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Brief Communication |
Correspondence: 1Corresponding Author: Elizabeth A. Giuliano, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri–Columbia, 379 East Campus Drive, Columbia, MO 65211, e-mail: GiulianoE{at}missouri.edu
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Abstract |
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Key Words: DNA extraction • horse • immunohistochemistry • moon blindness • periodic ophthalmia • real-time polymerase chain reaction (PCR)
Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide, and economic losses associated with ERU are significant.7 The disease is initiated by a single uveitis episode, which is followed by waxing and waning episodes of uveitis. It is the chronic and recurrent nature of the disease that differentiates ERU from the primary ocular and systemic forms of equine uveitis. Leptospira interrogans has been implicated as an etiologic agent in some cases of ERU. Horses experimentally or naturally infected with L. interrogans were shown to develop clinical uveitis 1 to 2 years after infection.1,14 Numerous studies directly connected L. interrogans and ERU by identifying the bacteria in fresh aqueous and vitreous humor samples of affected horses. A recent study performed in Western Europe revealed 52.8% of equine vitreous humor samples with ERU were positive via culture for Leptospira and 71% were positive by PCR.15 Although extensive studies were performed in Western Europe, only 1 study that confirmed the presence of Leptospira organisms in ERU affected equine ocular tissue was performed in the United States. In that California study, Leptospira DNA was detected by PCR in fresh samples of aqueous humor from 70% of horses with clinical ERU and in 6% of normal control horses.5 Horses with ERU also have increased antibodies to Leptospira in vitreous humor compared with serum antibodies; this suggests that there is a persistent ocular infection with Leptospira.16 Cross-reactivity among antigens of equine cornea, lens, and leptospires has been documented by using immunoblotting techniques.12 Research results suggest that local immune mechanisms also contribute to the pathogenesis of equine recurrent uveitis.9 The findings of predominantly CD4+ lymphocytes, increased interleukin (IL) 2 and interferon-
, and decreased IL-4 are suggestive of a cell mediated (Th1) immune response.6 The autoimmune nature of ERU was demonstrated experimentally by the induction of recurrent uveitis in horses after injection of interphotoreceptor retinoid-binding protein and S-angiten.3,4 The purpose of this study was to determine the frequency of detection of L. interrogans DNA and antigen in fixed equine ocular tissues affected with end-stage ERU by using real-time PCR and immunohistochemistry. Our hypothesis was that Leptospira DNA and antigen would be detectable in fixed archival equine ocular tissue affected with ERU.
Samples were fixed in 10% neutral buffered formaldehyde, dehydrated in ascending concentrations of ethanol and xylene, and routinely embedded in paraffin. Sections of 5-micron thickness were prepared from tissue blocks. Thirty globes were obtained from 30 separate horses. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes with ERU. A positive control sample of L. interrogans-infected porcine kidney was also included. Ocular histopathologic evaluation to confirm the presence or absence of ERU in experimental and control groups was performed by a board-certified veterinary pathologist (R.R.D.). DNA extraction was performed on paraffin-embedded, formalin-fixed equine ocular sections. The sample of Leptospira-infected porcine kidney was also extracted and served as a positive control for DNA extraction. Two individual 5-micron sections from each tissue specimen were deparaffinized in xylene, rehydrated in descending concentrations of ethanol, rinsed with 100% ethanol, centrifuged at 12,000 x g, and air-dried. Tissue was digested overnight with proteinase Ka at 56°C. Extraction of DNA was performed by using the MagneSil genomic fixed tissue systema according to the manufacturer's instructions. Samples of DNA with a final volume of 25 µL were stored at –20°C until analyzed. To ensure an adequate total DNA concentration in the samples for further experiments, a DNA quantification was performed on all equine ocular samples. The primersb F (forward) 5'-GGA GCA GAC ATC AAA TAA GTA GG-3' and R (reverse) 5'-CCA TAA CCA TCA CCA TGA TAG G-3' were used to target the ubiquitous equine membrane associated transporter gene.10 A product size of 100 bp was targeted and SYBR greenc was used to monitor and quantify double-stranded DNA levels. Real-time quantitative PCR was performed on a Stratagene Mx4000d by using SYBR green reagents.c The PCR reaction contained 5 µl of template in a final volume of 20 µl. Each primer was used at a final concentration of 200 nM. The amplification protocol consisted of 15 min at 95°C followed by 40 cycles of amplification (denaturation at 94°C for 15 sec, annealing at 60°C for 1 min, and extension at 76°C for 30 sec), after which the reaction was stopped (95°C for 2 min) and melted (55–96°C), with plate readings every 1°C. All samples were subjected to real-time PCR for Leptospira. The primersb and probesb for this PCR were previously described13 and were obtained from alignments of available Leptospira 16S rDNA partial sequences obtained from the GenBank nucleotide sequence database. The PCR primers F 5'-CCC GCG TCC GAT TAG-3' and R 5'-TCC ATT GTG GCC GRA GAC AC-3' were located between the positions 191 and 278 of the Leptospira 16S ribosomal subunit gene (GenBank accession no. DQ991474), with an expected product size of 87 base pairs (bp). The probe, 5'- (FAM)CTC ACC AAG GCG ACG ATC GGT AGC-3'(3IABlkFQ), had the fluorescent reporter dye, 6-carboxy-fluorescein (FAM) located at its 5' end and the quencher Iowa Black FQ located at the 3' end and corresponds to positions 225–248 of GenBank accession no. DQ991474. The PCR was performed on a Stratagene Mx4000d and was carried out by using 5 µL of sample DNA, extraction control (porcine kidney), or known positive and negative DNA controls added to the 45 µL mastermix,e providing final concentrations of 200 nM of each primer and 200 nM of the FAM-IABlkFQ labeled probe, as previously described.13 Immunohistochemistry for L. interrogans was also performed on all samples. Paraffin-embedded, formalin-fixed equine ocular sections and the section of Leptospira-infected porcine kidney were positioned on slides, hydrated, and placed in Tris buffer for at least 5 min before staining. Slides were treated with 3% H2O2 for 15 min, washed, treated with proteinase K for 5 min, washed, subjected to Protein Blockf for 5 min, and drained. Deparaffinized sections were incubated in rabbit anti-Leptospira cocktail antibodyg (a mixture of antibodies against L. interrogans serovars pomona, hardjo, icterohaemorrhagiae, canicola, grippotyphosa, and bratislava) at a 1 : 1,600 concentration for 30 min. Negative controls were treated with rabbit immunoglobulin G antibodyh at a 1 : 1,000 dilution for 30 min. Subsequently, sections were incubated with a biotinylated secondary antibodyi for 20 min and a streptavidin-biotin complex reagenti for 20 min, both at room temperature, followed by a Tris buffer rinse. Development proceeded for 5 min with NOVA Red.j NOVA Red chromogen is readily discernible from black-brown melanin pigment present in equine globes. Slides were counterstained in Mayer hematoxylin and azure blue, then dehydrated, and cover-slipped. Additional controls were also included via omission of the primary antibody or both primary and secondary antibodies. The staining intensity and distribution of Leptospira antigen was evaluated by a masked board-certified veterinary pathologist (J.R.T.). Immunostaining from an average of 3 high-power (200x) microscopic fields was assessed by using the following semiquantitative grading scale: grade 0, none or occasional positive cells only; grade 1, sparse positive cells; grade 2, moderate positive cells; and grade 3, intense positive-staining cells.
The presence of amplifiable DNA was confirmed on all equine ocular tissue samples after extraction by performing PCR for an equine genomic target. Amplifiable DNA was identified in 29 of 30 samples after initial extraction with 1 normal control sample being negative. This sample was extracted a second time, and DNA PCR was repeated, confirming that amplifiable DNA was present in the second extraction and in the final extraction of 30 of 30 samples. Real-time PCR evaluation for Leptospira DNA was negative for all equine ocular samples and for the negative DNA control. The extraction control (porcine kidney) and positive DNA control were positive, showing amplification of the 87-bp target Leptospira product. Immunohistochemistry indicated that 2 of 10 eyes from the ERU affected experimental group and the section of Leptospira-infected porcine kidney exhibited positive immunoreactivity. Positive results were repeatable on multiple sections. Of the positive horses in the ERU group, grade 2 focal immunoreactivity was noted in the corneal epithelium of 1 horse and in the uveal tract (inflammatory cells within the ciliary body) of another horse. A representative photomicrograph is shown in Figures 1A and 1B. The control sample of porcine kidney exhibited multifocal areas of mixed grade 2 and grade 3 immunoreactivity. Positive samples were confirmed by repeating immunohistochemistry on a minimum of 3 sections from the same sample. The 8 additional samples from the experimental (ERU) group were negative, as were all samples from control groups and the negative controls (Fig. 1C). Statistical evaluation with the Fisher exact test revealed no significant difference (P = 0.1) in the detection of Leptospira antigen between the experimental (ERU) group and the control groups.
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a. Promega Corporation, Madison, WI.
b. Integrated DNA Technologies Inc., Coralville, IA.
c. Qiagen QuantiTect SYBR Green PCR Kit, Valencia, CA.
d. Stratagene Inc., La Jolla, CA.
e. TaqMan Universal PCR Mastermix Mix, Applied Biosystems, Foster City, CA.
f. DakoCytomation, Carpenteria, CA.
g. National Veterinary Services Laboratories, Aimes, IA.
i. LSAB2 System-HRP, DakoCytomation, Carpenteria, CA.
j. Vector Laboratories, Burlingame, CA.
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