Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane

Objectives

This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.

Procedures

A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.

Results

Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.

Conclusions

This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

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.     

Bacteroides sp. as a cause of anaerobic keratitis in a pony

This Case Report describes anaerobic infection of the conjunctiva and cornea in a pony. A 13‐year‐old pony was admitted for evaluation following suspected ocular trauma. Bacterial conjunctivitis and keratitis were diagnosed based on clinical appearance of the eye and cytological evaluation of a corneal scraping. The pony responded poorly to topical therapy, and enucleation was performed. Culture of the corneal scraping yielded Corynebacterium sp. and Bacteroides sp. Anaerobic keratitis and conjunctivitis is uncommonly reported in horses, but should be suspected when there is a history of ocular trauma or skin disease, or when a malodorous ocular discharge is identified.     

The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6

TLR2 recognises bacterial lipopeptides and lipoteichoic acid, and forms heterodimers with TLR1 or TLR6. TLR2 is relatively well characterised in mice and humans, with published crystal structures of human TLR2/1/Pam3CSK4 and murine TLR2/6/Pam2CSK4. Equine TLR4 is activated by a different panel of ligands to human and murine TLR4, but less is known about species differences at TLR2. We therefore cloned equine TLR2, TLR1 and TLR6, which showed over 80% sequence identity with these receptors from other mammals, and performed a structure-function analysis. TLR2/1 and TLR2/6 from both horses and humans dose-dependently responded to lipoteichoic acid from Staphylococcus aureus, with no significant species difference in EC50 at either receptor pair. The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species’ TLRs do not significantly affect ligand recognition. Species differences were seen between the responses to Pam2CSK4 and Pam3CSK4 at TLR2/1. Human TLR2/1, as expected, responded to Pam3CSK4 with greater potency and efficacy than Pam2CSK4. At equine TLR2/1, however, Pam3CSK4 was less potent than Pam2CSK4, with both ligands having similar efficacies. Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning. These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections.     

In vivo confocal microscopy of the normal equine cornea and limbus

Objective  To describe morphologic features, pachymetry and endothelial cell density of the normal equine cornea and limbus by in vivo confocal microscopy.
Animals studied  Ten horses without ocular disease.
Procedure  The central and peripheral corneas were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module using a combination of automated and manual image acquisition modes. Thickness measurements of various corneal layers were performed and endothelial cell density determined.
Results  Images of the constituent cellular and noncellular elements of the corneal epithelium, stroma, endothelium, and limbus were acquired in all horses. Corneal stromal nerves, the subepithelial nerve plexus, and the sub-basal nerve plexus were visualized. Cells with an appearance characteristic of Langerhans cells and corneal stromal dendritic cells were consistently detected in the corneal basal epithelium and anterior stroma, respectively. Median central total corneal thickness was 835 μm (range 725–920 μm) and median central corneal epithelial thickness was 131 μm (range 115–141 μm). Median central endothelial cell density was 3002 cells per mm² (range 2473–3581 cells per mm²).
Conclusions  In vivo corneal confocal microscopy provides a noninvasive method of assessing normal equine corneal structure at the cellular level and is a precise technique for corneal sublayer pachymetry and cell density measurements. A resident population of presumed Langerhans cells and corneal stromal dendritic cells was detected in the normal equine cornea. The described techniques can be applied to diagnostic evaluation of corneal alternations associated with disease and have broad clinical and research applications in the horse.     

Amniotic membrane transplantation for corneal surface reconstruction after excision of corneolimbal squamous cell carcinomas in nine horses

OBJECTIVE: The purpose of this study was to evaluate the usefulness and effectiveness of permanent amniotic membrane transplantation as an adjunctive treatment to superficial keratectomy alone or combined with strontium-90 irradiation for treatment of equine corneolimbal squamous cell carcinoma (SCC) to decrease corneal scarring and recurrence rate. STUDY: The retrospective case study included 11 horses (n = 12 eyes) diagnosed and treated for ocular SCC that involved the limbus and cornea. Nine of those horses (n = 9 eyes) were treated between 2002 and 2006, with superficial lamellar keratectomy alone or combined with strontium-90 irradiation and followed by placement of a permanent amniotic membrane graft in the surgical defect. The level of scarring (i.e. the clarity of the cornea) resulting with the use of amniotic membrane was subjectively compared to cases where a permanent bulbar conjunctival graft was performed following keratectomy combined with strontium-90 irradiation or cryotherapy (n = 3 eyes). Recurrence was defined as the postoperative and postirradiation regrowth of SCC in the same site and globe. RESULTS: The nine horses that received an amniotic membrane graft after keratectomy alone or combined with irradiation showed a minimal level of scarring in a cornea that regained a greater transparency in comparison to the horses that were treated with a bulbar conjunctival graft. All of the horses that received an amniotic membrane graft had 226 +/- 218 days of follow-up without tumor recurrence (mean +/- SD), ranging from 21 days to 778 days. CONCLUSIONS: The combination of superficial keratectomy alone or associated with beta-irradiation and permanent amniotic membrane transplantation is an effective treatment of corneal or corneolimbal SCC in horses. The placement of an amniotic membrane material represents an alternative surgical procedure to bulbar conjunctival grafts, especially if there is a lack of bulbar conjunctiva tissue available after tumor resection or if a particularly large corneal resection is necessary. The amniotic membrane is incorporated into the corneal defect and seems to create noticeably much less scarring than a corneal defect covered by bulbar conjunctiva.     

Presumed primary ocular lymphangiosarcoma with metastasis in a miniature horse

A 7‐year‐old, 153.0‐kg American Miniature mare presented for evaluation of keratoconjunctivitis of the right eye (OD). A superior palpebral conjunctival mass and stromal keratitis were diagnosed. The incisional biopsy diagnosis was a presumptive corneal hemangiosarcoma. Transpalpebral enucleation was performed, and histopathologic evaluation confirmed angiosarcoma of the conjunctiva, cornea, and extraocular muscles. The horse developed progressive epistaxis and orbital swelling following surgery. A systemic workup was performed 3 months after enucleation, revealing regrowth within the orbit and marked cranial cervical lymphomegaly, suggestive of metastasis. Humane euthanasia was performed, and necropsy confirmed a locally invasive periorbital tumor with metastasis to the submandibular tissue, submandibular lymph node, and thoracic inlet. Histopathologic evaluation of necropsy specimens revealed polygonal to spindle neoplastic cells lining neoplastic vascular channels lacking erythrocytes. Immunohistochemically, the neoplastic cells labeled strongly positive for PROX‐1, vimentin, CD‐31, VEGF, weakly positive for factor VIII‐related antigen, and negative for collagen IV. Based on the clinical, histological, and immunohistochemical features of this tumor, a primary ocular lymphangiosarcoma with metastasis was diagnosed.     

Aqueous humor and plasma concentrations of a compounded 0.2% solution of terbinafine following topical ocular administration to normal equine eyes

Objective To determine the transcorneal penetration and systemic absorption of a compounded 0.2% terbinafine solution following repeated topical administration to normal equine eyes. Sample population Six healthy adult horses with normal ocular examinations. Procedures One eye of each horse received 0.2 mL of a compounded 0.2% terbinafine solution every 4 h for seven doses. During the 1 h following administration of the final dose, multiple peripheral blood samples were obtained, and a single aqueous humor (AH) sample was collected at the end of the hour. AH and plasma concentrations of terbinafine were determined using high pressure liquid chromatography (HPLC). Stability of the formulation was assessed with HPLC analysis over a 14‐day time period. Results Terbinafine was not detected in the AH or plasma of any horse at any time point. No signs of ocular irritation or systemic toxicity were noted in any horse at any time point. The solution was stable over 14 days. Conclusion Topical ocular administration of compounded 0.2% terbinafine solution does not result in detectable AH or plasma levels following administration to normal equine eyes, suggesting its use for deep corneal or intraocular fungal infections in equine ophthalmology may be limited.     

Canine amniotic membrane transplantation for corneal reconstruction after the excision of dermoids in dogs

Transplantation of canine amniotic membrane (AM) in conjunction with a third eyelid flap was performed after the removal of large dermoids by keratectomy and conjunctivectomy on 7 eyes of 7 dogs. Corneal epithelialization was completed within 2 weeks after the transplantation. Five eyes attained normal transparency of the cornea within 5 weeks. Slight pigmentation of the bulbar conjunctiva at the limbus was observed in 1 dog that had pre-existing pigmentary keratitis. Neovascularization and scarring of the cornea and impaired vision were not found in any dogs at 8 weeks after the transplantation. In conclusion, transplantation of canine AM can promote corneal healing after the excision of large dermoids in dogs.     

Application of superficial keratectomy and soft contact lens for the treatment of symblepharon in a cat: a case report

A 7-month-old intact female Persian cat was diagnosed with symblepharon accompanied by epiphora, brownish ocular discharge, and ocular discomfort in the left eye. Superficial keratectomy (SK) was performed to remove adhesions between the conjunctiva and cornea. To prevent re-adhesion after SK, the detached conjunctival tissue was sutured to the corneal limbus, and a soft contact lens (SCL) was inserted and a partial temporary tarsorrhaphy was performed. The SCL and tarsorrhaphy sutures were maintained for 22 days, and symblepharon did not recur 347 days postoperatively. SK combined with SCL is a relatively easy and cost-effective surgical option for feline symblepharon.     

Corneal hemangiosarcoma in a cat

A 10 year-old castrated male Domestic Short-hair cat with a history of chronic bilateral keratitis was referred for assessment of a red, elevated mass involving the left cornea. The rapid growth of the mass, over a month period in combination with pronounced vascularization and invasion of the corneal surface suggested an aggressive inflammatory or neoplastic process. Following keratectomy, the lesion was diagnosed histopathologically as a hemangiosarcoma. The tumor recurred locally within 3 weeks and enucleation was performed. Histopathologic examination of the globe confirmed the diagnosis and did not reveal infiltration of the limbus and conjunctiva. No signs of local recurrence or metastatic disease have been observed 18 months following enucleation. To the authors' knowledge this is the first case of primary corneal hemangiosarcoma described in the feline species.     

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.     

Immunological characterization of the equine airway epithelium and of a primary equine airway epithelial cell culture model

Our understanding of innate immunity within the equine respiratory tract is limited despite growing evidence for its key role in both the immediate defense and the shaping of downstream adaptive immune responses to respiratory disease. As the first interface to undergo pathogen invasion, the respiratory epithelium is a key player in these early events and our goal was to examine the innate immune characteristics of equine respiratory epithelia and compare them to an in vitro equine respiratory epithelial cell model cultured at the air-fluid interface (AFI). Respiratory epithelial tissues, isolated epithelial cells, and four-week old cultured differentiated airway epithelial cells collected from five locations of the equine respiratory tract were examined for the expression of toll-like receptors (TLRs) and host defense peptides (HDPs) using conventional polymerase chain reaction (PCR). Cultured, differentiated, respiratory epithelial cells and freshly isolated respiratory epithelial cells were also examined for the expression of TLR3, TLR9 and major histocompatibility complex (MHC) class I and class II using fluorescence-activated cell sorting (FACS) analysis. In addition, cytokine and chemokine profiles from respiratory epithelial tissues, freshly isolated respiratory epithelial cells, and cultured, differentiated, epithelial cells from the upper respiratory tract were examined using real-time PCR. We found that respiratory epithelial tissues and isolated epithelial cells expressed TLRs 1-4 and 6-10 as well as HDPs, MxA, 2'5' OAS, β-defensin-1, and lactoferrin. In contrast, epithelial cells cultured at the AFI expressed TLRs 1-4 and 6 and 7 as well as MxA, 2'5' OAS, β-defensin-1, but had lost expression of TLRs 8-10 and lactoferrin. In addition, MHC-I and MHC-II surface expression decreased in epithelial cells cultured at the AFI compared to isolated epithelial cells whereas TLR3 and TLR9 were expressed at similar levels. Lastly, we found that equine respiratory epithelial cells express an array of pro-inflammatory, antiviral and regulatory cytokines and that after four weeks of in vitro growth conditions, equine respiratory epithelial cells cultured at the AFI retained expression of GM-CSF, IL-10, IL-8, TGF-β, TNF-α, and IL-6. In summary, we describe the development of an in vitro equine respiratory epithelial cell culture model that is morphologically similar to the equine airway epithelium and retains several key immunological properties. In the future this model will be a used to study equine respiratory viral pathogenesis and cell-to-cell interactions.     

Conjunctivitis and ulcerative keratitis secondary to conjunctival plant foreign bodies in a herd of alpacas (Lama Pacos)

Purpose To describe six cases of conjunctivitis and ulcerative keratitis secondary to plant foreign bodies from weed‐contaminated grass hay which were lodged within the temporal conjunctival fornix in a herd of alpacas. Methods A total of 21 alpacas from the same farm developed blepharospasm and mucopurulent ocular discharge. Six of the 21 were evaluated at the University of Tennessee College of Veterinary Medicine within a 2‐week period. The six animals evaluated received an ophthalmic examination including slit‐lamp biomicroscopy, fluorescein staining, and topical corneal anesthesia for foreign body removal when necessary. Results Six animals had conjunctivitis characterized by hyperemia and mucopurulent discharge. Five animals had superficial ulcerative keratitis with intense corneal neovascularization and edema. In all cases, the keratitis was located at the temporal limbus. Seven plant foreign bodies were observed and were removed following topical corneal anesthesia in five animals. The plant seeds were identified as three common weed species found in orchard grass hay. Complete resolution of all clinical signs was achieved with foreign body removal and medical therapy. Conclusions Fan‐shaped temporal corneal ulcers may signify the presence of conjunctival foreign bodies in alpacas. Plant foreign bodies should be considered in addition to ocular infection when multiple animals of a herd are affected as outbreaks may occur with weed‐contaminated hay.     

Treatment of Advanced Squamous Cell Carcinomas Involving the Equine Cornea

Corneal squamous cell carcinomas with surface areas of more than 2.0 cm were diagnosed in 26 eyes of 25 horses. The depth of the lesion into the corneal stroma and involvement of the ocular adnexa were important determinants for surgical treatment. Corneal tumors that extended into adjacent palpebral conjunctiva, eyelid, or orbit were not amenable to complete excision, while those that involved only cornea and bulbar conjunctiva were treated surgically by keratectomy and beta radiation. Initial treatment was keratectomy followed by beta radiation on 24 eyes and enucleation of two eyes. Twenty keratectomy patients remained tumor free after long-term follow-up. One was lost to follow-up and three recurred. The recurrences were retreated with subsequent cure (one horse), enucleation (one horse), or euthanasia (one horse).     

一例法斗犬眼角膜皮样囊肿切除及结膜瓣遮盖术

角膜皮样囊肿是一种长在人或动物的角膜、结膜、角巩膜等处被覆毛发的异常皮肤样组织。在人和动物均可见,动物中尤其是在牛和犬中较为常见。角膜皮样囊肿可以引发眼睑痉挛、眼分泌物增加、结膜炎、角膜炎和角膜溃疡等,严重者可导致角膜穿孔和眼球坏死。通过角膜切除术或结膜切除术,切除角膜皮样囊肿并对症治疗的方法来治疗眼部角膜皮样囊肿。以下是在2022年3月接诊的一例幼年法斗犬患有右侧眼角膜皮样囊肿,对患犬实施浅层角膜切除术切除右侧角膜皮样囊肿并用结膜瓣遮盖的手术方法进行治疗,术后恢复良好。