Impact of Equine Ocular Surface Squamous Neoplasia on Interactions between Ocular Transcriptome and Microbiome.

Ocular surface squamous neoplasia (OSSN) represents the most common conjunctival tumor in horses and frequently results in vision loss and surgical removal of the affected globe. Multiple etiologic factors have been identified as contributing to OSSN progression, including solar radiation exposure, genetic mutations, and a lack of periocular pigmentation. Response to conventional treatments has been highly variable, though our recent work indicates that these tumors are highly responsive to local immunotherapy. In the present study, we extended our investigation of OSSN in horses to better understand how the ocular transcriptome responds to the presence of the tumor and how the ocular surface microbiome may also be altered by the presence of cancer. Therefore, we collected swabs from the ventral conjunctival fornix from 22 eyes in this study (11 with cytologically or histologically confirmed OSSN and 11 healthy eyes from the same horses) and performed RNA sequencing and 16S microbial sequencing using the same samples. Microbial 16s DNA sequencing and bulk RNA sequencing were both conducted using an Illumina-based platform. In eyes with OSSN, we observed significantly upregulated expression of genes and pathways associated with inflammation, particularly interferon. Microbial diversity was significantly reduced in conjunctival swabs from horses with OSSN. We also performed interactome analysis and found that three bacterial taxa (Actinobacillus, Helcococcus and Parvimona) had significant correlations with more than 100 upregulated genes in samples from animals with OSSN. These findings highlight the inflammatory nature of OSSN in horses and provide important new insights into how the host ocular surface interacts with certain microbial populations. These findings suggest new strategies for the management of OSSN in horses, which may entail immunotherapy in combination with ocular surface probiotics or prebiotics to help normalize ocular cell and microbe interactions.

Intralesional interferon alpha-2b as a novel treatment for periocular squamous cell carcinoma in horses.

OBJECTIVE: To determine the safety and efficacy of perilesional human recombinant interferon alpha-2b (IFNα2b) for treatment of periocular squamous cell carcinoma (PSCC) in horses. ANIMALS STUDIED: Eleven horses (12 eyes) with PSCC were enrolled in this prospective clinical study with owner consent. PROCEDURES: Systemically healthy horses were included in the study following confirmation of PSCC via biopsy. Every two weeks for a maximum of six treatments, horses were sedated and perilesional injection of IFNα2b (10 million IU) was performed. Tumors were measured prior to each injection and at one, three, and 12 months after treatment completion. A greater than 50% reduction in tumor size was considered positive response to treatment (i.e., partial or complete response). Development of anti-IFNα2b antibodies was assessed using serum samples obtained after treatment initiation and compared with treatment responses. Antibody concentrations were analyzed using a mixed model. Statistical significance was considered p < 0.05. RESULTS: Each horse received four to six perilesional injections of IFNα2b. Five of 12 eyes (4/11 horses) responded to treatment. Two of five eyes showed complete resolution of gross PSCC. No systemic adverse effects were seen. Local swelling occurred during treatment protocol in 6/11 horses but resolved without intervention. All horses developed serum anti-IFNα2b antibodies. There was no evidence of statistical difference in antibody concentration between responders and non-responders. CONCLUSIONS: Perilesional administration of IFNα2b was found to be well-tolerated in horses with PSCC, and induced tumor regression in 42% of treated eyes. Treatment failure appears unrelated to the development of IFNα2b antibodies.

Nanoparticle ocular immunotherapy for herpesvirus surface eye infections evaluated in cat infection model.

Ocular herpes simplex type 1 (HSV-1) infections can trigger conjunctivitis, keratitis, uveitis, and occasionally retinitis, and is a major cause of blindness worldwide. The infections are lifelong and can often recrudesce during periods of stress or immune suppression. Currently HSV-1 infections of the eye are managed primarily with anti-viral eye drops, which require frequent administration, can cause irritation, and may take weeks for full resolution of symptoms. We therefore evaluated the effectiveness of an ocular immune activating nanoparticle eye drop as a novel approach to treating HSV-1 infection, using a cat feline herpesvirus -1 (FHV-1) ocular infection model. In vitro studies demonstrated significant induction of both type I and II interferon responses by the liposome-dual TLR 3/9 agonist nanoparticles, along with suppression of FHV-1 replication. In cats with naturally occurring eye infections either proven or suspected to involve FHV-1, ocular nanoparticle treated animals experienced resolution of signs within several days of treatment, including resolution of keratitis and corneal ulcers. In a cat model of recrudescent FHV-1 infection, cats treated twice daily with immune nanoparticle eye drops experienced significant lessening of ocular signs of infection and significantly fewer episodes of viral shedding compared to control cats. Treatment was well-tolerated by all cats, without signs of drug-induced ocular irritation. We concluded therefore that non-specific ocular immunotherapy offers significant promise as a novel approach to treatment of HSV-1 and FHV-1 ocular infections.

Novel ocular immunotherapy induces tumor regression in an equine model of ocular surface squamous neoplasia.

Ocular surface squamous neoplasia (OSSN) is the major cause of corneal cancer in man and horses worldwide, and the prevalence of OSSN is increasing due to greater UVB exposure globally. Currently, there are no approved treatments for OSSN in either species, and most patients are managed with surgical excision or off-label treatment with locally injected interferon alpha, or topically applied cytotoxic drugs such as mitomycin C. A more broadly effective and readily applied immunotherapy could exert a significant impact on management of OSSN worldwide. We therefore evaluated the effectiveness of a liposomal TLR complex (LTC) immunotherapy, which previously demonstrated strong antiviral activity in multiple animal models following mucosal application, for ocular antitumor activity in a horse spontaneous OSSN model. In vitro studies demonstrated strong activation of interferon responses in horse leukocytes by LTC and suppression of OSSN cell growth and migration. In a trial of 8 horses (9 eyes), treatment with topical or perilesional LTC resulted in an overall tumor response rate of 67%, including durable regression of large OSSN tumors. Repeated treatment with LTC ocular immunotherapy was also very well tolerated clinically. We conclude therefore that ocular immunotherapy with LTC warrants further investigation as a novel approach to management of OSSN in humans.

Ocular penetration of oral acetaminophen in horses.

BACKGROUND: Acetaminophen (paracetamol) is increasingly used to treat painful conditions in horses but its ocular penetration has not been studied. OBJECTIVES: To determine whether orally administered acetaminophen penetrates the aqueous humour of the normal equine eye and report an aqueous humour:serum acetaminophen concentration ratio in horses. STUDY DESIGN: In vivo experiment. METHODS: Six privately owned horses with normal ophthalmic examinations weighing 568 ± 65 kg (mean ± standard deviation) and aged 11 ± 4 years were given 20 mg/kg acetaminophen orally every 12 h for a total of six doses. Physical exam parameters were recorded prior to, during, and after the dosing period. One hour after the final dose, horses were sedated and simultaneous aqueous humour and serum samples were collected and analysed for acetaminophen concentrations and selected eicosanoids. An aqueous humour:serum acetaminophen concentration ratio was calculated. A second aqueous humour sample was taken and analysed for eicosanoid concentrations 3 months after acetaminophen dosing. Physical exam data were compared between time points using a mixed model analysis (significance p < 0.05). RESULTS: Acetaminophen was detected in both serum and aqueous humour of all horses and mean ± standard deviation aqueous humour:serum acetaminophen concentration ratio was 44.9 ± 15.9%. No significant changes in physical exam parameters occurred during or after dosing. Eicosanoids were not detected in aqueous humour at any sampling point. MAIN LIMITATIONS: Presence of acetaminophen in the aqueous humour may not relate to clinical effect. A therapeutic level of acetaminophen has not been determined in horses, and the absence of ocular inflammation does not reflect conditions in which acetaminophen may be used. CONCLUSIONS: Acetaminophen readily penetrates the aqueous humour of the normal equine eye after consecutive oral dosing. Further study is required to determine whether acetaminophen is useful in the treatment of ocular pain and inflammation.

Preliminary evaluation of safety and migration of immune activated mesenchymal stromal cells administered by subconjunctival injection for equine recurrent uveitis.

Introduction: Equine recurrent uveitis (ERU), an immune mediated disease characterized by repeated episodes of intra-ocular inflammation, affects 25% of horses in the USA and is the most common cause of glaucoma, cataracts, and blindness. Mesenchymal stromal cells (MSCs) have immunomodulatory properties, which are upregulated by preconditioning with toll-like receptor agonists. The objective was to evaluate safety and migration of TLR-3 agonist polyinosinic, polycytidylic acid (pIC)-activated MSCs injected subconjunctivally in healthy horses prior to clinical application in horses with ERU. We hypothesized that activated allogeneic MSCs injected subconjunctivally would not induce ocular or systemic inflammation and would remain in the conjunctiva for >14 days. Methods: Bulbar subconjunctiva of two horses was injected with 10 × 106 pIC-activated (10 µg/mL, 2 h) GFP-labeled MSCs from one donor three times at two-week intervals. Vehicle (saline) control was injected in the contralateral conjunctiva. Horses received physical and ophthalmic exams [slit lamp biomicroscopy, rebound tonometry, fundic examination, and semiquantitative preclinical ocular toxicology scoring (SPOTS)] every 1-3 days. Systemic inflammation was assessed via CBC, fibrinogen, and serum amyloid A (SAA). Horses were euthanized 14 days following final injection. Full necropsy and histopathology were performed to examine ocular tissues and 36 systemic organs for MSC presence via IVIS Spectrum. Anti-GFP immunohistochemistry was performed on ocular tissues. Results: No change in physical examinations was noted. Bloodwork revealed fibrinogen 100-300 mg/dL (ref 100-400) and SAA 0-25 µg/mL (ref 0-20). Ocular effects of the subjconjucntival injection were similar between MSC and control eyes on SPOTS grading system, with conjunctival hypermia, chemosis and ocular discharge noted bilaterally, which improved without intervention within 14 days. All other ocular parameters were unaffected throughout the study. Necropsy and histopathology revealed no evidence of systemic inflammation. Ocular histopathology was similar between MSC and control eyes. Fluorescent imaging analysis did not locate MSCs. Immunohistochemistry did not identify intact MSCs in the conjunctiva, but GFP-labeled cellular components were present in conjunctival phagocytic cells. Discussion: Allogeneic pIC-activated conjunctival MSC injections were well tolerated. GFP-labeled tracking identified MSC components phagocytosed by immune cells subconjunctivally. This preliminary safety and tracking information is critical towards advancing immune conditioned cellular therapies to clinical trials in horses.

Investigation of the effects of orally administered trazodone on intraocular pressure, pupil diameter, physical examination variables, and sedation level in healthy equids.

OBJECTIVE: To investigate the effects of orally administered trazodone on intraocular pressure (IOP), pupil diameter measured in the vertical plane (ie, vertical pupil diameter [VPD]), selected physical examination variables, and sedation level in healthy equids. ANIMALS: 7 horses and 1 pony. PROCEDURES: Food was withheld for 12 hours prior to drug administration. After baseline (time 0) sedation scoring, physical examination, and measurement of IOP and VPD, equids received 1 dose (approx 6 mg/kg) of trazodone orally. Examination and measurement procedures were repeated 0.5, 1, 2, 4, 8, 12, and 24 hours after drug administration. Blood samples were collected at each time point for analysis of plasma trazodone concentrations. Repeated-measures analysis was used to compare examination results between downstream time points and baseline. RESULTS: 7 of 8 equids had mild sedation from 0.5 to 8 hours after treatment; compared with baseline values, mean IOP was significantly lower from 0.5 hours to 8 hours, mean VPD was significantly smaller at 0.5 hours, and mean rectal temperature was significantly lower from 1 to 8 hours after drug administration. Adverse effects (signs of excitement in 1 equid and sweating in 4) were self-limiting and considered minor. Mean maximum plasma concentration of trazodone was 1,493 ng/mL 0.75 hours after administration, and terminal half-life of the drug was 9.96 hours. CONCLUSIONS AND CLINICAL RELEVANCE: The described oral dose of trazadone elicited sedation with a few self-limiting adverse effects in the study sample. Drug effects on IOP and VPD may alter ocular examination findings. Further investigation is warranted prior to use of trazodone for sedation in equids, particularly those with ophthalmic conditions.

Unfolding the diagnosis of subspectacular fluid opacity in a corn snake (Pantherophis guttatus).

OBJECTIVE: To present the results of clinical, surgical, and histopathologic procedures and how these were compared with the initial presumptive clinical diagnosis in a corn snake (Pantherophis guttatus) presenting with subspectacular fluid opacity; and to improve upon currently established surgical enucleation techniques in the snake. ANIMAL STUDIED: An 8-month-old corn snake was presented for enlarged globe OD. PROCEDURES: The following diagnostics were performed: systemic and ophthalmic examinations, complete blood count, cytology and culture of subspectacular fluid, and histopathology of enucleated globe and spectacle. Enucleation was performed in a routine fashion with the addition of a porcine small intestinal submucosa bioscaffold graft (SISplus™; Avalon Medical, Stillwater, MN), sutured over the orbit. RESULTS: Systemic examination revealed signs of maxillary stomatitis. Ophthalmic examination revealed semitransparent fluid in the subspectacular space. Complete blood count was unremarkable. Cytology of fluid obtained via subspectacular centesis was acellular, and culture grew Clostridium perfringens, which was consistent with the clinical suspicion of right maxillary stomatitis. Histopathology of the enucleated globe revealed spectaculitis, characterized by regional heterophilic inflammation, and no evidence of lymph dissection in the (peri)ocular tissues. The final diagnosis was a subspectacular abscess. Follow-up revealed that the SIS graft provided excellent healing and cosmesis of the surgical site. CONCLUSIONS: While there are reports of lymphatic fluid dissection between skin layers during ecdysis, which can result in an opaque spectacle, the fluid opacity in this case was attributed to a subspectacular abscess secondary to an ascending oral infection. Addition of biological wound dressing may contribute to positive post-enucleation outcome in the snake.

Surgically induced astigmatism in canines following sutured dorsonasal vs dorsotemporal clear corneal incisions.

OBJECTIVES: To investigate use of the Pentacam® HR for evaluation of surgically induced corneal astigmatism (SIA) in canines undergoing bilateral phacoemulsification and determine differences between dorsonasal and dorsotemporal clear corneal incisions. ANIMALS: Client-owned canines undergoing bilateral phacoemulsification. PROCEDURES: Patients received anterior segment imaging pre-operatively, immediately post-operatively, and 2-4 months post-operatively (follow-up). Total corneal refractive power was used to determine SIA. Surgically induced astigmatism was compared between right and left eyes, representing dorsotemporal and dorsonasal incisions, respectively. Repeated measures analyses were used between time points and paired t test compared SIA between eyes. RESULTS: Complete imaging series were obtained for seven patients. Follow-up imaging occurred at a median of 112 days (range 60-132 days) post-operatively. For repeated measures analyses, significant differences were found between pre- and immediate post-operative values (P < 0.01), and between immediate post-operative and follow-up values (P < 0.01). There was no significant difference between pre-operative and follow-up values. Surgically induced astigmatism was significantly different between right and left eyes, with values of 2.01 ± 1.24 D and 3.05 ± 1.58 D at 3 mm radius (P < 0.05), and 2.04 ± 1.18 D and 3.06 ± 1.27 D at 4 mm radius (P < 0.05) for dorsotemporal and dorsonasal incisions, respectively. CONCLUSIONS: Preliminary investigation revealed improvement of corneal SIA 2-4 months post-operatively, but development of significantly more SIA in dorsonasal vs dorsotemporal incisions. This prompts consideration of patient or microscope rotation to create a more dorsotemporal incision when possible.

Ocular Manifestations of Systemic Disease in the Horse.

Many systemic diseases have ocular manifestations. In some cases, ocular abnormalities are the most obvious or first recognized sign of disease that prompts veterinary evaluation. In other cases, the systemic disease leads to secondary ocular changes that might lead to loss of vision or globe if not addressed. Therefore, recognition of ocular abnormalities that might result from systemic diseases is an essential skill for the equine practitioner. This article provides practitioners with information regarding the most common systemic diseases of horses in North America that have ocular manifestations, organized by ocular signs.

Eosinophilic keratitis in 46 eyes of 27 horses in the Mid-Atlantic United States (2008-2012).

OBJECTIVE: To review the signalment, clinical characteristics, treatment, and outcome of equine EK cases in the Mid-Atlantic United States; to evaluate the effects of topical or systemic corticosteroid treatment, oral cetirizine treatment and secondary corneal infection on disease duration; and to evaluate the association between corticosteroid and cetirizine treatment and likelihood of recurrence. ANIMALS: Twenty-seven horses (47 eyes) diagnosed with EK from 2008 to 2012. PROCEDURE: Retrospective medical record review followed by phone interview to obtain recurrence data. RESULTS: Average age of affected horses was 8.2 years, SD 5.8 years. Eleven of 27 horses (41%) were diagnosed with EK in July. Twelve horses (44%) had been affected in previous years. Time to resolution averaged 3.7 months, SD 2.3 months. Ten horses (18 eyes) were treated with systemic dexamethasone, with a significantly shorter time to resolution, P = 0.03, averaging 2.23 months, SD 1.13 months, relative to horses not so treated, averaging 4.20 months, SD 1.47 months. Secondary infection led to a significant increase in time to resolution, P = 0.03, average 4.1 months, SD 1.7 months, relative to horses without secondary infection, average 3.0 months, SD 1.5 months. All eyes were visual at resolution. Horses treated with cetirizine were less likely to have recurrence during the follow-up period (1/13, or 8%) relative to horses not so treated (8/14, or 57%). CONCLUSIONS: Eosinophilic keratitis has a seasonal occurrence in summer in the Mid-Atlantic United States. Systemic but not topical corticosteroid treatment may decrease therapy duration. Treatment with cetirizine may be associated with a decreased risk of recurrence.

Combined excision and intralesional bevacizumab for sebaceous carcinoma of the eyelid in an Amur tiger (Panthera tigris altaica).

An 18-year-old zoo-kept female Amur tiger presented with an approximately 5 mm diameter lateral canthal eyelid mass in the left eye which grossly appeared red and irregular. The mass was completely excised via lateral canthoplasty. Histopathologic evaluation was consistent with a diagnosis of sebaceous cell carcinoma, which is a potentially aggressive cutaneous neoplasm. The sebaceous carcinoma recurred within 3 months and slowly increased in size until a second surgical excision was performed 9 months following the first surgery. The second surgical excision was combined with intralesional injection of 10 mg of the antiangiogenic drug bevacizumab. Histology confirmed the diagnosis. The tiger was euthanized 16 months postoperatively for reasons unrelated to, and without recurrence of, the eyelid neoplasm. At postmortem, no gross periocular or metastatic lesions were noted, and histopathology of the lateral canthus provided no evidence of recurrence. Surgical excision combined with intralesional bevacizumab treatment induced life-long resolution of the sebaceous carcinoma. Bevacizumab treatment may be associated with the regression of periocular angiogenic proliferative conditions, including neoplasia, by inhibiting angiogenesis.

Presumed immune-mediated hemolytic anemia in two foals with Rhodococcus equi infection.

OBJECTIVE: To describe the clinical presentation, case management, and outcome in 2 foals with Rhodococcus equi infection associated with presumptive severe immune-mediated hemolytic anemia. SERIES SUMMARY: Two foals diagnosed with R. equi pneumonia on the basis of tracheal wash cultures, thoracic radiographs, and thoracic ultrasonography were concurrently diagnosed with hemolytic anemia. Both foals required whole blood transfusions, and were treated with the antimicrobial combination of rifampin and a macrolide (eg, clarithromycin, erythromycin, or azithromycin). Dexamethasone was used to prevent further hemolysis in both foals, and to treat acute lung injury/acute respiratory distress syndrome in 1 of the foals. Both foals survived, and required prolonged antimicrobial therapy. NEW OR UNIQUE INFORMATION PROVIDED: Although extra-pulmonary disorders are commonly diagnosed in foals infected with R. equi, hemolytic anemia is rarely described. Dexamethasone is considered the treatment of choice for immune-mediated hemolytic anemia, but may be contra-indicated in foals with severe bacterial infections. In these foals, a relatively low dose and short duration of dexamethasone was utilized in an attempt to minimize immune suppression, although early discontinuation in 1 foal precipitated a second hemolytic crisis.

Effect of treatment with a topical ophthalmic preparation of 1% nalbuphine solution on corneal sensitivity in clinically normal horses.

OBJECTIVE: To assess the effect of treatment with a topical ophthalmic preparation of 1.2% nalbuphine solution on corneal sensitivity in clinically normal horses. ANIMALS: 8 horses. PROCEDURES: Baseline corneal touch threshold (CTT) was measured (defined as the mean filament length [mm] at which a consistent blink response was elicited) for both eyes of each horse by use of a Cochet-Bonnet aesthesiometer. Subsequently, 0.2 mL of 1.2% nalbuphine solution was instilled in 1 randomly selected eye of each horse, and 0.2 mL of artificial tears solution was instilled in the contralateral eye (control treatment). For all 8 horses, CTT of each eye was measured within 1 minute following nalbuphine or artificial tears administration and every 15 minutes thereafter for 60 minutes. For 5 of the 8 horses, CTT was also measured in both eyes at 120 minutes. Changes in CTT values from baseline over time were assessed, as were differences between treated and control eyes. RESULTS: At any time point, corneal sensitivity following nalbuphine treatment did not differ significantly from control treatment findings. Mean CTTs for nalbuphine-treated and control eyes were 38.8 and 37.9 mm, respectively. In both groups, CTT was significantly lower than baseline value at 15, 45, 60, and 120 minutes. No tearing or redness developed in any eye treated with nalbuphine. CONCLUSIONS AND CLINICAL RELEVANCE: Topical administration of ophthalmic 1% nalbuphine solution had no effect on corneal sensitivity in clinically normal horses. The topical ocular treatment was not associated with local irritation.

Evaluation of duration of corneal anesthesia induced with ophthalmic 0.5% proparacaine hydrochloride by use of a Cochet-Bonnet aesthesiometer in clinically normal horses.

OBJECTIVE: To measure duration of corneal anesthesia and time and degree of maximal anesthetic effect of 0.5% proparacaine hydrochloride by use of a Cochet-Bonnet aesthesiometer in horses. ANIMALS: 10 clinically normal adult horses. PROCEDURES: Baseline corneal touch threshold (CTT) was measured in millimeters for 1 randomly selected eye of each horse by use of the aesthesiometer by applying the filament to the cornea at maximum length (60 mm) and decreasing in 5-mm increments until a consistent blink response was elicited. Following baseline CTT measurement, 0.2 mL of 0.5% proparacaine hydrochloride was instilled in the selected eye. The CTT was measured within 1 minute following proparacaine administration and every 5 minutes thereafter for 60 minutes. A mixed-model ANOVA with tested eye varying between subjects and measurement time varying within subject was used to test for main effects and any interaction between these factors. A contrast between means of baseline and each subsequent CTT identified the duration of corneal anesthesia as the time at which there was no difference from baseline. Maximal anesthetic effect occurred at the time with the lowest mean CTT. RESULTS: Duration of corneal anesthesia achieved by use of proparacaine was 25 minutes, and maximal anesthetic effect occurred within 5 minutes, although CTT never went to 0 in any horse at any time. CONCLUSIONS AND CLINICAL RELEVANCE: Duration of corneal anesthesia in horses was shorter than in dogs, and degree of maximal effect was less than in cats and dogs, most likely because of increased sensitivity of the equine cornea, compared with corneal sensitivity in those species.

Infections of the head and ocular structures in the horse.

Infectious conditions of the equine head are commonly encountered in clinical practice. Pathogenic bacterial, viral, and fungal organisms may localize in the extensive nasal passages, paranasal sinuses, and guttural pouches, creating a range of clinical signs and conditions that can be severe enough to lead to unexpected fatality. Renewed interest in equine dentistry has led to a greater recognition of dental disease that is associated with infection. This article focuses on bacterial and fungal infections of the main anatomic regions of the equine head, where advances in diagnosis and management have been made or consolidated in recent years. It also addresses recent advances made in the area of infectious equine corneal disease, including bacterial, viral, and fungal etiologies. Recent developments in equine recurrent uveitis as it relates to infectious diseases and ocular manifestations of systemic disease are also discussed.