Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous Cavity Leads to Recurrent Uveitis in Horses

Equine recurrent uveitis (ERU) is a disease known and feared for centuries, as it almost always leads to blindness even with careful and meticulous conservative treatment of the individual episodes of uveitis. In about one-third of horses, both eyes are affected, often necessitating euthanasia. A link between ERU and leptospiral infection has been suspected for nearly 80 years. Vitreous lavage (vitrectomy) can preserve vision in affected eyes. After surgery, no further episodes of uveitis occur in up to more than 95% of operated eyes. With routine performance of vitrectomies, numerous vitreous samples could be used for further investigations. Intraocular anti-Leptospira antibody production was proven, leptospires could be cultured from the vitreous samples, and the LipL32 gene could be detected in the vitreous samples by PCR. Thus, there was convincing evidence of a chronic intraocular leptospiral infection, which can be eliminated most reliably by vitrectomy. Recently, it has been shown that the intraocular leptospires produce biofilm in the equine vitreous. Biofilm formation explains not only the success of vitrectomy, but also the survival of leptospires in the vitreous cavity for many years despite the presence of high intraocular antibody titers and immunocompetent cells, as well as the high tolerance to antibiotics

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

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

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

BackgroundCataracts 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.MethodsThirty-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).ResultsIn 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 p ConclusionsIn 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

Immune responses to retinal autoantigens and peptides in equine recurrent uveitis

PURPOSE. To test the hypothesis that autoimmune mechanisms are involved in horses in which equine recurrent uveitis (ERU) develops spontaneously. METHODS. Material obtained from horses treated for spontaneous disease by therapeutic routine vitrectomy was analyzed for total IgG content and IgG specific for S-Antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). The cellular infiltrate of the vitreous was analyzed by differential counts of cytospin preparations and flow cytometry using equine lymphocyte-specific antibodies. Antigen-specific proliferation assays were performed comparing peripheral blood lymphocytes (PBLs) with vitreal lymphocytes by stimulation with S-Ag and several S-Ag-and IRBP-derived peptides. RESULTS. The total IgG content of specimens from horses with ERU was very high with great variability among the investigated samples (11.5 Ϯ 8.0 mg). Autoantibodies to S-Ag or IRBP or both were found in 72% of vitreous specimens from horses with uveitis. The leukocyte infiltrates (up to 2 ϫ 10 8 cells per sample) were dominated by lymphocytes (Ͼ90%) in most cases (22/32). Flow cytometry showed that more than 50% of these cells were CD4 ϩ T cells. In vitro stimulation of vitreal lymphocytes, but not of PBL, showed a strong proliferative response to peptides derived from S-Ag or IRBP in 9 of 12 patients. CONCLUSIONS. In the eyes of horses with ERU, IgG antibodies and autoreactive T cells specific for retinal antigens were detected. These results strongly support the hypothesis that ERU is an autoimmune-mediated disease and is highly similar to recurrent uveitis in humans in both clinical and immunologic parameters. (Invest Ophthalmol Vis Sci. 2001;42:393-398

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