Subconjunctival bone marrow‐derived mesenchymal stem cell therapy as a novel treatment alternative for equine immune‐mediated keratitis: A case series

Equine immune‐mediated keratitis (IMMK) leads to increased corneal opacity and inflammation secondary to an alteration of the local immune system. Bone marrow‐derived mesenchymal stem cells (BM‐MSC) have been shown to modulate the immune system by downregulating inflammation. Four horses with unilateral IMMK poorly responsive to traditional medical treatments underwent novel, autologous subconjunctival BM‐MSC therapy. Bone marrow was harvested and processed as previously described for equine orthopedic disease. Horses received autologous subconjunctival BM‐MSC injections approximately every 3‐4 weeks for 1‐5 treatments total. Horses were maintained on their current medical treatment regimen throughout the BM‐MSC treatment period. Three horses had a positive response to therapy as demonstrated by an increase in corneal clarity, a decrease in neovascularization and a reduction in surface irregularity. One horse was nonresponsive to therapy. These experimental results demonstrate the safety and potential efficacy of an innovative solution for IMMK.     

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.     

Septic funiculitis caused by Streptococcus equi subspecies equi infection with associated immune‐mediated haemolytic anaemia

A 2‐year‐old Quarter Horse gelding presented for anaemia, icterus, depression and intermittent colic 2 weeks after routine castration. Bilateral septic funiculitis with Streptoccocus equi ssp. equi with secondary immune‐mediated haemolytic anaemia were diagnosed. A blood transfusion was required to facilitate general anaesthesia for surgical excision of the septic funiculitis. Antibiotic therapy was provided initially with chloramphenicol and later enrofloxacin. Immunosuppressive therapy was provided with dexamethasone and later azathioprine. The horse responded well to treatment and was discharged 8 weeks after presentation. Streptococcus equi ssp. equi should be considered in cases with septic funiculitis and the potential for a secondary immune‐mediated haemolytic anaemia exists with this bacterial species.     

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.     

Expression of Toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva

Objective Human corneal cells have detectable levels of TLRs 1‐10. TLRs 2 and 4 are the major corneal receptors, recognizing the PAMPs associated with fungal invasion in humans. The conjunctiva and limbus contain TLRs 2, 4, and 9. Our purpose was to determine the expression of TLRs 2, 3, 4, 6, 9, and MD‐2 in the normal equine cornea, conjunctiva, and limbus. Methods Corneal, limbal, and conjunctival tissues were collected from seven euthanized horses having no evidence of ocular disease. RNA extraction with DNase‐1 digestion was performed followed by RT‐PCR to determine expression of TLRs 2, 3, 4, 6, 9, and MD‐2. Products were resolved by electrophoresis on 1.5% agarose gels and visualized using ethidium bromide staining. Results Expression of TLRs 2, 3, 4, 6, 9, and MD‐2 was present in the cornea, limbus, and conjunctiva of each horse, except one horse, where TLR3 expression was unable to be demonstrated in the dorsal and ventral conjunctiva. Conclusions Confirming the expression of TLRs in equine ocular tissues is an initial step in identifying how they play a role in infectious keratitis, particularly fungal. The results further support the use of equine ocular tissues as a model for human fungal keratitis. Studies of the TLR expression together with their cytokine profile induced during equine fungal keratitis may help further clarify the pathogenesis of the disease and possibly lead to the development of new treatment protocols for both equines and humans.     

C‐reactive protein concentration in dogs with primary immune‐mediated hemolytic anemia

Background: Canine primary immune-mediated hemolytic anemia (IMHA) is associated with a high-mortality rate. C-reactive protein (CRP) is the most important acute-phase protein in dogs and may have value as a marker of prognosis or response to treatment in IMHA. Objective: The objectives of this study were to evaluate serum CRP concentration in dogs with primary IMHA at presentation and during treatment, to assess potential differences based on survival time, and to compare CRP with other laboratory parameters of inflammation and prognosis. Methods: Inclusion criteria for primary IMHA were anemia (PCV<0.30 L/L), a positive Coombs' test or persistent autoagglutination of erythrocytes, and the exclusion of underlying diseases by other diagnostic tests. Dogs were divided into 2 groups based on survival: dogs that were still alive 14 days after start of treatment (group 1) and dogs that died or were euthanized before day 14 (group 2). Serum CRP concentration, a CBC, and a biochemistry profile were performed on days 0, 3, 8, and 14. Serum CRP also was determined in 25 clinically healthy dogs. Results: CRP concentration in the 25 clinically healthy dogs ranged from 0–8.9 μg/mL (median 2.2 μg/mL). Thirty dogs were diagnosed with primary IMHA, 24 in group 1 and 6 in group 2. On day 0, CRP concentration in dogs in both groups (median 224 μg/mL) was increased above the reference interval. In group 1 dogs, median CRP concentration was 242 μg/mL on day 0, 69 μg/mL on day 3, 35 μg/mL on day 8, and 2 μg/mL on day 14. In group 2 dogs, median CRP concentration was 194 μg/mL on day 0, 119 μg/mL on day 3, and 41 μg/mL on day 8; only 1 dog in group 2 survived to day 8. There was a significant correlation between CRP and total WBC concentrations on days 0 and 3 (r=−.598, P=.003). Conclusions: Serum CRP concentration was markedly increased in dogs with primary IMHA. CRP concentration did not differ based on patient survival, but might be a marker for long-term monitoring of these patients.     

Clinical findings and progression of 10 cases of equine ulcerative keratomycosis (2004–2007)

Ulcerative keratomycosis is a serious sight‐threatening disease of horses and the veterinary literature is replete with cases of poor visual outcome following this condition. During a 3 year period, 10 horses were treated for confirmed keratomycosis at the Veterinary Teaching Hospital (VTH) of the University of Cordoba (Spain). Ulcerative keratomycosis accounted for an average of 8.62% of the total equine ophthalmic admissions during this time and an average of 33.3% of horses were diagnosed with infectious keratitis. Fungi were diagnosed using cytology (n = 4) and/or culture (n = 8) and histopathology (n = 1). Aspergillus sp. was the most commonly isolated fungus. Medical therapy alone or combined medical and surgical treatment was utilised for therapy depending on the clinical condition. Miconazole 1% was the most common topical antifungal therapy employed. Median duration of treatment was 73.12 days. Records were evaluated to determine visual outcome and globe survival.     

The use of amniotic membrane transplantation for ocular surface reconstruction: a review and series of 58 equine clinical cases (2002–2008)

Amniotic membrane transplantation (AMT) is an effective clinical therapy for reconstruction of the ocular surface in human and veterinary patients. Amnion is avascular and strong, contains antiangiogenic and antiinflammatory properties and growth factors, and has properties that prevent or decrease fibrosis in healing tissue. Indications for its use are steadily growing and include grafting to replace diseased, missing or excised tissue, patching to support diseased tissue during the healing process and as a substrate for the expansion of epithelial cells for transplantation to the cornea. AMT through a combination of mechanical and biologic factors can preserve the integrity of the globe, optimize the visual outcome, and minimize scarring in severely diseased corneas.     

Storage‐associated artefact in equine muscle biopsy samples

Reasons for performing study: Muscle biopsy is increasingly used in equine veterinary practice for investigating exertional, inflammatory or immune mediated myopathies and unexplained muscle atrophy. Although formalin‐fixed samples are often used, for complete evaluation, fresh‐frozen tissue is required. Freezing muscle in veterinary practice is impractical: samples sent to specialist laboratories for processing are therefore susceptible to delays, potentially leading to artefact and compromising histological interpretation. Hypothesis: Altered temperature, duration and hydration status influence the severity of storage‐induced artefact in equine muscle. Methods: Skeletal muscle obtained immediately post euthanasia was divided into 6 independent samples from each of 8 horses. One sample per horse was frozen immediately in isopentane precooled in liquid nitrogen. Additional samples were stored in conditions designed to mimic possible situations encountered in practice, including increased storage times, temperature and hydration status. Following storage, stored samples were frozen as before. Cryosections were stained using haematoxylin and eosin and ranked for artefact on 2 occasions by 2 blinded observers. The best samples were processed subsequently with a panel of routine stains and immunolabelled for collagen V to enable the measurement of minimum fibre diameters. Results: Both prolonged storage and increased hydration resulted in more storage‐associated artefact. Samples stored for 24 h chilled on dry gauze were ranked higher than those stored on damp gauze; however, a panel of routinely‐used histochemical staining techniques was unaffected by chilled 24 h storage. There was no significant effect of storage on mean fibre diameter; however, both chilled dry and damp storage for 24 h caused a significant increase in fibre‐size variability. Conclusion and potential relevance: Caution should be exercised when interpreting fibre size profiles in shipped samples. Equine muscle biopsy samples are optimally shipped in dry gauze, sealed in plastic containers and shipped on ice packs to be processed within 24 h and can thus be interpreted by the receiving laboratory with minimal artefact.     

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.