Equine recurrent uveitis with PCR detection of Leptospira spp. in aqueous humor

Rebecca Egan, Davor Ojkic

Animal Health Laboratory, University of Guelph, Guelph, ON

AHL Newsletter 2023;27(1):23.

Equine recurrent uveitis (ERU), also known as periodic ophthalmia or moon blindness, is the most common cause of blindness in horses.  Clinically, it presents as recurrent episodes of uveitis at random intervals and with increasing severity, and the Appaloosa has a known breed predisposition.  The exact etiology and pathogenesis of this condition has not been fully elucidated, although there is evidence to suggest that leptospiral infection is a common inciting factor in this disease.  It has also been suggested that an immune-mediated reaction may trigger autoimmune disease due to cross-reactivity between leptospiral antigens and endogenous ocular antigens.  In one study by Faber et al., PCR testing and culture were performed on samples of aqueous humor from globes with a clinical diagnosis of ERU.  It was reported that 21 of the 30 samples had PCR detection of Leptospira DNA in the aqueous humor, and culture of 6 of 21 samples isolated leptospires.  Interestingly, 1 of the globes sampled from the 16 control horses with no current or historical evidence of uveitis was also positive by PCR.  Serum was collected from these horses to evaluate for the presence of antibodies against five leptospira serovars, and it was found that the serologic results did not correlate well with the detection of Leptospira DNA or isolation of organisms from the aqueous humor.  There were two aqueous samples that were positive by PCR and culture with negative serology, and none of the cultured isolates were serologically reactive with the serovars used in the microagglutination test (MAT), and this led the authors to speculate that these results might reflect infection with a different leptospiral serovar.

 Between 2013 and 2022, a total of 391 equine samples were submitted for MAT to the Animal Health Laboratory for various reasons (Table 1).  Among 173 samples with available history, 76.9% were from horses with ophthalmic issues (e.g., uveitis, cataract, blindness).  All samples were tested for the presence of agglutinating antibodies against 7 Leptospira interrogans serovars: L. Autumnalis, L. Bratislava, L. Canicola, L. Grippotyphosa, L. Hardjo, L. Icterohaemorrhagiae, and L. Pomona.  The positivity rate (titer of 100 or higher for at least one serovar) was 82.7% for samples with ophthalmic history.  Among positive samples, 39.9% had antibody titers of 800 or higher.  Samples submitted for other reasons had a comparable positivity rate of 76.9%; however, the occurrence of titers of 800 or higher was at a much lower rate of 10%.  MAT may not be suitable for specifically identifying the infecting serovar because cross-reactivity may occur due to previous exposure and the characteristics of the test; however, in 110 submissions in which the single highest titer was determined, the most frequent were L. Pomona (33), L. Autumnalis (31) and L. Bratislava (28).

The Animal Health Laboratory recently received an enucleated eye from a 5-year-old female Warmblood horse.  The patient had been assessed by an ophthalmologist and a clinical diagnosis of ERU was made. A blood sample had been submitted for Leptospira MAT at another laboratory, and results were negative; however, a false negative result is possible, if Leptospira spp. antigen of the infecting serovar was not present in the test panel.  The severe panuveitis and band keratopathy in this horse did not improve with treatment, conferring a guarded long-term prognosis for vision in this eye; therefore, enucleation was elected.

Table 1: Summary of equine serum MAT testing, 2013-2022.

The enucleated globe was submitted to the AHL in formalin, and upon arrival at the lab, a sample of aqueous humor was collected via needle aspiration.  Gross examination revealed a distinct localized region of corneal opacity at the medial aspect of the cornea, consistent with the clinically diagnosed keratopathy.  Dissection of the globe identified a focally adherent inflammatory membrane along the posterior aspect of the ciliary body.  Microscopic examination confirmed the presence of lesions typical of ERU, including lymphoplasmacytic inflammation in the ciliary body stroma, proteinaceous membrane with inflammatory cells along the posterior aspect of ciliary body processes (Fig. 1), and hypereosinophilic linear inclusions in the cytoplasm of non-pigmented ciliary body epithelium (Fig. 2). Immunohistochemistry performed on a section of the globe with ciliary body did not detect any immunoreactivity for leptospiral antigen.  The aqueous humor was submitted for real-time PCR testing which detected the presence of Leptospira spp., and sequencing was applied in an attempt to identify the serovar.  Based on partial SecY gene analysis, the sample was determined to be 99.5% similar to L. Grippotyphosa and 99.7% to L. Mozdok, and was thus not sufficient to confirm the exact identity of the serovar, as Leptospira genotyping results usually attain a 100% match or sequence homology.   AHL

Figure 1. Microscopic section of the eye (H&E, 1.25x) demonstrating lymphoplasmacytic inflammation in the ciliary body stroma (*) and proteinaceous membrane with inflammatory cells along the posterior aspect of ciliary body processes (arrow). Cornea= C, filtration angle= FA.

Figure 2.  Microscopic section of eye (H&E, 20x) demonstrating lymphoplasmacytic inflammation within the epithelium ciliary body epithelium (>) and proteinaceous membrane with inflammatory cells along the posterior aspect (arrow).  Inset (H&E, 40x): Hypereosinophilic linear inclusions in the cytoplasm of non-pigmented ciliary body epithelium.

References

1. Allbaugh RA. Equine recurrent uveitis: A review of clinical assessment and management. Eq Vet Ed. 2017;29(5):279-288.

2. Frellstedt L. Equine recurrent uveitis: A clinical manifestation of leptospirosis. Eq Vet Ed. 2009;21(10):546-552.

3. Dubielzig RR, Ketring K, McLellan GJ, Albert DM. Chapter 9: The uvea. In: Veterinary Ocular Pathology. Dubielzig RR, Ketring K, McLellan GJ, Albert DM, eds. WB Saunders, 2010:258-260.

4. Wilcock B, Njaa B. Special senses. In: Jubb, Kennedy, and Palmer's Pathology of Domestic Animals, 6th ed. Maxie MG, ed. Elsevier, 2016;vol 1:455-456.