Phacoemulsification Combined with Pars Plana Vitrectomy: Outcome in Horses with Acquired Cataracts Associated with Uveitis.

BACKGROUND: Cataracts resulting from equine recurrent uveitis (ERU) or other forms of uveitis are usually associated with rapid progression. ERU is the most common ocular disease cause of blindness and cause of cataracts in horses. The necessity for the posterior capsulorhexis (PC) during phacoemulsification (PE) is controversial. This study aimed to evaluate vision and complications after PE combined with pars plana vitrectomy (PPV) in horses with uveitis-associated cataracts and compare the PE technique with and without posterior capsulorhexis. METHODS: Thirty-two eyes of 28 horses with uveitis-associated cataracts aged 14 months to 19.6 years were treated with PE-PPV under identical conditions. Twenty-three eyes of 21 horses were affected by an ERU-associated (ERU group), and nine eyes of 7 horses were affected by cataracts related to uveitis with pathogenesis different to ERU (non-ERU group). PE-PPV was performed in 12 eyes of 10 horses (PC group) and 20 eyes of 18 horses without posterior capsulorhexis (NPC group). Follow-up examination was performed at a mean of 1.7 ± 1.8 years postoperatively (range: 1 month-6.4 years). RESULTS: In the period up to 1 month postoperatively, 17/20 (85%) NPC-eyes and 8/12 (67%) PC-eyes (total: 25/32 [78%]) were visual. From 1-6 months postoperatively, 16/20 (80%) NPC-eyes and 7/12 (58.3%) PC-eyes (total: 23/32 [72%]), and from 6-12 months, 7/11 (63.6%) NPC-eyes and 3/8 (37.5%) PC-eyes (total: 10/19 [52.6%]) were visual. From 12-18 months postoperatively, 3/7 (42.9%) NPC-eyes and 2/9 (22.2%) PC-eyes (total: 5/16 [31.3%]), and from 18-24 months, 3/8 (37.5%) NPC-eyes and 1/8 (12.5%) PC-eyes (total: 4/16 [25%]) were visual. After 24 months postoperatively, 2/7 (28.6%) NPC-eyes and 1/8 (12.5%) PC-eyes (total: 3/15 [20%]) were visual. Despite the higher number of visual eyes in the NPC group at each time point, differences were not significant. No obvious differences regarding postsurgical vision were observed between the ERU- and non-ERU groups at each time point. In the overall population, a significant decrease in the number of eyes with postoperative active uveitis was observed during the follow-up examinations (p < 0.001). A significant increase in the number of eyes that were blind due to retinal detachment was observed in the overall patient population as the examination period progressed (p < 0.001). Retinal detachment was the sole long-term cause of blindness. CONCLUSIONS: In horses diagnosed with uveitis-associated cataracts and treated with PE-PPV, no persistent active uveitis was observed in the present study during follow-up examinations. However, the proportion of eyes that were blind due to retinal detachment increased. Whilst PE-PPV may prevent postsurgical persistent active uveitis and remove lens opacity, the prognosis for a visual outcome is guarded. A superior outcome in postsurgical vision was observed in the NPC group. However, caution is required when interpreting these results due to several factors that affect the independent comparison of the surgical groups.

Analysis of 1840 Equine Intraocular Fluid Samples for the Presence of Anti-Leptospira Antibodies and Leptospiral DNA and the Correlation to Ophthalmologic Findings in Terms of Equine Recurrent Uveitis (ERU)-A Retrospective Study.

In the equine clinic of the LMU in Munich, therapeutic vitrectomies have been routinely performed in horses for three decades. The vitreous samples obtained during vitrectomies were usually tested for anti-Leptospira antibodies and for more than 20 years also by PCR for leptospiral DNA. If the indication for surgery was ophthalmologically inconclusive, an aqueous humor was collected preoperatively and examined for evidence of leptospiral infection. In this study, medical records from 2002 to 2017 were analyzed. Records for 1387 eyes affected by equine recurrent uveitis (ERU) and 237 eyes affected by another type of uveitis met the inclusion criteria. A total of 216 samples from healthy eyes were used as controls. In 83% of intraocular samples from ERU eyes, antibody titers of 1:100 or higher were detectable by microscopic agglutination test (MAT). Similarly, 83% of intraocular samples had anti-Leptospira antibodies detected by ELISA. In 72% of the intraocular specimens, leptospiral DNA was detectable by PCR. No antibodies were detectable in the samples from eyes with another type of uveitis or in the samples from healthy eyes. A PCR was positive in only one sample from a healthy eye. These results with a very high number of intraocular specimens demonstrate the great importance of an intraocular leptospiral infection for ERU. It can be concluded that for a reliable diagnosis of intraocular leptospiral infection or to reliably exclude an infection multiple tests should be applied.

Infectious Uveitis in Horses and New Insights in Its Leptospiral Biofilm-Related Pathogenesis.

Uveitis is a sight-threatening eye disease in equids known worldwide that leads to considerable pain and suffering. By far the most common type of uveitis in Germany and neighboring countries is classical equine recurrent uveitis (ERU), which is caused by chronic intraocular leptospiral infection and is the main cause of infectious uveitis in horses. Other infectious causes are extremely rare and are usually clinically distinguishable from ERU. ERU can be treated very effectively by vitreous cavity lavage (vitrectomy). For proper indications of this demanding surgery, it is necessary to differentiate ERU from other types of uveitis in which vitrectomy is not helpful. This can be conducted on the basis of anamnesis in combination with ophthalmologic findings and by aqueous humor examination. During vitrectomy, vitreous material is obtained. These vitreous samples have historically been used for numerous etiologic studies. In this way, a chronic intraocular leptospiral infection has been shown to be the cause of typical ERU and, among other findings, ERU has also been recognized as a biofilm infection, providing new insights into the pathogenesis of ERU and explaining some thus far unexplainable phenomena of ERU. ERU may not only have transmissible aspects to some types of uveitis in humans but may also serve as a model for a spontaneously occurring biofilm infection. Vitreous material obtained during therapeutically indicated vitrectomy can be used for further studies on in vivo biofilm formation, biofilm composition and possible therapeutic approaches.

Detection of Anti-LipL32 Antibodies in Serum Samples from Horses with Chronic Intraocular Infection with Leptospira spp.

Equine recurrent uveitis (ERU) is typically caused by chronic intraocular leptospiral infection in warm-blooded horses in central Europe. The most effective therapy for leptospiral-induced ERU is the surgical removal of diseased vitreous (vitrectomy). Since vitrectomy is a highly specialized and invasive surgery, the indication must be determined very carefully. In order to obtain evidence of intraocular leptospiral infection by laboratory diagnostics in questionable leptospiral ERU-cases, sampling of aqueous humor is required, because serum tests using microscopic agglutination test (MAT) are too unspecific. The SNAP Lepto is a cross-species rapid test for the detection of anti-Lipl32 antibodies that has a high sensitivity (0.97) and specificity (1.00) for the detection of anti-leptospiral antibodies using aqueous humor or vitreous samples, which is comparable to MAT. To evaluate sensitivity and specificity of SNAP Lepto using serum, serum samples from 90 horses with confirmed leptospiral ERU and from 103 ocularly healthy horses were tested by both MAT and SNAP Lepto. Sensitivity was similar for both tests (0.82 vs. 0.79), but specificity was lower for MAT (0.52 vs. 0.95). Sensitivity and specificity are therefore lower in serum samples compared to intraocular samples, however, the SNAP Lepto is far superior to MAT and suitable as a screening method using equine serum.

In Vivo Biofilm Formation of Pathogenic Leptospira spp. in the Vitreous Humor of Horses with Recurrent Uveitis.

Equine recurrent uveitis (ERU) causes painful inflammatory attacks and oftentimes blindness in the affected eyes. The disease is considered a late sequela of systemic leptospirosis. The most effective therapy is the surgical removal of the vitreous (vitrectomy), which is not only therapeutic, but provides vitreous material that can be assessed diagnostically. For example, the lipL32 gene, culturable Leptospira spp., and anti-Leptospira antibodies have all been detected in vitreous samples obtained from eyes with chronic ERU. Despite this clear evidence of leptospiral involvement, the systemic administration of antibiotics in infected horses is ineffective at resolving ERU. This syndrome of chronic recurrent inflammation, which is unresponsive to antibiotic therapy, combined with apparent bacteria evading the immune response, is consistent with a biofilm-associated infection. The purpose of this study, therefore, was to detect the in vivo biofilm formation of Leptospira spp. in vitreous samples collected during vitrectomy and examined using a Warthin-Starry silver stain and immunohistochemistry. All known steps of biofilm formation were visualized in these samples, including individual Leptospira spp., leptospiral microcolonies and dense roundish accumulations of Leptospira spp. In many instances spirochetes were surrounded by an extracellular substance. Taken together, data from the present study show that ERU is a biofilm-associated intraocular leptospiral infection, which best explains the typical clinical course.

Measurement of Endurance of Untrained Five-Year-Old and Six-Year-Old Horses Raised in Multi-age Herds on Pasture.

The objective of this study was to explore how the endurance of five-year-old horses, kept out on pasture all their life and ridden for the first time well into their fifth year of age, developed within one year and compared to that of six-year-old horses raised under the same conditions and to other horses. Horses were submitted to a standardized exercise test (SET) to calculate their v4 (velocity run under defined conditions inducing 4 mmol/L of blood lactate concentration (LA)) and v180 (velocity run under defined conditions inducing a heart rate of 180 beats/min). The test consisted of up to five consecutive intervals at increasing speed until the blood LA of a horse increased above 4 mmol/L. The blood LA measured after each interval was plotted exponentially against running speed to derive v4 from the blood lactate-running speed relationship, and the mean heart rate during the intervals was plotted linearly against running speed to derive v180 from the heart rate-running speed relationship. The following were examined: (1) the development of v4 and v180 of five-year-old horses within one year through measurements in September and in the following July and September; the comparison of endurance variables (2) between five-year-olds and six-year-olds; (3) between six-year-olds on consecutive years; and 4) between six-year-olds and foreign horses. The results showed that: (1) there were no changes of either variable within one year (repeated measures ANOVA P > .05); (2) there were no significant differences between five-year-olds and six-year-olds (one-way ANOVA P > .05); (3) no significant differences between six-year-old groups (one-way ANOVA; P > 05); and (4) foreign horses had higher v4 and v180 values than six-year-olds (one-way ANOVA; P = .0001 and P = .003, respectively). There was no significant relationship between v4 and v180 (P > .05; r2 = 0.02). In conclusion, one additional year on pasture in multiage herds did not increase the endurance variables of five-year-old horses. Thus, the endurance appeared to be consolidated in these horses at the age of five years, and additional training seems to be necessary to increase it.

Effect of retrobulbar nerve block on heart rate variability during enucleation in horses under general anesthesia.

OBJECTIVE: Analysis of any effect of retrobulbar block during ocular surgery on heart rate variability and oculocardiac reflex. STUDY DESIGN: Prospective study. ANIMAL STUDIED: Horses (n = 16) undergoing eye enucleation due to chronic ophthalmologic diseases. PROCEDURE: Eye enucleation was performed under general anesthesia. The horses were randomly assigned to the first (inhalation anesthesia only, n = 10) or second group (inhalation and local retrobulbar anesthesia, n = 6). The retrobulbar block was performed using 12 mL of mepivacaine hydrochloride 2%. ECG data were taken by a Telemetric ECG before, during, and after surgery. Heart rate variability was analyzed in the time domain as mean heart rate, mean beat-to-beat interval duration, and standard deviation of continuous beat-to-beat intervals. The frequency domain analysis included the low- and high-frequency components of heart rate variability and the sympathovagal balance (low/high frequency). The low frequency represents mainly sympathetic influences on the heart, whereas high frequency is mediated by the parasympathetic tone. RESULTS: All horses without a retrobulbar block showed a significant decrease in the heart rate during traction on the globe and pressure on the orbital fat pad for homoestasis (P = 0.04). Simultaneously, high-frequency power, as an indicator of vagal stimulation, increased significantly. High-frequency and low-frequency power in the retrobulbar block group increased in five horses, and heart rate decreased in only one horse. Both were not significant within the group, but there was a significant difference between both groups relating to the incidence of heart rate decrease occurring at globe traction. CONCLUSIONS AND CLINICAL RELEVANCE: Heart rate variability is a sensitive, non-invasive parameter to obtain sympathovagal stimulations during general anesthesia. The retrobulbar block can prevent heart rate decrease associated with initiation of the oculocardiac reflex.

Magnetic resonance imaging of pulp in normal and diseased equine cheek teeth.

A retrospective analysis of 619 upper and lower cheek teeth from 62 horses was performed. Based on clinical findings, as well as radiographic and magnetic resonance (MR) imaging findings, the teeth were classified into five groups. There were 20 teeth with abnormal MR imaging signals as well as clinical alterations and 599 healthy teeth. Using MR imaging, the appearance of pulp in diseased and disease-free teeth was compared, and the appearance of abnormal pulp was studied. Subsequently, the ability of MR imaging to diagnose pulpitis and pulp necrosis in teeth with normal external appearance was investigated. In horses with clinically verified dental disease, abnormal MR imaging findings were confirmed in the pulp of all affected teeth. An enlarged blurred pulp image with a lower signal intensity was observed only in clinically diseased teeth and was a reliable criterion for diagnosing dental disease on MR imaging. On the other hand, partial or complete absence of pulp in all MR imaging sequences was observed in both diseased and nondiseased teeth. These data demonstrate that pulp changes in equine cheek teeth can be evaluated using MR imaging.

Simplified method to measure glucocorticoid metabolites in faeces of horses.

Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3alpha,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3alpha,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.

Retinal Mueller glial cells trigger the hallmark inflammatory process in autoimmune uveitis.

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. To elucidate such processes, we studied changes in the total retinal proteome of ERU-diseased horses compared to healthy controls. Severe changes in the retinal proteome were found for several markers for blood-retinal barrier breakdown and whose emergence depended upon disease severity. Additionally, uveitic changes in the retina were accompanied by upregulation of aldose 1-epimerase, selenium-binding protein 1, alpha crystallin A chain, phosphatase 2A inhibitor (SET), and glial fibrillary acidic protein (GFAP), the latter indicating an involvement of retinal Mueller glial cells (RMG) in disease process. To confirm this, we screened for additional RMG-specific markers and could demonstrate that, in uveitic retinas, RMG concomitantly upregulate vimentin and GFAP and downregulate glutamine synthetase. These expression patterns suggest for an activated state of RMG, which further downregulate the expression of pigment epithelium-derived factor (PEDF) and begin expressing interferon-gamma, a pro-inflammatory cytokine typical for T-helper 1 cells. We thus propose that RMG may play a fatal role in uveitic disease progression by directly triggering inflammatory processes through the expression and secretion of interferon-gamma.

Identification and functional validation of novel autoantigens in equine uveitis.

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.

The uveitogenic potential of retinal S-antigen in horses.

PURPOSE: To investigate the uveitogenic potential of retinal S-antigen (S-Ag) in horses. METHODS: Horses were immunized subcutaneously with S-Ag or BSA as control antigen, emulsified in complete Freund's adjuvant. Simultaneously, Bordetella pertussis was given intravenously. Antigen specific T- and B-cell responses were analyzed in a 3-day interval. Disease development was judged clinically and histopathologically. Two identical booster immunizations were given every 4 weeks to test induction of recurrences. RESULTS: T- and B-cell responses specific for S-Ag were observed in all immunized horses but were absent in control animals. However, uveitis developed in only one of five animals. Reimmunization with S-Ag did not lead to a uveitic relapse in this horse. All other horses of the S-Ag- and BSA-treated groups neither showed any signs of uveitis, nor had inflammatory infiltrates of the inner eye. CONCLUSIONS: In contrast to interphotoreceptor retinoid-binding protein (IRBP), S-Ag is a weak autoantigen in horses. Even though S-Ag immunization leads to the activation of autoreactive T- and B-cells, infiltration of the inner eye and induction of uveitis are controlled in most horses.

[Diagnostic validity of palpation in horses with back problems]. / Diagnostische Validität der Palpation bei Pferden mit Rückenproblemen.

There is a regularly high proportion of x-ray findings indicating a pathological deterioration of the equine vertebral column which do not correlate with the clinical symptoms. Therefore, palpation remains one of the most important clinical examination methods to determine whether or not a horse is suffering from back pain. The aim of this study was to check the validity of palpation results of horses with an assumed back problem. The palpation results of 167 horses were evaluated in retrospect: If the palpation proceeds under following conditions, positive palpation results agree with an primary back problem in about 100% of cases: 1. Hind limb lameness, being the most likely reason for misleading positive palpation results, has to be ruled out. 2. Only reproducible painful behavior is allowed to be interpreted as positive. Under these conditions a negative palpation result is correct in about 72% of cases. For the examiner it is important to keep in mind that an apparent Kissing Spine-Syndrome can exist without being palpable. By means of clinical examination it is not always possible to distinguish between painful and normal reactions. In doubtful cases diagnosis can be made by a "diagnostic therapy", or better by scintigraphy.

Uveitis in horses induced by interphotoreceptor retinoid-binding protein is similar to the spontaneous disease.

Equine recurrent uveitis (ERU) is an inflammatory eye disease with high similarity to uveitis in man. It is the only spontaneous animal model for uveitis and the most frequent eye disease in horses affecting up to 10% of the population. To further investigate the pathophysiology of ERU we now report the establishment of an inducible uveitis model in horses. An ERU-like disease was elicited in seven out of seven horses by injection of interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. Control horses did not develop uveitis. The disease model is characterized by a highly reproducible disease course and recurrent episodes with an identical time course elicited in all horses by repeated IRBP injections. The histology revealed the formation of lymphoid follicle-like structures in the eyes and an intraocular infiltration dominated by CD3(+) lymphocytes, morphological patterns typical for the spontaneous disease. Antigen-specific T cell proliferation of PBL was monitored prior to clinical uveitis and during disease episodes. An initial T cell response to IRBP-derived peptides was followed by epitope spreading to S-antigen-derived peptides in response to subsequent immunizations. Thus, horse experimental uveitis represents a valuable disease model for comparative studies with the spontaneous disease and the investigation of immunomodulatory therapeutic approaches after onset of the disease.