Sung G. Kim

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.

Detection of Equine Herpesvirus-1 in Nasal Swabs of Horses by Quantitative Real-Time PCR

BACKGROUND Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples. OBJECTIVES To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses. ANIMALS Fifteen horses experimentally infected with EHV-1. METHODS Experimental study: Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Mareks disease virus as an internal control. qPCR was performed. RESULTS The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI (P< .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86-100) and 27% (95% CI: 20-35). CONCLUSIONS AND CLINICAL IMPORTANCE We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C(T) values are provided as well as justification of a minimum 10-day quarantine period.

Evaluation of a vectored equine herpesvirus type 1 (EHV-1) vaccine expressing H3 haemagglutinin in the protection of dogs against canine influenza

In 2004, canine influenza virus (CIV) was identified as a respiratory pathogen of dogs for the first time and found to be closely related to H3N8 equine influenza virus (EIV). We generated a recombinant vectored vaccine that expresses H3 of a recent isolate of EIV using equine herpesvirus type 1 (EHV-1) as the delivery vehicle. This EHV-1 vectored vaccine exhibited robust and stable EIV H3 expression and induced a strong influenza virus-specific response in both mice and dogs upon intranasal or subcutaneous administration. Furthermore, upon challenge with the recent CIV isolate A/canine/PA/10915-07, protection of vaccinated dogs could be demonstrated by a significant reduction in clinical sings, and, more importantly, by a significant reduction in virus shedding. We concluded that the EHV-1/H3 recombinant vector can be a valuable alternative for protection of dogs against clinical disease induced by CIV and can significantly reduce virus spread.

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.

Outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony

OBJECTIVE To describe clinical and virological findings of an outbreak of ocular disease attributed to naturally-acquired primary canine herpesvirus-1 (CHV-1) infection in a closed domestic dog colony. ANIMALS STUDIED Twenty-seven 10- to 16-week-old laboratory Beagles. PROCEDURE Complete ophthalmic examinations were performed and ocular samples collected for CHV-1 polymerase chain reaction and virus isolation. RESULTS The prevalence of ocular morbidity was 100% in examined dogs. Lesions were restricted to the ocular surface and included bilateral conjunctivitis (100% of dogs); punctate, dendritic, or geographic ulcerative keratitis (26% of dogs); and non-ulcerative keratitis (19% of dogs). Conjunctival petechiae were detected in 22% of dogs. Punctate and dendritic corneal ulcers were frequently organized into discrete groups or linear arrangements. Non-ulcerative keratitis appeared clinically as a perilimbal ring of superficial corneal vascularization and leukocyte infiltration. CHV-1 was detected in ocular samples by polymerase chain reaction or virus isolation in all dogs sampled. CONCLUSIONS In susceptible populations of domestic dogs, CHV-1 may be associated with outbreaks of highly contagious ocular infection in the absence of concurrent overt systemic disease. This naturally-acquired outbreak of CHV-1 infection provides an opportunity to report the spectrum and prevalence of ocular lesions associated with primary ocular CHV-1 infection in dogs. Conjunctivitis was the most frequent ocular lesion detected. Ulcerative and non-ulcerative keratitis were less prevalent and of variable clinical appearance. Dendritic ulcerative keratitis, a classic and relatively specific ocular lesion associated with alphaherpesvirus infection, was detected in < 20% of dogs.

Disseminated Canine Herpesvirus-1 Infection in an Immunocompromised Adult Dog

An 8-year-old, spayed-female Golden Retriever was evaluated at the Cornell University Hospital for Animals for complaints of lethargy, inappetence, and fever 4 days after treatment with doxorubicin. Fifteen weeks before this presentation, the dog had generalized lymphadenomegaly. Ultrasonography revealed diffuse mesenteric lymphadenomegaly, a mildly enlarged liver with multiple small hypoechoic nodules, and small hypoechoic splenic nodules. Lymph node biopsy revealed high-grade lymphoma consistent with a B-cell lineage. The diagnosis was World Health Organization stage IV B-cell lymphoma. Treatment with a CHOP-based protocol was initiated. The dog achieved a complete remission by week 2 of the protocol. During weeks 2 through 15 of the chemotherapy protocol, the dog had nausea, vomiting, and prolonged inappetence, and two documented bouts of afebrile neutropenia, which necessitated treatment delays, dose reductions of vincristine and doxorubicin, as well as symptomatic therapy (metoclopramide, maropitant citrate, and cyproheptadine) and intermittent hospitalization. Approximately 15 weeks after the start of chemotherapy, the dog received the 3rd treatment of doxorubicin (1mg/kg IV). She was discharged with maropitant citrate (4mg/kg PO q24h), cyproheptadine (0.5mg/kg PO q12h), and metoclopramide (0.5mg/kg PO q8h). Despite pre-emptive supportive care, the dog developed persistent anorexia and lethargy. Three days after treatment with doxorubicin she was normothermic, lethargic, and mildly dehydrated. There was anemia (PCV 36%; reference range, 37–55%), leukopenia (5.6 10 cells/mL; reference range, 5.7–14.2 10 cells/mL), lymphopenia (0.1 10 cells/mL; reference range, 0.9–4.7 10 cells/ mL), and thrombocytopenia (168 10 cells/mL; reference range, 186–545 10 cells/mL). The dog was treated with lactated Ringer’s Solution, SQ (1,000mL) and maropitant citrate (1mg/kg SQ), and was discharged with instructions to continue metoclopramide, oral maropitant, and mirtazepine (0.5mg/kg PO q24h). Fluids were dispensed to be continued at home (lactated Ringer’s Solution, 500mL SQ q24h). Four days after treatment with doxorubicin, the dog had worsening lethargy, persistent inappetence, and shaking. She was depressed, dehydrated, and febrile (40.41C). There was progressive nonregenerative anemia (HCT 33%; reference range, 41–60%), neutropenia (2.5 10 cells/uL; reference range, 2.7–9.4 10 cells/uL), and elevation in serum ALP (418U/L; reference range, 12–122) and ALT (1,514U/L; reference range, 25–106 U/L) activity. The dog was treated with PlasmaLyte-A (120mL/kg/day IV), supplemented with potassium chloride (10 mEq KCl/L), ampicillin sodium/sulbactam sodium (22mg/kg IV q8h), enrofloxacin (10mg/kg q24h), famotidine (0.5mg/kg IV q12h), and maropitant sodium (1mg/kg SQ q24h). On hospitalization day 2, she remained febrile (40.51C) and lethargic. Mild chemosis and mucoid discharge were noted in both eyes and melena was present. The dog was anemic (PCV 23%). Metronidazole (10mg/kg IV q12h) was administered. Fresh frozen plasma (10mL/kg) and packed red blood cells (10mL/kg) were infused IV separately over 4 hours. On hospitalization day 3, the dog remained febrile (40.51C) and depressed. Petechiae and ecchymoses were noted in her oral cavity, inner pinnae, and ventrum. There was neutropenia (0.1 10 cells/mL) and thrombocytopenia (7 10 cells/mL). Thoracic radiographs revealed consolidation of the right middle lung lobe and air bronchograms. Cytology of an endotracheal lavage revealed suppurative inflammation with both intracellular and extracellular, Gram-negative bacteria through aerobic culture revealed only fastidious organisms. Nebulization with sterile saline was instituted 4 times daily. Because of the persistent pyrexia, ampicillin sodium/ sulbactam sodium and enrofloxacin were discontinued and cefazolin (22mg/kg IV q8h) and amikacin (20mg/ kg IV q24h) were administered. Additional fresh frozen plasma was administered (10mL/kg). There was persistent elevation of serum ALP (1,545U/L), and ALT (992U/L) activity. Total bilirubin was also elevated (0.9mg/dL; reference range 0.0–0.3mg/dL). Abdominal ultrasonography revealed mild hepatomegaly. Complete ophthalmic examination, including slit-lamp biomicroscopy and indirect ophthalmoscopy, revealed bilateral severe ulcerative conjunctivitis associated with conjunctival From the Departments of Clinical Sciences (Malone, Ledbetter, Rassnick), Biomedical Sciences (Russell), and Population Medicine and Diagnostic Sciences (Kim), College of Veterinary Medicine, Cornell University, Ithaca, NY. Corresponding author: Dr Erin K. Malone, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; e-mail: ekm24@cornell.edu. Submitted September 23, 2009; Revised February 11, 2010; Accepted March 1, 2010. Copyright r 2010 by the American College of Veterinary Internal Medicine 10.1111/j.1939-1676.2010.0512.x Abbreviations:

The effect of topical ocular corticosteroid administration in dogs with experimentally induced latent canine herpesvirus-1 infection

Recurrent herpes simplex virus-1 (HSV-1) ocular infection is a frequent cause of morbidity and blindness. Factors that trigger viral reactivation are poorly understood and the role of topical ocular corticosteroid administration in the development of recurrent HSV-1 ocular disease is not clear. Clinical reports and epidemiological studies suggested topical corticosteroids may reactivate latent HSV-1 and result in recrudescent ocular disease; however, experimental studies to establish this causal relationship produced inconsistent results. The previous experimental studies were performed by infecting unnatural host species with HSV-1 and aspects of viral behavior and reactivation within these animals may differ from the host for which the virus is adapted. The purpose of the study reported here was to determine if topical ocular corticosteroid administration results in viral reactivation and recrudescent ocular disease in a host-adapted pathogen animal model of HSV-1 recurrent ocular disease. Canine herpesvirus-1 (CHV-1) is a corticosteroid-sensitive alphaherpesvirus that is biologically related to HSV-1 and induces similar ocular lesions in canids during recurrent infection. A randomized, masked, placebo-controlled, crossover study was performed. Primary ocular CHV-1 infection was experimentally induced in mature specific pathogen-free beagles by topical ocular inoculation and the presence of reactivatable latency was later confirmed by administration of an immunosuppressive dosage of systemic corticosteroid to the dogs. Twelve months following experimental CHV-1 reactivation, dogs were administered either topical ocular prednisolone acetate (1.0% ophthalmic suspension, one drop in both eyes, four times daily) or placebo (artificial tear solution, one drop in both eyes, four times daily) for 28 days. After a 14 day washout period, the treatment groups were reversed and study agents administered for an additional 28 days. Ophthalmic examinations, in vivo ocular confocal microscopy, real-time quantitative CHV-1 polymerase chain reaction assays, and CHV-1 serum neutralization antibody titers were performed at regular intervals throughout the study. Viral reactivation was not detected in dogs administered topical ocular prednisolone or placebo as determined by clinical ocular disease recrudescence, in vivo ocular confocal microscopic findings, ocular viral shedding, and serologic response. Similar to other animal models of recurrent HSV-1 ocular infection, the behavior of latent CHV-1 in dogs may differ from HSV-1 in humans; however, results of the present study suggest administration of topical ocular prednisolone at the evaluated drug concentration, dosing frequency, and treatment duration is not likely to result in detectable reactivation of latent CHV-1 in experimentally infected dogs. This may be attributed to insufficient systemic absorption of locally administered corticosteroid to reactivate latent virus and produce recurrent disease. Crystalline corneal opacities that were apparently not associated with viral reactivation were detected by clinical examination and in vivo confocal microscopy in two dogs during topical ocular prednisolone administration. The crystalline keratopathy may have resulted from corneal degeneration associated with metaherpetic disease, corticosteroid administration, or a combination of both factors.

Frequency of spontaneous canine herpesvirus-1 reactivation and ocular viral shedding in latently infected dogs and canine herpesvirus-1 reactivation and ocular viral shedding induced by topical administration of cyclosporine and systemic administration of corticosteroids

OBJECTIVE To determine the frequency of spontaneous canine herpesvirus-1 (CHV-1) reactivation and ocular viral shedding in latently infected dogs and the effect of topical ocular administration of cyclosporine. ANIMALS 8 mature Beagles with experimentally induced latent CHV-1 infection. PROCEDURES Following induction of primary ocular CHV-1 infection, the presence of reactivatable CHV-1 latency was confirmed by systemically administering prednisolone to the dogs. Dogs were then monitored for 36 weeks via clinical examination and conjunctival sample CHV-1 PCR assay performed at 4-day intervals and CHV-1 virus neutralization antibody assay performed at 2-week intervals. During weeks 16 to 32, dogs were administered 0.2% cyclosporine ointment in both eyes twice daily and blood cyclosporine concentrations were monitored. During weeks 33 to 36, the presence of reactivatable CHV-1 latency was reconfirmed via systemic administration of prednisolone. RESULTS Reactivation of latent CHV-1 was not detected via clinical examination or viral shedding during the initial 32 weeks, including before and during topical ocular administration of cyclosporine, and there were no significant differences in CHV-1 virus neutralization titer increases between the study periods. Blood cyclosporine concentrations were less than assay detection limits in all dogs on the sampling days. Systemic administration of corticosteroids repeatedly resulted in ocular disease and viral shedding. CONCLUSIONS AND CLINICAL RELEVANCE Spontaneous CHV-1 reactivation did not occur frequently in latently infected mature dogs, and this was not altered by topical ocular administration of cyclosporine. This characteristic may be a factor contributing to the lower frequency of recurrent herpetic ocular disease in dogs relative to other host species and their associated alphaherpesviruses.