Recurrent uveitis is a leading cause of blindness in horses in the United States.1 Recurrent uveitis in horses is characterized by recurrent bouts of active ocular inflammation followed by variable-length periods of quiescence.1 These painful recurrent bouts of inflammation continue and, in many horses, result in blindness.
Recurrent uveitis in horses is considered an immune-mediated disease2–6; however, the cause of the initiation and recurrence of the inflammatory episodes is unknown. One theory to explain recurrent inflammation in affected horses is that a T-cell–mediated helper T-cell type 1 response3,4 is initiated by ocular inflammation, including bacterial infections, but the recurrent bouts of inflammation are associated with immune response to various ocular retinal autoantigens, such as Santigen, interphotoreceptor binding protein, and cellular retinaldehyde-binding protein.2,5,7 Results of a recent study6 indicate that intermolecular and intramolecular epitope spreading to S-antigen occurs in horses immunized with interphotoreceptor binding protein and in horses with naturally occuring ERU. The spontaneous inflammatory response in the eye occurs when the new antigen epitope is recognized by the immune system of the horse.6
Another theory to explain recurrent inflammation in affected horses proposes that inciting antigens become established in the ocular tissues and their continued presence causes periodic episodes of inflammation; results of recent studies8,9 indicate that Leptospira organisms may be one of the antigens. Although study findings from California,8 New York,10 and Europe9 have implicated Leptospira organisms as the cause of ERU in horses, results of a recent study11 from the midwestern United States did not, suggesting a regional difference in the pathogenesis of ERU in horses. Furthermore, vaccination against Leptospira organisms does not appear to alter the course of disease in horses with ERU,12 whereas anti-inflammatory and immunosuppressive medications lessen or eliminate recurrent episodes of uveitis in affected horses.13,14 Therefore, the role of bacteria in the pathogenesis of ERU in horses remains unknown.
Results of a recent study15 on herpes simplex virus, varicella zoster virus, and Toxoplasma gondii indicate that analysis of intraocular antibody production (ie, calculating the GWC) combined with real-time PCR analysis for organismal DNA is the most sensitive method for comprehensive diagnosis of the role of organisms in the pathogenesis of uveitis. The purpose of the study reported here was to determine the role of intraocular bacterial infections in the pathogenesis of ERU in horses from the southeastern United States by evaluating eyes of affected horses for bacterial DNA by use of real-time PCR assay and detection of intraocular production of antibodies against Leptospira spp (by calculating GWC).
Materials and Methods
Animals—The use of horses in this study adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and was reviewed and monitored by the North Carolina State University Institutional Animal Care and Use Committee. For client-owned animals, informed consent was obtained from all owners prior to participation in the study.
Adult horses with no abnormal findings on ophthalmic examinations and complete vaccination and deworming schedules, which were donated to North Carolina State University, were used as control horses for the collection of aqueous humor, vitreous humor, and blood samples (group 1; n = 24). Horses (group 2; n = 52) with naturally occuring ERU from the southeastern United States (ie, from the states of North Carolina, Virginia, South Carolina, and Georgia) were evaluated in this study. Horses were considered to have ERU and included in this study if they had the following: a > 3-month history of disease, documented episodes of recurrent inflammation, clinical signs consistent with ERU (ie, ≥ 1 of the following: aqueous flare, hypopyon, corpora nigra atrophy, iris hyperpigmentation and fibrosis, vitreal cellular infiltrate, and retinal degeneration), lack of corneal inflammatory disease, and a complete evaluation by a veterinary ophthalmologist.1,14,16 Horses had active inflammatory disease, as indicated by the presence of aqueous flare, and were not treated within 14 days of sample collection with topical or systemic corticosteroids or antimicrobials. A third group (group 3; n = 17) of horses had ocular inflammation that was not associated with ERU. Horses with acute uveitis (ie, trauma-induced or primary uveitis of < 14 days duration; n = 4), infectious keratitis (melting corneal ulcer, 2; fungal keratitis, 2), and other inflammatory diseases (corneal or limbal squamous cell carcinoma, 3; primary glaucoma, 3; primary cataract, 1; immune-mediated keratitis, 1; uveal melanoma, 1) were included in this group.
Sample collection and processing—For evaluation for the presence of bacterial DNA in the vitreous and aqueous humor of clinically normal horses (group 1 subset A; n = 12) or horses with ERU (group 2 subset A; 28), vitreous humor and aqueous humor were aspirated from eyes that were immediately enucleated after euthanasia. Horses were euthanatized by IV injection of a barbiturate euthanasia solution. Aqueous humor was collected by inserting a 27-gauge needle through the limbus into the anterior chamber, then slowly aspirating approximately 1 mL of aqueous humor. Vitreous humor was then collected by inserting an 18-gauge needle 1 cm caudal to the limbus and aspirating 2 mL of vitreous humor from the central vitreal body. Samples were placed into sterile containers and frozen at −80°C until further processing. Time from sample collection to freezing was within 30 minutes for all samples.
For evaluation of anti-Leptospira antibody titers in clinically normal horses (group 1 subset B; n = 12), horses with ERU (group 2 subset B; 24), and horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation; group 3; 17), aqueous humor (1 mL) was aspirated after aseptic surgical preparation of the eye in either standing horses or in horses under general anesthesia. Samples were placed into sterile containers and frozen at −80°C until further processing. In these horses, blood was collected from a jugular vein immediately prior to collection of aqueous humor. Serum was separated routinely, and the serum samples were frozen at −80°C until further processing. Time from sample collection to freezing was within 30 minutes of collection for all samples.
Real-time PCR analysis—Broad-range ribosomal RNA gene sequence PCR method (ie, real-time PCR) was used to detect bacterial DNA in aqueous humor and vitreous humor from clinically normal horses of group 1A (n = 12) and horses with ERU of group 2A (28). The DNA was extracted from ocular samples by use of a standard phenol-chloroform extraction protocol.17 The DNA was quantified by spectrophotometry. The real-time PCR method was used as previously described.18–20 Brilliant SYBRa green dye was used for PCR amplification. Each sample was assayed in triplicate within a single run. Genomic DNA (200 pg) was added to the reaction mixture. The amplification protocol was as follows: 1 cycle at 95°C for 5 minutes for initial denaturation, then 50 cycles at 95°C for 2 minutes followed by annealing at 60°C for 1 minute and extension at 72°C in a thermal cycler.b Primers consisted of p201(GAG GAA GGI GIG GAI GAC GT) and p1370 (AGI CCC GIG AAC GTA TTC AC) creating an amplicon of 216 to 217 bp. This universal primer set was chosen because the amplicon was small, allowing for efficient amplification, and because it had the ability to amplify ≥ 96% of bacterial DNA, including known ocular pathogens and spirochetes.18–20 Test sample results were compared with a standard curve of 16S ribosomal DNA ranging from 109 to 101 DNA copies.
For all amplification runs, control target DNA, Escherichia coli (from 108 to 100) copies, was concurrently assayed to establish the standard curve for quantifying copy numbers of target present. Sensitivity of the assay was ≥ 0.1 pg of bacterial DNA. Negative controls, composed of nontemplate control and DNA that did not harbor the target gene, were included in each amplification run. Randomly chosen aqueous and vitreous humor samples were spiked (or purposefully inoculated) with known amount of E coli to confirm that each experiment was valid. To control for the presence of DNA in each sample, each sample underwent traditional, nonquantitative PCR analysis for G3PDH, a housekeeping gene, resulting in a 452-bp product. The primer sequences were (forward G3PDH primer) 5′-ACC G3PDH ACA GTC CAT GCC ATC AC-3′ and (reverse G3PDH primer) 5′-ATG TCG TCG TTG TCC CAC CAC CT-3′. Samples were randomly sequenced to confirm that appropriately sized bands were G3PDH.
Leptospiral antibody titers and GWC—Because results of PCR analysis did not detect bacterial DNA from group 1B horses, antibody titers against Leptospira spp (MAT)c were determined in aqueous humor and serum samples from a second group of horses with ERU (group 2B; n = 24). These results were compared with aqueous humor and serum leptospiral antibody titers in clinically normal horses (group 1B; n = 12) and horses with non-ERU inflammation (group 3; 17). All aqueous humor and serum samples were tested for antibodies against Leptospira interrogans serovars Pomona, Grippotyphosa, Icterohemorrhagiae, Canicola, Hardjo, and Autumnalis, as described previously,21 and were considered reactive when > 50% of the leptospiral antigen agglutinated at the screening dilution of 1:100. Final antibody titers of samples were determined by assay of sample serial dilutions.21
The GWC, or C-value, for each leptospiral serovar for each horse was determined by dividing the aqueous humor antibody titer value by the serum leptospiral antibody titer value. A GWC > 1 was suggestive evidence of intraocular antibody production22,23 and considered confirmatory when the C-value was > 3.15,24
Statistical analysis—Comparisons were made of the number of positive antibody titer results and C-values among the 3 groups of horses by use of a 2-way frequency (contingency) table with the Pearson (C2) and likelihood-ratio (G2) statistics, whereas actual values of antibody titers and C-values were compared by use of an ANOVA and the Tukey test. A commercial software programd was used for all analyses. Values of P b 0.05 were considered significant.
Results
Detection of bacterial DNA—Bacterial DNA was not detected in aqueous humor or vitreous humor of horses with ERU or in samples from clinically normal horses by use of real-time PCR assay. Amplification of G3PDH was successful in all test samples. Results of negative and positive control samples were as expected (data not shown).
Anti-Leptospira antibody titers and GWC—In clinically normal horses (group 1B), 2 of 12 were seropositive for Leptospira organisms (serovars Grippotyphosa and Icterohemorrhagiae at antibody titers of 1:200 and 1:400, respectively). However, antibodies against Leptospira organisms were not detected in aqueous humor of clinically normal horses.
In horses with non-ERU inflammation (group 3), 3 of 17 were seropositive for Leptospira organisms (serovars Icterohemorrhagiae, Pomona, and Grippotyphosa at antibody titers of 1:200, 1:400, and 1:800, respectively), and 3 of 17 (not all the same horses as the seropositive horses) had positive MAT results for the detection of antibodies against Leptospira organisms in aqueous humor (serovars Icterohemorrhagiae, Pomona, and Grippotyphosa at antibody titers of 1:200, 1:400, and 1:3,200, respectively). Two of the 17 horses, both with acute primary uveitis, had C-values > 1: one had a C-value of 2 for serovar Icterohemorrhagiae, and 1 had a C-value of 4 for serovar Grippotyphosa.
In horses with ERU (group 2B), 10 of 24 (41.7%) were seropositive for Leptospira organisms, with 3 of the 10 horses being seropositive for 2 serovars (Pomona, antibody titers of 1:200 and 1:800 [n = 2]; Grippotyphosa, titers of 1:200 to 1:800 [5]; Icterohemorrhagiae, titer of 1:200 [4]; Canicola, titer of 1:200 [1]). Six of the 24 (25%) horses had positive MAT results for the detection of antibodies against Leptospira organisms in aqueous humor, with 1 horse having positive MAT results for 2 serovars (Pomona, antibody titer of 1:200 [n = 1]; Grippotyphosa, titer of 1:200 to 1:1,600 [5]; Icterohemorrhagiae, titer of 1:200 [1]). Three of the 24 horses (12.5%) had C-values > 1: 2 had a C-value of 2 for serovar Grippotyphosa, and 1 had a C-value of 8 for serovar Grippotyphosa.
Although a higher percentage of horses with ERU were seropositive for Leptospira organisms (10/24; 41.7%), compared with horses with non-ERU inflammation (3/17 horses) or clinically normal horses (2/12 horses), these differences were not significantly different (Pearson C2 P = 0.11). Horses with ERU were also not significantly more likely to have positive MAT results for the detection of antibodies against Leptospira organisms in aqueous humor than clinically normal horses or horses with non-ERU inflammation. Furthermore, there were no significant differences in the number of C-values > 1 among the 3 groups. In these calculations, only the higher antibody titer or C-value was used in horses that had 2 positive results for antibodies against Leptospira organisms (in group 2B, 3 horses with ERU had 2 positive results for the detection of antibodies in serum, and 1 horse had 2 positive MAT results for the detection of antibodies in aqueous humor).
The most commonly detected antibody in serum and aqueous humor was against serovar Grippotyphosa, and this serovar was most commonly associated with a C-value > 1 (Table 1). Antibodies reacting to serovars Icterohemorrhagiae and Pomona were also detected, but less frequently. Antibodies against serovar Icterohemorrhagiae were associated with a positive C-value (C-value > 1) in 1 horse with non-ERU inflammation (group 3), whereas antibodies against serovar Grippotyphosa were associated with a positive C-value in 4 horses (3 horses with ERU and 1 horse with non-ERU inflammation).
List of the detection of antibodies against leptospiral serovars in serum and aqueous humor samples of horses from the southeastern United States.
| Horses* | Leptospira interrogans serovars | ||
|---|---|---|---|
| Seropositive (No. of horses) | +AH (No. of horses) | C-value > 1 (No. of horses) | |
| With ERU (n = 24) | Pomona (3) Grippotyphosa (5) Icterohaemorrhagiae (4) Canicola (1) | Pomona (1) Grippotyphosa (5) Icterohaemorrhagiae (1) | Grippotyphosa (3) |
| With non-ERU inflammation (n = 17) | Pomona (1) Grippotyphosa (1) Icterohaemorrhagiae (1) | Pomona (1) Grippotyphosa (1) Icterohaemorrhagiae (1) | Grippotyphosa (1) Icterohaemorrhagiae (1) |
| Clinically normal (n = 12) | Grippotyphosa (1) Icterohaemorrhagiae (1) | None | None |
Some horses had positive results for 2 serovars.
+AH = Positive MAT results for the detection of antibodies against Leptospira organisms in aqueous humor.
Discussion
In this study of horses with ERU from the southeastern United States, there was no bacterial DNA detected by use of real-time 16S ribosomal PCR assay in affected eyes of horses with ERU (group 2A). No significant difference was found in serum antibody titers, aqueous humor antibody titers, or positive C-values (ie, C-value > 1) for Leptospira organisms between clinically normal horses (group 1B), horses with ERU (group 2B), and horses with non-ERU inflammation (group 3). In fact, most horses were seronegative for Leptospira organisms, and there were few positive C-values. Only 2 horses, 1 horse with ERU and 1 horse with non-ERU inflammation, had definitive intraocular production of antibodies against Leptospira organisms (ie, C-value > 3).15,24 Although the sample size of the groups in this study was small and the statistical power of these tests was low, the results of this study support a hypothesis that bacterial infections, such as those caused by Leptospira spp, may help initiate ERU in some, though not all, horses, but the continued presence of the organisms may not play a direct role in the pathogenesis of recurrent disease.
Because several bacterial infections have been associated with ERU in horses (eg, Borrelia spp, Streptococcus spp, Rhodococcus spp, Leptospira spp),1 the first goal of this study was to determine whether evidence of bacterial DNA could be found in eyes from horses with ERU. A sensitive method, with universal primers for the detection of bacterial DNA, was used in this study. The 16S ribosomal RNA gene contains regions of highly conserved sequences that are common among all bacteria interspersed with sequences that can differentiate organism species.25 Primers that are complementary to conserved sequences of the 16S gene and that flank variable regions can be used to amplify a portion of ribosomal RNA or its complementary ribosomal DNA. The PCR product can then be sequenced to provide a unique identifier for the bacteria present in the specimen.25 The primer set used in this study was demonstrated to detect 23 common bacterial pathogens across the genera, including spirochetes, with a sensitivity of 0.01 pg of bacterial DNA.18 Furthermore, this primer sequence allows amplification of ≥ 96% of more than 1,200 bacteria, as determined by inspection of their 16S ribosomal sequences.18 A recent analysis of the leptospiral 16S ribosomal sequences determined that all recognized species of Leptospira organisms should be identified by use of a standardized 16S ribosomal RNA gene sequencing with universal primers.26
Detecting bacterial DNA by use of 16S ribosomal DNA typing is an accepted, common, and rapid method for determining the cause of intraocular inflammation.27–30 Because of the large number of potential pathogens in uveitis, amplification and sequencing of 16S ribosomal DNA to permit the identification of specific bacteria is preferred over developing a large assortment of specific pathogen primers.28 However, the sensitivity of different primer sets may differ among bacterial species, and there have been reports of falsenegative PCR analysis results. For example, in a recent paper by Chiquet et al,30 it was determined that the correlation of PCR analysis results with bacterial culture results was high (> 94%), but PCR analysis did fail to detect Staphylococcus and Streptococcus spp. Although the sensitivity of the method and primer set used in this study has been demonstrated to be high in most bacteria, including spirochetes,18–20 it is possible that some bacteria in our samples may not have been detected. Although the goal of this study was to determine whether bacterial DNA (but not specifically leptospiral DNA) could be detected in affected eyes of horses with ERU, the sensitivity of the primer set used in this study for Leptospiral spp is also not known. However, in a recent study11 of horses with ERU from the midwestern United States, no detectable leptospiral DNA was found in fixed ocular tissues by use of real-time PCR assay with specific leptospiral 16S primers, thus supporting the findings of this study.
Although thought to be specific,15,30 the sensitivity of real-time PCR 16S ribosomal DNA or MAT and GWC in determining the presence of bacterial DNA or antibodies, respectively, in the eye is not known. There have been numerous studies in human and veterinary medicine on the use of GWC to help determine intraocular antibody production and thus provide definitive diagnosis of toxoplasmosis chorioretintitis,15,22,31 herpesvirus uveitis,15,23 and varicella zoster virus infection.15,32 The sensitivity of the GWC has been estimated at between 50% and 70% for human toxoplasmosis with similar sensitivity as that for a positive diagnosis by use of aqueous humor PCR methods.15,33 Because of the lack of antibody response in nonimmunocompetent hosts, which may result in a negative GWC, or lack of organism DNA in the eye in some stages of disease that may result in a negative PCR analysis result, the use of both GWC and PCR is suggested to be the most sensitive method to determine the role of bacteria in uveitis.15,33–35 In a study36 of active leptospiral uveitis in humans, 72% had positive aqueous humor MAT results, and 80% were positive for leptospiral DNA by PCR analysis. Results of that study also revealed that a combination of MAT and PCR improves diagnosis of the disease. Therefore, the use of MAT and GWC for evaluation of antibodies against Leptospira organisms and the use of real-time PCR analysis for the detection of bacterial DNA were likely both sensitive and specific for determining whether bacteria have a role in pathogenesis of ERU in horses. Furthermore, in this study, a conservative 1:100 leptospiral antibody titer (in aqueous humor and serum) and C-value (> 1) were considered positive to suggest intraocular antibody production. However, other studies15,24,32,34,35 suggest a more stringent C-value of > 3 or 4 to definitively demonstrate intraocular antibody production. Immunoblotting for local specific IgG and IgA may have increased the sensitivity of detection of leptospiral antibody production in the eye34,35; however, the numerous serovars that required testing in this study would have made this technique difficult.
In horses with non-ERU inflammation (group 3) in this study, 2 of 4 horses with acute primary uveitis had C-values > 1. These horses with acute uveitis (< 2 weeks in duration) did not have ERU, by definition.1,14,16 It is not known what percentage of horses with acute uveitis proceed to become horses with ERU; however, on the basis of field and experimental studies,37–39 the percentage may be high. The finding that primary uveitis involved intraocular antibody production (C-values > 1) in 2 horses in this study, but rarely in horses with ERU, supports the theory that acute primary uveitis caused by leptospiral infections may proceed to ERU through a molecular mimicry and autoimmune process.
The results of this study regarding the role of bacteria in horses with ERU differ from 2 recent studies from other geographic areas (California8 and Germany9). In the California study, 21 of 30 (70%) horses with uveitis had detectable leptospiral DNA in aqueous humor, and 6 of these 21 had positive leptospiral culture results, but GWC was not performed, nor was the presence of Leptospira organisms or DNA in affected eyes of horses with non-ERU inflammation compared. However, similar to our study but unlike others,10,40 the California study did not find correlation between serum anti-Leptospira antibody titers and uveitis. In the German study, leptospiral antibody titers were performed on serum and vitreous humor samples from horses with uveitis, but not aqueous humor, as is the standard in the GWC calculation.15,22–24,32,34,35 Furthermore, PCR analysis was not performed, and Leptospira organisms or DNA was not evaluated in affected eyes of horses with non-ERU inflammation. Nonetheless, > 76% of horses with ERU had Leptospira organisms detected in vitreous humor, and there was evidence of intraocular production of antibodies against Leptospira organisms.9 Furthermore, 52% of these horses had positive vitreal leptospiral culture results. The disparate results of the study reported here and these previous reports may stem from regional geographic differences in the prevalence of Leptospira infections and possibly from differences in the diagnosis of ERU in horses. Also, in this study, samples were tested only for serovars that are known to be prevalent in the southeastern United States and have been previously associated with uveitis, but not all known serovars. Therefore, some positive results may have been missed, such as L interrogans serovars Australis and Sejroe, which have been associated with uveitis in horses in Germany.41
It is possible that horses from group 2A that were euthanatized because of ERU may have been biased toward advanced stage and duration of disease, compared with horses in group 2B and group 3. Results of a recent study13 in humans with uveitis indicate that bacterial DNA (T gondii) may be detected later in the course of the disease (> 3 weeks' duration) but that intraocular antibody production (against herpes simplex virus, varicella zoster virus, and T gondii) is found throughout the course of the disease.13 The ideal time for detection of bacterial DNA in horses with uveitis is not known; however, the antibody titers are expected to be elevated during active inflammatory disease if a Leptospira organism is involved.9
Once the inflammatory process has been initiated, possibly by leptospiral infection or other ocular disease, there may be a general loss of ocular immune tolerance as a result of the presence of an inflammatory process. In this environment, activated antigen–presenting cells in the uveal tract can effectively present autoantigens to the adaptive immune system, and cross-reaction between infectious agents and self-antigens can develop into autoimmune disease.42 Leptospira organisms (lipoprotein fragments and DNA) have been demonstrated to be particularly cross-reactive with equine ocular tissues.43–47 When cross-reactivity occurs and self-antigens are recognized and processed, the specific subcellular site, or epitope, is processed within cytoplasmic endosomes to ultimately present to T cells. Autoantigen cleavage in these endosomes can create neoepitopes, either from novel conformations or realignment of protein sequences.42 This process can lead to intramolecular epitope spreading, which is the development of autoantibodies against additional components of the autoantigen.6 Similarly, autoantibodies specific for 1 antigen may bind to apoptotic bodies or other similar autoantigens produced or recognized during inflammatory processes, leading to activated antigen–presenting cell uptake of this material and reamplification of inflammatory response (ie, intermolecular epitope spreading). This reamplification of the inflammatory response to these neoepitopes likely explains the recurring nature of inflammation in many autoimmune diseases, including ERU and immunemediated keratitis in horses.6
The results of this study, along with the findings that recurrent episodes of inflammation in affected horses can be suppressed with pharmacologic14 or mechanical48 suppression of helper T-cell function in the horse, support the theory that the recurrent episodes of inflammation in horses with ERU are a result of an immune-mediated response. The results of this study, and that of a recent publication of horses with ERU in the midwestern United States,11 suggest that continued presence of bacteria or organism components does not contribute to the perpetuation of disease in horses with ERU from some geographic areas, such as the southeastern United States.
Abbreviations
| ERU | Equine recurrent uveitis |
| G3PDH | Glyceraldehyde 3-phosphate dehydrogenase |
| GWC | Goldmann-Witmer coefficient |
| MAT | Microscopic agglutination test |
QPCR Master Mix, Stratagene, La Jolla, Calif.
Mx3000P QPCR thermal cycler, Stratagene, La Jolla, Calif.
Microscopic agglutination testing, Animal Health Diagnostic Center, Cornell University, Ithaca, NY.
JMP 5.1, SAS Institute Inc, Cary, NC.
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