, glucagon, and somatostatin in pancreatic islets of horses with and without insulin resistance.

OBJECTIVE To assess insulin, glucagon, and somatostatin expression within pancreatic islets of horses with and without insulin resistance. ANIMALS 10 insulin-resistant horses and 13 insulin-sensitive horses. PROCEDURES For each horse, food was withheld for at least 10 hours before a blood sample was collected for determination of serum insulin concentration. Horses with a serum insulin concentration < 20 µU/mL were assigned to the insulin-sensitive group, whereas horses with a serum insulin concentration > 20 µU/mL underwent a frequently sampled IV glucose tolerance test to determine sensitivity to insulin by minimal model analysis. Horses with a sensitivity to insulin < 1.0 × 10-4 L•min-1•mU-1 were assigned to the insulin-resistant group. All horses were euthanized with a barbiturate overdose, and pancreatic specimens were harvested and immunohistochemically stained for determination of insulin, glucagon, and somatostatin expression in pancreatic islets. Islet hormone expression was compared between insulin-resistant and insulin-sensitive horses. RESULTS Cells expressing insulin, glucagon, and somatostatin made up approximately 62%, 12%, and 7%, respectively, of pancreatic islet cells in insulin-resistant horses and 64%, 18%, and 9%, respectively, of pancreatic islet cells in insulin-sensitive horses. Expression of insulin and somatostatin did not differ between insulin-resistant and insulin-sensitive horses, but the median percentage of glucagon-expressing cells in the islets of insulin-resistant horses was significantly less than that in insulin-sensitive horses. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that, in insulin-resistant horses, insulin secretion was not increased but glucagon production might be downregulated as a compensatory response to hyperinsulinemia.

INVESTIGATION OF ENROFLOXACIN-ASSOCIATED RETINAL TOXICITY IN NONDOMESTIC FELIDS.

Enrofloxacin is known to cause retinal toxicity in domestic cats. The hallmark lesion of enrofloxacin-associated retinal toxicity in domestic cats is thinning of the outer nuclear layer of the retina. Enrofloxacin is commonly used to treat bacterial infections in nondomestic felids because of its action against a wide spectrum of bacteria and the ability for it to be given orally. No previous studies have investigated the potential retinal toxicity of enrofloxacin in nondomestic felids. This retrospective study evaluated 81 eyes from 14 lions ( Panthera leo ) and 33 tigers ( Panthera tigris ) that had been enucleated or collected postmortem. The thickness of the outer nuclear retina was assessed in two separate sites in each eye by counting the rows of nuclei and by using digital image analysis software to determine the area of the nuclei at each site. Medical records were reviewed to determine the enrofloxacin dose for each cat. Cats that had not received enrofloxacin (n = 11) were compared with treated animals (n = 36). The outer nuclear layer thickness or area in treated versus untreated cats was not significantly different. Additionally, no clinical blindness was reported in any of the cats. This study showed no evidence of enrofloxacin-associated thinning of the outer nuclear layer in the lions and tigers evaluated, suggesting that enrofloxacin can be used safely in these animals.

Evaluation of the role of the cyclooxygenase signaling pathway during inflammation in skin and muscle tissues of ball pythons (Python regius).

OBJECTIVE To determine degrees of production of cyclooxygenase (COX)-1 and -2 and other mediators of inflammation in noninflamed and inflamed skin and muscle tissues in ball pythons (Python regius). ANIMALS 6 healthy adult male ball pythons. PROCEDURES Biopsy specimens of noninflamed skin and muscle tissue were collected from anesthetized snakes on day 0. A 2-cm skin and muscle incision was then made 5 cm distal to the biopsy sites with a CO2 laser to induce inflammation. On day 7, biopsy specimens of skin and muscle tissues were collected from the incision sites. Inflamed and noninflamed tissue specimens were evaluated for production of COX-1, COX-2, phosphorylated protein kinase B (AKT), total AKT, nuclear factor κ-light-chain-enhancer of activated B cells, phosphorylated extracellular receptor kinases (ERKs) 1 and 2, and total ERK proteins by western blot analysis. Histologic evaluation was performed on H&E-stained tissue sections. RESULTS All biopsy specimens of inflamed skin and muscle tissues had higher histologic inflammation scores than did specimens of noninflamed tissue. Inflamed skin specimens had significantly greater production of COX-1 and phosphorylated ERK than did noninflamed skin specimens. Inflamed muscle specimens had significantly greater production of phosphorylated ERK and phosphorylated AKT, significantly lower production of COX-1, and no difference in production of COX-2, compared with production in noninflamed muscle specimens. CONCLUSIONS AND CLINICAL RELEVANCE Production of COX-1, but not COX-2, was significantly greater in inflamed versus noninflamed skin specimens from ball pythons. Additional research into the reptilian COX signaling pathway is warranted.

Effect of laser treatment on first-intention incisional wound healing in ball pythons (Python regius).

OBJECTIVE: To evaluate effects of laser treatment on incisional wound healing in ball pythons (Python regius). ANIMALS: 6 healthy adult ball pythons. PROCEDURES: Snakes were sedated, a skin biopsy specimen was collected for histologic examination, and eight 2-cm skin incisions were made in each snake; each incision was closed with staples (day 0). Gross evaluation of all incision sites was performed daily for 30 days, and a wound score was assigned. Four incisions of each snake were treated (5 J/cm(2) and a wavelength of 980 nm on a continuous wave sequence) by use of a class 4 laser once daily for 7 consecutive days; the other 4 incisions were not treated. Two excisional skin biopsy specimens (1 control and 1 treatment) were collected from each snake on days 2, 7, 14, and 30 and evaluated microscopically. Scores were assigned for total inflammation, degree of fibrosis, and collagen maturity. Generalized linear models were used to investigate the effect of treatment on each variable. RESULTS: Wound scores for laser-treated incisions were significantly better than scores for control incisions on day 2 but not at other time points. There were no significant differences in necrosis, fibroplasia, inflammation, granuloma formation, or bacterial contamination between control and treatment groups. Collagen maturity was significantly better for the laser-treated incisions on day 14. CONCLUSIONS AND CLINICAL RELEVANCE: Laser treatment resulted in a significant increase in collagen maturity at day 14 but did not otherwise significantly improve healing of skin incisions.

Expression of epidermal growth factor receptor and human epidermal growth factor receptor 2 in periocular squamous cell carcinomas of horses.

OBJECTIVE: To determine whether epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are expressed in periocular squamous cell carcinomas (SCCs) of horses. SAMPLE: Biopsy specimens of SCCs from 46 horses. PROCEDURES: Pathology records were searched retrospectively for biopsy specimens of periocular SCCs obtained from horses. Slides of the specimens were reviewed histologically to confirm the SCC diagnosis and stained for EGFR and HER2 by immunohistochemical methods. For both EGFR and HER2, the immunohistochemical staining intensity and percentage of stain-positive cells on the slides were determined. RESULTS: 43 of 46 (93%) SCCs were immunoreactive for EGFR. The median score for EGFR staining intensity was 4 (range, 2 to 12), and the median number of mitotic figures was 8 mitotic figures/10 hpfs (range, 0 to 34 mitotic figures/10 hpfs). Mitotic index was not correlated with the percentage of EGFR stain-positive cells or staining intensity. Of the 43 EGFR-immunoreactive SCCs, 38 had stain present primarily in the cytoplasm and 5 had stain equally distributed between the cytoplasm and cell membranes. Thirty-five of 46 (76%) SCCs were immunoreactive for HER2. Mitotic index was not correlated with the percentage of HER2 stain-positive cells or staining intensity. Of the 35 HER2-immunoreactive SCCs, the stain was present primarily in the cytoplasm and 7 had stain equally distributed between the cytoplasm and cell membranes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that most periocular SCCs of horses expressed EGFR and HER2, which suggested that equine periocular SCCs might respond to treatment with EGFR inhibitors.

Detection of papillomavirus in equine periocular and penile squamous cell carcinoma.

Squamous cell carcinoma (SCC) is the most common tumor arising in the periocular and penile areas of horses. Both ultraviolet radiation and papillomaviruses have been implicated in the pathogenesis of SCC in various species, including the horse. This retrospective study used polymerase chain reaction (PCR) to detect papillomavirus DNA in archival biopsy samples from equine periocular and penile SCC from 3 different geographic areas (northeast, southeast, and central United States). Forty-two periocular SCCs were tested; none contained papillomavirus DNA. Twenty-two penile SCCs were tested, and papillomavirus DNA was identified in 10 (43%) cases. Sequencing of the PCR products revealed homology with Equus caballus papillomavirus 2 (EcPV-2). No geographic distribution in the detection of papillomavirus was identified. Penile SCCs were significantly more likely to be papillomavirus positive than the periocular SCCs (P < 0.001). The role of papillomavirus in the development of penile SCC requires further investigation. The differing pathogeneses of periocular and penile SCC suggest that the tumors may respond differently to treatment.

Evaluation of delivery agents used for introduction of small interfering RNAs into feline corneal cells.

OBJECTIVE: To evaluate agents used for delivery of small interfering RNAs (siRNAs) into feline corneal cells, toxicity of the delivery agents, and functionality of anti-feline herpesvirus 1 (FHV-1)-specific siRNA combinations. SAMPLE: Feline primary corneal cells and 19 six-month-old colony-bred cats. PROCEDURES: siRNA delivery into corneal cells via various delivery agents was evaluated via flow cytometric detection of labeled siRNAs. Cellular toxicity was evaluated with a proliferation assay. Functionality was tested via quantitative reverse transcriptase PCR assay, plaque assay, and flow cytometry. In vivo safety was evaluated with an ocular scoring method following topical application of delivery agents containing siRNAs into eyes. Corneal biopsy specimens were used to assess safety and uptake of siRNAs into corneal cells. RESULTS: Use of 3 delivery agents resulted in > 95% transfection of primary corneal cells. Use of a peptide for ocular delivery yielded approximately 82% transfection of cells in vitro. In cultured corneal cells, use of the siRNA combinations resulted in approximately 76% to 89% reduction in FHV-1-specific mRNA, 63% to 67% reduction of FHV-1-specific proteins in treated cells, and 97% to 98% reduction in FHV-1 replication. The agents were nonirritating in eyes, caused no substantial clinical ocular signs, and were nontoxic. Histologically, corneal epithelium and stroma were normal in treated cats. However, none of the agents were effective in delivering siRNAs into the corneal cells in vivo. CONCLUSIONS AND CLINICAL RELEVANCE: The tested anti-FHV-1-specific siRNAs could potentially be used as a treatment for FHV-1 if a successful means of in vivo delivery can be achieved.