Introduction
Canine herpesvirus-1 (CHV-1) and feline herpesvirus-1 (FHV-1) are closely related alphaherpesviruses that share similar pathogeneses and produce similar clinical ocular lesions to herpes simplex virus (HSV-1) infections in human patients, including punctate and dendritic ulcerative keratitis.1,2,3,4 Dendritic corneal ulcers are classic ocular lesions associated with CHV-1 and FHV-1 infection.5,6 Dogs and cats infected with CHV-1 or FHV-1, respectively, can have substantial ocular morbidity1,2,3,4 that may be painful and potentially vision-threatening.7,8 Furthermore, both CHV-1 and FHV-1 establish lifelong latent infections that are frequently associated with recurrent ocular infections and recrudescent disease.9,10
Dogs and cats with naturally acquired infections with CHV-1 or FHV-1, respectfully, represent unique models for human ocular HSV-1 infection and have been used for comparative studies of basic viral pathogenesis and therapeutics.11,12,13,14 In veterinary and human medicine, the use of in vivo confocal microscopy (IVCM) permits noninvasive, real-time, spatial sectioning of tissues at the cellular level and provides high resolution and magnification imaging of all corneal layers on a single plane.15,16 In human medicine, IVCM features of dendritic keratitis associated with HSV-1 infection have been described.17,18 In veterinary medicine, IVCM features of clinically normal corneas of dogs and cats have been reported,19,20,21,22 whereas IVCM features in dogs and cats with naturally acquired active herpetic keratitis have not. Therefore, the objective of the study reported here was to describe the IVCM features of the corneal epithelium and stroma in dogs and cats with naturally acquired herpetic dendritic ulcerative keratitis. Our findings will advance the understanding of the basic pathophysiologic processes of herpetic keratitis in dogs and cats and may contribute to improved recognition of ocular disease associated with active viral infection or the sequelae of these infections resulting from structural corneal damage. In addition, the results of this study could further support the appropriateness of naturally occurring CHV-1 or FHV-1 infections as models of ocular disease caused by HSV-1 infection in people.
Materials and Methods
Animals and general ophthalmic examinations
All protocols were approved by the Animal Care and Use Committee of Cornell University (#2008-0089, Ithaca, NY) and were conducted in accordance with the statement by the Association for Research in Vision and Ophthalmology for the Use of Animals in Ophthalmic and Vision Research.23 Animals were grouped as affected (client-owned dogs or cats with CHV-1 or FHV-1 dendritic keratitis) or nonaffected (specific-pathogen-free laboratory dogs and cats).
Affected group—Client-owned dogs and cats with naturally acquired CHV-1 or FHV-1 dendritic keratitis, respectively, examined at the Cornell University Hospital for Animals between January 1, 2016, and December 31, 2019, were eligible for the study. For inclusion, the etiologic diagnosis of herpetic keratitis in each dog or cat had to have been confirmed with virus isolation, PCR assay, or both performed on ocular swab specimens. Animals were excluded from the study if they had received antiviral medication prior to IVCM examination. Before IVCM examination, each animal underwent a complete ophthalmic examination, including slit-lamp biomicroscopy (Kowa SL-17; Kowa Co), indirect ophthalmoscopy, Schirmer I tear testing, and corneal application of fluorescein and rose bengal stains. Digital photographs of each animal’s corneal lesions were obtained.
Nonaffected group—Specific-pathogen-free laboratory Beagles (n = 10) and cats (10) that did not have herpetic keratitis and had no abnormalities detected on ophthalmic examinations were included in the nonaffected group. These animals originated from established CHV-1 and FHV-1 free colonies and were born and maintained in strict bioisolation facilities. Each dog or cat was seronegative for antibodies against CHV-1 or FHV-1, respectively, and results were negative for PCR assay for CHV-1 or FHV-1, respectively, performed on an ocular swab specimen. Additionally, each nonaffected animal underwent a complete ophthalmic examination identical to that performed on each animal in the affected group.
IVCM examinations
The IVCM examinations of corneas were performed with a laser scanning in vivo confocal microscopy system (Heidelberg Retina Tomograph II-Rostock Cornea Module; Heidelberg Engineering) coupled with a 63 × objective (Carl Zeiss Meditec AG) and 400-µm field lens. The confocal microscope was mounted to an electric lift table and the chin and forehead rests were removed to facilitate objective positioning against the corneal surface of each dog and cat examined. Animals were positioned in sternal recumbency on a padded table or allowed to remain standing on the table surface. The IVCM examinations were performed with gentle manual restraint after the application of a single drop of topical ophthalmic anesthetic (proparacaine hydrochloride 0.5% ophthalmic solution.
Several drops of contact gel (GenTeal tear gel; Novartis Pharmaceuticals Corp) were applied to the front of the microscope lens and ocular surface. A sterile, single-use polymethylmethacrylate cap (TomoCap; Heidelberg Engineering) mounted on the microscope lens was positioned perpendicular to, and in slight contact with, the corneal surface. All examinations were performed by a single microscopist (ECL). Full-thickness images of corneal lesions were captured with a combination of manual and automated image acquisition modes. Multipoint imaging was performed over the center and around the periphery of each corneal lesion. For animals in the nonaffected group, full-thickness examination of the axial, paraxial, and peripheral areas of the cornea was performed systematically for both eyes. Following the examinations, digitized IVCM images of the cornea were further analyzed for pathological features. Specific IVCM findings that were recorded for each eye were the presence or absence of abnormal epithelial cell morphologies, epithelial leukocytes, Langerhans cells, anterior stromal dendritic cells, hyperreflective keratocyte nuclei with visible cytoplasmic processes, inflammatory cells in the anterior stroma, and abnormal corneal nerve morphologies.
Statistical analysis
Results were compared for dogs in the affected versus nonaffected groups and for cats in the affected versus nonaffected groups. The Mann-Whitney rank sum test and the Fisher exact test were used to evaluate age and sex similarities, respectively, between the affected and nonaffected groups on the basis of species. The IVCM examination findings were summarized as proportions and 95% CIs (determined with the Agresti-Coull method), and the Fisher exact test was used to compare results for examined eyes with versus without herpetic keratitis (affected group vs nonaffected group), further subgrouped by species. For the statistical analysis of IVCM findings, eyes were considered independent outcomes because the clinical and IVCM findings in eyes of individual animals bilaterally affected may not always be consistent. Thus, an animal with bilateral involvement would have had results from each affected eye included separately in the analysis of IVCM findings. All analyses were performed with available software (SPSS Statistics version 27; IBM Corp). Values of P ≤ 0.05 were considered significant.
Results
Affected dogs
There were 6 client-owned dogs with naturally acquired CHV-1 dendritic ulcerative keratitis in the affected group. The median age was 8.9 years (range, 0.8 to 12.2 years) and included 4 castrated males and 2 spayed females. The dogs included 2 mixed-breed dogs and 1 each of Australian Cattle Dog, American Cocker Spaniel, Shih Tzu, and Yorkshire Terrier.
Clinical ocular disease—All dogs in the affected group had unilateral ocular lesions, with the affected cornea having had a single dendritic ulcer (n = 4) or multiple dendritic ulcers (2; Figures 1 and 2). Ocular swab specimens were submitted for CHV-1 PCR assay alone (n = 2), virus isolation alone (2), or both (2). Clinical diagnosis of CHV-1 was confirmed by positive results for CHV-1 PCR assay (n = 3), virus isolation (2), or both (1). Five dogs had ≥ 1 concurrent ocular disease in the same eye as was affected with herpetic keratitis and included keratoconjunctivitis sicca (n = 3), primary glaucoma (2), or immature cataract (1), alone or in combination. Each of the concurrent ocular diseases had been diagnosed previously and was receiving topical ophthalmic treatment (2% cyclosporine solution [n = 3], artificial tear gel [2], 0.1% diclofenac sodium solution [1], dorzolamide-timolol solution [1], alone or in combination). No dogs in the affected group had known systemic diseases or were receiving any systemic medications when they underwent IVCM examination.
Clinical digital photograph (A) and in vivo confocal microscopy photomicrographs (B through F) of the cornea of a 7-year-old castrated male American Cocker Spaniel dog with canine herpesvirus-1 dendritic ulcerative keratitis in the affected group examined at the Cornell University Hospital for Animals between January 1, 2016, and December 31, 2019. A—A single large dendritic corneal ulcer (green arrow) stained with fluorescein stain is present in the axial portion of the cornea. B and C—In corneal regions immediately adjacent to the area of dendritic ulceration, abnormal epithelial cell morphologies present include round, small, hyperreflective cells (white arrows) intermixed with elongated, enlarged, hyperreflective cells (black arrows). D—Punctate, hyperreflective opacities (blue arrows) are diffusely distributed in the cornea. E—Langerhans cells (white arrowheads) are diffusely distributed in the cornea. F—In the corneal stroma, abnormal nerve morphologies of the subbasal nerve plexus include tortuous, beaded, and discontinuous nerves (yellow arrows). B through F—Bars = 50 µm.
Citation: American Journal of Veterinary Research 82, 11; 10.2460/ajvr.82.11.903
Clinical digital photograph (A) and in vivo confocal microscopy photomicrographs (B through F) of a cornea of an 0.8-year-old castrated male mixed-breed dog in the affected group described in Figure 1. A—A single large dendritic corneal ulcer (green arrow) stained with fluorescein stain is present in the axial portion of the cornea. B—The center of the dendritic ulcer appears as a hyporeflective region (asterisks) that contains amorphous material. C and D—Immediately adjacent to the area of dendritic ulceration, abnormal epithelial cell morphologies, including round, small, hyperreflective cells (white arrows) and elongated, enlarged, hyperreflective cells (black arrows), are present, and there are large accumulations of amorphous, hyperreflective material (gray arrowheads) in the superficial epithelial layer. E—Punctate, hyperreflective opacities (blue arrows) and inflammatory cells (black arrowheads) are present in all layers of the epithelium. F—Langerhans cells and anterior stromal dendritic cells (white arrowheads) are present in the basal epithelium and anterior stroma, respectively. B through F—Bars = 50 µm.
Citation: American Journal of Veterinary Research 82, 11; 10.2460/ajvr.82.11.903
IVCM findings—Epithelial abnormalities identified with the use of IVCM were similar in all examined eyes of dogs in the affected group (Figures 1 and 2; Table 1). The center of the dendritic ulcers appeared as hyporeflective dark regions primarily devoid of the normal epithelial layers. The dark areas of epithelial erosion often contained hyperreflective amorphous debris and what appeared to be individual hyperreflective sloughed epithelial cells. The epithelium immediately adjacent to the areas of dendritic ulceration had markedly abnormal morphology. These abnormal cellular morphologies included round, relatively small, hyperreflective cells intermixed with elongated, enlarged, hyperreflective cells. Adjacent to the areas of ulceration and within all layers of the epithelium were punctate, hyperreflective opacities and occasional inflammatory cells. In addition, larger accumulations of hyperreflective material were infrequently observed in the superficial epithelial layers adjacent to the corneal ulcers. Langerhans cells and anterior stromal dendritic cells were commonly encountered in the basal epithelium and anterior stroma, respectively, and were diffusely distributed in the corneas of all affected eyes.
Frequency distribution and 95% CIs of various abnormalities identified on in vivo confocal microscopy (IVCM) of corneas with herpetic dendritic ulcerative keratitis in the 6 client-owned dogs with canine herpesvirus-1 (all with unilateral ocular disease; 6 eyes examined) and 10 client-owned cats with feline herpesvirus-1 (7 with unilateral and 3 with bilateral ocular disease; 13 eyes examined) that were evaluated at the Cornell University Hospital for Animals between January 1, 2016, and December 31, 2019 and that comprised the affected group. None of the abnormalities listed were detected on IVCM of the corneas of the 10 dogs (20 eyes examined) and 10 cats (20 eyes examined) in the nonaffected group.
| Abnormal IVCM findings for corneas | Affected dogs | Affected cats | ||
|---|---|---|---|---|
| No. of eyes with the IVCM finding (n = 13) | 95% CI (%) | No. of eyes with the IVCM finding (n = 13) | 95% CI (%) | |
| Abnormal epithelial cell morphologies | 6 | 0.56-1.0 | 13 | 0.73-1.0 |
| Epithelial leukocytes | 6 | 0.56-1.0 | 13 | 0.73-1.0 |
| Langerhans cells | 6 | 0.56-1.0 | 13 | 0.73-1.0 |
| Anterior stromal dendritic cells | 6 | 0.56-1.0 | 13 | 0.73-1.0 |
| Activated keratocytes | 4 | 0.3-0.91 | 7 | 0.29-0.77 |
| Stromal leukocytes | 3 | 0.19-0.81 | 8 | 0.35-0.82 |
| Abnormal corneal nerve morphologies | 3 | 0.19-0.81 | 6 | 0.23-0.71 |
Stromal abnormalities identified with the use of IVCM were less consistently present in the examined eyes of dogs in the affected group and included hyperreflective keratocyte nuclei with visible cytoplasmic processes in 4 eyes and scattered inflammatory cells in the anterior stroma in 3 eyes. Abnormal nerve morphologies were present in the subbasal and subepithelial nerve plexuses in 3 eyes, and the nerves appeared tortuous, beaded, and in some cases discontinuous (Figure 1).
Nonaffected dogs
The specific-pathogen-free laboratory Beagles consisted of 5 sexually intact males and 5 sexually intact females that were all 18 months of age when they underwent IVCM examination. Sex distribution did not differ significantly (P = 0.6) between dogs in the affected and nonaffected groups; however, affected dogs were significantly (P = 0.03) older than nonaffected dogs. No dogs in the nonaffected group had a concurrent ocular disease or known systemic disease, and none were receiving any medications when they underwent IVCM examination.
IVCM findings—The IVCM findings in all 20 eyes of the 10 dogs in the nonaffected group were similar to previously published descriptions of clinically normal corneas of dogs as observed with IVCM.19,20,21,22 None of the morphological abnormalities identified in the eyes of dogs with CHV-1 keratitis were identified in the corneal epithelium in dogs of the nonaffected group, and the proportion of eyes with epithelial cell abnormalities was significantly (P < 0.001) higher for the examined eyes of dogs in the affected group (6/6) versus the nonaffected group (0/20). Additionally, Langerhans cells and anterior stromal dendritic cells were not detected in any of the 20 corneas of dogs in the nonaffected group, which was significantly (P < 0.001) less than the proportion of eyes examined in dogs of the affected group that had each of the cellular abnormalities identified (6/6). Similarly, hyperreflective keratocyte nuclei with visible cytoplasmic processes, inflammatory cells in the anterior stroma, and abnormal corneal nerve morphologies were not present in any of the 20 eyes examined for dogs in the nonaffected group but were identified for significantly (P = 0.001, P = 0.008, and P = 0.008, respectively) higher proportions (4/6, 3/6, and 3/6, respectively) in the examined eyes of dogs in the affected group.
Affected cats
There were 10 client-owned cats with naturally acquired FHV-1 dendritic ulcerative keratitis in the affected group. The median age was 6.9 years (range, 1.1 to 15.8 years) and included 7 castrated males and 3 spayed females. The cats were reported as either domestic shorthair (n = 9 cats) or Burmese (1).
Clinical ocular disease—Seven cats had unilateral ocular lesions, and 3 cats had bilateral ocular lesions; thus, there were 13 examined eyes for the cats in the affected group. Of these 13 eyes, 8 had multiple dendritic corneal ulcers, 5 had single dendritic corneal ulcers (Figures 3 and 4). In addition to dendritic corneal ulcerations, 3 eyes of cats in the affected group concurrently had multiple punctate corneal ulcerations. Ocular swab specimens were submitted for FHV-1 PCR assay for 8 cats and virus isolation for the remaining 2 cats. Diagnostic confirmation of FHV-1 was achieved with positive results for PCR assay (n = 8 cats) or virus isolation (2). None of the cats in the affected group had a concurrent or preexisting ocular disease in the same eyes with herpetic keratitis. Additionally, none of the cats in the affected group were receiving topical ophthalmic or systemic medications when they underwent IVCM examination. Furthermore, no cats had known systemic disease.
Clinical digital photograph (A) and in vivo confocal microscopy photomicrographs (B through F) of a cornea of a 14-year-old spayed female domestic shorthair cat with feline herpesvirus-1 dendritic ulcerative keratitis in the affected group described in Figure 1. A—There are multiple dendritic and punctate corneal ulcers (green arrows) stained with fluorescein stain. B—The center of a punctate ulcer appears as a hyporeflective region (asterisk) that contains amorphous material and sloughing epithelial cells. In the corneal regions immediately adjacent to the area of ulceration, there are abnormal epithelial cell morphologies including round, small, hyperreflective cells (white arrows) intermixed with elongated, enlarged, hyperreflective cells (black arrows). C and D—Adjacent to the corneal ulcers in all layers of the epithelium are amorphous, hyperreflective material (gray arrowheads) and hyperreflective opacities (blue arrows) that are sometimes organized into ring configurations. E—Inflammatory cells (black arrowheads) are scattered in all layers of the epithelium. F—Abnormal nerve morphologies are present within the subbasal nerve plexus and include enlarged, tortuous, and beaded nerves (yellow arrows). B through F—Bars = 50 µm.
Citation: American Journal of Veterinary Research 82, 11; 10.2460/ajvr.82.11.903
Clinical digital photograph (A) and in vivo confocal microscopy photomicrographs (B through F) of a cornea of an 8-year-old castrated male domestic shorthair cat with feline herpesvirus-1 dendritic ulcerative keratitis in the affected group described in Figure 1. A—A single large dendritic corneal ulcer (green arrow) stained with rose bengal stain is present in the axial portion of the cornea. B through E—The center of the dendritic ulcer appears as a hyporeflective region (asterisks). In the corneal regions adjacent to the area of ulceration, there are abnormal epithelial cell morphologies, including round, small, hyperreflective cells (white arrows) intermixed with elongated, enlarged, hyperreflective cells (black arrows). In the epithelium adjacent to the corneal ulcer, there are large amorphous (gray arrowheads) and punctate (blue arrows) hyperreflective opacities. F—In the anterior stroma, there are hyperreflective keratocyte nuclei with visible cytoplasmic processes (visible in the entire field of the photomicrograph). B through F—Bars = 50 µm.
Citation: American Journal of Veterinary Research 82, 11; 10.2460/ajvr.82.11.903
IVCM findings—On IVCM examination, all examined eyes for cats in the affected group had epithelial abnormalities (Figures 3 and 4) similar to those identified for dogs in the affected group. As identified in affected dogs, the center of the dendritic ulcers in affected cats appeared as hyporeflective regions that occasionally contained hyperreflective amorphous debris and individual hyperreflective sloughed epithelial cells. The epithelium immediately adjacent to the areas of dendritic ulceration possessed similar abnormal cellular morphologies as those observed in dogs of the affected group. In 10 of the 13 examined eyes of affected cats, these regions of atypical epithelial cells also extended for several millimeters away from the ulcer border, which was a finding not observed in the affected dogs. In the affected cats, these abnormal cellular morphologies included round, small, hyperreflective cells and a distinct separate population of elongated, enlarged, hyperreflective cells. Similar to findings for the affected dogs, immediately adjacent to the dendritic ulcers and in all layers of the epithelium were punctate, hyperreflective opacities and occasional inflammatory cells. However, unlike in the affected dogs, these opacities and cells were sometimes organized into circular ring configurations in the affected cats. Also similar to the affected dogs, all examined eyes for cats in the affected group had Langerhans cells and anterior stromal dendritic cells frequently and diffusely distributed in the corneas.
In 3 eyes of cats in the affected group, punctate corneal ulcerations appeared as roughly circular hyporeflective depressions or pits in the corneal epithelium surrounded by a ring of hyperreflective epithelial cells (Figure 3). With IVCM, it was possible to determine that some of these punctate ulcers were only partial-thickness epithelial defects and lined posteriorly by thin layers of epithelial cells.
Similar to findings in the affected dogs, stromal abnormalities identified in examined eyes of affected cats were less consistently present. Scattered inflammatory cells were present in the anterior stroma of 8 of the 13 eyes examined for the affected cats. Hyperreflective keratocyte nuclei with visible cytoplasmic processes were present in 7 of the 13 cat eyes (Figure 4). Abnormal nerve morphologies were present in the subbasal and subepithelial nerve plexuses in 6 of the 13 examined eyes for cats in the affected group and included nerves that appeared enlarged, tortuous, beaded, and rarely discontinuous (Figure 3).
Nonaffected cats
The specific-pathogen-free laboratory cats included 5 sexually intact males and 5 sexually intact females that were all 6 months of age when they underwent IVCM examination. Sex distribution was not significantly (P = 0.6) different between the affected and nonaffected cats; however, affected cats were significantly (P < 0.001) older than nonaffected cats. No cats in the nonaffected group had concurrent ocular diseases or known systemic disease, and none were receiving any medications when they underwent IVCM examination.
IVCM findings—The IVCM findings in all 20 eyes of the 10 cats in the nonaffected group were similar to previously published descriptions of clinically normal corneas in cats as observed with IVCM.19,20,21,22 None of the morphological abnormalities in the eyes of cats with FHV-1 keratitis were identified in the corneal epithelium of cats in the nonaffected group; thus, the proportion of eyes with epithelial cell abnormalities was significantly (P < 0.001) higher for the examined eyes of cats in the affected group (13/13) versus the nonaffected group (0/20). Similar to our findings for affected versus nonaffected dogs, the proportion of eyes in which Langerhans cells and anterior stromal dendritic cells were detected was significantly (P < 0.001) less for cats in the nonaffected group (0/20) versus the affected group (13/13). Furthermore, hyperreflective keratocyte nuclei with visible cytoplasmic processes, inflammatory cells in the anterior stroma, and abnormal corneal nerve morphologies were not present in any of the 20 examined eyes of cats in the nonaffected group but were identified for significantly (P < 0.001, P < 0.001, and P = 0.002, respectively) higher proportions (7/13, 8/13, and 6/13) for the examined eyes of cats in the affected group.
Discussion
Descriptions of IVCM features of HSV-1 keratitis in human patients include those from investigations on changes in corneal innervation and other corneal morphological features during nonepithelial HSV-1 keratitis or after the resolution of active herpetic keratitis.24,25,26,27,28,29,30,31 However, to our knowledge, reports specifically describing IVCM features of active HSV dendritic keratitis are limited and include a single case report18 and a case series17 of 4 clinical patients. The findings in those previous reports17,18 of HSV-1 keratitis are largely similar to what was detected in the affected dogs and cats of the present study. Specifically, the irregular epithelial cell morphologies and the presence of epithelial inflammatory cells and presumptive antigen-presenting cells in the epithelium and stroma were analogous. Infected epithelial cells, epithelial cell necrosis, and then the resultant inflammatory response likely produced the abnormalities detected with IVCM. Nerve alterations described in the subbasal and subepithelial nerve plexuses of a human patient18 were similar to those we observed in some of the cats and dogs of the affected group in the present study. Keratocyte abnormalities identified in the affected animals of the present study were less uniform than in human patients with HSV keratitis and appeared morphologically more consistent with activated keratocytes than stromal edema as was postulated in the previous IVCM studies of human patients.32,33 Virus and host species differences as well as examination at different stages of the active viral infection could have accounted for some of the variations in the stromal and nerve changes observed in the present study and previous reports.
Cytologic and histopathologic evaluations for humans with HSV dendritic keratitis revealed swollen, rounded corneal epithelial cells, giant multinucleated epithelial cells, and mixed population of leukocytes in the lesions.34,35,36 Elongated epithelial cells surrounded the dendrites and were orientated parallel to the lesions, with abnormal epithelial cells identified up to 2 mm distal to the dendritic ulcerations in 1 study.34 Comparable cytologic alterations, and the desquamation of corneal epithelial cells, are reported in rabbits with experimental HSV dendritic keratitis.34,37 Histopathologic and cytologic corneal alterations in cats with experimental FHV-1 keratoconjunctivitis included the presence of rounded epithelial cells, macrophages, and neutrophils.6 It was likely that similar cellular changes in the cornea corresponded to the abnormalities observed by IVCM in the present study.
Recurrent episodes of HSV keratitis in human patients are associated with morphological alterations in the corneal subbasal and subepithelial nerve plexuses that can be detected by IVCM. These changes are associated with the loss of corneal sensation and include reductions in nerve density, total nerve numbers, and nerve branch density.25,29,30 Similar alterations of the subbasal and subepithelial nerve plexuses were identified by IVCM, histopathology, and immunohistochemistry in a dog with neurotrophic keratitis that developed after recovery from severe CHV-1 keratitis, and immunohistochemical evaluation of the dog’s corneas with antineurotublin antibody demonstrated marked hyperinnervation of the stroma and increased numbers of morphologically abnormal neurites.38 In that dog, the abnormal nerve morphologies were characterized by a normal dichotomous branching pattern of stromal innervation superimposed with large numbers of excessively tortuous fibers and moderate‐to‐large numbers of filamentous, straight, and thin immature neurites.38 The abnormalities identified in the nerve plexuses of affected animals in the present study could have represented precursors to the more chronic alterations seen in the dog of the previous report.38 Similar nerve plexus changes are also speculated to occur in cats following FHV-1 keratitis.39
Limitations of the present study include the small sample size and the frequent presence of concurrent ocular diseases in the examined dogs. Most of the dogs with CHV-1 dendritic ulcerative keratitis in the present study had a concurrent ocular disease that might have affected findings on IVCM examination. In addition, the medical treatment administered for these concurrent ocular diseases could have impacted the IVCM examination findings in undetermined ways. However, the dogs in the affected group were typical representations of dogs with CHV-1 keratitis because the virus most frequently develops in dogs receiving medical treatment for other ocular conditions.1 Although it is not possible to exclude these other conditions as contributing to the corneal abnormalities observed with the use of IVCM, our findings in affected dogs and cats were remarkably similar even though none of the cats in the affected group had a concurrent ocular disease or were receiving ophthalmic or systemic medications when they underwent IVCM examination. Furthermore, previous studies evaluating IVCM examination findings in dogs with keratoconjunctivitis sicca40 and humans with glaucoma41,42 also report markedly different IVCM characteristics of these conditions than what was detected in the animals with viral keratitis in the present study.
Another limitation of the present study was that the nonaffected animals were significantly younger than the affected animals, and the impact of age on the evaluated IVCM characteristics is unknown. Additionally, the inclusion of specific-pathogen-free laboratory dogs and cats as a comparison population was selected to conclusively as possible demonstrate that the IVCM findings from animals with viral keratitis were not present in dogs and cats that were free of herpesvirus infection and that had clinically normal findings on ophthalmic examinations. The almost ubiquitous nature of latent CHV-1 and FHV-1 infections in the general dog and cat populations, combined with the lack of any available diagnostic assays that reliably detect latent infection in all individuals, render it difficult to fully determine whether a specific dog or cat is free of CHV-1 and FHV-1 infection. Furthermore, we recognize that evaluation of the contralateral cornea in animals with unilateral ocular lesions could be of interest as a separate study. For instance, recent IVCM studies26,43 evaluating unilateral ocular HSV-1 infection in human patients have detected several corneal abnormalities in the contralateral uninfected eyes26,43; however, in the present study, only the clinically diseased eyes in animals of the affected group were evaluated so as to help prevent any adverse impacts on the healthy eyes of these client-owned animals. Examination of a larger population of animals with bilateral ocular lesions would be useful to help determine the similarity and dependency of the IVCM findings between eyes of individual animals.
Herpetic dendritic ulcerative keratitis in dogs and cats is associated with numerous microanatomic corneal abnormalities that can be detected by IVCM. Epithelial and stromal changes identified by IVCM in the present study were similar for dogs with CHV-1 keratitis and cats with FHV-1 keratitis. The detected morphological corneal abnormalities are also comparable to those described for humans with HSV dendritic keratitis and provide further evidence of the analogous properties and suitability of CHV-1 and FHV-1 infections in dogs and cats, respectively, as models of ocular HSV-1 infection in people.
References
- 1. ↑
Ledbetter EC. Canine herpesvirus-1 ocular diseases of mature dogs. N Z Vet J. 2013;61(4):193–201.
- 3. ↑
Ledbetter EC, Riis RC, Kern TJ, Haley NJ, Schatzberg SJ. Corneal ulceration associated with naturally occurring canine herpesvirus-1 infection in two adult dogs. J Am Vet Med Assoc. 2006;229(3):376–384.
- 4. ↑
Gould D. Feline herpesvirus-1: ocular manifestations, diagnosis and treatment options. J Feline Med Surg. 2011;13(5):333–346.
- 5. ↑
Ledbetter EC, Kim SG, Dubovi EJ, Bicalho RC. Experimental reactivation of latent canine herpesvirus-1 and induction of recurrent ocular disease in adult dogs. Vet Microbiol. 2009;138(2):98–105.
- 6. ↑
Nasisse MP, Guy JS, Davidson MG, Sussman WA, Fairley NM. Experimental ocular herpesvirus infection in the cat. Sites of virus replication, clinical features and effects of corticosteroid administration. Invest Ophthalmol Vis Sci. 1989;30(8):1758–1768.
- 7. ↑
Gervais KJ, Pirie CG, Ledbetter EC, Pizzirani S. Acute primary canine herpesvirus-1 dendritic ulcerative keratitis in an adult dog. Vet Ophthalmol. 2012;15(2):133–138.
- 9. ↑
Miyoshi M, Ishii Y, Takiguchi M, et al. Detection of canine herpesvirus DNA in the ganglionic neurons and the lymph node lymphocytes of latently infected dogs. J Vet Med Sci. 1999;61(4):375–379.
- 10. ↑
Townsend WM, Jacobi S, Shih-Han T, Kiupel M, Wise AG, Maes RK. Ocular and neural distribution of feline herpesvirus-1 during active and latent experimental infection in cats. BMC Vet Res. 2013;9:185.
- 11. ↑
Maes R. Felid herpesvirus type 1 infection in cats: a natural host model for alphaherpesvirus pathogenesis. ISRN Vet Sci. 2012;2012:495830.
- 12. ↑
Stiles J, Guptill-Yoran L, Moore GE, Pogranichniy RM. Effects of lambda-carrageenan on in vitro replication of feline herpesvirus and on experimentally induced herpetic conjunctivitis in cats. Invest Ophthalmol Vis Sci. 2008;49(4):1496–1501.
- 13. ↑
Pennington MR, Ledbetter EC, Van de Walle GR. New paradigms for the study of ocular alphaherpesvirus infections: insights into the use of non-traditional host model systems. Viruses. 2017;9(11):349.
- 14. ↑
Ledbetter EC, Kice NC, Matusow RB, Dubovi EJ, Kim SG. The effect of topical ocular corticosteroid administration in dogs with experimentally induced latent canine herpesvirus-1 infection. Exp Eye Res. 2010;90(6):711–717.
- 15. ↑
Jalbert I, Stapleton F, Papas E, Sweeney DF, Coroneo M. In vivo confocal microscopy of the human cornea. Br J Ophthalmol. 2003;87(2):225–236.
- 16. ↑
Patel DV, McGhee CN. Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review. Clin Experiment Ophthalmol. 2007;35(1):71–88.
- 17. ↑
Yokogawa H, Kobayashi A, Mori N, Sugiyama K. Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy. Clin Ophthalmol. 2015;9:1771–1777.
- 18. ↑
Martone G, Alegente M, Balestrazzi A, et al. In vivo confocal microscopy in bilateral herpetic keratitis: A case report. Eur J Ophthalmol. 2008;18(6):994–997.
- 19. ↑
Kafarnik C, Fritsche J, Reese S. In vivo confocal microscopy in the normal corneas of cats, dogs and birds. Vet Ophthalmol. 2007;10(4):222–230.
- 20. ↑
Kafarnik C, Fritsche J, Reese S. Corneal innervation in mesocephalic and brachycephalic dogs and cats: assessment using in vivo confocal microscopy. Vet Ophthalmol. 2008;11(6):363–367.
- 21. ↑
Strom AR, Cortes DE, Thomasy SM, Kass PH, Mannis MJ, Murphy CJ. In vivo ocular imaging of the cornea of the normal female laboratory Beagle using confocal microscopy. Vet Ophthalmol. 2016;19(1):63–67.
- 22. ↑
Reichard M, Hovakimyan M, Wree A, et al. Comparative in vivo confocal microscopical study of the cornea anatomy of different laboratory animals. Curr Eye Res. 2010;35(12):1072–1080.
- 23. ↑
Statement for the use of animals in ophthalmic and vision research. The Association for Research in Vision and Ophthalmology. Accessed January 1, 2016. https://www.arvo.org/About/policies/statement-for-the-use-of-animals-in-ophthalmic-and-vision-research/
- 24. ↑
Hamrah P, Sahin A, Dastjerdi MH, et al. Cellular changes of the corneal epithelium and stroma in herpes simplex keratitis: an in vivo confocal microscopy study. Ophthalmology. 2012;119(9):1791–1797.
- 25. ↑
Moein HR, Kheirkhah A, Muller RT, Cruzat AC, Pavan-Langston D, Hamrah P. Corneal nerve regeneration after herpes simplex keratitis: A longitudinal in vivo confocal microscopy study. Ocul Surf. 2018;16(2):218–225.
- 26. ↑
Müller RT, Pourmirzaie R, Pavan-Langston D, et al. In vivo confocal microscopy demonstrates bilateral loss of endothelial cells in unilateral herpes simplex keratitis. Invest Ophthalmol Vis Sci. 2015;56(8):4899–4906.
- 27. ↑
Cottrell P, Ahmed S, James C, et al. Neuron J is a rapid and reliable open source tool for evaluating corneal nerve density in herpes simplex keratitis. Invest Ophthalmol Vis Sci. 2014;55(11):7312–7320.
- 28. ↑
Rosenberg ME, Tervo TM, Muller LJ, Moilanen JAO, Vesaluoma MH. In vivo confocal microscopy after herpes keratitis. Cornea. 2002;21(3):265–269.
- 29. ↑
Nagasato D, Araki-Sasaki K, Kojima T, Ideta R, Dogru M. Morphological changes of corneal subepithelial nerve plexus in different types of herpetic keratitis. Jpn J Ophthalmol. 2011;55(5):444–450.
- 30. ↑
Hamrah P, Cruzat A, Dastjerdi MH, et al. Corneal sensation and subbasal nerve alterations in patients with herpes simplex keratitis: an in vivo confocal microscopy study. Ophthalmology. 2010;117(10):1930–1936.
- 31. ↑
Mocan MC, Irkec M, Mikropoulos DG, Bozkurt B, Orhan M, Konstas AGP. In vivo confocal microscopic evaluation of the inflammatory response in non-epithelial herpes simplex keratitis. Curr Eye Res. 2012;37(12):1099–1106.
- 32. ↑
Jordan C, Patel DV, Abeysekera N, McGhee CNJ. In vivo confocal microscopy analyses of corneal microstructural changes in a prospective study of collagen cross-linking in keratoconus. Ophthalmology. 2014;121(2):469–474.
- 33. ↑
Hovakimyan M, Guthoff R, Reichard M, Wree A, Nolte I, Stachs O. In vivo confocal laser-scanning microscopy to characterize wound repair in rabbit corneas after collagen cross-linking. Clin Exp Ophthalmol. 2011;39(9):899–909.
- 34. ↑
Maudgal PC, Missotten L. Histopathology of human superficial herpes simplex keratitis. Br J Ophthalmol. 1978;62(1):46–52.
- 36. ↑
Kim JH, Ko MK, Shin JC. Infectivity of basal epithelial cells in herpetic dendritic epithelial keratitis. Korean J Ophthalmol. 1997;11(2):84–88.
- 37. ↑
Park HJ, Ko MK. Impression cytology of herpetic simplex keratitis in rabbits. Korean J Ophthalmol. 2005;19(2):96–100.
- 38. ↑
Ledbetter EC, Marfurt CF, Dubielzig RR. Metaherpetic corneal disease in a dog associated with partial limbal stem cell deficiency and neurotrophic keratitis. Vet Ophthalmol. 2013;16(4):282–288.
- 39. ↑
Uhl LK, Saito A, Iwashita H, Maggs DJ, Mochel JP, Sebbag L. Clinical features of cats with aqueous tear deficiency: a retrospective case series of 10 patients (17 eyes). J Feline Med Surg. 2019;21(10):944–950.
- 40. ↑
Leonard BC, Stewart KA, Shaw GA, et al. Comprehensive clinical, diagnostic, and advanced imaging characterization of the ocular surface in spontaneous aqueous deficient dry eye disease in dogs. Cornea. 2019;38(12):1568–1575.
- 41. ↑
Ranno S, Fogagnolo R, Rossett L, Orzalesi N, Nucci P. Changes in corneal parameters at confocal microscopy in treated glaucoma patients. Clin Ophthalmol. 2011;5:1037–1042.
- 42. ↑
Martone G, Frezzotti P, Tosi GM, et al. An in vivo confocal microscopy analysis of effects of topical antiglaucoma therapy with preservative on corneal innervation and morphology. Am J Ophthalmol. 2009;147(4):725–735.e1.
- 43. ↑
Chirapapaisan C, Muller RT, Sahin A, et al. Effect of herpes simplex keratitis scar location on bilateral corneal nerve alterations: an in vivo confocal microscopy study. Br J Ophthalmol. 2020.
