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

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Bettina Wollanke and Hartmut Gerhards

Submitted: January 5th, 2022 Reviewed: March 16th, 2022 Published: May 13th, 2022

DOI: 10.5772/intechopen.104527

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Abstract

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.

Keywords

  • equine recurrent uveitis (ERU)
  • pathogenic Leptospira spp.
  • biofilm formation
  • vitreous cavity
  • intraocular specimens
  • intraocular antibody production
  • leptospiral culture
  • real-time PCR targeting LipL32

1. Introduction

Equine recurrent uveitis (ERU) occurs in mules and horses and is a disease that has been known for a long time. From about the beginning of time-counting, ancient writings have described symptoms that are consistent with today’s definition of ERU. Since the end of the nineteenth century and the beginning of the twentieth century, more and more detailed descriptions of this disease have been published [1]. In earlier times, the working power of the horse was quite crucial for the survival of men [2]. Not only during war, but also for the cultivation of the fields, for the transport of people and freight as well as for serving as living motors in preindustrial times, people were dependent on horses and mules.

Without horses, the development of mankind would not have been possible to the extent that has been achieved in the past centuries. All the more the health maintenance of the horses was of paramount importance [3]. The recurrent and painful episodes of uveitis led to reduced performance and not infrequently to blindness and thus often to unserviceability of the affected horses. For this reason, equine recurrent uveitis has preoccupied many generations of owners and veterinarians [3, 4]. There are the most diverse historical treatment approaches and theories about the causes of this disease [3, 5].

Among many causes that had not been confirmed, wet pastures and flooding as well as heritability were discussed [5, 6, 7]. An infectious etiology has been suspected for over 100 years, although Leptospiraspp. were not known at that time [3]. Since the first description of Weil’s disease in humans, “eye complications” were known to be associated with this disease [8]. A first description of leptospires was given in 1915 [9]. At that time, the identification of Leptospiraspp. was made in Japan and Germany at approximately the same time [10].

After a link between leptospiral infections and uveitis had been established in human medicine, the Swiss ophthalmologist Gsell and coworkers studied aqueous humor from equine ERU eyes and described for the first time a link between ERU (then called “moon blindness” or “periodic ophthalmia”) and leptospiral infection [11]. Since then, there have been numerous investigations addressing the leptospiral etiology of ERU.

Because antibody detection in intraocular fluids was relatively common [11, 12, 13, 14, 15, 16, 17], but uveitis bouts typically do not become apparent until months or even years after the acute systemic infection [18, 19, 20, 21, 22], it was assumed that the infection was a trigger of ERU, but the bacteria were no longer present when the uveitis attacks started [18, 23]. In addition, a culture of Leptospiraspp. from equine intraocular samples failed many times [12, 16, 22, 24, 25, 26, 27, 28]. For this reason, ERU has also been considered by some authors to be an “autoimmune” disease [29, 30, 31].

Different causes of uveitis can occur in horses just like in other species [30, 32, 33]. However, in equine uveitis associated with painful recurrent episodes causing the typical ocular changes, chronic intraocular leptospiral infection has been found to be the cause [34, 35]. Therefore, the term “ERU” will be used hereafter to refer to leptospiral-induced recurrent uveitis.

It was not until the routine use of vitrectomy (irrigation of the vitreous chamber) in horses [36, 37, 38] and the resulting ability to obtain intraocular specimens from eyes affected with ERU [39], that the importance of leptospiral etiology in ERU was confirmed [34, 35, 40, 41, 42, 43, 44].

Only recently it was recognized that recurrent uveitis in horses is a biofilm-mediated disease [45]. The ERU has many aspects that had raised questions and been incomprehensible before the discovery of biofilm formation of pathogenic Leptospiraspp. in the vitreous chamber. However, knowing the characteristics of chronic and biofilm-associated infections, the pathogenesis of ERU can now be better understood [33].

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2. Incidence and clinical course of ERU

Leptospiral-induced uveitis is not only in horses a late consequence of systemic infection [18, 34, 46], but also human leptospiral uveitis often occurs a long time after the acute infection [11, 46, 47, 48, 49, 50, 51]. A causal relationship between uveitis and a previous leptospiral infection is often difficult to recognize when uveitis occurs, because systemic leptospirosis is predominantly inapparent in horses [19, 52] and can also be inapparent in humans [53].

ERU affects quite a lot of horses. In the United States, where there are many leopard coat pattern horses (Appaloosas), it has been reported that up to 25% of horses are affected and lose vision in one or both eyes during the course of the disease [30]. However, in that study, leopard coat pattern uveitis (Section 4.), which accounts for a large proportion of affected horses in the United States, was also classified as ERU. In other studies, the percentage of horses affected with ERU ranges from 7 to 10% [54, 55, 56], with up to one-third of the horses suffering from the disease on both sides [3, 34, 57]. The attacks of uveitis in both eyes often do not start at the same time, but with a time delay of several months up to about 2 years [34].

The first episodes of uveitis are usually noticed in younger horses between 4 and 6 years of age [34]. More rarely, however, horses can still develop ERU up to over 20 years of age. Foals up to 6 months of age typically do not develop ERU. When uveitis occurs in foals younger than 6 months, it is typically septicemia-associated and bilateral, e.g., in the course of rhodococcosis [58, 59, 60, 61].

In ERU, recurrent episodes of uveitis occur in unpredictable intervals and oftentimes not, as the former term “periodic ophthalmia” suggests, periodically. The interval between episodes of uveitis can be less than 2 weeks and up to more than a year. In most cases, ERU episodes are associated with blepharospasm, epiphora, and photophobia, so the owner notices the eye disease and seeks veterinary advice. The severity of uveitis also varies greatly from horse to horse. Sometimes very mild episodes occur, which subside after 1–2 days. Other ERU attacks are so severe that after one or two attacks, the eye may already show significant and irreversible changes and in the worst case may even lose vision. In most horses, the clinically quiescent intervals between episodes of uveitis become shorter over time, and at the same time the uveitis bouts become more severe.

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3. Clinical signs of ERU

Descriptions of the ophthalmologic findings in ERU have been given repeatedly and in broad agreement [3, 30, 32, 34, 62, 63, 64]. Acute attacks are usually painful or even very painful. Affected horses are depressed, show decreased appetite, can have a moderate rise in body temperature, severe blepharospasm, serous and later sero-mucous lacrimation, and more or less swollen eyelids. These symptoms, although typical, are not pathognomonic and can also occur with other eye lesions.

Ocular examination in horses is the easiest and most informative when a simple handheld (direct) ophthalmoscope with bright light source is used. The handheld ophthalmoscope can be used as a focal light source, magnifying glass, and slit lamp, and is most crucial for examining the posterior segment of the eye (posterior lens surface, vitreous cavity, and fundus). Since serum in horses is yellowish in color, aqueous humor and vitreous humor in acute uveitis (“leakage”) are also jaundiced. Ophthalmic examination typically reveals the following findings during an acute ERU episode:

  • Reddening of the conjunctiva

  • Low-grade diffuse corneal haziness

  • Incipient circular vascularization of the cornea

  • Jaundiced aqueous humor with positive Tyndall effect, usually also fibrin in the anterior chamber of the eye

  • Miosis and only delayed and often incompletely medically achievable mydriasis

  • Diffuse haziness of the vitreous humor

  • Ocular hypotension (intraocular pressure often <10 mmHg)

In the inflammation-free interval, after mild ERU episodes and meticulous conservative treatment, sometimes no definite changes can be detected in early stages of the disease. However, when multiple ERU attacks have occurred, pathologic changes become increasingly apparent that are also evident during the clinically quiescent phase of the disease:

  • Gradually increasing atrophy of the globe (if necessary, the inner anterior-posterior diameter can be measured by ultrasound; the difference is definite as from ≥2 mm side-to-side difference)

  • Delayed pupillary response to light, drug-induced mydriasis also only achievable with delay

  • In mydriasis, otherwise hidden posterior synechiae or iris residuals may be detected on the anterior lens capsule

  • Diffuse vitreous opacification may still be recognizable in the inflammation-free interval (in some cases only evident by comparison with the other eye, and if the fundus on the diseased side is less clear compared with the other side)

  • Vesicular cataract, typically in the periphery of the posterior lens capsule

  • Dense vitreous deposits, initially visible only in mydriasis and typically located high dorsally close to the ciliary body; in the course of the disease, these deposits become more pronounced and can eventually also be seen in the center of the vitreous cavity, many times in combination with a murky yellowish discoloration of the liquefied vitreous

  • In more advanced stages of the disease, moderate to severe bulbar atrophy or even phthisis, cataract, lens luxations, and retinal detachment may occur

In 3% of ERU cases, the inflammation occurs primarily in the posterior segment of the eye [65]. Hardly any pain is evident in these horses, and this form of ERU is sometimes detected only as an incidental finding during routine examinations or purchase examinations of horses. Only rarely do very observant owners notice a change in the fundus reflex of the diseased eye and call a veterinarian. In most cases, however, iritis occurs in the course of the disease, which then leads to the typical and easily recognizable pain symptoms. Depending on the changes that have already occurred in the posterior segment of the eye, the prognosis for preservation of vision is often guarded at this point. Sometimes these horses are not presented to the veterinarian until “sudden” blindness due to cataract formation or retinal detachment has occurred.

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4. Differential diagnosis

A significant and strinkly common type of uveitis not caused by leptospires occurs in leopard coat pattern horses [65, 66]. This type of uveitis is strikingly common in leopard coat pattern horses. In contrast to ERU, leopard coat pattern uveitis progresses insidiously and does not present as recurrent painful episodes of uveitis. In the literature, it is therefore often referred to as “insidious uveitis,” but not distinguished from ERU. Other forms of uveitis may be phacogenic, traumatic, tumor-associated, septicemia-associated, or triggered by other infectious causes such as parasites (Micronema(syn. Halicephalobus) deletrixor Sertariaspp.) or, e.g., staphylococci [33]. In addition, a chronic iritis similar to Fuchs’ heterochromia iritis in humans occurs in horses [33, 67]. In most cases, all these forms of uveitis can be relatively clearly differentiated from ERU based on the clinical picture and/or the course of the disease (Table 1) [33].

HistoryTypical ophthalmologic findingsTherapyPrognosis
ERUmost times recurring episodes of painful uveitis attackssee Section 3.acute uveitis: topical atropine and dexamethasone, systemic nonsteroidal anti-inflammatory drugs, see Section 6.
quiet interval: see Section 7. and 8.
unfavorable, if exclusively conservative therapy is given, usually increasing cloudiness of the transparent media; good for prevention of recurrences with vitrectomy; good for permanent preservation of vision if vitrectomy is performed before ERU has caused irreversible intraocular damage
Leopard coat pattern uveitisno obvious painful uveitis attacks, “suddenly” noticed impaired vision or cloudiness of the eyelens pathology (initially or very early in the course of the disease), cataract formation, lens (sub-) luxation, sometimes glaucoma, sometimes atrophy of the globeso far no etiologic therapy possible, only symptomatic treatment (anti-inflammatory, cyclosporine devices, combatting an elevated intraocular pressure)unfavorable, often enucleation is required; if both eyes are affected, euthanasia may be indicated
Phacogenic uveitisoften mild uveitis, rarely severe uveitis attacks, sometimes presentation only because of cataract formationlesions of the lens capsule, sometimes lens fragments in the anterior chamber or in the vitreous chamber, mild or moderate amount of fibrin in the anterior chamber, posterior synechiae (sometimes sealing defects in the anterior lens capsule)conservative therapy see section 6.
phacoemulsification might be considered in selected cases
depending on the degree of leakage of lens proteins and the course of the disease good to unfavorable; phacoemulsification: guarded (risk of retinal detachment)
Traumatic uveitissudden onset of eyelid swelling, no previous uveitis attacks observed; in some cases: observed head and/or globe traumasometimes accompanying lesions of the eyelids or the cornea, often sero-hemorrhagic uveitis, sometimes hyphema or even hemophthalmusif no corneal defects: see section 6., in case of corneal defects: no corticosteroids until the cornea is fluorescein-negative; if the fibrin and especially blood are not decreasing within about 10 days: injection of fibrinolytics (e.g., urokinase) or mechanical removal of the inflammatory products and the blood coagulum (e.g. using a small vitrectomy-cutter)good if there has been no damage to lens and retina and no severe damage to cornea and sclera
Chronic iritis, similar to Fuchs’ heterochromic iritis in humansno painful attacks, most often presented because of corneal cloudiness, rarely because of pigment loss of the irisoften endothelial precipitates and circumscribed depigmentations in the iris, increasing corneal edema, usually slow progression of the disease over years, glaucoma might finally occurno etiologic, but only symptomatic therapy possibleguarded concerning long-term vision, sometimes enucleation required
Uveitis caused by severe keratitis or corneal infections (kerato-uveitis)often very painful, increasing corneal opacification, no satisfying response to treatmentdeep corneal ulcer or outlined round dense opacities, sometimes annular opacities (ulcus serpens)corneal debridement, in case of severe weakening of the cornea and imminent rupture: suturing of a conjunctival flap into the lesion; in case of corneal rupture: corneal transplantdepending on the stage of the disease, the extent of the infection and the pretreatment (topical or systemic nonsteroidal drugs and especially corticosteroids worsen the prognosis) good to unfavorable
Uveitis accompanying septicemiauveitis, most often in foals <6 months, rarely adult horsestypical acute uveitis, comparable to an acute ERU-attack, but often both eyes involved, presence of a severe general infection (e.g. rhodococcosis)treatment of acute uveitis (Section 6.) and treatment of the septicemiagood if septicemia can be treated successfully and if conservative treatment achieves mydriasis; usually no further uveitis attacks occur
Uveitis caused by intraocular parasitic infectionschronic severe (kerato-) uveitis, not responding to therapycloudy cornea, depending on the causative parasite parasites might be visible in the anterior chamber or seen in the ultrasound examinationtreatment of acute uveitis (section 6.), but usually uveitis is not responding; if parasites are visible in the anterior chamber: surgical removal may be discussed; enucleation might be indicatedfor preserving vision: unfavorable
for preserving the globe: guarded to unfavorable
Uveitis caused by intraocular tumorspresentation because of (mild) uveitis, impaired vision and / or corneal or intraocular opacitiesmost often iris-melanoma, causing keratitis and sometimes glaucoma, other tumors occur less frequently (e.g. medulloepithelioma or malignant lymphoma); uveitis can be caused mechanically and immunologicallysymptomatic treatment as long as possible, if the intraocular tumor causes chronic pain or if the tumor is going to spread enucleation is requiredunfavorable for preserving vision and for preserving the globe
Endophthalmitisdisturbed general condition, fever, severe eyelid swellingphlegmon of the eyelids, purulent epiphora, circular deep corneal vascularization, severe yellow-green corneal opacityin case of very early stages: lavage of the vitreous cavity; otherwise enucleationfor vision: unfavorable
for preserving the globe: guarded to unfavorable

Table 1.

Different types of uveitis in horses, symptoms, therapy, and prognosis.

Sometimes recurrent keratitis is misinterpreted as ERU, as some types of keratitis can also cause painful with miosis and responds to the same conservative therapy as ERU. However, in keratitis cases, medical dilation of the miotic pupil results usually more rapidly and completely than in ERU. In recurrent keratitis, however, the changes that almost always are evident in ERU after several episodes of uveitis, even in the inflammation-free interval, are absent.

If an ocular disease is clinically not clearly assignable to an etiology (e.g., “recurrent keratitis” or “uveitis of unknown cause”), it is possible to take aqueous humor during the inflammation-free interval [33]. In horses, approximately 1 ml of aqueous humor can be safely collected and then used for laboratory tests [33, 68, 69, 70]. To investigate for the presence of ERU, testing for both anti-Leptospiraantibodies and by PCR for, e.g., LipL32 is advisable [35, 71, 72, 73]. For scientific questions, a leptospiral culture can additionally be performed [34, 35]. Depending on the laboratory findings, a decision can then be made on the further course of action. In case of positive leptospiral findings, vitrectomy is indicated. With negative leptospiral laboratory findings, a leptospiral infection of the vitreous cavity can be excluded with a high probability. These horses would not benefit from vitrectomy—except to remove vitreous opacities that impair vision. In this case, however, a preoperative aqueous humor examination would be superfluous—just as in the case of unequivocal findings in terms of ERU.

For the detection of intraocular anti-Leptospiraantibodies, the microscopic agglutination test (MAT) is used in most cases. The MAT is highly sensitive and specific when examining aqueous humor or vitreous samples [34, 35, 40]. In addition, other antibody tests can be used, which are either commercially available or available as in-house ELISA tests. Specific anti-Leptospiraimmunoglobulin class A (IgA) antibodies are particularly reliable for detecting intraocular leptospiral infection [72, 74]. Another well-suited test is the SNAP Lepto, which detects anti-LipL32 antibodies and is neither immunoglobulin-specific nor serovar-specific. It can be used for samples from different species. With its easy handling and the result visible within 10 minutes, this is a very useful test with a sensitivity and specificity comparable to MAT for intraocular specimens [73, 75]. In contrast to MAT, which is too unspecific for serum testing, SNAP Lepto is well qualified as a screening method even when serum is tested [71].

Antibody detections are equally reliable in vitreous and aqueous humor samples [70, 76, 77]. Both PCR and leptospiral culture are somewhat more reliable when testing vitreous humor samples compared with testing aqueous humor samples [34, 35, 78]. However, the collection of a vitreous sample is disproportionately risky and should be rejected for a preoperative diagnosis, because the aqueous humor analysis is overall very informative [33]. In rare cases, e.g., no anti-Leptospiraantibodies are detectable in the aqueous humor, but at the same time the PCR yields a positive result. In routine diagnostics, culture has been largely replaced by the much faster and less expensive PCR.

If time is not an issue, but economic reasons have to be taken into account, a reasonable approach for the examination of aqueous humor samples is to first perform an on-site rapid test for the detection of anti-Leptospiraantibodies. If this test is negative, the MAT can be commissioned externally if necessary. If the MAT is also negative, further antibody tests (e.g., specific in-house ELISA tests) and a PCR can be performed. The more laboratory tests are performed, the fewer “false negatives” can be expected, but the higher the costs for laboratory diagnostics will be.

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5. Interpretation of intraocular antibodies

In eyes with a history of recurrent inflammations, but without clear evidence of ERU, and thus without aqueous or vitreous humor opacities, protein levels are typically not elevated. If protein levels in intraocular fluids are not elevated, leakage from the blood can be excluded. In these cases, even very low MAT titers are indicative of intraocular antibody production. The authors consider a MAT result of 1:50 as sufficient indication for vitrectomy in these cases. In eyes with obvious aqueous humor and vitreous opacities, however, the diagnosis of ERU is usually unambiguous even without aqueous humor examination. In cases of doubt, the Goldmann-Witmer coefficient can be used to differentiate leakage from intraocular antibody production [79]. It is crucial that not only the intraocular and the serum titer are evaluated, as it often could be read lately [80, 81, 82, 83, 84, 85, 86], but that—as described by Goldmann and Witmer—a reference value is determined both in the aqueous humor and in the serum. Any other antibody titer (e.g., tetanus) can be used as a reference value, provided that antibodies are present in the serum. Alternatively, the total IgG content or, if necessary, even the total protein content can be used as a reference value [33, 34].

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6. Therapy of acute uveitis

Acute ERU is treated in the same way as any other equine uveitis [30, 32, 57, 58]. First of all, it is important to achieve mydriasis to avoid posterior synechiae and resulting cataract formation. Atropine is the drug of choice for this purpose and can be used as of 1–2% eye drops or eye ointment. Since the ophthalmic ointment adheres slightly better and acts more protracted, ointment is preferable, if available.

Atropine should initially be given several times daily or even hourly until the pupil dilates. Thereafter, the intervals can be adjusted to the pupil width and often considerably prolonged. Systemic side effects associated with the topical use of 1–2% atropine in horses do not play a significant role in the authors’ experience and after having treated thousands of horses over a 30-year period. Colic, e.g., due to an impaction or a meteorism, can occur in any hospitalized horse, not just ophthalmic patients. By feeding mash and monitoring the fecal consistency, an impaction can be detected early and countermeasures (e.g., administration of laxatives) can be taken to avoid more serious colic.

Apart from mydriasis, anti-inflammatory treatment is important. Topical application of ophthalmic ointments containing dexamethasone is particularly effective, provided the corneal epithelium is intact. If corneal defects are present, topical corticosteroids must not be given.

In addition, the administration of a nonsteroidal anti-inflammatory drug (NSAID) orally is indicated. Only in exceptional situations and in case of very significant diffuse vitreous opacification, systemic administration of prednisolone (1 mg/kg per os) for several days may be considered additionally. In these particularly severe cases with significant diffuse vitreous opacification, adjunctive therapy with a systemically given antibiotic, e.g., enrofloxacin [87], can also be performed, to eliminate at least part of the intraocular bacteria—even if this does not completely eliminate the infection [88].

Other measures accompanying the therapy are keeping the horse in a dark place and resting in the stall or just light exercise until the acute inflammation has subsided. If it is not possible to keep the horse in the dark, wearing a light absorbing mask can be considered.

6.1 Brief historical overview of the development of conservative treatment of uveitis in horses valid today (without treatment proposals that did not prove successful or were even questionable from an animal welfare point of view)

  • Topical atropine has been recognized as an essential therapeutic mydriatic for equine uveitis as early as 1821 and has been considered a standard treatment for ERU in textbooks since 1842 [89]

  • Topical cocaine has been recommended for the control of pain since the beginning of the last century [90]

  • Salicylic acid preparations have been included among the treatment options for uveitis since 1922 [91]

  • Corticosteroids have been used both parenterally [92] and topically [93] to treat uveitis since the middle of the last century

  • In addition to eye drops and ointments, subconjunctival injections with corticosteroids [94, 95], later also with cocaine and atropine, were suggested to intensify the local effect

  • Systemic administration of nonsteroidal anti-inflammatory drugs (NASIDs) has also been part of the standard treatment of acute uveitis in horses since their approval for veterinary use in the late 1970s (flunixin meglumine and phenylbutazone) [96, 97]

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7. Vitrectomy during the quiet intervals

The most effective treatment for ERU is vitrectomy (removal of diseased vitreous and irrigation of the vitreous cavity). This surgery is performed exclusively in intervals without acute inflammation. Mechanical removal of the vitreous opacities caused by inflammation and accessible vitreous parts very reliably and permanently eliminates the leptospires in the biofilm. Postoperatively, up to 98% of eyes remain free of recurrences when surgery is performed properly [98]. If, exceptionally, further episodes of inflammation occur after surgery, a second vitrectomy can, if necessary, permanently eliminate the infection and prevent further episodes.

Vitrectomy as a vision-preserving procedure is a demanding surgery, having a relatively long learning curve. Prerequisites for successful performing vitrectomies are solid training, availability of for equine ophthalmo-surgery optimized, custom-made instrumentation and equipment as well as careful and intensive perioperative examination and conservative treatment. Any complication may have devastating consequences and can lead to blindness or even enucleation. Only rarely eyes that are already blind undergo surgery in order to prevent both future painful uveitis attacks and removal of the globe, which is cosmetically unsightly.

In order to perform vitrectomy with minimal complications, an experienced team (surgeon, sterile and nonsterile assistant, skilled anesthesiologist) is required, as well as expensive equipment and instruments specially adapted to the dimensions of the horse’s eye. For this reason, only a few specialized equine clinics perform vitrectomies to date. In clinics in which vitrectomy is performed as a routine procedure, it is a quick (total anesthesia time is about 40 minutes, the surgical instrument is in the eye <10 minutes) and relatively safe procedure with a very good prognosis [38, 39, 89, 99].

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8. Other treatment options for ERU

Apart from vitrectomy, other treatment options have been described, of which two in particular are favored in recent publications. One consists of an intravitreal gentamicin injection. However, the recommended dosage for this purpose (4–6 mg) [80, 100, 101] is 3–4 times the drug concentration that was found to be “safe” with regard to retinal toxicity in experimental studies [102]. So far, there are no long-term results after these injections and the number of horses treated in this way is still limited. Surprisingly, gentamicin injection is not recommended exclusively for equine eyes with intraocular leptospiral infection; other forms of uveitis are also treated with this injection. Improvement after the intravitreal injection is also thought to result from the antibiotic gentamicin having immunomodulatory effects [103].

The second therapeutic option described since the turn of the millennium is the deep intra- or subscleral implantation of a cyclosporine device [104, 105, 106, 107]. These implants lead to less frequent and milder episodes of uveitis over a period of up to about 2 years. However, the uveitis does not stop completely, and if the effect wears off, a new implant may have to be inserted. Like gentamicin injection, implantation of cyclosporine devices is performed independently of leptospiral infection in the vitreous cavity. Only individual authors differentiate and use the implants exclusively when no leptospiral infection is detectable [86]. Attention should also be paid to the drug law in its current version, which currently prohibits the import of cyclosporine devices, at least in the EU [108].

However, neither gentamicin injection nor implantation of cyclosporine devices can remove the dense vitreous floaters that often lead to impaired vision. Over time, these deposits also often adhere to the posterior capsule of the lens and, just like extensive posterior synechiae, can lead to a cataract formation.

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9. Results of the examination of intraocular specimens

In the literature, before the introduction of vitrectomy to the therapeutic measures against ERU, there were only very sporadic reports of cultural detection of leptospires in intraocular specimens from eyes affected with ERU [109, 110]. Numerous investigators failed to obtain cultural evidence of leptospires, casting doubt on chronic intraocular leptospiral infection. It was rather assumed that although leptospires somehow trigger ERU, the inflammations are not subsequently maintained by the presence of the pathogen [22, 24, 63, 68, 111, 112].

Vitrectomy was initially performed to remove vitreous opacities. The aim was to improve vision in the eyes affected by ERU [36, 37]. However, it soon became apparent that vitrectomy was surprisingly effective in preventing further episodes of uveitis. Therefore, more and more horses were sent to the clinic for vitrectomy.

It was only with the routine performance of vitrectomy that it had become possible to examine numerous vitreous samples from horses suffering from ERU. The peculiarity was that the samples were predominantly from eyes still able to see at an early stage of the disease. By collecting the first milliliters aspirated from the vitreous cavity before opening the intraocular infusion line, it was possible to use undiluted vitreous material for investigations. The results of these examinations, in turn, provided insights into which ocular findings were associated with leptospiral infection and which were not. It was also shown that the prognosis in terms of postoperative absence of recurrences was best when eyes with an intraocular leptospiral infection were treated by vitrectomy [98]. In this way, on the other hand, the indication for vitrectomy was optimized.

With careful assessment of the indication for vitrectomy and examination of undiluted vitreous specimens, MAT titers of 1:100 or higher were detected in 382 of 426 vitreous samples (90%) examined [34, 35]. In some MAT-negative specimens, specific anti-Leptospiraantibodies (especially immunoglobulin class A) could be detected by an in-house ELISA [74]. Leptospires were culturally detected in 189 of the undiluted vitreous samples from 358 eyes (53%) affected with ERU [34, 35]. The positive cultures had been obtained only after optimization of the sampling technique and immediate sterile inoculation into a transport medium for mailing to a laboratory. The sensitivity of PCR is in between culture and antibody detection. In 70–77% of vitreous samples from eyes affected by ERU, the PCR result was positive [35, 73, 75, 113].

In Germany and neighboring countries, infections with leptospires of the serogroup Grippotyphosa are dominating, accounting for about 80% of intraocular infections in horses suffering from ERU. Infections with leptospires of the Australis serogroup account for about 13–14% of intraocular infections. Less frequently, leptospires of the serogroups Pomona, Sejroe, and Javanica were also detected in the vitreous samples from ERU eyes [34, 35, 114].

Vitreous samples obtained during vitrectomies from eyes affected by ERU were also used for histological and ultrastructural studies. It has been shown that the leptospires in the vitreous of eyes affected with ERU are surrounded by a homogeneous layer, which is lacking the leptospires from culture [115]. This homogeneous layer surrounding the leptospires could be extracellular matrix. In another study, in addition to phagocytosed leptospires, dense roundish structures were detected in vitreous material from eyes affected with ERU [116]. Some of these roundish structures had been phagocytosed, but others of these structures were so large that phagocytosis was impossible. These dense round structures could represent mature leptospiral biofilm constructs.

In 1971, Williams reported on immunologically mediated tissue damage in cases of equine uveitis [22]. However, autoimmune reactions that can be detected at the same time as the leptospiral infection [117, 118, 119, 120, 121] must be autoimmune phenomena accompanying the infection, since they cease as soon as the infection has been eliminated [33, 35]. Thus, there is no evidence of autoimmune disease following ERU.

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10. Pathogenic Leptospiraspp. and biofilm

Since many chronic infections are associated with biofilm formation, it has long been suspected that leptospires also form biofilm in vivo. In in vitro studies, biofilm formation of pathogenic Leptospiraspp. was observed [122], and a detailed description of the three-dimensional structure of these biofilms was given [123]. The main focus with regard to in vivo biofilm formation was on small rodents, which are considered the main vectors of pathogenic leptospires and are chronic shedders. Following experimental infections, evidence of biofilm formation in the proximal renal tubules had been observed [124, 125]. Recently, there was also a description of in vivo biofilm formation in naturally infected rats [126]. At about the same time, biofilm formation of leptospires in vitreous samples from eyes affected with ERU could be demonstrated by immunohistochemistry (IHC) [45].

11. Characteristics of biofilm infections in ERU

Recurrent episodes of uveitis and the concomitant intraocular persistence of leptospiral infection over a long period of time meet the criteria of a biofilm infection [127, 128] very well [129].

The infection primarily affects the vitreous cavity. Possibly following the vitreous clearance, leptospires (more rarely) can also enter the anterior chamber of the eye and be detected there [33, 34, 35, 130, 131]. However, the infection obviously remains limited to the eye, there is no evidence of further spreading. As with other local biofilm infections, IgA antibodies are of particular importance in diagnostics [72, 132, 133, 134, 135].

One criterion of biofilm infections is the difficult cultural detection of the causative pathogen and ERU meets this criterion. Despite urgent suspicion of leptospiral infection in ERU (high intraocular antibody titers, intraocular antibody production), however, cultural detection of leptospires is demanding and often failed [24, 25, 26, 136].

In the vitreous of horses suffering from ERU, there are not only high antibody titers, but also immunocompetent cells (besides lymphocytes, especially plasma cells, macrophages, and granulocytes) [116, 137, 138]. The epithelium of the ciliary body shows many plasma cells in eyes affected by ERU [139]. In the area of the ciliary body and the iris root, even lymph follicles develop during the course of ERU, which contain B lymphocytes in the center [30, 140, 141]. Nevertheless, the immune system fails to eliminate the infection from the large vitreous chamber of the horse.

Leptospiraspp. could be visualized in vitreous samples from eyes affected with ERU by immunohistochemistry (IHC). The infecting bacteria are bound to each other, and extracellular matrix could be demonstrated around and in between the bacteria [33, 45, 115, 142]. Leptospiraspp. could be demonstrated in planktonic forms as well as in smaller and larger cell aggregates and in larger biofilm structures [33, 45].

Leptospires localized in the vitreous chamber show high tolerance to antibiotics. The first cultures were performed with samples from the entire lavage fluid collected during vitrectomy [41, 42, 44]. In the lavage fluid, the vitreous material was diluted about 10-fold and the lavage fluid contained 0.08 mg gentamicin/ml. This concentration had been shown to be 100 times the minimum inhibitory concentration (MIC) for WHO strains of pathogenic leptospires in vitro [143]. Cultures with these vitreous samples were less frequently positive than in later studies performed with undiluted vitreous samples [34, 35, 88], but nevertheless several culture sets eventually became positive after further inoculations and thus dilution of the antibiotic concentration [41, 42, 44].

Similar results were found in a study in which horses had been treated preoperatively intravenously with enrofloxacin. In the undiluted vitreous samples obtained at vitrectomy, the enrofloxacin content was above the MIC. Compared with the control group, in which more than 50% of the cultures were positive for pathogenic Leptospiraspp., only 30% of the cultures in the group treated with enrofloxacin were positive. Thus, although the probability of a positive culture had been reduced with antibiotic treatment, reliable elimination of the infection was not achieved.

12. Discussion

ERU with persistent intraocular leptospiral infection over a long period of time meets all criteria of an infection associated with biofilm formation. The most likely route by which leptospires enter the vitreous cavity during acute systemic infection is by the fenestrated capillaries in the Pars plicata region of the ciliary body [30, 33]. In the healthy vitreous with its collagen fibers and viscosity, there are ideal conditions for the formation of leptospiral biofilm (Figure 1) [129].

Figure 1.

Schematic illustration of the discussed pathogenesis of equine recurrent uveitis (ERU) caused by a leptospiral biofilm infection in the vitreous chamber. Each uveitis bout leads to increasing damage to the intraocular structures. 1. Infection of horses withLeptospiraspp. may occur on humid and muddy pastures or by drinking from standing waters. The bacteria can enter the blood stream via intact mucous membranes (e.g., oral cavity) or small skin lesions (e.g., on the legs). 2. Leptospira spp. most probably enter the vitreous chamber (VC) via the fenestrated capillaries of the pars plicata of the ciliary body (CB). 3.Leptospiraspp. within the vitreous chamber attach to each other and to vitreous fibers, starting biofilm production. 4. Transmission electron microscopy using a vitreous sample from an ERU eye:Leptospiraspp. are surrounded by extracellular matrix (reprint of [115] courtesy of Schluetersche specialized media GmbH, Hanover, Germany). 5. MostLeptospiraspp. are protected within the biofilm, single planktonic bacteria are in the vitreous chamber. 6. Vitreous samples from ERU eyes, containing visible inflammatory products (“vitreous floaters”); the yellow color indicates increased permeability of the blood-ocular barrier. 7. Threshold exceeded, immune privilege of the eye temporarily suspended, clinically apparent uveitis bout (left: Epiphora and blepharospasm; right: Much fibrin in the anterior chamber).

The vitreous body is 98% water and contains a collagen fiber scaffold. It has been shown that plant fibers in rice fields are important sites for biofilms [144]. The vitreous fibers [138] might also serve as “surfaces” to which Leptospiramay adhere and start biofilm production. Furthermore, viscous media promote biofilm production of Leptospiraspp. [145], and healthy vitreous humor is such a viscous substance. With the collagen fiber scaffold and viscous consistency, the vitreous thus represents an ideal medium for biofilm formation of Leptospiraspp. [33, 45, 129, 145].

Another factor to consider is that the vitreous cavity of the horse has a volume of approximately 28 ml, making it a large immunologic niche [34]. In addition, there is the immune privilege of the eye [146, 147], which effectively suppresses the immune defense. In this way, pathogenic Leptospiraspp. can remain clinically unnoticed in the eye for a long time. The latency period can be many months or several years. It probably varies with individual factors of the host, the amount of Leptospiraspp. in the vitreous, and possibly the leptospiral serovar involved.

Only after months or years, when a threshold is exceeded due to gradual multiplication of the leptospires and increase of immune reactions despite the ocular immune privilege, a uveitis attack with disturbance of the blood-aqueous barrier or blood-ocular barrier becomes apparent [33, 34]. The immune response that occurs in conjunction with the inflammation likely results in the elimination of some planktonic bacteria. Other bacteria in the biofilm outlast the inflammatory bout. After the inflammation subsides under antiphlogistic treatment and with the help of intraocular immunosuppressive mechanisms, a clinically apparently inflammation-free interval occurs, which, however, does not represent a totally quiescent phase immunologically [137].

There are reports, and some own experiences seem to support this, that episodes of uveitis can be triggered by exposure to stressful situations (e.g., competitions, long-distance transport, change of stables, general anesthesia and major surgery). It is conceivable that endogenous cortisol release in stressful situations further reduces the immune defense in the eye (in addition to the ocular immune privilege). This in turn might increase the number of planktonic Leptospiraspp. in the vitreous cavity after a stress situation and lead to contact with the uvea—which then causes an exaggerated immune reaction resulting in a uveitis attack.

A gradual spread of biofilm structures in the vitreous cavity could explain that ERU episodes occur at shorter intervals and become more severe over time. In addition, there are immune reactions that fail to eliminate the leptospires but may result in damage to the ocular structures adjacent to the vitreous chamber. One example is neutrophil extracellular traps (NETs), which have been detected in vitreous samples from eyes affected by ERU [148]. These NETs are formed by granulocytes to remove pathogens too large for phagocytosis [149]. A disadvantage of the formation of NETs is that tissue-damaging substances are also secreted, which in turn promote an inflammatory reaction of the surrounding tissue [150, 151], which in ERU cases is the uvea.

The high MAT titers in eyes affected by ERU certainly also play a crucial role in the course of the disease, as they promote agglutination of planktonic leptospires. However, since complete elimination of the bacteria is usually not possible, this agglutination can also be the starting point for new biofilm formation. During agglutination, leptospiral aggregates are formed, extracellular matrix is produced after surface contact of bacteria with each other, and thus new biofilm structures can be built. In this way, the agglutinating antibodies could accelerate the biofilm formation of pathogenic Leptospiraspp. [33].

High levels of serum amyloid A (SAA) [152] and the formation of AA amyloid [153, 154] were detected in intraocular samples from eyes affected with ERU. The formation of amyloid is a good explanation for the fact that the dense vitreous floaters in ERU fail to resolve, but instead increase as the disease progresses. Besides the collagen fibers of the vitreous scaffold, the NETs and the amyloid fibers provide additional fiber structures that could be used for biofilm formation. The formation of NETs and biofilm promote each other [155, 156]. Similar to what has been described for otitis media [157], these numerous fibers could be incorporated into the biofilm and help to reinforce the biofilm scaffold, so that therapeutically only mechanical removal is promising.

With knowledge of the successful cultivation of leptospires from vitreous specimens that contained an active level of gentamicin or enrofloxacin above the MIC, it is questionable whether intraocular gentamicin injections, which are performed therapeutically by some veterinarians, provide lasting success. Biofilms can increase tolerance to antibiotics up to 1000-fold compared with planktonic bacteria [158, 159]. The described improvement of eyes suffering from ERU after gentamicin injection could be due to the fact that planktonic bacteria are eliminated. However, it is questionable whether the bacteria in the biofilm can really be eliminated by the injection. It could also be that the structure and composition of the biofilm change accordingly, so that the bacteria survive protected in the biofilm and then lead to ERU relapses again after some time. With the therapeutically used cyclosporin-devices, spread of the leptospiral biofilm in the vitreous cavity could even be favored, since immune reactions of the host, including those directed against the bacterial pathogen, are suppressed.

In vivo biofilm formation has also been described for other spirochetes. In human medicine, for example, chronic Lyme disease with its various organ manifestations plays an important role [160, 161]. In patients with Lyme disease, in vivo biofilm formation was shown to be associated with the long-term persistence of Borreliaspp. [162], and biofilms were found to contain both Borreliaspp. and Chlamydiae [163]. For example, alginates have been found in biofilms of Borrelia[164]. Alginates induce a distinct immune response [165] and result in the biofilm being more pathogenic than the planktonic bacteria. For lymphocytoma [166] and Alzheimer’s disease [167, 168], there are detailed descriptions of biofilm formation and indications for improved treatment options. In Alzheimer’s disease, Borreliabacteria in planktonic form do not appear to cause noticeable harm. Here, too, it is the biofilms that create the pathology [167]. Biofilm formation and approaches to improve therapy have also been demonstrated following experimental Borreliainfections of mice as a model for Lyme disease [169].

The composition of leptospiral biofilms in the vitreous cavity in ERU is still largely unknown. Neither alginates nor curli fibers (bacterial amyloid) could be detected in the in vitro Leptospirabiofilms [123]. The in vitro biofilms of Leptospiraspp. consisted predominantly of extracellular DNA. However, the composition of in vivo biofilms of leptospires could be quite different [170]. It is possible that further analysis of the leptospiral biofilms in the vitreous cavity of horses suffering from ERU may provide further information on how to disperse these biofilms in a manner that is as tissue (retina, lens capsule) compatible as possible. This could provide new insights for the treatment of other biofilm-associated infections that are also relevant to human medicine.

13. Conclusions

ERU is a spontaneously occurring intraocular leptospiral biofilm infection. For centuries, only symptomatic conservative treatment was possible, which has become increasingly effective with the availability of modern anti-inflammatory drugs. However, even the most potent anti-inflammatory treatment could not prevent recurrences of uveitis, which led to gradual damage and even destruction of the affected globe. It was not until the introduction of vitrectomy in equine ophthalmology that causative therapy had become possible. Samples containing leptospiral biofilm can easily be collected in the course of therapeutic vitrectomy. Not only can these samples be used for laboratory diagnostics regarding intraocular leptospiral infection, but further studies can be performed on the composition of the biofilm. There could be significant differences between the composition of the biofilm formed in vitro and that formed in vivo, as host tissues (here: vitreous material and collagen fibrils) and interactions with the host immune system (e.g., agglutinating antibodies, macrophages, granulocytes, NETs, fibrin, and amyloid) influence the composition of the biofilm. ERU provides possibilities for investigation of an in vivo biofilm infection without the need for animal experiments and, thus, could serve as a naturally occurring entity for further research.

References

  1. 1. Braun D. Die Geschichte der Erforschung und Behandlung der “periodischen Augenentzündung” des Pferdes im deutschsprachigen Raum von 1750-1950 [Doctoral Thesis]. Munich, Germany: Ludwig-Maximilians-University (LMU); 1994
  2. 2. Paglia DT, Miller PE, Dubielzig RR. James Wardrop and equine recurrent uveitis. Archives of Ophthalmology. 2004;122:1218-1223. DOI: 10.1001/archopht.122.8.1218
  3. 3. Bayer J. Mondblindheit oder periodische Augenentzündung. In: Bayer J, editor.Handbuch der Tierärztlichen Chirurgie und Geburtshilfe. 2nd ed. Vol. V. Wien, Oesterreich & Leipzig, Germany: Wilhelm Braumüller; 1906. pp. 427-484
  4. 4. Zündel A. Die Mondblindheit der Pferde. Der Gesundheitszustand der Hausthiere in Elsass-Lothringen vom 1. April 1880 bis 1. April 1881. Zeitschrift für vergleichende Augenheilkunde. 1882;I:153
  5. 5. Fröhner E. Die Mondblindheit oder periodische Augenentzündung. In: Fröhner E, editor.Kompendium der speziellen Chirurgie für Tierärzte. 4th ed. Stuttgart, Germany: Ferdinand Enke Verlag; 1910. pp. 31-35
  6. 6. Kellner R. Ein Beitrag zu Erbfehlerstudien. Deutsche Landwirtschaftliche Tierzucht. 1934;38:209-211
  7. 7. Schwarznecker G. Die periodische Augenentzündung im Saargebiet. Zeitschrift für Veterinärkunde 1892;IV:1-10
  8. 8. Weil A. Über eine eigenthümliche mit Milztumor, Icterus und Nephritis einhergehende acute Infectionskrankheit. Deutsches Archiv für Klinische Medizin. 1886;39:209-232
  9. 9. Köhler W. Weilsche Krankheit. In: Gerabek WE, Haage BD, Keil G, Wegner W, editors.Enzyklopädie Medizingeschichte. Berlin, Germany: De Gruyter; 2005. p. 1470
  10. 10. Uhlenhut P, Fromme W. Experimentelle Untersuchungen über die sogenannte Weilsche Krankheit. Medizinische Klinik. 1915;46
  11. 11. Gsell O, Rehsteiner K, Verrey F. Iridocyclitis als Spätfolge von leptospirosis Pomona (Schweinehüterkrankheit). Agglutinin- und Lymphozytose-Befund im Kammerwasser [Iridocyclitis as a late consequence of leptospirosis Pomona (porter's disease): Agglutinin and lymphocytosis in the aqueous humor]. Ophthalmologica. 1946;112:320-334. DOI: 10.1159/000300399
  12. 12. Heusser H. Zur Ätiologie der periodischen Augenentzündung. Schweizer Archiv für Tierheilkunde. 1952;94:296-306
  13. 13. Witmer R, Löhrer J, Wiesmann E. Zur Ätiologie, Diagnose und Therapie der periodischen Augenentzündung (p. A.) des Pferdes. Schweizer Archiv für Tierheilkunde. 1953;95:419-439
  14. 14. Rossi P, Kolochine-Erber B. Iridocyclites des équidés et leptospiroses. Revue de Pathologie Générale et de Physiologie Clinique. 1954;54:432-477
  15. 15. Witmer R. Periodic ophthalmia in horses. American Journal of Ophthalmology. 1954;37:243-253. DOI: 10.1016/0002-9394(54)91570-6
  16. 16. Kemenes F, Surjan J, Vizy L. Leptospira as the cause of periodic ophthalmia in horses. The Veterinary Bulletin. 1961;31:12
  17. 17. Bürki F, Egli P, Wiesmann E. Experimentelle Infektion von Pferden mit Leptospira pomona. Berliner und Munchener Tierarztliche Wochenschrift. 1963;76:265-269
  18. 18. Lowe RC. Equine uveitis: a UK perspective. Equine Veterinary Journal. Supplement. 2010;37:46-49. DOI: 10.1111/j.2042-3306.2010.tb05634.x
  19. 19. Zwierzchowski J. Klinik und Therapie der Leptospirosen der Haus- und Nutztiere. In: Kathe J, Mochmann H, editors.Leptospiren und Leptospirosen. Vol. I. Jena, Germany: Gustav Fischer Verlag; 1967. pp. 79-137
  20. 20. Roberts SJ. Sequelae of leptospirosis in horses on a small farm. Journal of the American Veterinary Medical Association. 1958;133:189-194
  21. 21. Bolte HF. Uveitis, a Sequela to Experimentally Induced Leptospira Pomona Infection in the Shetland Pony. West Lafayette, IN, USA: Purdue University; 1966
  22. 22. Williams RD, Morter RL, Freeman MJ, Lavignette AM. Experimental chronic uveitis. Ophthalmic signs following equine leptospirosis. Investigative Ophthalmology. 1971;10:948-954
  23. 23. Sillerud CL, Bey RF, Ball M, Bistner SI. Serologic correlation of suspected Leptospira interrogans serovar Pomona-induced uveitis in a group of horses. Journal of the American Veterinary Medical Association. 1987;191:1576-1578
  24. 24. Bryans JT. Studies on equine leptospirosis. The Cornell Veterinarian. 1955;45:16-50
  25. 25. Williams RD. The presence and duration of persistence of Leptospira Pomona in Equine Ocular Tissues following experimentally induced systemic infection [Master of Science Thesis]. West Lafayette, IN, USA: Purdue University; 1968
  26. 26. Bohl E, Ferguson L. Leptospirosis in domestic animals. Journal of the American Veterinary Medical Association. 1952;121:421-428
  27. 27. Tomasek V. Etiology and treatment of equine periodic ophthalmia. Veterinarski Glasnik. 1954;8:243
  28. 28. Morter RL, Herschler RC, Fessler JF, Lavignette A. Experimental equine leptospirosis (Leptospira pomona). Proceedings, Annual Meeting of the United States Animal Health Association. 1964;68:147-152
  29. 29. Deeg CA, Ehrenhofer M, Thurau SR, Reese S, Wildner G, Kaspers B. Immunopathology of recurrent uveitis in spontaneously diseased horses. Experimental Eye Research. 2002;75:127-133. DOI: 10.1006/exer.2002.2011
  30. 30. Gilger BC, Hollingsworth SR. Diseases of the uvea, uveitis, and recurrent uveitis. In: Gilger BC, editor.Equine Ophthalmology. 3rd ed. Wiley Blackwell: Ames, IA, USA; 2017. pp. 369-415
  31. 31. Williams RD. Equine Uveitis: A model system for study of immunologically mediated tissue injury [PhD Thesis]. West Lafayette, IN, USA: Purdue University; 1973
  32. 32. Gerhards H, Wollanke B. Uveitis bei Pferden-Diagnose und Therapie. Pferdeheilkunde. 2001;17:319-329. DOI: 10.21836/PEM20010402
  33. 33. Wollanke B, Gerhards H, Ackermann K. Infectious uveitis in horses and new insights in its leptospiral biofilm-related pathogenesis. Microorganisms. 2022;10(2):387. DOI: 10.3390/microorganisms10020387
  34. 34. Wollanke B. Die Equine Rezidivierende Uveitis (ERU) Als Intraokulare Leptospirose. Habilitation [Thesis]. Munich, Germany: Ludwig-Maximilians-University (LMU); 2002. DOI: 10.13140/RG.2.2.16979.50720
  35. 35. Wollanke B, Gerhards H, Brem S, Meyer P, Kopp H. Ätiologie der equinen rezidivierenden Uveitis (ERU): Autoimmunkrankheit oder intraokulare Leptospireninfektion [Etiology of equine recurrent uveitis (ERU): autoimmune disease or intraocular leptospiral infection]. Pferdeheilkunde. 2004;20:327-340. DOI: 10.21836/PEM20040403
  36. 36. Werry H, Gerhards H. The surgical therapy of equine recurrent uveitis. Tierärztliche Praxis. Ausgabe G, Grosstiere/Nutztiere. 1992;20:178-186
  37. 37. Werry H, Gerhards H. Technique and indications for surgical treatment of equine recurrent uveitis. Pferdeheilkunde. 1991;7:321-331. DOI: 10.21836/PEM19910602
  38. 38. Gerhards H, Wollanke B. Surgical treatment of equine recurrent uveitis: Trans-pars-plana vitrectomy in horses. In: Gilger BC, editor.Equine Ophthalmology. 1st ed. Elsevier Saunders: St. Louis, MO, USA; 2005. pp. 314-319
  39. 39. Gerhards H, Wollanke B, Brem S. Vitrectomy as a diagnostic and therapeutic approach for equine recurrent uveitis (ERU). In: Proceedings of the 45th Annual Convention of the American Association of Equine Practitioners (AAEP). Albuquerque, NM, USA; 1999. pp. 89-93
  40. 40. Wollanke B, Rohrbach BW, Gerhards H. Serum and vitreous humor antibody titers in and isolation of Leptospira interrogans from horses with recurrent uveitis. Journal of the American Veterinary Medical Association. 2001;219:795-800. DOI: 10.2460/javma.2001.219.795
  41. 41. Wollanke B, Gerhards H, Brem S, Wolf E, Kopp H, Meyer P. Leptospiral aetiology of equine recurrent uveitis (ERU). Results of studies on vitreous and serum samples. Tierärztliche Praxis. Ausgabe G, Grosstiere/Nutztiere. 2000;28:153-158
  42. 42. Brem S, Gerhards H, Wollanke B, Meyer P, Kopp H. 35 leptospira isolated from the vitreous body of 32 horses with recurrent uveitis (ERU). Berliner und Münchener Tierärztliche Wochenschrift. 1999;112:390-393
  43. 43. Wollanke B, Gerhards H, Brem S, Kopp H, Meyer P. Intraocular and serum antibody titers to Leptospira in 150 horses with equine recurrent uveitis (ERU) subjected to vitrectomy. Berliner und Münchener Tierärztliche Wochenschrift. 1998;111:134-139
  44. 44. Brem S, Gerhards H, Wollanke B, Meyer P, Kopp H. Demonstration of intraocular leptospira in 4 horses suffering from equine recurrent uveitis (ERU). Berliner und Münchener Tierärztliche Wochenschrift. 1998;111:415-417
  45. 45. Ackermann K, Kenngott R, Settles M, Gerhards H, Maierl J, Wollanke B. In vivo biofilm formation of pathogenic Leptospira spp. in the vitreous humor of horses with recurrent uveitis. Microorganisms. 2021;9:1915. DOI: 10.3390/microorganisms9091915
  46. 46. Verma A, Stevenson B. Leptospiral uveitis - there is more to it than meets the eye! Zoonoses and Public Health. 2012;59(Suppl. 2):132-141. DOI: 10.1111/j.1863-2378.2011.01445.x
  47. 47. Moro F. Les Uvéites leptospirosiques. Documenta Ophthalmologica. 1960;14:383-398
  48. 48. Shukla D, Rathinam SR, Cunningham ET Jr. Leptospiral uveitis in the developing world. International Ophthalmology Clinics. 2010;50:113-124. DOI: 10.1097/IIO.0b013e3181d2df58
  49. 49. Rathinam SR. Ocular leptospirosis. Current Opinion in Ophthalmology. 2002;13:381-386. DOI: 10.1097/00055735-200212000-00007
  50. 50. Rathinam SR, Rathnam S, Selvaraj S, Dean D, Nozik RA, Namperumalsamy P. Uveitis associated with an epidemic outbreak of leptospirosis. American Journal of Ophthalmology. 1997;124:71-79. DOI: 10.1016/s0002-9394(14)71646-0
  51. 51. Pappachan JM, Mathew S, Thomas B, Renjini K, Scaria CK, Shukla J. The incidence and clinical characteristics of the immune phase eye disease in treated cases of human leptospirosis. Indian Journal of Medical Sciences. 2007;61:441-447. DOI: 10.4103/0019-5359.32929
  52. 52. Ellis WA. Animal leptospirosis. Current Topics in Microbiology and Immunology. 2015;387:99-137. DOI: 10.1007/978-3-662-45059-8_6
  53. 53. Sivakumar R, Balakrishnan V, Gowri P, Visalakshi J. Leptospiral uveitis: Usefulness of clinical signs as diagnostic predictors. Ocular Immunology and Inflammation. 2018;26:569-576. DOI: 10.1080/09273948.2016.1217341
  54. 54. Henriksen ML, Dwyer AE, Krarup Nielsen R, Bäcklund S, Dahlmann Christensen N, Holberg Pihl T. Ocular abnormalities in the Icelandic horse with a focus on equine recurrent uveitis: 112 Icelandic horses living in Denmark and 26 Icelandic horses living in the United States. Veterinary Ophthalmology. 2021. DOI: 10.1111/vop.12961 [Online ahead of print]
  55. 55. Szemes P, Gerhards H. Study on the prevalence of equine recurrent uveitis in the Cologne-Bonn area. Praktische Tierarzt. 2000;81:408-420
  56. 56. Alexander CS, Keller H. Ätiologie und Vorkommen der periodischen Augenentzündung des Pferdes im Raum Berlin [Etiology and occurrence of periodic eye inflammation of horses in the area of Berlin]. Tierärztliche Praxis. 1990;18:623-627
  57. 57. Tóth J, Hollerieder J, Sótoni P.Augenheilkunde beim Pferd - Lehrbuch und Atlas. Stuttgart, Germany: Schattauer GmbH; 2010
  58. 58. Barnett KC. Uveitis. In: Barnett KC, Crispin SM, Lavach JD, Matthews AG, editors.Equine Ophthalmology - an Atlas and Text. 2nd ed. Edinburgh, UK: Saunders; 2004. pp. 191-197
  59. 59. Blogg J, Barton M, Graydon R, Cust R. Blindness caused by Rhodococcus equi infection in a foal. Equine Veterinary Journal. 1983;15:25-26
  60. 60. Tarancón I, Leiva M, Jose-Cunilleras E, Ríos J, Peña T. Ophthalmologic findings associated with Rhodococcus equi bronchopneumonia in foals. Veterinary Ophthalmology. 2019;22:660-665. DOI: 10.1111/vop.12637
  61. 61. Barr BS. Rhodococcus equi pneumonia in a foal. The Veterinary Clinics of North America. Equine Practice. 2006;22:239-246. DOI: 10.1016/j.cveq.2005.12.022
  62. 62. Cross RS. Equine periodic ophthalmia. The Veterinary Record. 1966;78:8-13. DOI: 10.1136/vr.78.1.8
  63. 63. Gelatt KN, Peiffer RL Jr, Gwin RM, Williams LW. The status of equine ophthalmology. Journal of Equine Medicine and Surgery. 1977;1:13-19
  64. 64. Heusser H. Die Periodische Augenentzündung, Eine Leptospirose? Schweizer Archiv für Tierheilkunde. 1948;90:287-312
  65. 65. Baumgart A, Gerhards H. Characteristics of uveitis in horses with leopard coat color and potential use of cyclosporine a in its therapy in Germany. Pferdeheilkunde. 2014;30:626-632
  66. 66. Baumgart A. Cyclosporin a Und Dessen möglicher Einsatz Bei der Tigerschecken-Uveitis [Cyclosporine a and its Potential Use in Leopard Coat Pattern Uveitis] [Doctoral Thesis]. Munich, Germany: Ludwig-Maximilians-University (LMU); 2014. Available from:https://edoc.ub.uni-muenchen.de/16899/1/Baumgart_Alessandra.pdf
  67. 67. Pinto NI, McMullen RJ Jr, Linder KE, Cullen JM, Gilger BC. Clinical, histopathological and immunohistochemical characterization of a novel equine ocular disorder: Heterochromic iridocyclitis with secondary keratitis in adult horses. Veterinary Ophthalmology. 2015;18:443-456
  68. 68. Halliwell RE, Hines MT. Studies on equine recurrent uveitis. I: Levels of immunoglobulin and albumin in the aqueous humor of horses with and without intraocular disease. Current Eye Research. 1985;4:1023-1031. DOI: 10.3109/02713688509003347
  69. 69. Gelatt KN. Ophthalmic examination and diagnostic procedures. In: Gelatt KN, editor.Veterinary Ophthalmology. Philadelphia, PA, USA: Lea and Febiger; 1981. pp. 219-221
  70. 70. Wollanke B, Gerhards H. Differential diagnosis of equine recurrent uveitis: The importance of a paracentesis of the anterior chamber and aqueous analysis. In: Proceedings of the International Society of Veterinary Ophthalmology (IVO) Pre-Congress Programme of the 30th WSAVA Congress Mexico City, Mexico, 9th – 10th May 2005. 2005
  71. 71. Geiger T, Gerhards H, Wollanke B. Detection of anti-LipL32 antibodies in serum samples from horses with chronic intraocular infection with Leptospira spp. Pathogens. 2021;10:1325. DOI: 10.3390/pathogens10101325
  72. 72. Loibl JK, Gerhards H, Brem S, Wollanke B. Improving the laboratory diagnosis of leptospiral uveitis in horses by using an indirect ELISA for the detection of antibodies against Leptospira spp. in intraocular samples. Pferdeheilkunde Equine Medicine. 2018;34:267-277. DOI: 10.21836/PEM20180308
  73. 73. Wollanke B, Geiger T, Gerhards H. Evaluation of “SNAP® Lepto”-ELISA and comparison with MAT and PCR results for diagnosis of leptospiral uveitis in horses using intraocular samples. Pferdeheilkunde Equine Medicine. 2018;34:508-516. DOI: 10.21836/PEM20180601
  74. 74. Loibl JK. Immunologische und mikrobiologische Untersuchungen zur intraokular persistierenden Leptospireninfektion bei Pferden mit rezidivierender Uveitis [Doctoral Thesis]. Munich, Germany: Ludwig-Maximilians-University (LMU); 2009. Available from:https://edoc.ub.uni-muenchen.de/10508/1/Loibl_Julia.pdf
  75. 75. Geiger T. Evaluation of a Rapid ELISA Test (SNAP-Lepto®) for the Detection of Antibodies against LipL32 in Serum and Intraocular Specimen Material from Horses as a Diagnostic Tool for Equine Recurrent Uveitis [Doctoral Thesis]. Munich, Germany: Ludwig-Maximilians-University (LMU); 2019. Available from:https://edoc.ub.uni-muenchen.de/24938/1/Geiger_Tobias_Xaver.pdf
  76. 76. Gesell S, Wollanke B, Brem S, Gerhards H. Vergleich der Antikoerpertiter gegen Leptospiren in Kammerwasser- und Glaskoerperproben bei Pferden mit rezidivierender Uveitis. In: Proceedings of the 19th DVG-Fachtagung Pferdekrankheiten. Hanover, Germany; 2006. pp. 239-241
  77. 77. Eule JC, Wagner B, Leibold W, Deegen E. Occurrence of various immunoglobulin isotopes in horses with equine recurrent uveitis (ERU). Berliner und Münchener Tierärztliche Wochenschrift. 2000;113:253-257
  78. 78. Sauvage AC, Monclin SJ, Elansary M, Hansen P, Grauwels MF. Detection of intraocular Leptospira spp. by real-time polymerase chain reaction in horses with recurrent uveitis in Belgium. Equine Veterinary Journal. 2019;51:299-303. DOI: 10.1111/evj.13012
  79. 79. Goldmann H, Witmer R. Antibodies in the aqueous humor. Ophthalmologica. 1954;127:323-330. DOI: 10.1159/000301976
  80. 80. Fischer BM, McMullen RJ Jr, Reese S, Brehm W. Intravitreal injection of low-dose gentamicin for the treatment of recurrent or persistent uveitis in horses: Preliminary results. BMC Veterinary Research. 2019;15:29. DOI: 10.1186/s12917-018-1722-7
  81. 81. Himebaugh N, Gilger B. Role of Leptospira spp. testing and ocular examination in horses with equine recurrent uveitis: A retrospective study of 63 horses. Equine Veterinary Education. 2021:1-10. DOI: 10.1111/eve.13543
  82. 82. Gilger BC. Association of acute leptospirosis with systemic disease and uveitis in horses. Equine Veterinary Education. 2018;30:137-138. DOI: 10.1111/eve.12693
  83. 83. Gerding JC, Gilger BC. Prognosis and impact of equine recurrent uveitis. Equine Veterinary Journal. 2016;48:290-288. DOI: 10.1111/evj.12451
  84. 84. Gilger BC, Salmon JH, Yi NY, Barden CA, Chandler HL, Wendt JA, et al. Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States. American Journal of Veterinary Research. 2008;69:1329-1335. DOI: 10.2460/ajvr.69.10.1329
  85. 85. Allbaugh R. Equine recurrent uveitis: A review of clinical assessment and management. Equine Veterinary Education. 2017;29:279-288. DOI: 10.1111/eve.12548
  86. 86. Voelter K, Vial Z, Pot SA, Spiess BM. Leptospiral antibody prevalence and surgical treatment outcome in horses with equine recurrent uveitis (ERU) in Switzerland. Veterinary Ophthalmology. 2020;23:648-658. DOI: 10.1111/vop.12767
  87. 87. Divers TJ, Irby NL, Mohammed HO, Schwark WS. Ocular penetration of intravenously administered enrofloxacin in the horse. Equine Veterinary Journal. 2008;40:167-170. DOI: 10.2746/042516408x255972
  88. 88. Popp MK, Gerhards H, Wollanke B. Enrofloxacinkonzentrationen im Glaskörper und im serum an equiner rezidivierender uveitis (ERU) erkrankter Pferde nach wiederholter intravenöser Verabreichung [Enrofloxacin concentrations in the vitreous of horses with equine recurrent uveitis (ERU) after repeated intravenous administration]. Pferdeheilkunde. 2013;29:574-580. DOI: 10.21836/PEM20130501
  89. 89. Braun D. [History of the research and treatment of periodic ophtalmia (ERU) in horses in the German speaking area between l750 and 1950] Die Geschichte der Erforschung und Behandlung der "Periodischen Augenentzündung" des Pferdes im deutschsprachigen Raum von 1750-1950. Pferdeheilkunde. 1995;11:43-49. DOI: 10.21836/PEM19950104
  90. 90. Jakob H.Tierärztliche Augenheilkunde. Berlin, Germany: Verlagsbuchhandlung Richard Schoetz; 1920
  91. 91. Schleich G. §73 Die periodische, intermittierende Augenentzündung, die Mondblindheit des Pferdes, Iridochorioiditis recidiva equi, Moonblindness. In: Graefe A, Saemisch T, editors.Handbuch der gesamten Augenheilkunde. 2nd ed. Berlin, Germany: Tier-Augenheilkunde; Julius Springer Verlag; 1922. pp. 141-151
  92. 92. Unger HA. Allergie Und Herdinfekt in Ihren Beziehungen Zur Periodischen Augenentzündung der Pferde [Anaphylaxis, Allergy, and Herd Infection in their Relationships to Equine Periodic Ophthalmia] [Doctoral Thesis]. Zurich, Switzerland: University of Zurich; 1955
  93. 93. Eikmeier H. [corticosteroids in ophthalmology] das Cortison in der Augenheilkunde. Berliner und Münchener Tierärztliche Wochenschrift. 1955;68(83-85):110-112
  94. 94. Komar G, Szutter L. Die innere periodische Augenentzündung (“Mondblindheit”) der Pferde. In: Komar G, Szutter L, editors.Tieraerztliche Augenheilkunde. Berlin, Germany: Paul Parey; 1968. pp. 231-237
  95. 95. Schmidt V. Periodische Augenentzündung des Pferdes. In: Schmidt V, editor.Augenkrankheiten der Haustiere. 2nd ed. Jena, Germany: VEB Gustav Fischer Verlag; 1988. pp. 174-176
  96. 96. Bistner S, Shaw D. Uveitis in the horse. Compendium: Continuing Education for Veterinarians. 1980;2:535-543
  97. 97. Tobin T, Chay S, Kamerling S, Woods WE, Weckman TJ, Blake JW, et al. Phenylbutazone in the horse: A review. Journal of Veterinary Pharmacology and Therapeutics. 1986;9:1-25. DOI: 10.1111/j.1365-2885.1986.tb00008.x
  98. 98. Wollanke B, Gerhards H, Schinagl C. Results of 654 trans-pars plana vitrectomies of equine eyes with recurrent uveitis - follow-up until 18 years after surgery. Pferdeheilkunde Equine Medicine. 2021;37:204-214. DOI: 10.21836/PEM20210301
  99. 99. Winterberg A, Gerhards H. Longterm-results of pars-plana-vitrectomy in equine recurrent uveitis. Pferdeheilkunde. 1997;13:377-383. DOI: 10.21836/PEM19970409
  100. 100. Kleinpeter A, Göpfert A, Köhler E, Brehm W. Intravitreal injection of low-dose gentamicin for the treatment of ERU-affected horses. Tierärztliche Praxis. Ausgabe G, Grosstiere/Nutztiere. 2019;47:25-34. DOI: 10.1055/a-0816-7156
  101. 101. Launois T, González Hilarión LM, Barbe F, Leurquin C, Bihin B, Hontoir F, et al. Use of Intravitreal injection of gentamicin in 71 horses with equine recurrent uveitis. Journal of Equine Veterinary Science. 2019;77:93-97. DOI: 10.1016/j.jevs.2019.02.018
  102. 102. Zachary IG, Forster RK. Experimental intravitreal gentamicin. American Journal of Ophthalmology. 1976;82:604-611. DOI: 10.1016/0002-9394(76)90549-3
  103. 103. Neumann M, Ohnesorge B. The intravitreal gentamicin-injection for treatment of the equine recurrent uveitis (ERU) – Status quo. Pferdeheilkunde Equine Medicine. 2021;37:302-311. DOI: 10.21836/PEM20210312
  104. 104. Gilger BC, Wilkie DA, Clode AB, McMullen RJ Jr, Utter ME, Komaromy AM, et al. Long-term outcome after implantation of a suprachoroidal cyclosporine drug delivery device in horses with recurrent uveitis. Veterinary Ophthalmology. 2010;13:294-300. DOI: 10.1111/j.1463-5224.2010.00807.x
  105. 105. Gilger BC, Salmon JH, Wilkie DA, Cruysberg LP, Kim J, Hayat M, et al. A novel bioerodible deep scleral lamellar cyclosporine implant for uveitis. Investigative Ophthalmology & Visual Science. 2006;47:2596-2605. DOI: 10.1167/iovs.05-1540
  106. 106. Gilger BC, Wilkie DA, Davidson MG, Allen JB. Use of an intravitreal sustained-release cyclosporine delivery device for treatment of equine recurrent uveitis. American Journal of Veterinary Research. 2001;62:1892-1896. DOI: 10.2460/ajvr.2001.62.1892
  107. 107. Gilger BC, Malok E, Stewart T, Horohov D, Ashton P, Smith T, et al. Effect of an intravitreal cyclosporine implant on experimental uveitis in horses. Veterinary Immunology and Immunopathology. 2000;76:239-255. DOI: 10.1016/s0165-2427(00)00219-1
  108. 108. Wollanke B, Gerhards H. Cyclosporine a (CsA) concentrations in aqueous and vitreous humour samples and clinical and ophthalmological findings in 16 equine eyes after implantation of a sustainedrelease CsA delivery device. Pferdeheilkunde Equine Medicine. 2021;37:234-242. DOI: 10.21836/PEM20210304
  109. 109. Hartwigk H, Stoebbe E. Kultureller Nachweis von Leptospiren bei Hund und Pferd. Berliner und Munchener Tierarztliche Wochenschrift. 1952;65:212-214
  110. 110. Gelatt K. Ophthalmoscopic studies in the normal and diseased ocular fundi of horses. Journal of the American Animal Hospital Association. 1971;7:158-167
  111. 111. Kalsow CM, Hainworth SA, Russell NS, Dwyer AE. Retinal autoimmunity in leptospiral associated uveitis. Investigative Ophthalmology & Visual Science. 1998;39:S782
  112. 112. Kemenes F, Tamas L. Ist die fibrinöse Iridozyklitis der Pferde eine Leptospirose. Acta Veterinaria. 1952;2:327-336
  113. 113. Wollanke B, Gerhards H, Kaufmann S. Untersuchungen zur Beteiligung von Borrelien an der Ätiologie der equinen rezidivierenden Uveitis (ERU). Pferdeheilkunde Equine Medicine. 2017;33:447-451. DOI: 10.21836/PEM20170504
  114. 114. Hartskeerl RA, Goris MG, Brem S, Meyer P, Kopp H, Gerhards H, et al. Classification of leptospira from the eyes of horses suffering from recurrent uveitis. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health. 2004;51:110-115. DOI: 10.1111/j.1439-0450.2004.00740.x
  115. 115. Niedermaier G, Wollanke B, Hoffmann R, Brem S, Gerhards H. Detection of leptospira in the vitreous body of horses without ocular diseases and of horses with equine recurrent uveitis (ERU) using transmission-electron microscopy. DTW. Deutsche Tierarztliche Wochenschrift. 2006;113:418-422
  116. 116. Brandes K, Wollanke B, Niedermaier G, Brem S, Gerhards H. Recurrent uveitis in horses: Vitreal examinations with ultrastructural detection of leptospires. Journal of Veterinary Medicine. A, Physiology, Pathology, Clinical Medicine. 2007;54:270-275. DOI: 10.1111/j.1439-0442.2007.00921.x
  117. 117. Degroote RL, Deeg CA. Immunological insights in equine recurrent uveitis. Frontiers in Immunology. 2020;11:609855. DOI: 10.3389/fimmu.2020.609855
  118. 118. Deeg CA, Hauck SM, Amann B, Pompetzki D, Altmann F, Raith A, et al. Equine recurrent uveitis--a spontaneous horse model of uveitis. Ophthalmic Research. 2008;40:151-153. DOI: 10.1159/000119867
  119. 119. Deeg CA, Hauck SM, Amann B, Kremmer E, Stangassinger M, Ueffing M. Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis. Molecular Immunology. 2007;44:3291-3296. DOI: 10.1016/j.molimm.2007.02.027
  120. 120. Deeg CA, Marti E, Gaillard C, Kaspers B. Equine recurrent uveitis is strongly associated with the MHC class I haplotype ELA-A9. Equine Veterinary Journal. 2004;36:73-75. DOI: 10.2746/0425164044864651
  121. 121. Deeg CA, Kaspers B, Gerhards H, Thurau SR, Wollanke B, Wildner G. Immune responses to retinal autoantigens and peptides in equine recurrent uveitis. Investigative Ophthalmology & Visual Science. 2001;42:393-398
  122. 122. Ristow P, Bourhy P, Kerneis S, Schmitt C, Prevost MC, Lilenbaum W, et al. Biofilm formation by saprophytic and pathogenic leptospires. Microbiology. 2008;154:1309-1317. DOI: 10.1099/mic.0.2007/014746-0
  123. 123. Thibeaux R, Soupé-Gilbert ME, Kainiu M, Girault D, Bierque E, Fernandes J, et al. The zoonotic pathogen Leptospira interrogans mitigates environmental stress through cyclic-di-GMP-controlled biofilm production. npj Biofilms and Microbiomes. 2020;6:24. DOI: 10.1038/s41522-020-0134-1
  124. 124. Brihuega B, Samartino L, Auteri C, Venzano A, Caimi K. In vivo cell aggregations of a recent swine biofilm-forming isolate of Leptospira interrogans strain from Argentina. Revista Argentina de Microbiología. 2012;44:138-143
  125. 125. Yamaguchi T, Higa N, Okura N, Matsumoto A, Hermawan I, Yamashiro T, et al. Characterizing interactions of Leptospira interrogans with proximal renal tubule epithelial cells. BMC Microbiology. 2018;18:64. DOI: 10.1186/s12866-018-1206-8
  126. 126. Santos AAN, Ribeiro PDS, da França GV, Souza FN, Ramos EAG, Figueira CP, et al. Leptospira interrogans biofilm formation in Rattus norvegicus (Norway rats) natural reservoirs. PLoS Neglected Tropical Diseases. 2021;15:e0009736. DOI: 10.1371/journal.pntd.0009736
  127. 127. Parsek MR, Singh PK. Bacterial biofilms: An emerging link to disease pathogenesis. Annual Review of Microbiology. 2003;57:677-701. DOI: 10.1146/annurev.micro.57.030502.090720
  128. 128. Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cellular Microbiology. 2009;11:1034-1043. DOI: 10.1111/j.1462-5822.2009.01323.x
  129. 129. Geißler P, Wollanke B. Biofilm formation in persistent infections and its role in the pathogenesis of equine recurrent uveitis (ERU) – A literature review. Pferdeheilkunde Equine Medicine. 2021;37:225-233. DOI: 10.21836/PEM20210303
  130. 130. Faber NA, Crawford M, LeFebvre RB, Buyukmihci NC, Madigan JE, Willits NH. Detection of Leptospira spp. in the aqueous humor of horses with naturally acquired recurrent uveitis. Journal of Clinical Microbiology. 2000;38:2731-2733. DOI: 10.1128/jcm.38.7.2731-2733.2000
  131. 131. Polle F, Storey E, Eades S, Alt D, Hornsby R, Zuerner R, et al. Role of intraocular Leptospira infections in the pathogenesis of equine recurrent uveitis in the southern United States. Journal of Equine Veterinary Science. 2014;34:1300-1306
  132. 132. Bjarnsholt T, Jensen P, Fiandaca MJ, Pedersen J, Hansen CR, Andersen CB, et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatric Pulmonology. 2009;44:547-558. DOI: 10.1002/ppul.21011
  133. 133. Høiby N, Bjarnsholt T, Moser C, Bassi GL, Coenye T, Donelli G, et al. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014. Clinical Microbiology and Infection. 2015;21(Suppl. 1):1-25. DOI: 10.1016/j.cmi.2014.10.024
  134. 134. Pedersen SS, Shand GH, Hansen BL, Hansen GN. Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres. APMIS. 1990;98:203-211
  135. 135. Aanaes K, Johansen HK, Poulsen SS, Pressler T, Buchwald C, Høiby N. Secretory IgA as a diagnostic tool for Pseudomonas aeruginosa respiratory colonization. Journal of Cystic Fibrosis. 2013;12:81-87. DOI: 10.1016/j.jcf.2012.07.001
  136. 136. Halliwell RE, Brim TA, Hines MT, Wolf D, White FH. Studies on equine recurrent uveitis. II: The role of infection with Leptospira interrogans serovar Pomona. Current Eye Research. 1985;4:1033-1040. DOI: 10.3109/02713688509003348
  137. 137. Roth T, Brandes K, Gerhards H, Giving E, Wollanke B. Histologische Untersuchungen des Glaskörpers bei Pferden mit equiner rezidivierender Uveitis. Pferdeheilkunde. 2014;30:512-520. DOI: 10.21836/PEM20140501
  138. 138. Niedermaier G, Wollanke B, Hoffmann R, Matiasek K, Gerhards H. Depiction of the structure of the vitreous body in horses without ocular diseases and in horses with equine recurrent uveitis (ERU) using transmission electron microscopy. DTW. Deutsche Tierarztliche Wochenschrift. 2006;113:211-217
  139. 139. Dubielzig R, Render J, Morreale R. Distinctive morphologic features of the ciliary body in equine recurrent uveitis. Veterinary and comparative ophthalmology. 1997;7:163-167
  140. 140. Gilger BC, Deeg CA. Equine recurrent uveitis. In: Gilger BC, editor.Equine Ophthalmology. 2nd ed. Maryland Heights, MO, USA: Elsevier Saunders; 2011. pp. 317-349
  141. 141. Romeike A, Brügmann M, Drommer W. Immunohistochemical studies in equine recurrent uveitis (ERU). Veterinary Pathology. 1998;35:515-526. DOI: 10.1177/030098589803500606
  142. 142. Ackermann K. Untersuchungen von Glaskörperproben aus an equiner rezidivierender Uveitis (ERU) erkrankten Augen im Hinblick auf Leptospiren und deren Biofilmbildung [Doctoral Thesis]. Munich, Germany: Ludwig-Maximilians-University (LMU); 2021. Available from:https://edoc.ub.uni-muenchen.de/28756/7/Ackermann_Kerstin.pdf
  143. 143. Brem S. Personal information, State Office for Health and Food Safety, 85764 Oberschleissheim, Germany. Unpublished data. 1996
  144. 144. Vinod Kumar K, Lall C, Vimal Raj R, Vedhagiri K, Vijayachari P. Molecular detection of pathogenic leptospiral protein encoding gene (lipL32) in environmental aquatic biofilms. Letters in Applied Microbiology. 2016;62:311-315. DOI: 10.1111/lam.12533
  145. 145. Trueba G, Zapata S, Madrid K, Cullen P, Haake D. Cell aggregation: A mechanism of pathogenic Leptospira to survive in fresh water. International Microbiology. 2004;7:35-40
  146. 146. Grisanti S. Immune privilege of the eye. Der Ophthalmologe. 1998;95:124-135. DOI: 10.1007/s003470050250
  147. 147. Zhou R, Caspi RR. Ocular immune privilege. F1000 Biology Reports. 2010;2(1):3. DOI: 10.3410/b2-3
  148. 148. Fingerhut L, Ohnesorge B, von Borstel M, Schumski A, Strutzberg-Minder K, Mörgelin M, et al. Neutrophil extracellular traps in the pathogenesis of equine recurrent uveitis (ERU). Cell. 2019;8:1528. DOI: 10.3390/cells8121528
  149. 149. Branzk N, Lubojemska A, Hardison SE, Wang Q , Gutierrez MG, Brown GD, et al. Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nature Immunology. 2014;15:1017-1025. DOI: 10.1038/ni.2987
  150. 150. Tan C, Aziz M, Wang P. The vitals of NETs. Journal of Leukocyte Biology. 2021;110:797-808. DOI: 10.1002/jlb.3ru0620-375r
  151. 151. Hänsch GM. Host defence against bacterial biofilms:“Mission impossible”? International Scholarly Research Notices. 2012;2012:853123. DOI: 10.5402/2012/853123
  152. 152. Waldner J, Gerhards H, Wollanke B. Investigations into the occurrence of serum amyloid a in the equine eye. Pferdeheilkunde Equine Medicine. 2018;34:461-467. DOI: 10.21836/PEM20180508
  153. 153. Linke RP, Brandes K, Cielewicz MB, Gerhards H, Wollanke B. Ocular leptospiral infection leads to ciliary induction and local AA-amyloidosis in horses. Amyloid. 2019;26:127-128. DOI: 10.1080/13506129.2019.1584100
  154. 154. Ostevik L, de Souza GA, Wien TN, Gunnes G, Sørby R. Characterization of amyloid in equine recurrent uveitis as AA amyloid. Journal of Comparative Pathology. 2014;151:228-233. DOI: 10.1016/j.jcpa.2014.04.007
  155. 155. Thanabalasuriar A, Scott BNV, Peiseler M, Willson ME, Zeng Z, Warrener P, et al. Neutrophil extracellular traps confine Pseudomonas aeruginosa ocular biofilms and restrict brain invasion. Cell Host & Microbe. 2019;25:526-536.e524. DOI: 10.1016/j.chom.2019.02.007
  156. 156. Papayannopoulos V. Neutrophils facing biofilms: The Battle of the barriers. Cell Host & Microbe. 2019;25:477-479. DOI: 10.1016/j.chom.2019.03.014
  157. 157. Tsuprun V, Shibata D, Paparella MM, Cureoglu S. Formations of host Fibers and Bacteria in human temporal bones with otitis media. Otology & Neurotology. 2021;42:e949-e957. DOI: 10.1097/mao.0000000000003126
  158. 158. Donlan RM, Costerton JW. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews. 2002;15:167-193. DOI: 10.1128/cmr.15.2.167-193.2002
  159. 159. Nielsen SM, Nørskov-Lauritsen N, Bjarnsholt T, Meyer RL. Achromobacter species isolated from cystic fibrosis patients reveal distinctly different biofilm Morphotypes. Microorganisms. 2016;4:33. DOI: 10.3390/microorganisms4030033
  160. 160. Di Domenico EG, Cavallo I, Bordignon V, D'Agosto G, Pontone M, Trento E, et al. The emerging role of microbial biofilm in Lyme Neuroborreliosis. Frontiers in Neurology. 2018;9:1048. DOI: 10.3389/fneur.2018.01048
  161. 161. Rudenko N, Golovchenko M, Kybicova K, Vancova M. Metamorphoses of Lyme disease spirochetes: Phenomenon of Borrelia persisters. Parasites & Vectors. 2019;12:237. DOI: 10.1186/s13071-019-3495-7
  162. 162. Sapi E, Kasliwala RS, Ismail H, Torres JP, Oldakowski M, Markland S, et al. The long-term persistence of Borrelia burgdorferi antigens and DNA in the tissues of a patient with Lyme disease. Antibiotics. 2019;8(4):183. DOI: 10.3390/antibiotics8040183
  163. 163. Sapi E, Gupta K, Wawrzeniak K, Gaur G, Torres J, Filush K, et al. Borrelia and Chlamydia can form mixed biofilms in infected human skin tissues. European Journal of Microbiology and Immunology. 2019;9:46-55. DOI: 10.1556/1886.2019.00003
  164. 164. Sapi E, Theophilus PA, Pham TV, Burugu D, Luecke DF. Effect of RpoN, RpoS and LuxS pathways on the biofilm formation and antibiotic sensitivity of Borrelia Burgdorferi. European Journal of Microbiology & Immunology. 2016;6:272-286. DOI: 10.1556/1886.2016.00026
  165. 165. Høiby N, Ciofu O, Bjarnsholt T. Pseudomonas aeruginosa biofilms in cystic fibrosis. Future Microbiology. 2010;5:1663-1674. DOI: 10.2217/fmb.10.125
  166. 166. Sapi E, Balasubramanian K, Poruri A, Maghsoudlou JS, Socarras KM, Timmaraju AV, et al. Evidence of In vivo existence of Borrelia biofilm in Borrelial Lymphocytomas. European Journal of Microbiology & Immunology. 2016;6:9-24. DOI: 10.1556/1886.2015.00049
  167. 167. Allen HB. A novel approach to the treatment and prevention of Alzheimer's disease based on the pathology and microbiology. Journal of Alzheimer's Disease. 2021;84:61-67. DOI: 10.3233/jad-210429
  168. 168. Senejani AG, Maghsoudlou J, El-Zohiry D, Gaur G, Wawrzeniak K, Caravaglia C, et al. Borrelia burgdorferi Co-localizing with amyloid markers in Alzheimer's disease brain tissues. Journal of Alzheimer's Disease. 2022;85(2):889-903 DOI: 10.3233/jad-215398
  169. 169. Feng J, Li T, Yee R, Yuan Y, Bai C, Cai M, et al. Stationary phase persister/biofilm microcolony of Borrelia burgdorferi causes more severe disease in a mouse model of Lyme arthritis: Implications for understanding persistence, post-treatment Lyme disease syndrome (PTLDS), and treatment failure. Discovery Medicine. 2019;27:125-138
  170. 170. Bjarnsholt T, Whiteley M, Rumbaugh KP, Stewart PS, Jensen P, Frimodt-Møller N. The importance of understanding the infectious microenvironment. The Lancet Infectious Diseases. 2022;22(3):e88-e92. DOI: 10.1016/s1473-3099(21)00122-5

Written By

Bettina Wollanke and Hartmut Gerhards

Submitted: January 5th, 2022 Reviewed: March 16th, 2022 Published: May 13th, 2022