Microorganisms 10 00387 v2

microorganisms
Review
Infectious Uveitis in Horses and New Insights in Its Leptospiral
Biofilm-Related Pathogenesis
Bettina Wollanke * , Hartmut Gerhards and Kerstin Ackermann
Equine Clinic, Ludwig-Maximilians-University, 80539 Munich, Germany; [email protected] (H.G.);

[email protected] (K.A.)
* Correspondence: [email protected]
Abstract: Uveitis is a sight-threatening eye disease in equids known worldwide that leads to con-
siderable pain and suffering. By far the most common type of uveitis in Germany and neighboring
countries is classical equine recurrent uveitis (ERU), which is caused by chronic intraocular leptospiral
infection and is the main cause of infectious uveitis in horses. Other infectious causes are extremely
rare and are usually clinically distinguishable from ERU. ERU can be treated very effectively by
vitreous cavity lavage (vitrectomy). For proper indications of this demanding surgery, it is necessary
to differentiate ERU from other types of uveitis in which vitrectomy is not helpful. This can be
conducted on the basis of anamnesis in combination with ophthalmologic findings and by aqueous
humor examination. During vitrectomy, vitreous material is obtained. These vitreous samples have
historically been used for numerous etiologic studies. In this way, a chronic intraocular leptospiral
infection has been shown to be the cause of typical ERU and, among other findings, ERU has also
been recognized as a biofilm infection, providing new insights into the pathogenesis of ERU and
explaining some thus far unexplainable phenomena of ERU. ERU may not only have transmissible
aspects to some types of uveitis in humans but may also serve as a model for a spontaneously

occurring biofilm infection. Vitreous material obtained during therapeutically indicated vitrectomy
Citation: Wollanke, B.; Gerhards, H.;
can be used for further studies on in vivo biofilm formation, biofilm composition and possible
Ackermann, K. Infectious Uveitis in therapeutic approaches.
Horses and New Insights in Its
Leptospiral Biofilm-Related Keywords: equine recurrent uveitis (ERU); Leptospira spp.; biofilm infection; amyloid; neutrophil
Pathogenesis. Microorganisms 2022, extracellular traps; Goldmann–Witmer coefficient; local antibody production; aqueous and vitreous
10, 387. https://doi.org/10.3390/ samples; vitrectomy
microorganisms10020387
Academic Editor: Gianfranco Donelli
Received: 31 December 2021 1. Introduction

Accepted: 3 February 2022
As in other species, uveitis in horses can be caused by various etiologies. Equine
Published: 7 February 2022
recurrent uveitis (ERU) is the main form of uveitis occurrence worldwide and is still the
Publisher’s Note: MDPI stays neutral most common cause of blindness in equids [1–8]. The incidence of uveitis is 7–10% in
with regard to jurisdictional claims in Germany [9,10] and is reported to be 2–25% in the United States [5]. More than half of
published maps and institutional affil- horses suffering from chronic uveitis will develop unilateral or bilateral blindness over
iations. time if only conservative therapy is performed [1,5,11], and many horses even have to be
euthanized in the course of the disease [12]. For these reasons, uveitis in horses also has
significant economic importance to the horse industry [2,4].
The term “ERU” is not used consistently in the literature. Many authors also refer to
Copyright: © 2022 by the authors.
the chronic insidious uveitis of leopard coat pattern horses as “ERU” [5,7,13,14], whereas
Licensee MDPI, Basel, Switzerland.
other publications distinguish between classic “ERU” and “leopard coat pattern uveitis”,
This article is an open access article
distributed under the terms and
not only because of the ophthalmological findings and the course of the disease, but also
conditions of the Creative Commons
because the etiology is different for each type of uveitis [15,16]. In horses suffering from
Attribution (CC BY) license (https://
recurrent episodes of typical uveitis attacks in central Europe, an intraocular leptospiral
creativecommons.org/licenses/by/ infection is almost always detectable [16–21]. Herein, the term ERU will therefore be used
4.0/).
Microorganisms 2022, 10, 387. https://doi.org/10.3390/microorganisms10020387 https://www.mdpi.com/journal/microorganisms

Microorganisms 2022, 10, 387 2 of 60
exclusively for leptospiral-induced uveitis, which is the main representative of infectious

uveitis in horses.
Often, previous reports, the patient’s signalement (e.g., age, breed, and coat color),
the general examination (evidence of general disease, possible septicemia), and ophthal-
mologic examinations will indicate which form of uveitis is involved [22]. By far the most
common types are equine recurrent uveitis (ERU) [1,5,7,11,12,15,16,23–29] and “leopard
coat pattern uveitis” [5,12,15,30]. Less commonly, possible etiologies include traumatic
uveitis [5,18], phacogenic uveitis [18,31], chronic iritis (similar to human Fuchs’ hete-
rochromia iritis) [18,32], kerato-uveitis in severe corneal disease and co-reaction of the
uvea [5], septicemia-accompanying uveitis [18,19,28,33–40], uveitis induced by intraocular
tumors [5], and endophthalmitis caused by pyogenic bacteria [5]. Very rarely, therapy-
resistant uveitis results from infection with Halicephalobus (syn. Micronema) deletrix [41,42].
Parallel testing of numerous equine uveitis patients for Borrelia spp., Toxoplasma, her-
pesviruses, and bornavirus revealed no evidence that these pathogens cause recurrent
uveitis in horses [35,43–45]. Glaucoma may occur as a sequela of uveitis or may cause
uveitic irritation only in the course of the disease, but it is usually an independent clinical
picture and has very different underlying causes [5,18,19,46–48].
A genetic predisposition to uveitis has long been suspected [29,49–51] and has further
been investigated for both Appaloosas (belonging to leopard coat pattern horses) [1,7,52–54]
and warm-blooded horses [55,56]. Genetic factors predisposing to uveitis include immune
system characteristics (e.g., the composition of major histocompatibility complexes (MHCs),
immunocompetence with respect to defense against infectious agents, or favoring an
autoaggressive response) as well as the anatomic configuration of the eye, particularly of
the lens [3,5,18].
Due to the discovery of intraocular biofilm formation of leptospires in ERU eyes [57],
the aim of this review was to differentiate infectious uveitis from noninfectious uveitis, to
characterize leptospiral induced ERU, and to highlight the possible pathogenesis of ERU
with respect to intravitreal in vivo leptospiral biofilm production. Realization that ERU
is a biofilm infection explains many characteristics of ERU that have been mysteries until
recently: the recurrent episodes of uveitis, the unsatisfactory efficacy of antibiotics, the
failure of the immune response to eliminate the pathogen, the accompanying autoimmune
phenomena, and the difficulties in detecting the causative agent.
2. Equine Leptospirosis
Serologic surveys indicate that infection with leptospires is common in horses, but
systemic leptospirosis is almost always subclinical and therefore is unnoticed [58,59]. The
main carriers are small rodents, which excrete pathogenic Leptospira spp. in their urine
as renal carriers and thus contaminate the environment, including horses’ drinking water
and food [59,60]. In particular, water from standing sources and wet or swampy pastures
present a risk for equine infections [60–62]. Oral mucous membranes, conjunctiva, nasal
mucous membranes, and skin lesions are considered sites of entry for the pathogens [59,63].
Numerous leptospiral serovars from different serogroups can lead to infections in
horses [58,59,64,65]. The dominant serovar in Germany and many neighboring countries is
Grippotyphosa, followed by the serovar Bratislava [19,66–69]. In countries further east and
in the United States, infections with serovars from the Pomona serogroup are described in
particular [19,58,63]. The geographical region in which the horses are located and which
carriers shedding leptospires are present is decisive for which serovar leads to infection [13].
In rare cases where horses develop clinically apparent leptospirosis during systemic
infection, the symptoms are similar to those of other species, such as humans or dogs
(among others: disturbed general condition, fever, anemia, jaundice, hemoglobinuria, and
impaired renal function). Clinically apparent courses mainly, but not exclusively, affect
foals and young horses [40,59,63,70–73].
Leptospirosis should also be considered in abortions [70,74–78]. After leptospiral-
induced abortion, leptospires are detectable in placenta and fetal tissue [75,79–82]. In
addition, leptospires appear to be detectable in the uterus for at least several months after
these abortions, although serum tests for anti-Leptospira antibodies are of no value in this
context [83].
After leptospires are eliminated from well-vascularized structures, they can survive in
immune-privileged sites (e.g., proximal renal tubules and vitreous cavity) for extended peri-
ods of time [63], and in the eye for many years [19,57]. Studies have shown that leptospires
may also be detectable in the kidneys of healthy horses that have been slaughtered [84–86]
and that between 8% and 35% of horses without an impaired general condition (e.g., mares
after abortion, horses after flooding events, and horses with ERU) can excrete leptospires in
the urine [64,87,88]. The importance of horses as shedders (e.g., via urine or placenta) for
the infection of other horses is difficult to assess thus far.
The numerically and economically most important sequela of leptospirosis in horses is
ERU, which, similarly to leptospiral uveitis in humans [89–95], is a late sequela of systemic
infection, and typically occurs many months to years after acute infection [13,18,19,58,96–103].
3. Historical and Preliminary Remarks on the Etiology of ERU

Symptoms of uveitis in equids have been known and feared for centuries. Especially
at times when people were still dependent on the labor of horses for working the fields,
locomotion, and conflict, their welfare was of paramount importance. Diseases and the
blindness of horses played a significant economic role. In this context, symptoms of
uveitis in horses are said to have been described by Pliny the Elder (23 to 79 AD) in his
Historia Naturalis. Later, a description of uveitis symptoms was found, among others, in
the Mulomedicina Chironis (an equine medical treatise from the 4th century AD, book II,
chap. 77, VII) (citation in [104]). The well-known Scottish human surgeon James Wardrop
successfully competed for a prize offered by the Board of Agriculture in 1819 for the best
“Essay on the Diseases of the Eye of the Horse, and on Their Treatment” [105,106]. Since
the end of the 19th and the beginning of the 20th century, very characteristic descriptions of
recurrent uveitis bouts in horses have been published repeatedly, and different infectious
causes for this equine ocular disease have been postulated by various authors (numerous
citations in [107–110]).
As early as the end of the 19th century, the occurrence of recurrent uveitis in
horses was associated with keeping horses in humid regions, summers with high pre-
cipitation, clayey and marshy soil, and floods [111–113], which was confirmed in later
publications [50,51,58,103,114–117].
In the beginning of the 20th century, Bayer (Vienna, Austria), in his detailed chapter on
moon blindness or periodic eye inflammation of his book Augenheilkunde (Ophthalmology),
among other things, details the causes of the disease and discusses its possible heredity. At
that time, this had been resolutely asserted by some authors, and just as resolutely denied
by others. Bayer summarizes the knowledge of the international literature at that time
in such a way that the more frequent occurrence of the disease with descendants of ill
parent animals is to be explained with bad soil conditions of the respective studs, because
the descendants inevitably stayed on the same terrain as the parent animals. Where the
overall incidence of the disease is low, offspring are also less likely to contract the disease.
Bayer also reported on a breeding experiment with a stallion that was moonblind on one
side and a mare that was moonblind on both sides. The foals were born with healthy eyes
and did not become blind. Humid climates and marshy, boggy pasture soils or low-lying
horse pastures with flooded meadows, as well as “wet years”, favored the development
of ERU, whereas keeping horses in dry areas with limestone soils served to prevent the
disease [51].
In 1906, Bayer considered different infectious agents, mostly bacteriae, incorporated
by contaminated food and water, to be the main cause of ERU [51]. Notably, he had stated
that ERU had to be considered an infectious disease. It should be termed “a non-purulent
panophthalmitis, induced by microorganisms”, although the infectious agent had not yet
been identified at that time. Bayer thought that some microorganisms could reside in an
encapsulated condition in the body or in the eye until being reactivated under favorable
circumstances. Furthermore, in an epidemic of “jaundice” described by Bayer, which had
been observed in Lorraine in 1874, symptoms consistent with acute leptospirosis and uveitis
in horses were observed.
However, a link between ERU and leptospiral infection was first described in 1946
by Gsell (Switzerland), when very high anti-Leptospira antibody titers were found in the
aqueous humor of a horse suffering from ERU [89]. Repeated detection of anti-Leptospira
antibodies in aqueous humor and vitreous fluids from ERU eyes supported an intraocular
leptospiral infection [118–123], and there were a few reports of the pathogen being cultured
from aqueous humor samples [25,124].
After both spontaneous [70,96–98,125] and experimental [97,100,101,126] infections
with leptospires, the occurrence of ERU has been described in up to 100% of horses over
the following months and years. However, many attempts to detect the pathogens in
intraocular specimens from affected horses have failed [96,101,118,122,126–130], and ERU
can occur months to years after the systemic infection. It was finally assumed that leptospi-
ral infections somehow trigger ERU, but the pathogens themselves are no longer present
when episodes of uveitis occur, and that ERU is a postinfectious uveitis, which is triggered
by various mechanisms and immune reactions [96,101,128,131–134]. This autoimmune
pathogenesis is still held by many authors, possibly also because, especially in the United
States, no differentiation is made between “ERU” and uveitis in horses with leopard coat
patterns. In the United States, many Appaloosas, which belong to leopard coat pattern
horses, and which predominantly do not suffer from infectious uveitis, are included in the
investigations (Section 4.2).
A connection between leptospiral infections and ERU is now generally accepted by
most equine ophthalmologists; however, for a long time, it remained unclear how a systemic
leptospiral infection could lead to uveitis attacks years later [1,3,5,13,14,63,135–137]. The
apparent absence of the pathogens from the eyes is irritating and seems to lead to a
commingling of various immune responses [6,101,133,138–144] with an “autoimmune
disease” [5,145].
In particular, a research group around Parma has intensively studied cross-reactions,
immunologic mimicry, and specifically, the occurrence of similar epitopes of leptospires
and equine corneas [146–151]. Cross-reactions between leptospires and the uvea [126,152],
cross-reactions between leptospires and lens proteins [153], and epitope spreading have also
been reported [154]. In a subsequent study, at least for L. grippotyphosa, no cross-reactions
with proteins of the cornea, lens, vitreous, and retina could be detected [45].
Altogether, many of the immunological processes described for human autoimmune
diseases [155,156] have also been found in horses suffering from ERU [133,134,142,157–172].
Only a few authors considered that, in addition to autoimmune etiology, the permanent
presence of leptospires in an “atypical” form could also be considered as a cause of recurrent
episodes of uveitis [63,173,174]. In addition to the primary trigger for uveitis, environmental
conditions, and especially the individual genetic make-up, are considered important in the
development of ERU [175].
3.1. Leptospirosis and Autoimmune Disease

An autoimmune disease usually implies that no causative therapy is possible and
that symptoms can only be alleviated by medical therapy. It is also known that various
infectious agents can contribute to the development of autoimmune diseases, but that the
autoimmune-related inflammation eventually persists independently of the infection as
postinfectious autoimmunity [155,176–182].
In contrast, in ERU, further episodes of uveitis can be prevented very convincingly by
the removal of vitreous material and flushing the vitreous cavity (vitrectomy) [16]. How-
ever, because vitrectomy only removes vitreous material and leaves the intraocular retinal
antigens and lens untouched, the unusually high percentage of postoperative recurrence-
free cases can be explained only by removal of intraocular leptospiral infection in the
vitreous cavity [19]. An “autoimmune disease” would not explain the good prognosis of
vitrectomy [183]. Although irrigation of the vitreous cavity temporarily relieves autoim-
mune uveitis by removing the “immunologic memory” from the vitreous cavity [184–190];
in these cases, it does not have the lasting and significant effect that it has in ERU [16,191].
Thus, the autoimmune reactions in equine eyes are only immune phenomena accompa-
nying the causative leptospiral infection. Once the infection is eliminated, autoimmune
reactions do not cause any further problems.
Autoaggressive immune responses are detectable (e.g., autoantibodies and autoantigen-
specific T cells); therefore, ERU is nevertheless referred to as an “autoimmune disease”
in many papers. Thereby, it seems to be irrelevant what causes the autoimmune reac-
tions [163,169,192]. However, studies of the working group of Deeg [157–159,165,170,193]
were performed with samples from horses in which a chronic intraocular leptospiral in-
fection was present [17–20,194–199] and in which no further inflammation occurred after
vitrectomy [16], i.e., no further autoimmune disease occurred either. For this reason, it
would be preferable to refer to “autoimmune reactions accompanying infections” rather
than “autoimmune disease” [19,45].
One argument by supporters of the theory that ERU is an autoimmune disease is that
uveitis improves with treatment with corticosteroids and nonsteroidal anti-inflammatory
drugs [145,158,200]. However, this disregards the fact that, in principle, treatment with
corticosteroids and nonsteroidal anti-inflammatory drugs is indicated for almost every case
of uveitis, regardless of its etiology [189,201–206]. In human leptospiral uveitis, treatment
with corticosteroids is also indicated [92,207]. Thus, improvements in acute inflammatory
episodes under topical treatment with corticosteroids do not contradict the presence of a
chronic infection.
Immune responses which are independent of the etiology of uveitis always include
autoimmune responses, which are detectable in any disruption of the blood–ocular barrier.
The presence of pathogenic Leptospira spp. is a good explanation for the autoimmune reac-
tions detected in ERU eyes, which cease after elimination of the infection [16,19,208]. Au-
toimmune reactions are a well-known phenomenon in chronic infections, and there are sev-
eral mechanisms that can lead to autoimmune reactions in chronic infections [178,209–215].
4. Types of Equine Uveitis and Differential Diagnosis

In many cases of uveitis, the etiology, and thus, also the therapy, can be determined
based on the clinical and ophthalmologic examinations. However, sometimes it is only
during the course of the disease that it becomes clear which type of uveitis is present—or
additional laboratory tests, possibly also the examination of aqueous humor, are indicated
in order to decide on the further course of action. In rare cases, an etiological classification
of the uveitis is not possible.
4.1. Equine Recurrent Uveitis (ERU)

ERU is by far the most common form of uveitis in horses in central Europe. ERU is a
sero-fibrinous, although rarely sero-hemorrhagic inflammation of the uvea, which occurs
acutely, is chronically recurrent, and can lead to blindness due to progressive destruction of
the intraocular structures [28]. In about one-third of equine patients, both eyes are affected,
often with a time lag of a few weeks to about 2 years [18,51,216].
The disease has also been termed periodic ophthalmia, although it had long been
clear that, generally, the symptoms did not reoccur periodically. Often, very painful
episodes of uveitis occur at varying intervals, and the inflammation-free intervals between
episodes can be less than 2 weeks or up to more than 1 year. In most cases, recurrences
occur within several weeks to several months, the intervals between relapses usually
become shorter over time, and subsequent episodes of inflammation often increase in
severity [19,217,218]. The typical symptoms of ERU have been described repeatedly and in
broad agreement [5,11,18,23–28,107,219].
(a) Acute uveitis

The first two points are non-specific, but prompt horse owners to request a veterinary
examination:
• Epiphora, blepharospasm, and photophobia;
• Concomitant conjunctivitis;
• Gossamer to smoky corneal opacity (Supplementary Material S1, Figures S1 and S2);
• After a short time, circular vascular ingrowth into the cornea may begin (Supplemen-
tary Material S1, Figures S1–S4);
• Inflammatory products in the anterior chamber of the eye (positive Tyndall effect,
fibrin, leukocytes and/or erythrocytes) (Supplementary Material S1, Figures S3–S5);
• More rarely, also a hypopyon (Supplementary Material S1, Figure S3);
• Pain-related and often complete miosis; mydriasis can often only be achieved with a
striking delay, and possibly incompletely with medication (Supplementary Material S1,
Figures S1–S4);
• Diffuse vitreous haze (Supplementary Material S1, Figures S6 and S7);
• In rare cases, rubeosis iridis (Supplementary Material S1, Figure S8).
After mild inflammation and immediate rigorous conservative treatment of acute
uveitis, the eye may still look unchanged ophthalmoscopically in the inflammation-free
interval after the first episodes, or only very subtle changes may be left that are only noticed
by veterinarians experienced in equine ophthalmology and with a maximally dilated pupil.
The more uveitis episodes which have passed, the more changes can be seen in the course
of time. even in the inflammation-free interval; thus, the diagnosis “ERU” can even be
reliably made between acute inflammatory episodes. The findings therefore include:
(b) Chronic recurrent uveitis, inflammation-free interval
• Atrophy of the globe (often resulting in the development of a so-called “3rd corner of
the eye” occurring on the upper eyelid, Supplementary Material S1, Figure S9);
• Chronic keratitis;
• Posterior (more rarely, also anterior) synechiae, sometimes only iris residues on the
anterior lens capsule (Supplementary Material S1, Figures S10 and S11);
• Vesicular cataract, typically in the periphery of the posterior surface of the lens
(Supplementary Material S1, Figure S12);
• Cataract formation (cataract typically originating from the lens capsule: either as a
result of posterior synechiae or as a result of inflammatory products adhering to the
posterior surface of the lens) (Supplementary Material S1, Figures S8–S11);
• Vitreous liquefaction;
• Sometimes chronic diffuse vitreous haziness, which does not completely resolve in the
inflammation-free interval (Supplementary Material S1, Figures S10 and S13);
• Over time, increasing accumulation of dense cloudy to membranous inflammatory
deposits in the vitreous body (“floaters”), which are not resorbed in the inflammation-
free interval, typically starting high dorsal in the liquefied vitreous, floating with
eye-movements (Supplementary Material S1, Figure S13);
• Centrifugal retinal folds surrounding the optic nerve disc, bullous, planar, or complete
retinal detachments (Supplementary Material S1, Figures S14 and S15);
• More rarely also lens (sub-)luxations,
• In later stages, significant bulbar atrophy, corneal opacities due to collapsed anterior
chamber and extensive contact between iris and corneal endothelium, phthisis bulbi
(Supplementary Material S1, Figures S9 and S11);
• Very rarely: glaucoma (e.g., in later ERU stages after cataract formation and subsequent
subluxations or luxations of the lens).
In the case of an initial uveitis flare-up, blunt trauma is sometimes considered as a
differential diagnosis. After the acute uveitis has subsided, a careful ophthalmoscopic
examination should be performed in mydriasis to detect inflammatory deposits dorsally in
the vitreous. If vitreous deposits are present, there is strong evidence that the acute inflam-
mation was a first episode of ERU. If the eye is inconspicuous again after the episode has
subsided, the diagnosis of “ERU” may not be made unless another uveitis episode occurs.
In horses in Germany and neighboring countries, the symptoms described here are highly
specific for chronic intraocular leptospiral infection leading to the uveitis episodes [16–19].
In human Leptospira-induced uveitis, which, as in horses, is a late consequence of sys-
temic infection, ophthalmologic examination (and, among other findings, the presence of
vitreous opacities) is also considered very sensitive and specific to establish an etiologic
diagnosis [220].
Most eyes exhibit classical symptoms of ERU, with recurrent painful attacks. However,
in about 3% of eyes, chronic inflammation of the posterior segment (vitreous cavity, ciliary
body, and choroid) dominates the disease [15]. In these cases, uveitis causes less pain in
the beginning of the disease (until the anterior segment is also affected—which happens
in most eyes after a while), and eventually lead to blindness (retinal detachment and/or
cataract formation) before the owner notices any problem. These cases of intermediate
and/or posterior uveitis are also highly specific for leptospiral uveitis in horses without a
leopard coat pattern [17–19,217].
If repeated inflammations have occurred without leaving ophthalmoscopically rec-
ognizable sequelae, it is advisable to re-examine the horse in an acute episode, e.g., to
differentiate between recurrent keratitis and uveitis. Alternatively, an aqueous humor ex-
amination could be performed [132] (Section 7). If leopard coat pattern horses are presented
with symptoms consistent with ERU, aqueous humor analysis is also indicated. The results
can then be used to decide on further therapy [20,221]. In cases of positive leptospiral
findings, the prevention of recurrence can be achieved by vitrectomy [16,19,222].
ERU is by far the most common form of equine infectious uveitis and, with early and
correct treatment, now has a good prognosis for both permanent absence of recurrences
and preservation of vision [16,191].
4.2. “Leopard Coat Pattern”—Uveitis

The term “insidious ERU” is widely used for uveitis in leopard coat pattern horses,
but it is misleading and a contradiction in terms. In leopard coat pattern horses, because
there are no typical recurrent episodes of uveitis, but uveitis is distinguished by a persistent
low-grade intraocular inflammation with a gradual and cumulative destructive effect,
rather than outwardly painful episodes of uveitis [4,5,7,12,13,15,52,54,175], it would be
more appropriate to differentiate between “ERU” and “insidious uveitis” rather than use
the term “Insidious ERU”, which is an oxymoron. In addition to the fact that horses with
leopard coat patterns typically do not exhibit classic painful recurrent episodes of uveitis,
most often, there is no infectious etiology of their uveitis either [1,15,16,197,223]. For this
reason, and to clearly differentiate this form of uveitis from ERU, the term “leopard coat
pattern uveitis” was introduced [15].
However, in many studies in the United States, Appaloosas, as the main representative
of leopard coat pattern horses, comprise a large proportion of ERU patients [4,5,12]. There
are no painful uveitis attacks; therefore, the veterinarian is often only consulted when the
owners have noticed opacities of the cornea or lens, bulbar atrophy, or buphthalmos (due to
glaucoma), or when an impairment of vision is suspected. At this point, irreversible changes
are often present, and the further course of the disease can hardly be influenced [5,12].
Appaloosas not only have an eightfold increased risk of developing uveitis compared
with other horses [1,15,30], but the course of uveitis is also particularly unfavorable and
both eyes are usually affected, often meaning that both eyes lose vision in the course of the
disease [5,7,12,15,30].
A lens pathology is often the primary condition in leopard coat pattern horses
(Supplementary Material S1, Figure S16). Relatively often and early in the course of
the disease, a (sub-) luxation of the lens and/or a cataract occur [1,15,30,153,224–227].
Lens changes are often present at very early stages of disease (even without concomitant
synechiae). The lens changes occur without an antecedent painful iritis and without in-
flammatory products on the posterior surface of the lens; therefore, the etiology in these
patients could be autoimmune-related inflammation, e.g., due to protracted leakage of lens
proteins through an abnormally configured lens capsule (Section 4.4). Amyloids can be
formed by refolding of the lens proteins in the inflamed milieu [228], and amyloids are
detectable in leopard coat pattern horses in the chamber angle and trabecular meshwork,
especially in eyes with a lens pathology [224].
A relatively large number of horses with leopard coat patterns (Knabstruppers more
often than Appaloosas) develop glaucoma [30], some develop phthisis bulbi (Appaloosas
more often than Knabstruppers), and somewhat less frequently, vitreous opacities are also
present [5,7,12,13,30].
Sometimes, ophthalmoscopic findings are present in leopard coat pattern horses
(e.g., hypotony of the globe, synechiae, cataract, vitreous opacities) that cannot be clearly
differentiated from ERU. Since leopard coat pattern horses may also have intraocular leptospi-
ral infection in some cases, aqueous humor analysis may be indicated [15,16,30,198,199,223].
Only if the aqueous humor analysis shows evidence of a leptospiral etiology the infection
can be eliminated by vitrectomy, and by this, vision may be preserved permanently—
provided there is no additional leopard coat pattern uveitis [15].
4.3. Traumatic Uveitis

Both blunt trauma and injury to the sclera or cornea may be followed by concomitant
uveitis due to transient damage to the blood–ocular barrier and disturbance of the immuno-
logic balance in the eye [229]. The following inflammation is immediately downregulated
by the effective immunosuppressive mechanisms in the eye, and the basic immunosup-
pressive setting of the eye is restored as far as possible [139,230,231]. If corneal or scleral
injury is evident when uveitis occurs, the traumatic etiology of this uveitis is obvious.
After blunt trauma, significant hyphemia in the anterior chamber of the eye or lesions
around the eye or on the eyelid skin may be evidence of trauma. However, sometimes it is
difficult to decide whether the first noticed ocular inflammation is a result of trauma or a
severe sero-hemorrhagic uveitis in terms of ERU [5,28,232] (Supplementary Material S1,
Figure S17).
This makes no difference for the initial treatment of acute uveitis. If the uveitis
improves satisfactorily with conservative treatment, and if there is no ophthalmoscopic
evidence of a previous uveitis event after the inflammation has subsided, mydriasis has
been achieved, and the hemorrhagic effusion has resorbed, it is possible to simply wait and
observe the eye for another inflammation. If uveitis recurs, then trauma is unlikely to have
been the cause of inflammation and ERU can be assumed as the most likely diagnosis [28].
If the blood does not reabsorb satisfactorily from the anterior chamber, and therefore
either a fibrinolytic is injected or the blood has to be removed mechanically from the
anterior chamber, an aqueous humor sample can be taken during the procedure and tested
for anti-Leptospira antibodies and the LipL32 gene [28].
Rarely, foreign bodies remain in the eye after perforating injuries, which then either
leads to therapy-resistant uveitis or the uveitis immediately recurs after the discontinuation
of anti-inflammatory treatment. If the foreign body can be removed, this also resolves the
uveitis. In other cases (e.g., the foreign body cannot be removed or endophthalmitis has
developed after perforating bulbar trauma), enucleation is indicated [28].
4.4. Phacogenic Uveitis

In phacogenic uveitis, there is a leakage of lens proteins through the lens capsule,
resulting in an autoimmune reaction [226,233]. This can be the case both with a congenital
weakness of the lens capsule and as a result of trauma with damage to the lens capsule.
Often, the ophthalmologic findings are indicative of whether the condition is phacogenic
uveitis or not. Especially in phacogenic uveitis after trauma, the leakage of lens protein
is recognizable. In cases of congenital weakness of the lens capsule, the configuration
of the cataract also often indicates that the uveitis is not ERU. Congenital or juvenile
phacogenic uveitis is often milder than typical ERU attacks and may be self-limiting. If
bleb-like structures are visible in the center of the anterior surface of the lens, hernia-like
lens parenchyma protrudes here and iris residues may also adhere; this cannot reliably
be differentiated from ERU, because such changes may not only be the cause, but also
the consequence, of uveitis [5] (Supplementary Material S1, Figure S18). In these cases,
when in doubt, an aqueous humor analysis with regard to a leptospiral infection should be
considered [18,28].
4.5. Chronic Iritis, Similar to “Fuchs’ Heterochromic Iritis” in Humans

Chronic equine iritis, which resembles Fuchs’ heterochromic iritis described in human
medicine [234–236], is usually insidious–progressive and is not accompanied by painful in-
flammatory episodes. Horses are typically not referred until corneal edema has developed,
which is recognized by the owners. If the interior of the eye is still visible, irregular minor
depigmentation of the iris, and sometimes also posterior synechiae, may be recognizable
(Supplementary Material S1, Figure S19). A comparison with the second eye can reveal
a diffuse loss of the normally saturated iris coloration. In the posterior segment of the
eye (posterior surface of the lens, vitreous cavity and fundus), typically, no changes are
present. Chronic iritis causes precipitates to sediment on the corneal endothelium, some-
times leading to corneal edema without an increase in intraocular pressure. However, over
time, the iridocorneal angle and the trabecular meshwork may also become obstructed
with inflammatory products, resulting in an increase in intraocular pressure [18,32]. The
clinical course and the ophthalmologic findings enable differentiation from ERU in most
cases; thus, only rarely is aqueous humor analysis indicated. Compared with uveitis in
leopard coat pattern horses, the course of chronic iritis is milder and less devastating in
most cases.
4.6. Uveitis during Septicemia

In severe general infections, sero-fibrinous uveitis may occur [51], which then typ-
ically affects both eyes [237] (Supplementary Material S1, Figure S20). Most commonly,
this septicemia-accompanying uveitis occurs in foals with rhodococcosis [36,38,219,238].
However, it may also occur in neonates in which other bacteria are causative for sep-
ticemia [37,39,239,240]. Whether septicemia-associated uveitis is an immune-mediated
uveitis comparable to septicemia-associated non-septic and presumably immune-mediated
synovitis, or whether the pathogens reside in the eye and are eliminated from the eyes
as septicemia recedes, remains unclear without aqueous humor analysis [241,242]. In
any case, to the best of the authors’ knowledge, foals that survive septicemia are not ex-
pected to experience recurrent episodes of uveitis. Septicemia-associated uveitis, in which
pathogens may also enter the eye, is thus not an ERU. Foals up to 6 months of age affected
by uveitis typically have septicemic uveitis and no ERU. ERU only occurs in horses older
than 6 months of age [18].
There are also case reports of uveitis in the context of general infections with spirochaetes
in adult horses—especially in severe and fatal courses. These include Lyme disease [33,34],
as well as leptospirosis [40,70].
4.7. Uveitis Accompanying Intraocular Tumors

Intraocular tumors can lead to uveitis by mechanical irritation and/or immune responses.
In human ophthalmology, the term “pseudouveitis” was coined for tumors which clini-
cally mimic an inflammatory disease [243]. Intraocular tumors include melanomas [244–247],
medulloepitheliomas [248–253] (Supplementary Material S1, Figure S21), and rarely lym-
phomas [219,254–256]. Intraocular tumors can often be detected by ophthalmologic exami-
nations based on non-physiologic tissue formations; in cases of media opacity, sometimes
only with the help of ultrasound examination. The distinction from ERU is clear when
tumor tissue is visible or can be visualized by ultrasound. More rarely, general diseases
indicate a tumor and involvement of the eye in the form of more or less therapy-resistant
uveitis. This often results in considerable media opacities, in which etiological assignments
of the uveitis are not reliable. In cases of doubt, aqueous humor analysis is indicated to
exclude infectious uveitis and to perform cytological diagnostics.
4.8. Uveitis during Parasitic Infections

Relatively rarely, Halicephalobus (syn. Micronema) deletrix infections can cause therapy-
resistant uveitis [41,42,257]. If there is no satisfactory improvement on the known therapy
of acute uveitis, it is advisable to consider a general disease and, if necessary, to initiate
blood tests including electrophoresis. If micronematosis is suspected (clearly impaired
general condition, poor nutritional status, very high total protein in the blood, ß-peak in
electrophoresis), ultrasound examination of the kidneys and urinalysis would be indicated,
among other tests, because the urinary apparatus is often involved in this infection. Thus
far, there are only reports of fatal courses of this disease [258–279]. In early stages, and
when the general disease is not yet in the focus, uveitis caused by these nematodes may
not be reliably distinguished from ERU—even if the striking resistance to therapy of the
uveitis is not consistent with ERU (Supplementary Material S1, Figure S22). Therefore, in
cases of doubt, an aqueous humor analysis is indicated.
Additionally, infections with Setaria spp. are rarely described; they can lead to anterior
uveitis and corneal opacities [280]. Usually, the parasites are then visible in the anterior
chamber of the eye or can be visualized by ultrasound. Ophthalmologic symptoms are
easily distinguishable from ERU.
4.9. Uveitis Accompanying Severe Keratitis

In cases of severe keratitis or infections reaching deep into the cornea, concomitant
hypopyon uveitis may develop [5,219]. This may be a warning signal that endophthalmitis
may be developing, and subsequently, a surgical intervention is required. Corneal disease
dominates the ophthalmological findings, and, in most cases, this is easy to clinically
distinguish from ERU (Supplementary Material S1, Figure S23).
4.10. Endophthalmitis
Endophthalmitis is usually the result of bulbar lesions or deep and infected corneal
disease. More rarely, endophthalmitis is iatrogenically caused after intraocular surgery. En-
dophthalmitis caused by Staphylococcus spp. or other bacteria is accompanied by a highly
disturbed general condition and fever. In early stages, irrigation of the anterior chamber,
vitrectomy, and intraocular antibiotics can be used to try to eliminate the infection [281].
In advanced stages, enucleation is indicated. Visual function is lost; therefore, there is a
risk of ascending encephalitis and phthisis would develop within a short period of time
(Supplementary Material S1, Figure S24). Both the significant disturbance of the general
condition and the ophthalmologic findings enable easy differentiation from ERU [5,28,219].
4.11. Autoimmune Uveitis and Uveitis of Unknown Etiology

In rare cases, also in horses that have no leopard coat pattern, the cause of uveitis
cannot be determined. In these cases, aqueous humor testing is indicated to rule out an
infectious and treatable cause [16]. If no intraocular infection and no causative disease
for the uveitis is detectable, only symptomatic therapy is possible. After exclusion of
the described types of uveitis and an infectious etiology (including other bacteria than
Leptospira spp. and more exotic pathogens, such as viral, fungal, and protozoal infections
involving the uvea), the suspicion of an “autoimmune disease” might be justified in single
cases (Section 3.1).
5. Therapy
5.1. Acute Uveitis
Conservative therapy of acute uveitis—regardless of its etiology—always includes:
• Topical mydriatics, as often as needed for dilation of the pupil to prevent posterior
synechiae and thereby cataract formation (in equids, atropine is by far the most effec-
tive substance for this purpose, and to the best of the authors’ knowledge, atropine can
be used as frequently as needed without any concern (Supplementary Material S2));
• Topical treatment with corticosteroids to reduce inflammation (most effective are
ophthalmic ointments containing dexamethasone)—if corneal defects are present,
these are a contraindication for ophthalmic ointments containing corticosteroids,
because otherwise the development of serious corneal ulcers may be favored;
• Systemic administration of non-steroidal anti-inflammatory drugs;
• Palliative measures: the avoidance of bright light, limited exercise, or even stall rest;
• Systemic administration of antibiotics can be considered in severe cases when a bac-
terial infection is involved (e.g., hypopyon–keratitis or ERU with severe haziness of
intraocular fluids); thus, this is rarely indicated.
If the eye does not improve satisfactorily with this treatment, additional measures may
be required. If diffuse vitreous opacification is persistent and significant, systemic admin-
istration of prednisolone may be considered. In eyes in which mydriasis is not achieved
satisfactorily or in which the inflammatory products have not reabsorbed within 1–2 weeks,
surgical procedures may be required (e.g., synechiolysis, the injection of fibrinolytics into
the anterior chamber, or the mechanical removal of coagula from the anterior chamber of
the eye) [5,28,216,219].
5.2. Chronic Course of Uveitis

Both leopard coat pattern uveitis, which is chronically destructive and not accompa-
nied by painful episodes, and uveitis, which proceeds with recurrent painful episodes, lead
to progressive damage to the intraocular structures; therefore, the goal is always to contain
the inflammation as much as possible to prevent blindness or delay the process as long
as possible.
In leptospiral-induced ERU, vitrectomy, which was established in horses more than
30 years ago [282,283], is the most effective therapy [67,68,284–291], even if not 100% of
the vitreous humor can be removed (“subtotal vitrectomy”, because preventing retinal
detachment and damage to the posterior lens capsule are crucial). After successful surgery,
up to 97% of operated eyes do not develop recurrent uveitis episodes [2,16,191]. In this way,
vision can be preserved in many horses—provided that the operation is performed before
irreversible damage to the lens and/or retina has occurred [16,191,288]. Vitrectomy not
only stops ERU and often prevents blindness [2,191], but by removing vitreous opacities, it
can actually improve vision [16].
Vitrectomy is a highly specialized surgery that requires a high level of experience
and skill on the part of the surgeon and a well-trained surgical team for optimal results.
Any mishap by anyone involved and any malfunction of the equipment can be fatal to
vision or eyeball preservation. In addition, specific equipment, such as custom-made
instruments and reliable machines adapted to the dimensions of equine eyes are required,
which represents a substantial investment and is only worthwhile if larger numbers of
patients are to be expected. This could explain why vitrectomy in horses is still performed
predominantly in specialized ophthalmological clinics in Germany and has not yet gained
equal acceptance in other countries or continents [13,16].
In recent years, an injection of 4–6 mg gentamicin (which is really gentamicin sulfate,
but is dosed according to the gentamicin content), preferably a preservative-free solu-
tion [292], into the vitreous cavity has been described as a less demanding therapeutic
method for ERU [292–295]. This does not require specialized instruments and equipment,
and performing the injection is easier and less expensive than vitrectomy [296]. These injec-
tions are not only reported to be effective for leptospiral uveitis, but are also discussed to
reduce uveitis via immunomodulatory effects; therefore, surprisingly, gentamicin injections
are not recommended exclusively for infectious uveitis [292,296]. However, there are only
single studies on gentamicin injection in comparatively few patients, and there are still
scant conclusive data on long-term outcomes. Disturbingly, the relatively high gentamicin
dose administered exceeds concentrations largely considered “safe” to prevent retinal
damage by almost threefold (Supplementary Material S3). None of these studies included
ERG studies to monitor retinal function. In addition to intravitreal gentamicin injection,
the intravitreal injection of a third-generation cephalosporin has also been mentioned but
has not gained acceptance thus far [297].
Another treatment approach is systemic treatment with antibiotics that are effective
against leptospires and achieve a sufficient therapeutic level in the intraocular fluids. For
this purpose, the intravenous injection of enrofloxacin has been proposed [298]. In a later
study, it was shown that although this treatment results in therapeutic levels in the eye
(higher than the minimum inhibitory concentration (MIC) for leptospires), elimination of
leptospires from the eye is not reliable. Without enrofloxacin administration, leptospires
were culturally detected in 54% of vitreous samples from eyes affected with ERU; after
several days of intravenous enrofloxacin administration and with drug levels in vitreous
samples above the MIC, the culture was still positive in 30% of vitreous samples [196,299].
It can be concluded that systemic antimicrobial therapy, at least with enrofloxacin, is not
very effective in eliminating the organism from the eye [63].
Unlike in horses, leptospiral uveitis in humans can be self-limiting [207]. Although
recurrent episodes may occur and can be sight-threatening, leptospiral uveitis is reported
to respond well to the systemic administration of antibiotics, and generally has a good
prognosis [116,300,301]. Among other causes, this may be because the human vitreous
body has a volume of only about 4 mL, whereas that of the horse is about 28 mL. The
immunological niche is therefore much larger in horses. This might be an explanation
why the elimination of the infection is more problematic. It is also possible that the equine
vitreous is more susceptible to biofilm formation for other reasons.
In uveitis that does not have an infectious etiology, such as uveitis in leopard coat pat-
tern horses, only symptomatic anti-inflammatory treatment, not causal therapy, is possible.
In addition to the prolonged administration of NSAIDs and, if necessary, systemic corticos-
teroids, other therapeutic methods have been described to be effective locally. This includes
the intrascleral or suprachoroidal implantation of cyclosporine devices, which deliver
active substance over 1–2 years and counteract the intraocular inflammation [5,218,302].
Only a few publications distinguish between infectious and noninfectious uveitis and use
cyclosporine devices exclusively for noninfectious uveitis [68].
Cyclosporine implants, unlike vitrectomy, are only effective for about 2 years and may
then need to be replaced. In addition, the suppression of chronic uveitis with cyclosporine
devices is less effective than vitrectomy for ERU [191,218,291,302,303]. It must be considered
that the implants are not legally available in the European Union (any import into the EU
from non-EU countries is prohibited by law). Moreover, the implants had predominantly
been inserted incorrectly (episclerally instead of deep intrasclerally or subsclerally) in
horses that had nevertheless somehow received implants [303]. This could explain the lack
of effectiveness in several cases.
Other authors describe intravitreal [304] or suprachoroidal [8] injections of triamci-
nolone. However, thus far, there are only results from 36 eyes of 29 horses with a follow-up
of merely 3 months. Long-term therapy with acetylsalicylic acid (25 mg/kg 1 × daily),
which was recommended decades ago [11], has not yielded any long-term results, to the
best of the authors’ knowledge, and this therapeutic method has not gained acceptance.
Horses under treatment with anti-inflammatory drugs are not allowed to compete in
equestrian sports (doping regulations), on animal welfare grounds.
In leopard coat pattern horses, progression of the disease can be effectively prevented
by vitrectomy if only intraocular leptospiral infection is involved. In cases of significant
inflammatory vitreous deposits, vitrectomy may be considered—even independently of a
leptospiral infection in the eye—to improve vision and prevent the vitreous membranes
from attaching to the lens. However, a further course of the disease cannot be stopped by
this, only delayed. Otherwise, only symptomatic antiphlogistic and, if necessary, pressure-
lowering therapy, is possible. Here, cyclosporine implants, for example, would be suggested
to make the further course of the disease less severe and to delay blindness [218,302].
However, pharmaceutical regulations must be respected, because the import of cyclosporine
devices into the European Union is prohibited or may only be carried out with special
authorization [303].
6. Diagnostic Value of Testing Serum Samples for Anti-Leptospira Antibodies in

Horses Suffering from ERU and in Horses with Healthy Eyes
Many studies have assessed serum samples from horses with ERU and horses with
healthy eyes, and most often, the sera were analyzed by microscopic agglutination tests
(MATs) [1,9,17,19,43,305–307]; ELISA tests for the detection of anti-Leptospira antibodies
were used less frequently [194,195,198,308–310]. In some studies, no significant differences
were detectable between groups of these horses; however, in other studies, differences
were detected [1,9,43,135,305,306]. It is largely agreed, however, that serum assays using
MAT and various ELISA tests are inappropriate for diagnoses of intraocular leptospiral
infections in an individual animal [17–19,45,194,195,307,311–316]. Among other reasons,
this can be explained by the fact that leptospiral infections are endemic in many regions
and a high background level is present; thus, a large number of horses with healthy
eyes display MAT titers [17,19,84,85,307,313,315–326]. Therefore, as in human leptospiral
uveitis [327], MATs using sera are not diagnostic of leptospiral uveitis when only serum is
examined [17–19,45,307,308,314,328].
MAT titers detectable in serum are mainly influenced by the type of horse
stabling, its location, geographical region, climatic conditions, and age of the tested
horses [59,320,322,323,325,326,329–334]. Laboratory diagnostics (MAT titers considered
“positive” and serovars available) may have also influenced the results of these studies [308].
All of these factors are more decisive for the presence of agglutinating antibodies detectable
in MAT than the presence of leptospiral uveitis.
It should also be noted that ERU is a late consequence of systemic leptospirosis.
Episodes of uveitis often occur months and often even years after systemic infection;
therefore, MAT titers in the blood may have already decreased or may no longer be
detectable by the time the eye disease becomes evident. For chronic local infections in
immunologic privileged sites, blood sample testing is generally unreliable. This applies
not only to intraocular infections [95,335–339], but also to infections of the CNS or renal
tubules. For example, in various animal species in which indications of shedding were
detected by urinalysis (culture and/or PCR), very often, no antibody titer was detectable in
the blood by the MAT [340–342]. Furthermore, it is important to note that in about 10% of
189 ERU horses from which Leptospira spp. were cultured from vitreous samples, MAT titers
in the serum were negative (titer <1:100) [18,19]. MATs with serum are considered useful
for assessing the degree of infestation and the infection pressure of a livestock, and thus
also for identifying the risk of disease; therefore, it is not very meaningful for diagnostics in
individual animals [18,19,342]. If anti-Leptospira antibodies are present in serum samples,
one cannot be certain whether they are coincidental or related to an intraocular infection.
In a recent study, the detection of anti-LipL32 antibodies with an on-site rapid test
(SNAP Lepto, IDEXX company) has been shown to be a useful screening method to
diagnose chronic intraocular leptospiral infection [308]. This SNAP Lepto is serovar and
species non-specific, but it is a relatively inexpensive and easy to perform ELISA test that
provides a result in 10 min. LipL32 is present on the sample spot as well as in the conjugate
in this rapid test; thus, any anti-LipL32 antibody that has at least two binding sites can be
detected. This test allows veterinarians with less experience in ophthalmology to perform
blood tests in practice and thus check the probability of the presence of an intraocular
leptospiral infection. Specificity, positive predictive value, and kappa-value with respect
to the diagnosis of ERU are significantly higher for the SNAP Lepto than for the MAT
when serum is used. However, although diagnostics have improved with the detection
of antibodies directed against LipL32 in serum, testing of intraocular specimens is still the
most reliable for the detection of ERU [308].
7. Examination of Intraocular Specimens in Horses Suffering from ERU

To determine the etiology of uveitis, examinations of intraocular samples (aqueous
or vitreous humor) are much more informative than blood tests [18,19,58,335,343–345].
Intraocular specimens can generally be examined for various antibodies, by means of
PCR as well as cultures for various infectious agents. In addition, protein determinations,
electrophoresis, and cytological examinations (e.g., in the case of suspected tumors) are
also possible.
The collection and testing of aqueous humors can provide substantial information
for the diagnosis of unclear intraocular inflammation. This applies to infectious and some
noninfectious uveitis cases [346,347]. Therefore, in different species, the collection of aque-
ous humor, or even vitreous specimens and the analysis of these intraocular samples by
laboratory diagnostics, is often an excellent way not only to assign the uveitis etiologi-
cally [348–350], but also to carry out the best therapy [16,345]. Correct indications and
the adequate processing of ocular samples are decisive and determine the quality and
reliability of laboratory results [346].
Extractions of aqueous humors are less invasive and associated with fewer com-
plications than extractions of vitreous material, and aqueous humor examinations can
provide satisfactory information for many questions [16,199,337,347,351]. Aqueous humors
are also often informative for the diagnosis of posterior uveitis and allow for etiological
diagnoses [19,337,347,351–356].
In human medicine, only about 0.1–0.2 mL of aqueous humor can be safely col-
lected [344–346,357–359]. This is why the number of diagnostic tests that can be performed
with this small aqueous humor volume is limited. However, if the history and clinical
examinations have raised a certain suspicion, the low aqueous humor volume is often
sufficient for a specific diagnosis [347,352,360]. If further laboratory tests need to be carried
out and the small aqueous humor volume is not sufficient, intraocular sampling by vitrec-
tomy can be performed to obtain a larger sample volume [351,353,361–364]. Approximately
0.5--0.7 mL [345,365], or even 1–2 mL [366] of undiluted vitreous humor, may be carefully
aspirated at the beginning of a vitrectomy. More tests can then be performed, and there is a
better chance of determining the etiologic diagnosis of uveitis.
In horses, on the other hand, 1 mL of aqueous humor can be collected safely, so
that good preconditions are given to perform various laboratory tests [132,221,367]. The
reports in which intraocular samples from ERU eyes were used are far fewer in number
than studies using serum samples. One reason for this is probably that although the
collection of aqueous humor has been described and is also considered a safe method
under short anesthesia [132,221,222,367], many veterinarians hesitate to collect aqueous
humor in still-seeing eyes and, in addition, some ophthalmologic experience is needed
for paracentesis.
The number of intraocular specimens from ERU eyes examined in each case is also
very low in some reports from recent decades. Sometimes, only 1 or 2 aqueous humor
samples were examined [313,368]; in other reports, about 50 samples [316,328]; and in
additional studies, the sample size was in between [14,306]. Some studies describe aqueous
humor collection in horses under general anesthesia [132,221,222], but others describe the
procedure as occurring under sedation and local anesthesia [14,369]. Some investigators
differentiated the procedure and only performed anterior chamber puncture in eyes that
were already blind in standing position, and otherwise under general anesthesia [328].
However, the collection of intraocular samples from equine eyes that have not yet
suffered much damage is often difficult to justify if it does not result in a therapeutic
consequence. For this reason, many of the intraocular specimens reported to have been
examined in recent years were obtained from eyes that were blind and in the final stages
of ERU. Intraocular sampling was then performed after the enucleation of blinded eyes
or after euthanasia of the horses due to bilateral blindness [174,307,316,368,370]. By this

time, the eyes show considerable atrophy or even phthisis and recurrent inflammation
in the sense of ERU typically does not occur then. This means that intraocular samples
from eyes in the final stage of ERU are no longer representative of the condition in the eye
that existed during acute uveitis episodes. Reasons for the enucleation of blinded eyes
include an overly exposed conjunctiva that is chronically irritated, leads to secretion and
subsequent dermatitis, or an entropion that leads to persistent pain.
Reports of equine vitreous samples taken for diagnostic purposes are scarce;
samples were mostly obtained after the enucleation, euthanasia, or slaughter of the
horses [306,316,369]. It was not until vitrectomy was routinely performed, with vitre-
ous material as a waste product of surgery and available for examination, that equine
vitreous samples could be examined on a larger scale [16,18,19,66,69,315].
The greatest likelihood of detecting evidence of intraocular infection on the exam-
ination of intraocular specimens is generally whenever multiple testing procedures are
used [345]. For example, when an aqueous humor sample is collected from horses for a
preoperatively indicated diagnostic procedure, the sample should be examined as compre-
hensively as possible. Various antibody tests (MATs, in-house ELISA tests and SNAP Lepto)
and PCR should therefore be performed routinely, because in some samples, antibodies
are not detectable, but PCR is positive, and vice versa [19,66,68]. Since PCR has become
available, the expensive and often prolonged cultural detection of leptospires in intraocular
specimens is very rarely requested for routine diagnostics.
7.1. Breakthrough with Vitrectomy

Originally, the surgery was performed primarily to remove opacities from the vitreous
cavity and to improve vision [282,283]. However, it very soon became apparent that after
surgery, chronic recurrent inflammation also ceased in most eyes [67,68,284,286–289]. The
increasing experience with the clinical and ophthalmoscopic examination and the correct
indication, as well as the optimization of the operation itself—including the adaptation of
the instruments to the horse eyes and the CO2 laser-assisted sclerotomy—made vitrectomy
a relatively low-complication surgery with an astonishingly good prognosis. Indications
for vitrectomy could be made with increasing precision, as examinations of numerous
vitreous specimens and assignment of clinical findings to “Leptospira-positive eyes” have
led to increasingly accurate classification of ophthalmologic findings [2]. This, in turn,
has brought vitrectomy to the attention of horse owners and it has been requested with
increasing frequency [16,191,288].
Due to the routine surgery of ERU-affected eyes, sterilely obtained vitreous material
from thousands of eyes was available for various investigations. Vitrectomy was performed
exclusively in the inflammation-free intervals of the disease [2,191]. Most of the eyes had
suffered only two or three episodes of uveitis, and most of them were still visual [16].
Therefore, vitrectomy enabled the examination of numerous intraocular specimens from
eyes that had little damage and were still in a florid stage of the disease, unlike the end-stage
ERU eyes.
It was not until these numerous vitreous samples were examined that it became clear
that a chronic intraocular leptospiral infection was the cause of ERU in almost all horses
with typical clinical and ophthalmological signs [17,18]. In intraocular specimens obtained
from vitrectomized eyes sometime after surgery (e.g., horses euthanized for colic, fractures,
or other reasons), intraocular antibodies have been shown to continuously decrease over
time postoperatively, and culture and PCR are then negative (Supplementary Material S4,
Table S1 and Figure S25). It was concluded that the leptospiral infection is localized in
the vitreous cavity, and that vitrectomy, in which only vitreous material is removed, also
eliminates the infection [18,19,371]. The chronic leptospiral infection in the vitreous cavity
explains why vitrectomy is so highly effective in horses [16].
Initially, the entire volume of liquid collected during vitrectomies was used for anti-
body and pathogen detection. Although vitreous material in the surgical lavage fluid was
diluted approximately 10-fold, high antibody titers were often detectable using MAT [314].
In addition, 0.08 mg/mL gentamicin was added to the vitrectomy infusion solution for
the prophylaxis of postoperative endophthalmitis [45,191]. In vitro, this concentration was
100-fold higher than the MIC for standard WHO strains of pathogenic Leptospira spp. [372].
Despite this, Leptospira spp. could still be cultured from gentamicin-containing lavage
samples in some cases [373,374]. After optimization of the sampling technique, 2–3 mL of
undiluted vitreous material could be collected at the beginning of surgery [2,191], which
has significantly improved laboratory diagnostics with these samples, especially with
regard to the culture of Leptospira spp. [19].
Other investigators did not use undiluted vitreous samples but collected the samples at
the beginning of the surgery, so they were diluted as little as possible [66–68,288]. The vary-
ing results of the examination of the intraocular specimens (Supplementary Material S5,
Table S2) can be explained through specimen collection during vitrectomy, further handling
of the specimens, different laboratories with different serovars used for MAT, and the
respective test procedures requested. Presumably for budgetary reasons, often not all avail-
able tests were applied. In addition, some laboratories declared low titers as “negative”,
but did not take into account the specific knowledge from ophthalmology that any titer can
be an indication of an intraocular leptospiral infection. However, a crucial factor is also
the indication for vitrectomy (typical ERU) and the selection of suitable patients, because
critical inclusion criteria play a decisive role for the success of the surgery [16] as well as
for the intraocular fluid analysis [358].
Sometimes horses are referred for vitrectomy in which neither the history nor the
ophthalmoscopic findings clearly indicate an ERU, and thus, an indication for vitrectomy.
In these cases, it is indicated to first perform paracentesis of the anterior chamber of the
eye (Section 4.1) and to test the aqueous humor for anti-leptospiral antibodies (MAT and
ELISA tests) and for 16S rRNA and/or the LipL32 gene. If one of the above tests is positive,
there is an indication for vitrectomy [16,19,21,199]. On the other hand, if all tests come back
negative, the indication for vitrectomy must be critically reconsidered.
SNAP Lepto for the on-site detection of anti-LipL32 antibodies is not only suitable for
the analysis of serum [308] (Section 6), but also for the analysis of intraocular samples [199].
The SNAP Lepto result is available after 10 min; therefore, the horse can be left under
general anesthesia. If the SNAP Lepto result is positive, vitrectomy can be performed
immediately. This avoids another recovery, which can cause injury to horses, and poses
higher risks than general anesthesia itself. If the rapid test is negative, the horse must
recover, and the aqueous humor sample is sent to an external laboratory for further diag-
nostics (MAT and PCR) [199]. If then in the aqueous humor either anti-Leptospira antibodies
are detectable and/or the PCR is positive, vitrectomy must be performed under a second
general anesthesia.
Since the investigations of vitreous samples obtained during vitrectomies had brought
the leptospiral infection in ERU back into focus [17–19,45,314,373,374], and since the es-
tablishment of PCR for leptospiral diagnostics, investigations of intraocular samples with
regard to leptospiral infection have also been performed by other working groups that did
not have vitreous material obtained during vitrectomies at their disposal (Sections 7.2–7.6).
In parallel, vitreous material obtained during vitrectomies was used to intensively
investigate the (auto)immune reactions accompanying the infection [55,154,158,159,165,192]
(Section 3.1) and studies on drug transfer into the vitreous cavity could be performed
without the need for animal experiments [196,299,375].
7.2. Antibody Detection in Aqueous Humor and Vitreous Samples

As for the examination of serum samples, most often, MAT was used for antibody
detection for the examination of intraocular specimens [18,19,66–69,315]. In contrast to the
examination of serum samples, MAT is highly specific when examining intraocular samples.
The most common leptospiral serovar detected in equine eyes was L. grippotyphosa, but
several other serovars from different serogroups can also cause ERU [19,20,66,69,315,376].
In about 90% of undiluted vitreous samples from ERU eyes, agglutinating antibodies
are detectable by MAT (titer 1:100 or higher) [19,122,373]. Intraocular antibody titers
often considerably exceed serum titers (Supplementary Material S6, Tables S3–S5 and
Section 7.3). This phenomenon can also be observed without exact measurement of the titer
when evaluating the SNAP Lepto: the sample spot is usually stained considerably weaker
when examining serum compared with the examination of vitreous humor from the same
horse [308] (Supplementary Material S6, Figure S26).
In a study of 724 serum samples from horses affected with ERU, the highest MAT
titer measured in serum samples was 1:25,000 in a single horse (0.1%). In 426 vitreous
specimens obtained at vitrectomy, the highest MAT titer was 1:3,267,800. In total, 62 of the
426 vitreous samples (15%) had an MAT titer of 1:25,000 or higher [18] (Supplementary
Material S6, Table S4). However, when examining intraocular samples from healthy eyes,
positive results in MAT or PCR are only found in exceptional cases (Section 7.11). Therefore,
it cannot be ruled out that asymptomatic courses of intraocular leptospiral infection occur.
However, it is possible that the Leptospira-positive eyes could also have developed ERU
sometime in the future.
The sensitivity of leptospiral serology performed on intraocular samples from ERU
eyes can be further increased by complementary ELISA tests. In addition to antibod-
ies of immunoglobulin class G (IgG), IgA antibodies are of particular importance in in-
traocular infections [377–381], as well as in other local and especially biofilm-associated
infections [382–385]. On rare occasions, in-house ELISA tests were used in addition to
MAT, which were both immunoglobulin- and serovar-specific and enabled the detection
of IgA antibodies, and were highly sensitive for the detection of an intraocular infec-
tion [194,195,198,386]. However, these tests are not routinely available. Examination of
aqueous humor from ERU eyes in one study also revealed increased antibodies to LruC,
a protein from the outer membrane of pathogenic Leptospira spp. [174]. However, to the
best of the authors’ knowledge, no routine test is currently available for the detection of
this protein.
In recent years, the serovar- and species-unspecific SNAP Lepto has proven its value
for the examination of intraocular specimens (Sections 6 and 7.1). When examining in-
traocular specimens, the sensitivity and specificity of the SNAP Lepto are equivalent to
the MAT. SNAP Lepto and MAT provide predominantly, but not always, matching re-
sults when examining intraocular specimens [198,199]. High MAT titers correlate with a
more intense blue coloration of the sample spot in the SNAP Lepto [198]. In individual
intraocular samples (especially in very early ERU stages), only one of the two tests may
react positively. The immune response of the individual patient, as well as the stage of
the disease and different antibody dynamics (agglutinating antibodies versus anti-LipL32
antibodies), may play a role here. Therefore, in case of a negative result of the first test, it is
useful to perform another one for the detection of other anti-Leptospira antibodies [68,195].
Rarely, no antibodies at all are detectable in culture- and/or PCR-positive intraocular
specimens [17–19,57,66].
In aqueous humor and vitreous humor of healthy eyes of humans and animals, nor-
mally, no antibodies are present [18,19,199,386,387] and their protein contents are very low
(up to about 0.2 g/L) [43,119,189,192,344,388–391]. The average total protein content in
samples from eyes affected with ERU in one study was 6.7 g/L which, although higher
than that in healthy eyes, was, on average, only 1/10 of the total protein content of serum
samples [18] (Supplementary Material S6, Table S3).
Blood–Aqueous Barrier and Intraocular Protein Content

Whenever the uvea is irritated, the barrier between the blood and the intraocular
media (blood–aqueous barrier and/or blood–retinal barrier) is temporarily damaged and
the passage of serum components or even cells into the eye may occur [132,158,345,392,393].
Some studies have described the collection of aqueous humor from acutely inflamed equine
eyes [306,313,316,368]. In acute inflammation, the blood–ocular barrier is always disrupted,
and intraocular samples contain more proteins than in the inflammation-free interval [132].
It should also be taken into account that, after euthanasia or enucleation, the blood–ocular
barrier breaks down rapidly and the albumin content in aqueous humor and vitreous body
multiplies within a short period of time. This can lead to falsified results when examining
intraocular samples from euthanized horses or enucleated eyes [132].
Some authors point out that leakage from the serum into the eye persists for a long
time after an acute uveitis episode has subsided [101]. However, in the event of leakage,
the albumin content in the intraocular fluids would be expected to rise before the gamma
globulin content. In fact, however, the albumin content in vitreous samples obtained during
vitrectomies is comparatively low. In the serum samples, the albumin content is usually
only slightly below the gamma globulin content, but in the corresponding intraocular
samples, the albumin content is mostly very significantly below the gamma globulin
content. Sometimes the gamma globulin content is several times greater than the albumin
content. If, exceptionally, the albumin content is relatively high in an intraocular sample,
the antibody titer measured therein is typically so high that it cannot be explained by
leakage at all (Supplementary Material S6, Table S3).
Due to the highly effective clearance in the eye several days after trauma, no agglu-
tinating antibodies are detectable in the aqueous humor of horses with traumatic uveitis
and hyphemia, even if the serum of these horses reacts positively in MAT. In samples
from eyes with a persistent, although slight disturbance of the blood–aqueous barrier
(e.g., phacogenic uveitis, glaucoma, leopard coat pattern uveitis, and chronic heterochromic
iritis), typically, no anti-Leptospira antibodies are detectable in the aqueous humor either,
although MAT titers are present in the serum [15,16,18,19,45,69]. Furthermore, in horses
suffering from ERU, the testing of corresponding serum and intraocular samples often
reveals different serovars in MAT [18,45], so that even in these horses, no leakage may have
led to the antibody titers in the eye (Supplementary Material S6, Figure S27). In horses
in whose vitreous samples pathogenic Leptospira spp. could be detected by culture, the
serovar (in the case of multiple serovars, the one with the highest titer) detected in the
vitreous sample by MAT basically matched the serogroup of the culture. In contrast, MAT
titers of other serogroups often dominated in the corresponding serum samples [18,20]
(Supplementary Material S6, Figures S28 and S29).
Examination of numerous specimens from eyes affected with ERU has shown that
antibody titers measured in MAT increase with the severity of ERU. In particular, the de-
gree of diffuse vitreous opacification correlates positively with the titer level in intraocular
samples [18]. In contrast, MAT titers are typically rather low in eyes with little damage, in
which an aqueous humor examination may even be indicated before vitrectomy because
no clear ophthalmoscopic changes are present and the indication for vitrectomy is therefore
questionable. Based on decades of experience and thousands of test results, the authors
meanwhile consider MAT titers of 1:50 in aqueous humor samples from these ophthal-
moscopically unchanged eyes as “positive” and as an indication for vitrectomy, because
the protein content in these eyes, just as in healthy eyes, is typically <0.2 g/L [43]. Thus,
leakage with passage of antibodies from the blood into the eye can be almost completely
ruled out.
Notably:
• Vitrectomy is performed exclusively in the inflammation-free interval, not during an
acute attack of uveitis;
• A disturbed blood–ocular barrier especially plays a role during acute inflammation;
• Depending on the time of sampling and stage of ERU, the leakage of proteins from the
blood into the intraocular fluids is of minor importance at the time of surgery (quiet
interval and mostly an early stage of the disease);
• The immunoglobulin content in the serum is about ten times greater than the im-
munoglobulin content in the vitreous; nevertheless, the intraocular antibody titer is
usually higher (Supplementary Material S6, Tables S3–S5);
• The more significant the diffuse haziness in the vitreous, the higher the protein content
in the intraocular fluids, and the more obvious a “leakage” would be from the serum—
but, in fact, the antibody titer in intraocular samples is then usually also significantly
higher in relation to the antibody titer in the serum (Section 7.3) (Supplementary
Material S6, Table S3);
• Low MAT titers in intraocular samples are mainly present when the eye ophthalmo-
scopically shows hardly any or no changes in the sense of ERU (especially no diffuse
opacity of the intraocular fluids); then, the protein content in the intraocular samples
is also very low;
• Electrophoresis shows that the eye usually contains less albumin than the immunoglobu-
lin content would suggest; compared with serum samples, the albumin/immunoglobulin
ratio in intraocular samples from eyes affected by ERU is shifted in favor of the im-
munoglobulin content;
• In “non-ERU” uveitis, glaucoma and a few days after damage of the blood–ocular
barrier due to trauma with intraocular bleeding, typically, no intraocular anti-Leptospira
antibodies are detectable even if MAT titers are detectable in corresponding
serum samples.
7.3. Intraocular Antibody Production

Evidence for intraocular antibody production in ERU has already been provided by
the extremely high MAT titers detectable in intraocular fluids described in the early studies
(Section 3). At that time, the findings observed in horses served as a model for more
sophisticated diagnostics in infectious uveitis in humans and the detection of local antibody
production [394–396].
Intraocular antibody production is also supported by the detection of B cells in the
uvea of equine eyes affected with ERU, which are not detectable in the uvea of healthy
equine eyes [5,143,378,397]. In addition, the selective and marked increase in IgA content
in samples from ERU eyes compared with corresponding serum samples suggests local
antibody production in the eye [378].
The evaluation of intraocular antibody titers has been addressed several times since the
middle of the last century and has been applied to various infections. In human medicine,
toxoplasmosis plays an important role here [337,339,343,345,350,351,357,379,380,398–402],
as well as viral infections [345,358,380,403,404]. The detection of intraocular antibody
production for evidence of intraocular infection is easiest when intraocular antibody titers
are higher than those in the corresponding serum samples, because in this case, intraocular
antibody production is definitely present [313,395]. Otherwise, the intraocular titers could
not be higher as in serum samples of the same individual. If the antibody titer in the eye is
greater than the antibody titer in the blood, there may be intraocular antibody production,
but it could also be a breakdown of the blood–ocular barrier and leakage of antibodies from
the blood into the intraocular fluids. In this case, therefore, the protein content or another
parameter in the paired aqueous humor and serum samples must be taken into account
and set in relation to the respective antibody titer.
One possibility is to determine the total protein content in both samples [395]. Al-
though the protein fractions have a different composition in the eye compared with the
serum, the total protein value is still useful for calculations and is the simplest, cheapest,
and fastest method which is easily available everywhere. Instead of total protein content,
albumin content [344], total immunoglobulin content [335,343,395,400,403,405], total IgG
content [344,350,380,401,404,406,407], or antibodies against another pathogen in the intraoc-
ular sample and in the serum can be used as reference [344]. Antibodies against the second
pathogen must be present in the serum. If the ratio of the antibody titer in the intraocular
sample to the antibody titer in the serum (titer intraocular/titer serum) is at least fourfold
higher for the etiologically relevant pathogen in the eye than for the other pathogen, an
intraocular antibody production is also proven. The most commonly used reference for
antibody titers (specific IgG) is either the total globulin content or the total IgG content, if
the laboratory techniques are available (e.g., electrophoresis or (in-house) ELISA tests).
7.3.1. Calculation of the Goldmann–Witmer Coefficient (GWC)

When calculating the Goldmann–Witmer coefficient (GWC), it is sometimes misleading
that the “antibody titer” should be used, but actually, the reciprocal titer should be inserted
into the formula [18,19]. If this fact is not taken into account, “C” becomes smaller and
smaller the higher the intraocular titer is. The calculation of the GWC would be correct as
follows [18,19,335,343–345,391,400,408]:
GWC = ((reciprocal titer intraocular sample)/(IgG amount intraocular sample))

/((reciprocal titer serum sample)/(IgG amount serum sample))
(1)
= (reciprocal titer intraocular sample)/(IgG amount intraocular sample)
×(IgG amount serum sample)/(reciprocal titer serum sample)
Instead of the IgG content, the total immunoglobulin content or, if necessary, the
total protein content, could be used [395]. An example of the calculation could be as
follows using the total immunoglobulin content if the intraocular titer is 1:100 and the
serum titer is 1:200, and the serum immunoglobulin content is nine times greater than the
immunoglobulin content of the intraocular sample:
GWC = ((100)/(4 g per L))/(200/(36 g per L)) = (100)/4 × 36/200 = 4.5 (2)
7.3.2. Interpretation of the GWC

In previous studies [335,343,395], intraocular antibody production was basically as-
sumed at a coefficient >1 and then, to exclude errors and inaccuracies in the calculation,
a coefficient of 3 [344] or >3 [400,404,408,409], respectively, was suggested as certain evi-
dence for intraocular antibody production. In one publication, a coefficient of >8 was even
required [189]; however, this has not prevailed. In more recent publications, intraocular
antibody production is usually assumed from a GWC >3 [337,345,350]. When using the
GWC, however, it must be noted that a high antibody titer in serum in combination with
extensive breakdown of the blood–aqueous barrier may mask a positive coefficient, leading
to a false negative result [345,400]. In addition, it should be noted that even with positive
PCR or culture results, and especially in early stages of disease [380], sometimes there is no
immune response resulting in intraocular antibodies [17–19,57,66]. Therefore, a negative
GWC does not exclude intraocular infection [380,400,410].
When 426 undiluted equine vitreous samples obtained during vitrectomies and the
corresponding serum samples were examined, the MAT titers in paired samples were
compared without further breakdown of protein fractions. In 75.9% of the MAT titers
determined parallel in serum and vitreous samples, the titer in the vitreous was higher
than in the serum, so that intraocular antibody production was certainly present. In
10.3%, the MAT titers in the serum and the vitreous sample were the same which, with the
average 10-fold higher total protein content in the serum, also supports intraocular antibody
production, but would have to be verified in individual cases (e.g., with significant vitreous
opacification) by calculation of the GWC. Only in 13.8% of the paired samples was the titer
in the vitreous lower than in the serum. The mean MAT titer was 1:245 in the serum and
1:1680 in the vitreous sample [18] (Supplementary Material S6, Tables S4 and S5).
With 46 randomly selected corresponding vitreous and serum samples, electrophoresis
was additionally performed. In 3 of the 46 paired samples, MAT was negative in the serum
and positive only in the vitreous sample, so that intraocular antibody production was
definitely present. In 7 of the 46 samples, MAT was negative with the vitreous material
and positive only in the serum sample. Thus, no intraocular antibody production was
present here. In the remaining 36 paired samples, an MAT titer ≥1:100 was detectable in
both serum and vitreous, allowing calculation of the GWC. In 35 of the 36 paired samples
(97%) the GWC was >3; in 34 of the 36 paired samples (94%) the GWC was also >8;
and in 12 of the 36 paired samples (33%) the GWC was at least in the three-digit range,
once even at 1792 [18] (Supplementary Material S6, Table S3). Thus, MAT titers are often
several times higher in intraocular samples than in the corresponding serum samples
and intraocular antibody production is regularly supported by demonstration of high
Goldmann–Witmer coefficients.
7.3.3. Misleading Interpretation of the GWC in Recent Publications on Equine Ophthalmology

In many recent papers describing the examination of equine intraocular samples,
it appears that the γ-globulin fraction, the total globulin fraction, or the total protein
value or any other reference value were not considered for the evaluation of antibody
titers in the intraocular samples and the serum samples. The GWC is often cited, and the
original paper is also referred to sometimes. The GWC and the so-called “C-value” are
used synonymously. However, the calculation of the coefficient is not correct; simply, the
intraocular titer is divided by the serum titer in each case [4,14,68,137,292,307,316,411]. The
major problem with this simplified calculation is that it does not take into account the
protein content, which in eyes affected by ERU is, on average, only 10% [18,19,391] or even
less [390] of the protein content of the corresponding serum samples. Therefore, the result
of this “C-value” does not equal the GWC. If the GWC is calculated correctly, intraocular
antibody production can often clearly be detected even if the intraocular titer is lower than
the serum titer (Supplementary Material S6, Table S3). By calculating the “C-value” without
considering another reference value in the serum and intraocular fluid, the GWC is led
ad absurdum.
The citations given for a calculation of the GWC, or “C-value” are often inappropriate
or even wrong. In [14], for example, [412] is cited; in [410], however, no antibody deter-
minations were performed in the intraocular samples. In [292], [67] is cited, but in [66],
however, no antibody determinations were performed with the serum samples. For the cal-
culation of the “C-value”, reference is often made to other studies in which the calculation
is also not described, and further reference is made to other and often inappropriate papers
(Supplementary Material S7, Figure S30). The impression arises here that some things have
been adopted uncritically, too superficially, or even incorrectly from other studies, without
the authors having thoroughly explored the GWC.
Finally, the “C-value” is considered “suggestive” or “suspicious” for intraocular
antibody production in some papers with C > 1 [292,316]. In fact, however, the intraocular
titer cannot be due to leakage from the blood at all if it is higher than the serum titer.
Some publications consider C > 3 [292,316], C ≥ 4 [14,68], or even C > 4 [4,137,307] as
confirmatory for intraocular antibody production. For this interpretation of the “C-value”,
some papers are cited in which a consideration of the total immunoglobulin content or
total IgG content is correctly described for the calculation of the GWC (e.g., [344,350,395]).
The incorrect calculation of the GWC, in addition to the disease stage in which the sampled
eyes were assayed not always being suitable (“endstage”), may be decisive reason why
intraocular antibody production could only be detected in relatively few cases.
7.3.4. Application of the GWC

Correct calculation of the GWC, when sampling from appropriate eyes and using a
reference parameter in the serum and intraocular sample, reveals intraocular antibody
production in the vast majority of paired samples from horses affected with ERU. The
clinical diagnosis of “ERU” is very clear in most cases; therefore, a preoperative aqueous
humor examination is not routinely required prior to vitrectomy when the risk/benefit ratio
is considered but should only be performed when history and ophthalmologic findings do
not clearly indicate ERU. In case of a preoperatively performed aqueous humor analysis,
the GWC may be applied. Typically, however, these are eyes in a very early stage of
disease, in which the GWC can be (falsely) negative despite the presence of the pathogen.
Therefore, history, ophthalmologic findings, and GWC should not be evaluated in isolation,
but always as a whole. In addition to antibody tests, PCR should be performed with the
aqueous humor sample (Section 7.5), and culture of the pathogen (Section 7.4) may even
be attempted.
7.4. Cultural Detection of Pathogenic Leptospira spp.

The detection of the pathogens in eyes affected by ERU was only sporadically success-
ful (Section 3); therefore, the opinion had gained acceptance that leptospires are involved
in the etiology of ERU, but that the recurrent inflammatory episodes and the anti-Leptospira
antibodies present in intraocular specimens could no longer be explained by the presence of
the pathogens. Thus, frustrating culture attempts were abandoned, or at least not reported,
for some time.
Gaining convincing evidence of intraocular leptospiral infection was not possible
until the establishment of vitrectomy in horses. With the vitreous material produced as
a waste product of this operation, and the interest and commitment of S. Brem (head of
the leptospiral laboratory in Oberschleissheim at the time), the collection and transport
of samples could be optimized by successful cooperation. By taking 2–3 mL of undiluted
vitreous material at the beginning of the vitrectomy and immediate sterile inoculation
of vitreous material into a transport medium, pathogenic Leptospira spp. were cultured
in increasing numbers of these undiluted vitreous samples [17,373,374]. One horse had
suffered 21 apparently very well treated ERU episodes over 7 years (the eye was still visual),
and in the vitreous sample from this eye, leptospires were still culturally detectable [18].
Leptospira spp. could also be cultured from vitreous samples from other horses that had
suffered ERU relapses for many months or years.
With careful indication and optimized sampling, handling, and transport of vitreous
specimens, leptospires could be culturally detected in >50% of vitreous samples [18,19,196,387].
This relatively high percentage of successful cultures of leptospires is surprising because,
at the same time, anti-Leptospira anti-bodies were found by MAT in approximately 90%
of these vitreous samples from eyes with ERU [17–19]. This means that the agglutinating
antibodies had not resulted in the elimination of leptospires from the vitreous cavity and
had not thwarted the culture of leptospires from these vitreous samples. Statistically, the
chance for a positive culture was actually even greater the more substantial the diffuse
vitreous opacity and the higher the MAT titer in the vitreous samples [18].
Since the turn of the millennium, other research groups have reported again on
studies of intraocular specimens and the successful culture of leptospires from intraocular
specimens [69,315,328,412]. The percentages at which the cultures were successful range
from 16% to 21%. One reason for the varying results may be that, in some studies, aqueous
humor samples were used for culture and the probability for a positive culture is lower
when using aqueous humor compared with using vitreous material [18]. Some of the
vitreous material obtained during vitrectomies was diluted, and gentamicin was added
to the intraocular irrigation fluid [69], which also reduces the chance for a successful
culture [45]. Additionally, the handling of the specimens, the timing, the sterile processing
throughout, especially the inoculation into the culture medium immediately after collection,
and the presence of suitable transport media for shipping play a very decisive role here,
because leptospires are quickly overgrown by other bacteria and the culture can then not be
set up further, but the positive culture is sometimes only detectable after months [374,413].
Finally, the indication for vitrectomy is handled inconsistently in different clinics and
vitrectomy is not limited to typical ERU patients in all clinics [290].
Leptospiral serovars from different serogroups could be grown from ERU eyes, with
the majority of isolates belonging to the serogroup Grippotyphosa [17–19,69,315,373,374,376]
(Supplementary Material S6, Figures S28 and S29). Clinically, there was no detectable
association between the course or severity of ERU and any particular serovar [18,19].
Therefore, the serovar causative of ERU appears to be determined primarily by the location
of the horse and the vectors present there [58,59]. Uveitis has also been reported in humans
following infections with various leptospiral serovars [89,90].
The demanding and expensive culture of leptospires, which often only yields a pos-
itive result after months, has become replaced in practice by PCR. Only for scientific
questions, or if the owners want a stock-specific vaccine (Section 9), is the leptospiral
culture still requested.
7.5. PCR
With the establishment of PCR for pathogenic Leptospira spp., another test method has
been available since approximately the turn of the millennium, which is offered by almost
all laboratories and is more economical than culture. In addition, sample handling and
shipping are less complicated than for a culture of leptospires. PCR has proven to be a
very sensitive and specific testing option for intraocular specimens [337,345,350,351,414]].
Finally, the results for PCR are available much faster and very small sample volumes are suf-
ficient for an investigation. PCR has therefore significantly increased the informative value
of the examination of intraocular samples in both human [337,346,351,352,356,360,366,407]
and veterinary medicine [19,349,415] over the past decades.
In human medicine, the significance of PCR may be higher or lower than GWC de-
pending on the type of infectious uveitis, the stage of disease, and the patient’s immune
response. In immunocompromised human patients, PCR is superior, especially in diagnos-
ing viral infections [416]. In toxoplasmosis, on the other hand, the detection of intraocular
antibody production often plays a decisive role [416]. However, also in toxoplasmosis,
other factors such as the age of the patients, immune status, duration since the onset of
symptoms, and ophthalmologic findings influence the results of intraocular fluid sample
examinations [417]. Especially in early disease stages, PCR may be more reliable than GWC.
In principle, however, it is advisable to perform both the PCR and calculate the GWC in
order to detect an infection as reliably as possible [337,345,350,402].
With the development of PCR for the detection of 16S rRNA and the LipL32 gene
of pathogenic leptospires, this meant a great improvement not only for the diagnosis of
human leptospiral uveitis [91,94,207,327,418,419], but also for ERU. The demanding and
often not very successful culture of leptospires had been replaced by PCR in practice
and resulted in many positive detections in intraocular samples from horses suffering
from ERU [16,66,68,69,195,199,328,369,412]. In approximately 70% of undiluted vitrectomy
specimens from ERU eyes, LipL32 genes or 16S rRNA were detectable by PCR [19,198,199].
In horses with ERU, PCR is not as sensitive as antibody detection in intraocular
samples. Nevertheless, in some intraocular samples, only PCR reacts positively [19,57,66];
thus, PCR should be performed at least as a supplement if antibody tests are negative.
7.6. Comparison of Laboratory Results of Vitreous and Aqueous Humor Samples

Antibody titers in aqueous humor and vitreous samples are not significantly different
from one another [18,221,222,420]. For the detection of intraocular anti-Leptospira antibodies
in samples from eyes affected by ERU (about 90% positive), aqueous humor is easier to
obtain and is of equal value to a vitreous sample.
Both cultural detections of leptospires and PCR detections of the LipL32 gene or 16S
rRNA are slightly less sensitive when examining aqueous humor as compared with vitreous
samples [18,19,369], but diagnostic vitreous sampling would be disproportionately risky
because the additional increase in information is not very large. In addition, PCR often
yields positive results even when testing aqueous humor [18,19,222].
Therefore, the examination of aqueous humor is usually sufficient to diagnose ERU.
In human medicine, it is also recommended to examine aqueous humor at least initially,
because even in cases of posterior locations of inflammation, aqueous analyses often reveal
an infectious cause [337,345]. For viral infections, it has also been described for PCR that
aqueous and vitreous humor give comparable results [421].
Only in very rare cases, both antibody tests and PCR results of aqueous humor from
ERU eyes are negative for leptospires. It is possible that in individual cases, the examination
of a vitreous sample would then have yielded a positive result. If all tests are negative with
aqueous humor, the eyes typically are still normal ophthalmoscopically (if there are clear
findings in terms of ERU, examinations of intraocular specimens before vitrectomy would
not be indicated anyway—Sections 4.1 and 7.1). In these still largely healthy eyes, another
consequently treated ERU episode is not expected to have vision-threatening consequences
(Section 5.1). Especially in these ophthalmoscopically healthy eyes, taking a vitreous sample
would not be justifiable with regard to the associated risks.
7.7. Ultrastructural and Histological Examinations of Vitreous Specimens

Vitreous material obtained during vitrectomies was processed for electron microscopy
in a few studies. Transmission electron microscopy images reveal cells and vitreous struc-
tures from eyes affected by ERU [422,423]. These studies also succeeded in detecting
leptospires in the vitreous samples [424]. When comparing the Leptospira spp. that had
been present in eyes spontaneously infected with ERU and the Leptospira spp. from culture
that had been experimentally injected into the eyes of euthanized horses, it was morpho-
logically striking that only the leptospiral structures from the ERU eyes were coated by
a granular layer (Supplementary Material S8, Figure S31). This layer was suggested as
“masking” the bacteria from the immune system, possibly with host proteins.
In another study, phagocytosed leptospires were also visualized by transmission
electron microscopy [425]. In this investigation, numerous very dense and roundish
structures were present, which are not commented on in the publication (Supplementary
Material S8, Figure S32). These round dense structures are morphologically very similar
to the in vitro biofilm formation of leptospires and were exclusively detectable in vitreous
samples from eyes affected with ERU; therefore, they were subsequently also discussed as
biofilm structures. An older scanning electron microscopy image of a spherical structure
with individual fibril-like structures protruding from it, some of which have hooks at the
ends typical of leptospires, was retrospectively also discussed as a mature Leptospira biofilm
structure [57,426].
Histological examination of enucleated eyes affected by ERU demonstrated that ERU
leads to lympho-plasma cellular inflammation and the formation of lymphoid
follicles [100,101,114,397]. B cell clusters have been detected in the center of these folli-
cle [5,143,175], and the single-layered nonpigmented ciliary body epithelium contains
numerous plasma cells, among others [427]. The lymphoid follicles and plasma cells
are not present in healthy eyes and explain local antibody production in the eye. The
ciliary body epithelium also contains hyaline membranes, the microscopic findings of
which, along with the staining characteristics, indicated that the hyaline membrane has
ambiguous morphologic features with qualities of both amyloid and collagen (12–13 nm
filaments, Congo Red staining, spikes at 65 nm intervals, and blue staining with Masson’s
trichrome stain) [427]. In another study, these membranes were classified as amyloid [224]
(Supplementary Material S8, Figure S33).
Histological examinations of vitreous material are very rare. In “ERU-end-stage
eyes” [370], the vitreous body is at least significantly altered or, as a result of phthisis,
hardly or not at all present; thus, in these eyes, an examination of the vitreous body is not
possible. In vitreous samples obtained during vitrectomies, amyloid has been visualized in
the dense membranous floaters (Supplementary Material S8, Figures S34 and S35), which
could be classified as AA amyloid by immunohistochemistry [428]. The fact that amyloid
is detectable in the dense vitreous deposits in ERU eyes explains that these deposits are
not resorbed, but increase over the course of ERU and become progressively visible on
ophthalmoscopic examination during the clinically and ophthalmoscopically inflammation-
free interval. In a recent study, for the first time, Leptospira spp. could also be clearly and
reproducibly shown by immunohistochemistry in vitreous specimens obtained during
vitrectomies [57] (Section 8.3). However, immunohistochemistry is unsuitable for routine

diagnosis because it is extremely time-consuming and unreliable.
7.8. Cells and Cell Dynamics in Vitreous Specimens from ERU Eyes
In the mid-20th century, it was found that intraocular anti-Leptospira antibody titers
measured by MAT decrease during the inflammation-free interval between uveitis episodes,
and an acute ERU episode leads to an increase in intraocular antibody titers [115]. This is
consistent with the fact that cytology studies of 74 undiluted vitreous specimens obtained
during vitrectomies have shown that cell density in vitreous specimens is highest on the
8th to 16th day after acute uveitis and significantly decreases approximately 2 months after
an acute episode [429,430].
However, even in the inflammation-free interval, there are always more cells present
in eyes affected by ERU than in healthy eyes. The clinically quiet phase therefore does not
mean an immunological quiet phase, but only a subclinical chronic inflammation. It has
been suggested that the events between acute ERU relapses are controlled by T suppressor
cells, which is clinically interpreted as an “inflammation-free interval” [429,430].
Independently of the time of sampling, the cell density in the vitreous samples has
also been shown to correlate positively with the ophthalmologically determined degree
of diffuse vitreous haziness [429,430]. Differentiation of cells in uvea tissues and vitreous
samples from ERU eyes revealed that there were many lymphocytes and plasma cells in
both the uveal tissue [134,143,200] and vitreous samples. In the vitreous samples, further
characterization of the lymphocytes was repeatedly performed [169,193,431]. The presence
of TH-1 cells, which increase the effectiveness of phagocytosing and cytotoxic cells [134], is
also compatible with chronic intraocular leptospiral infection. In addition, autoreactive T
cells are always present, which are activated by inflammatory reactions during infections
(“bystander activation”) [210].
In eyes with high-grade vitreous haziness, it was noticeable that almost as many
macrophages were present as lymphocytes. Plasma cells were seen relatively frequently in
low-grade diffuse vitreous opacification. Cytological examination of vitreous samples was
performed both after cytocentrifugation and after the embedding of macroscopically visible
vitreous floaters. Regardless of the degree of vitreous haziness and the degree of vitreous
floaters, the percentage of lymphocytes was significantly higher after cytocentrifugation
(cytological examination) than in the histological examination. In contrast, the percentage
of macrophages and plasma cells was significantly higher in the histological examination
than after cytocentrifugation. It can be concluded that lymphocytes are mainly contained
in the liquid phase of the vitreous, macrophages, and plasma cells rather than in the
denser vitreous deposits which are visible ophthalmoscopically, and after vitrectomy, also
macroscopically in the samples. These findings may indicate that leptospires primarily
reside in the denser vitreous deposits [429,430].
7.9. Neutrophil Extracellular Traps (NETs)

Another phenomenon detected in vitreous samples from eyes affected by ERU is the
presence of neutrophil extracellular traps (NETs) [432]. NETs are externalized DNA fibers
from neutrophils which contain, among other substances, cytoplasmic proteins, chromatin,
and antimicrobial peptides [433,434]. Scanning electron microscopy revealed that NETs
contain smooth stretches with a diameter of 15–17 nm, globular domains of around 25 nm,
and aggregated fibers with diameters of up to 50 nm [435].
NETs are able to entrap and kill bacteria [435] and other pathogens [436]. NETs are
believed to be part of the innate immune system [437]. They can occur in all organs and are
abundant at sites of acute inflammation [438].
NETosis refers to an active cell death of neutrophils, which perish with the release
of NETs (“active cell death”) but can still kill bacteria through the excreted NETs even
after their cell death [439]. However, it has been shown that neutrophils can also form
NETs without dying in the process [440]. Some neutrophils only secrete mitochondrial
DNA, sometimes but not always resulting in cell death [441,442]. Meanwhile, distinctions
are made between “suicidal NETosis”, “vital NETs”, and “mitochondrial NETs” [441].
Components of NETs are non-specific, and a variety of stimuli can induce NET-release [443].
Among others, infections (e.g., presence of LPS) and pH changes (e.g., in inflammation)
can induce the release of NETs and NETosis [438].
The antimicrobial potential of NETs is positive for the host. However, because NETs
can also cause inflammation and tissue damage [441], not every minor milieu change or
small amount of LPS should lead to the release of NETs [440]. The formation of excessive
NETs and their tissue-damaging effects, caused both directly and via accompanying in-
flammatory reactions, have also been linked to the development of various autoimmune
diseases [438,441,443,444].
Most impressively, it has been shown that neutrophils can sense the size of microbial
antigens. For smaller antigens that can be phagocytosed, tissue-damaging NET formation
is suppressed. In contrast, for larger pathogens that cannot be phagocytosed, NETs are
released [436]. If, as suspected, the roundish structures on the electron micrographs of vit-
reous specimens from eyes affected with ERU (Supplementary Material S8, Figure S32) are
indeed leptospiral biofilm, these structures would undoubtedly be too large for phagocyto-
sis (“frustrated phagocytosis”). This would perfectly explain the NETs in the eyes affected
by ERU. In addition, NET formation promotes an increasing inflammatory response, the
inflammation attracts further leukocytes, and this, in turn, promotes tissue damage [445].
NETs, on the other hand, can also counteract inflammation and tissue damage.
They eliminate pathogens and serve as a physical barrier that not only prevents fur-
ther spread of bacteria or other pathogens, but also prevents tissue-damaging substances
(e.g., proteases) from making contact with the host tissue [435]. In the areas of the con-
junctiva and cornea, aggregated neutrophil extracellular traps prevent inflammation on
the neutrophil-rich ocular surface [434,446]. In corneal infections with biofilm-forming
Pseudomonas aeruginosa spp., NETs separate the biofilm from healthy tissue, for example,
and thus work against further expansion of the infection [447]. Possibly with respect to
the delicate balance in ocular immunity, “ocular neutrophils” are supposed to be less
inflammatory compared with neutrophils in the blood [446]. This may explain why there
are clinically inflammation-free intervals in ERU despite the presence of Leptospira spp. and
NETs in the vitreous cavity.
Another interesting point is that the biofilm formation of bacteria and the formation
of NETs promote one another. The bacteria “barricade” themselves in the biofilm, to be
protected from the host immune system. The neutrophils, on the other hand, produce NETs
to create a barrier between the biofilm and host tissue [447,448]. Fibrous material such as
NET and fibrin is suggested to prevent the clearance of bacteria by phagocytic cells, and
thus promotes the growth of bacterial clusters or biofilms. Fibrin and NETs are discussed
to be important for the recurrence and chronicity of otitis media in children [449].
7.10. AA Amyloid
Serum amyloid A (SAA) is generally an indicator of chronic inflammation and infec-
tion, and its determination in blood is now part of routine diagnostics. In horses, SAA
is considered to be the most sensitive and the only major acute-phase protein. The SAA
content can increase by a factor of up to 1000 in the case of tissue trauma [450]. SAA
increases within a few hours and also decreases rapidly when the inflammatory response
decreases [451]; therefore, SAA measurements are an excellent method to quickly check the
response to a therapy or antibiotic, for example.
In horses suffering from ERU, it has been shown that the eye disease has no effect on
the SAA content of the blood [452–454]. In a recent study, this fact was also described for
joint infections in horses [455]. Thus, local infection at immunoprivileged sites does not
reliably affect blood SAA levels.
In contrast, examinations of intraocular samples showed that the SAA content in the
vitreous samples obtained during vitrectomies of horses suffering from ERU is signifi-
cantly increased and the level of MAT titer in the vitreous samples correlates significantly
positively with the SAA content. In horses with leopard coat pattern uveitis, in which
anterior uveitis dominates the clinical picture, an increased SAA content was detectable,
especially in aqueous humor, but hardly in vitreous samples [453,454]. On the other hand,
a significantly increased SAA content in vitreous samples indicates an ERU. Thus, unlike
the comparable results in aqueous humor and vitreous samples when antibody titers are
measured, SAA does not appear to yield comparable results in both compartments. The
concentrations of SAA in eyes exhibiting non-uveitic related changes (e.g., keratitis, tumors,
and ERU partner-eyes) were generally below the detection limit [453,454].
Sustained overproduction of SAA is a prerequisite for the development of AA amyloido-
sis [456]. AA amyloid has been detected in intraocular specimens from ERU eyes—both in
the ciliary body area and in the “snowbanks” located on the ciliary body [224,427,457], and in
the dense vitreous floaters of ERU eyes [428] (Supplementary Material S8, Figures S33–S35).
AA amyloidosis can have different etiologies. A major cause is chronic infection [458,459].
The amyloid deposits that develop as a result of chronic infections may occur generalized or
localized [459]. In generalized AA amyloidosis, various organs are often affected (e.g., the
kidney, liver, spleen, and gastrointestinal tract) and renal insufficiency is a particularly
feared consequence in humans [456,459]. The most effective therapy is to eliminate the
cause of chronically elevated SAA levels because treating the underlying disease can lead to
subsequent reductions in acute-phase protein production, including the circulating serum
SAA levels. However, there are no common therapeutic strategies for AA amyloidosis due
to its diverse etiology [458].
There is no evidence of generalized amyloidosis in horses affected with ERU. AA
amyloid has exclusively been detected intraocularly to date [428]. Intraocular amyloid for-
mation also explains why, although diffuse opacification decreases in the inflammation-free
interval of ERU, the cloudy or membranous vitreous floaters which are visible ophthalmo-
scopically increase over time and do not resolve (Supplementary Material S1, Figure S13
and Supplementary Material S8, Figures S34 and S35).
These ophthalmoscopically visible vitreous floaters, which can most reliably be seen
using a direct hand-ophthalmoscope, provide strong evidence of ERU in non-leopard coat
pattern horses during the inflammation-free interval when the eyes are otherwise still
relatively undamaged. Even if there is no anterior uveitis or its sequelae and if no or hardly
any diffuse vitreous haziness is present, these dense and therefore vitreous floaters which
appear dark are easy to recognize in the ophthalmoscopic view [18,28,217].
However, in chronic infections, amyloid does not exclusively result from persistently
elevated SAA levels. Chronic infections are often associated with biofilm formation. Various
biofilm-forming bacteria, especially enterobacteriaceae such as Escherichia coli [460], are able
to produce microbial amyloid themselves. Amyloid is found in many naturally occurring
biofilms, and microbial amyloid can provide a support scaffold for biofilms [461]. In fact,
amyloid fibers are actually found in most naturally occurring biofilms [462]. Microbial
amyloid can occur in the form of fibrils and, in the case of aggregated fibrils, also in the form
of fibers [462,463]. These functional amyloid fibers (FAPs), which include “curli fibers”,
support the binding of bacteria to host proteins and promote biofilm formation [461,464].
In addition, the FAPs protect the bacteria in the biofilm from phagocytosis [465].
No FAPs were seen in in vitro studies of Leptospira biofilm formation [466], and to
the best of the authors’ knowledge, nothing is yet known about whether Leptospira spp.
are able to form FAPs in vivo. Independently, however, leptospires in the vitreous cavity
might take advantage of intraocular AA amyloid generated by immune reactions for their
biofilm scaffold. The amyloid fibrils and fibers, similarly to the vitreous collagen fibrils
and fibers, could serve as surfaces for the attachment of Leptospira spp. and thus promote
further biofilm formation as well as provide protection from the host immune system.
7.11. Samples from Healthy Eyes

Samples from clinically and ophthalmoscopically healthy equine eyes usually do not
show any signs of intraocular infection with Leptospira spp. [17,18,45,195,199,314]. Systemic
leptospirosis typically leads to clinical manifestations in the sense of ERU only after months
or years; therefore, evidence of leptospiral infection can be expected in individual cases
even in clinically healthy eyes. The incidence of ERU in some regions of Germany was
8–10% [9,10]; thus, in individual cases positive MAT and/or PCR results in intraocular
specimens from apparently healthy eyes can be expected if enough specimens are examined.
In individual studies, specimens from anamnestically and ophthalmoscopically healthy
eyes or in eyes that had disease other than uveitis were examined. An examination of
specimens from 366 healthy eyes failed to detect anti-Leptospira antibodies by MAT in any
of the intraocular specimens [122]. In another study, samples from 168 healthy eyes were
examined and anti-Leptospira antibodies were detectable in only 1 sample (by both MAT
and in-house ELISA). Culture yielded a negative result in all 168 sets. A total of 120 of these
samples were additionally tested for 16S rRNA by PCR, and in this assay, 6 of the samples
(5%) reacted positive. Control samples from 14 ERU-affected eyes all had anti-Leptospira
antibodies by both MAT and in-house ELISA, and with 8 of the 14 samples (57%), the
culture was positive [386,387].
In another study, no culture was attempted, but no MAT titers of 1:100 or higher were
obtained in any of the 120 intraocular samples (100 vitreous samples and 20 aqueous humor
samples), whereas a positive PCR result (detection of the LipL32 gene) was obtained in only
1 of the 120 intraocular samples [199].
The samples from healthy eyes were taken for ethical reasons only after euthanasia of
the [467,468] horses or after enucleation (for another reason than ERU); thus, the eyes could
not be observed further. It is therefore unknown whether the eyes from which the samples
with positive MAT or PCR results were taken would have developed ERU in the course of
the following months and years, or whether it had been an asymptomatic infection.
8. Bacterial Biofilm
Individual (planktonic) bacteria are relatively unprotected and vulnerable to an attack.
When it becomes “uncomfortable” for the bacteria, bacterial aggregates form, which can
barricade themselves in a matrix and thus survive under adverse environmental conditions,
both inside and outside a host. Biofilm is generally defined as “an aggregate of microbial
cells surrounded by a self-produced polymer matrix” [469]. There are both mono- and
polyspecies biofilms. Both in low-nutrient environments and in the presence of unfavorable
factors harmful to bacteria (e.g., pH, low temperature, lack of nutrients, oxygen tension,
UV light, antibiotics, host immune system, etc.), the biofilm represents an effective pro-
tective barrier [470]. Biofilms not only confer protection, but also exhibit a potential for
homeostasis, thereby reducing maintenance energy [460]. The in vivo biofilm can also
harness certain immune responses of the host and integrate them into the biofilm scaffold
(e.g., NETs and fibrin fibers) [449].
Bacteria in biofilm are relatively tolerant to antibiotics which is not only due to the
mechanical barrier that the biofilm represents; the ability of the antibiotics to diffuse through
the biofilm depends largely on the particular bacterial strain, the type of antibiotic, and the
growth conditions of the biofilm [471,472].
Depending on the pathogen species and the type of biofilm, it could be shown that
the bacteria, as long as they are hidden in the biofilm, have a tolerance to antibiotics up to
1000 times higher than the same planktonic pathogens [473,474]. After detachment from
the biofilm, the planktonic bacteria again exhibit normal sensitivity to antibiotics. For this
reason, the term “tolerance” is preferred for bacteria in the biofilm, because the planktonic
bacteria, after leaving the biofilm, exhibit the original sensitivity to antibiotics again and
have thus not developed “resistance” [475].
The first observations of biofilms, even though this term did not exist at the time, were
described in 1684 by Anthony van Leeuwenhoek for dental plaque [476,477]. The term
“biofilm” has been used for about 60 years [476]. Reports of aggregates of pseudomonads
classified as biofilm in cystic fibrosis (CF) patients [478], initial [479] and later updated [480]
definitions of biofilm, and implications of biofilm formation have indicated the importance
of infections associated with biofilm formation.
The growth of bacteria in biofilm initially appeared to be tied to surfaces, but it
was later shown that attachment to organic or inorganic surfaces is not an absolute re-
quirement for biofilm formation [477]. Biofilms can harbor different infectious agents
simultaneously [481]. Interestingly, bacteria are able to adjust their metabolic processes to
their environment. Bacteria within a biofilm show changes in gene expression, resulting
in phenotypic heterogeneity within the biofilm [467,482], which can be interpreted as a
specialization or division of labor [483].
For the regulation of processes in biofilms, cell-to-cell communication (quorum sens-
ing, QS) takes place [482,484,485]. QS is made possible by signaling molecules, which are
secreted to coordinate community behaviors and to defend against unfavorable environ-
mental conditions [467,468]. Within the biofilm scaffold are water-filled channels through
which nutrients, metabolites, and other secreted substances can be transported [466,485],
and through which QS is performed [483–485]. QS and the resulting adaptation of the
secretion systems of bacteria, as well as their expression levels of genes, are important
for cells to adjust to their environment. In this way, the biofilm matrix can become more
impermeable to antibiotics [468,472,486,487].
For an increasing number of infections, it has been recognized that biofilm forma-
tion of the causative pathogens is a major problem for sustained successful therapy; of-
ten, the infection appears eradicated with antibiotic treatment, but shortly after discon-
tinuing therapy, the infection may recur because bacteria have survived in the biofilm.
For infections, biofilm formation was defined as “A coherent cluster of bacterial cells
imbedded in a matrix—which are more tolerant to most antimicrobials and the host de-
fense, than planktonic bacterial cells” [476]. It is now believed that almost all bacteria
can form biofilms [484,488] and that 60% of all infections and 80% of chronic infections
in humans are biofilm-associated [485,489]. Biofilms in dental plaque [490–492] and in
CF [385,482,493,494] have been particularly well investigated.
Other biofilm-associated diseases include wound infections [495,496], infected im-
plants and bone sequestrum formation [497], osteomyelitis [498–500], chronic otitis [449,501],
infectious endocarditis [502], and borreliosis [503–505]. Whenever possible, mechanical or
surgical removal of the biofilm is considered the most effective therapy to eliminate the
infection [480,506,507]. When mechanical or surgical removal is not possible, it is critical
for successful therapy to know not only the environmental and in vitro biofilm formation,
but more importantly, the in vivo biofilm formation and microenvironment of the infection,
because the differences can be significant and crucial for successful therapy [508,509].
Depending on the phase of biofilm formation, different treatment strategies have been
proposed [510], such as substances dissolving the biofilm matrix [489,511] or disrupting
QS within biofilms [512–514]. Other approaches to attack biofilms include ultrasound-
targeted microbubble destruction under antibiotic administration (UTMD) [515], targeting
extracellular polymeric substances (EPS) (inhibiting EPS production or degrading EPS), or
the use of bacteriophages [468].
8.1. Steps of Biofilm Formation and Dissemination of Infection

After bacteria have come into contact with a surface or with other bacteria and irre-
versible adhesion has occurred, cell division begins so that aggregates of many bacteria are
formed. At the same time, EPS are secreted, which initially form a young biofilm matrix,
and later also constitute a major part of the biofilm matrix. The biofilm matures further to a
three-dimensional and well-organized entity. By means of QS, the biofilms mature in such
a way that cell density and composition of the matrix are adapted to the respective envi-
ronment of the bacteria. The matrix is continuously adapted to changing environmental
conditions by QS [460,475,483,485,509,516]. Some bacteria stabilize the biofilm scaffold by
self-produced amyloid fibers (curli fibers) (Section 7.10). In addition, fibrin fibers, NETs
and other structures can be integrated into the biofilm scaffold (Section 7.9).
The host’s immune defenses often struggle unsuccessfully to eliminate the bacte-
ria and biofilm. Antibiotics are also often unable to completely eradicate the infection.
However, even if the elimination of the infection does not succeed (e.g., “frustrated
phagocytosis” [383,445]), phagocytic enzymes are released, causing tissue damage around
the biofilm [480]. Spread of infection within an organ or throughout the host can occur
further along, both by individual planktonic bacteria emerging from the biofilm and by
shedding of biofilm parts [480].
One cause that leads to the release of bacteria from the biofilm or the detachment of
parts of the biofilm is an accumulation of metabolites in the biofilm which are toxic to the
bacteria within the biofilm. At some point, this leads bacteria within the biofilm to start the
self-dissolving program by secreting EPS-digesting hydrolases [468]. In particular, areas
located at the periphery of the biofilm appear to break open after bacterial proliferation and
production of saccharolytic enzymes, contributing to dissemination of the infection [485].
External influences that are favorable to the bacteria, such as a rise in temperature [517] or a
more nutrient-rich environment [518,519], are also perceived by the bacteria in the biofilm
and can trigger the release of bacteria from the biofilm. The planktonic cell–biofilm cycle
explains the chronic recurrent course of inflammation in biofilm infections [468].
Considering the difficulties in eliminating biofilm-associated infections, prophylaxis is
of great importance. In this context, the prophylactic administration of antibiotics during
certain operations appears in a different light, because the infectious agents can be elimi-
nated at an adequate level of action before biofilm formation can even begin [475,511,520].
8.2. Characteristics of Biofilm-Associated Diseases

Various characteristics of biofilm infections have repeatedly been described. The
following points are particularly significant [385,508,521,522]:
• The direct examination of infected tissue reveals bacteria living in cell aggregates or
microcolonies surrounded by an extracellular matrix;
• The infection is generally confined to a specific location or organ;
• The infection is impossible or difficult to eliminate using antibiotics, to which the
responsible organisms are sensitive when in their planktonic or free-living state;
• Often, no organism can be cultured despite a strong presumption of infection with the
pathogen of interest;
• Immune responses are ineffective, as evidenced by bacterial aggregates surrounded
by inflammatory cells within host tissue.
In CF, the detection of specific IgG antibodies in the blood has been shown to be useful
in the diagnosis of pneumonia, whereas the detection of specific IgA antibodies in the secre-
tion has been shown to be valuable in the diagnosis of sinus infection [384,494]. Similarly,
the detection of specific IgA antibodies in specimens from eyes affected with ERU is particu-
larly sensitive for the detection of chronic intraocular leptospiral infection [194,195]. Apart
from intraocular infections, chronic leptospiral infections have been described, especially
for proximal renal tubules, and have also been associated with biofilm formation [523–526].
In cattle, the detection of urinary IgA specific for leptospires has recently been described as
a “hallmark” to identifying reservoir hosts [527].
Further characteristics depend on the particular pathogen and the localization of the
infection. In CF, for example, alginates have been described as a particularly immunogenic
component of the Pseudomonas aeruginosa biofilm. In contrast, studies of a Leptospira biofilm
grown in vitro could not identify alginates as a major component [466].
Biofilm-associated infections cause chronic inflammation in the tissue surrounding
the biofilm and can also initiate autoimmune responses [528–531]. Inside the eye, any
disturbance of the immunologic balance leads to autoimmune reactions anyway. Thus,
the intraocular biofilm formation of leptospires is a good explanation for the infection-
associated autoimmune phenomena detectable in ERU.
8.3. Biofilm Formation of Leptospira spp.

Biofilm formation by leptospires has gained interest for about 20 years. Leptospira spp.
have been detected in dental care unit pipes [532], and it was found that Leptospira inter-
rogans serovar Canicola can survive in distilled water for more than 3 months, whereas
in a viscous medium, they can survive for over three times as long—whereby the viscous
medium had clearly favored the formation of cell aggregates within 48 h [533].
Biofilm formation was observed in vitro in distilled water (low-nutrient medium) in
different Leptospira interrogans strains [519], and morphologically demonstrated in different
serovars and strains of pathogenic Leptospira spp. [534]. Meanwhile, further details of the
morphology and composition of the leptospiral biofilm formed in vitro (Leptospira interro-
gans serogroup Pyrogenes serovar Manilae) are known [466]. In vitro studies of apathogenic
and pathogenic leptospires have shown that aggregates of leptospires form within 1 day,
and that biofilm formation begins within 2 days. Biofilm formations over 100 µm thick
can develop within 3 weeks [466,534]. The ultrastructure of the three-dimensional biofilm,
consisting of, e.g., L. interrogans with the organizational features therein (e.g., transport
channels), is quite impressive [466].
In in vitro biofilms of Leptospira interrogans serovar Laiit, it could be shown that after
the addition of nutrients (rabbit serum), free motile bacteria were increasingly found again
over the course of 2 h [519]. Thus, more favorable environmental conditions (here: the
addition of nutrients by rabbit serum) seem to be a signal for leptospires to leave the biofilm.
It has also been shown for different pathogenic Leptospira spp. that the bacteria within the
biofilm are more tolerant to antibiotics than the planktonic forms [535], although protection
by the biofilm appears to be less effective compared with what has been described for
Pseudomonas aeruginosa (Section 8).
In environmental multispecies biofilms obtained from rice fields, the LipL32 gene of
pathogenic leptospires was detected among other bacteria. Plant fibers in the standing
waters of rice fields are surfaces where biofilm formation is favored [481]. There are also
descriptions of environmental leptospiral biofilm formation that occurs in soil, which may,
in addition to rats and mice, contribute to the numerous human cases of leptospirosis
following floods [536,537]. Leptospira interrogans serovar Icterohaemorrhagiae can remain
infectious for at least 20 months under unfavorable environmental conditions [538], which
could be due to biofilm formation. Thus, Leptospira survival in the environment seems to
be favored by biofilm formation, which could contribute to disease transmission not only
after environmental disasters, but also among risk groups such as sewage and agricultural
workers [518,533,539].
In environmental biofilms, the coaggregation of leptospires and other bacteria has been
observed. These coaggregates have been discussed to be a key factor for the persistence
and survival of Leptospira spp. [540]. Furthermore, the observed coaggregation of different
Leptospira strains and Staphylococcus aureus might be important for leptospiral infections
via skin lesions [541].
In vivo biofilm formation of leptospires in the proximal renal tubules of chronic shed-
ders has been suspected for some time [523,534]; it has been demonstrated by electron
microscopy after experimental infections [524,525], and recently in naturally infected Rattus
norvegicus [526]. Experimental infections of mice were used to understand the in vivo
immune evasion [542]. Another experimental study demonstrated that antibiotics ad-
ministered within 1 day of infection work well, but the elimination of leptospires with
antibiotics 3 days after infection is considerably more problematic, which was linked to
biofilm formation in the proximal renal tubules [543].
Biofilm formation by leptospires as a pathogenic mechanism in ERU has been sus-
pected in recent years [21,426,544], and it could also be demonstrated morphologically [57,545]
(Figure 1). Previous work that did not yet use the term “biofilm” also showed evidence
of leptospiral biofilm formation in vitreous samples from eyes affected with ERU during
ultrastructural studies [424,425] (Supplementary Material S8, Figures S31 and S32).
Leptospirosis as a biofilm infection seems to be of great contemporary interest and has

been investigated by different research groups. In particular, biofilms produced in vivo by
leptospires need to be further analyzed and could yield interesting results. Presumably,
the composition of biofilms produced in vivo differs significantly from biofilms produced
in vitro, which may be crucial for their elimination.
Figure 1. Immunohistology (IHC) with a vitreous sample from an ERU eye. IHC was performed with
a polyclonal rabbit anti-L. grippotyphosa antibody from the Amsterdam UMC Leptospirosis Reference
Centre. The method used was described in [57]. All photographs in this figure are from different
slides from the same vitreous sample, which was PCR-positive for LipL32 and Subclade P1 (16S
RNA) and was MAT-positive (1:3200 L. grippotyphosa). (a) Single Leptospira spp., (b,c) small Leptospira
aggregates, and (d–f) large aggregates of Leptospira spp. and the extracellular matrix around and
in between the bacteria. The extracellular matrix indicates biofilm formation. Especially in images
(c,d,f) there seems to be abundant membrane blebbing. (Photographs: K.A.).
8.4. Vitreous Structure, ERU, and Biofilm

The vitreous, at least in the horse, represents an ideal site for the biofilm formation of
leptospires [426,544]:
• It consists of 98–99% water [11,546];
• Hardly any nutrients are present in the healthy vitreous body (Section 7.2);
• The collagen fiber scaffold in the vitreous body could serve as a surface, analogous to
plant fibers [481], where biofilm formation begins; the vitreous fibrils in the horse [422]
have a diameter of approximately 10–12 nm, as in other species [547], and several
fibrils can attach to each other to form fibers [546];
• Some vitreous areas are very viscous (hyaluronic acid and collagen fibers), which also
promotes biofilm formation [533];
• The equine vitreous represents an avascular “immunological niche” of approximately
28 mL [11], resulting in delayed and reduced host defense responses; thus, biofilm for-
mation can occur before the immune defense has any chance to eliminate
the infection;
• Intraocular immune privilege: the eye attempts to limit local immune and inflamma-
tory responses to preserve vision (and thus enables antigens and histo-incompatible
tissue to survive longer in the eye) [548,549], which also contributes to the delayed
and insufficient elimination of leptospires;
• As in the renal tubules, there are fenestrated capillaries in the area of the Pars plicata
of the ciliary body under the respective single-layered unpigmented and pigmented
epithelium [5], which can promote passage of the leptospires into the vitreous cavity
during hematogenous infection.
In the course of ERU, immune reactions occur, which are individually very different,
but typically lead to the invasion of cells (Section 7.8), intraocular antibody production
(Sections 7.2 and 7.3), and various autoimmune phenomena (Section 3.1). In addition to the
physiological collagen fiber scaffold, further fibrils and fibers are formed in the vitreous
body, which, in turn, can promote biofilm formation:
• Fibrin: the fibrin scaffold looks very similar to the collagen fiber scaffold of the vitreous
body by electron microscopy: see picture p. 10 in [550] (https://publications.rwth-
aachen.de/record/466223/files/466223.pdf; accessed on 5 December 2021);
• NETs (Section 7.9, vivid picture on: https://scitechdaily.com/are-overactive-immune-
cells-the-cause-of-COVID-19-deaths/; accessed on 5 December 2021);
• AA amyloid: amyloid fibrils are 8–15 nm in diameter [551], similar in thickness to
those of the vitreous scaffold, and amyloid fibrils can also assemble into fibers; the
curli fibers formed by some bacteria in the biofilm (Section 7.10) are only slightly
thinner (6–12 nm in diameter) [552].
It is conceivable that this network of fibrous material becomes integrated into the
leptospiral biofilm, making pathogen elimination increasingly impossible, similar to what
has been described for chronic recurrent otitis in children [449]. It is therefore clear why
vitrectomy is the most effective treatment option for typical ERU. Mechanical, or in this
case, substantial surgical removal of these biofilm-fiber conglomerates is the only way to
reliably eliminate the infection. In eyes, the infection is eliminated only in the context of
high-grade atrophy of the globe or phthisis; then, the space of the vitreous chamber is
considerably reduced and physiological conditions (e.g., the “immunological niche” and
healthy or viscous vitreous material) are no longer present in the eye.
9. Prophylaxis
Regarding the two decisive triggering factors of ERU, leptospiral infection and genetic
predisposition, starting points for a reasonable prophylaxis are also recognizable. Forty
years after the Royal Commission for Horse Breeding banned stallions with cataracts from
breeding, the incidence of ERU in the United Kingdom has dropped significantly [50].
Thus, with increasing knowledge of the genetic “make-up” associated with the incidence
of ERU, further breeding selection would be prudent.
In addition, attention should be paid to the prophylaxis of infection. The higher the in-
fection pressure in a herd, the higher the probability that many horses will become infected,
and later, some of these horses will develop uveitis (Section 6). Thus, if possible, control
of free-living small mammals should be implemented in the barn and its surroundings. If
possible, grazing should take place on pastures that are as dry as possible and watering
from stagnant water should be avoided in order to reduce the risk of infection.
Horses themselves can also shed leptospires after infection (Section 2), and could
therefore also be a source of infection for other horses, at least temporarily. Thus far, this
source of infection is generally considered to be of minor importance, and because of the
predominantly subclinical course of acute leptospirosis in horses, excretors would only be
detected by regular urine tests. However, this would be costly and time-consuming for
horse owners and is therefore usually not considered.
Finally, vaccination is another option [553]. No vaccine is currently available for

horses in Germany. Although a vaccine approved for dogs now covers, among oth-
ers, the two serovars most commonly found in horses in Germany (Grippotyphosa and
Bratislava [18–20]), the redesignation of the vaccine for use in horses is risky under phar-
maceutical law.
A legal option for the vaccination of horses is the production of a herd-specific vaccine.
Vaccine production would be useful if horses on a stud farm show an increased incidence
of recurrent uveitis. For this purpose, however, Leptospira spp. must first be cultured with
sample material from an animal from the farm (e.g., from small rodents or from an equine
intraocular sample). If an intraocular sample from a horse is used, the horse should have
been on the farm for at least two years before the first ERU bout.
When the culture is positive, it has to be enriched so that the production of a vaccine
is possible. Vitreous samples obtained during vitrectomies or chronically infected rodents
can be used for the culture. If the owners of the horses from a “problem stud farm” with
many horses suffering from ERU take the trouble and costs upon themselves, this approach
seems to be promising [19]. However, it is crucial that the vaccination takes place before
the systemic infection, i.e., ideally so that antibodies are present in the colostrum and the
foals are then vaccinated with the farm-specific leptospiral vaccine analogous to tetanus
prophylaxis. If the leptospires are already in the eye, or ERU attacks may even have already
occurred, vaccination is no longer useful [554].
In the United States, a vaccine for the serovar Pomona, which is dominant in many
regions here, has recently been approved [63]. Whether and how effectively this vaccination
prevents disease, whether the single serovar Pomona contained in the vaccine is sufficient,
and the level of acceptance for vaccination among horse owners, remains to be seen.
10. Discussion
After centuries in which ERU has plagued horse owners and veterinarians, a very
significant contribution to clarifying the etiopathogenesis of ERU has been made with the
confirmation of the causative chronic intraocular leptospiral infection [18,19] and, most
recently, with the demonstration of intraocular biofilm production [57]. ERU fulfils all
the characteristics of a biofilm infection, and many of the previously difficult-to-explain
characteristics of ERU are now evident:
• Clinically noticeable episodes of uveitis not until many months or even years after
systemic infection;
• Cultural leptospiral detection is difficult and has often failed;
• Remarkably, positive cultures, despite high intraocular antibody titers and (although
less frequently) antibiotic concentrations in vitreous samples that were well above the
MIC for planktonic leptospires;
• Persistence of leptospiral infection in the vitreous cavity for many years;
• Chronic recurrent inflammatory episodes at unpredictable intervals;
• “End-stage ERU” or phthisis: when no vitreous cavity is left, the immunological niche
and the infection are also eliminated, and no bacteria are detectable anymore [307,316];
• Outstanding success in terms of postoperative relapse-free outcomes after vitrectomy
(mechanical removal of the accessible parts of the vitreous is the best way to eliminate
chronic biofilm-associated infections) [480,506,507].
Although leptospires occasionally enter the anterior chamber of the eye, the vitre-
ous, with its gel-like consistency and collagen fibrils, has emerged as the seat of chronic
infection and biofilm formation. In morphological examinations of vitreous samples, the
ultrastructural formation of a biofilm is recognizable [424–426], and stages of biofilm forma-
tion in vitreous samples could be demonstrated immunohistologically (single leptospires,
microcolonies, larger dense roundish aggregates) [57]. During vitrectomy, only vitreous
material is removed, resulting in permanent elimination of the infection [16]. Both the
cultural detection of leptospires and detection of the LipL32 gene by PCR are somewhat
more
Microorganisms 2022, 10, x FOR PEER reliable
with vitreous samples compared with aqueous humor samples (Section
REVIEW 37 of 63 7.6).
A proposed etiopathogenesis of ERU is shown in Figure 2.
Figure 2. (1) Assumed infection route: humid pasture, field mice or other rodents, infection via
oral mucous membranes, conjunctiva or skin lesions of the distal limbs; infection usually clinically
inapparent [59]. (2) Hematogenous transport, possibly influenced by a high burden of Leptospira spp.
or immune-impaired horse: Leptospira spp. leave the fenestrated capillaries of the ciliary body (Pars
plicata) and settle in the vitreous chamber. The vitreous is a viscous and low-nutrient medium,
and there are collagen fibers within the vitreous body [546]. These are ideal conditions for biofilm
formation [544]. ♣ Ciliary body, Pars plicata, healthy equine eye (source: photographs courtesy
of Lisa Madlener). * Left: Vitreous material from a healthy equine eye, aspirated with a syringe
(photograph: H.G.). Right: Leptospira spp. from culture (WHO Grippotyphosa standard strain)
within vitreous fibers, transmission electron microscopy (source: photograph courtesy of Gabriele
Niedermaier). (3) Leptospira spp. adhering at vitreous collagenous fibers and sticking together,
which immediately induces the production of the extracellular matrix and represents the first step
of biofilm formation [485]. Often, “membrane blebbing” or outer membrane vesicles (OMVs) are
visible [533]. (4) Biofilm formation step 2 [485]: microcolony formation, increasing the extracellular
matrix. (5) Biofilm formation step 3 [485]: the development of dense round structures, which might
be mature biofilm complexes (arrows: possibly hooked ends of Leptospira spp.). It is unknown
how long the Leptospira spp. might stay inside these biofilm constructs, but certainly for several
months, and possibly for several years, which can be concluded from experimental and environmental
investigations [518]. # Suspected Leptospira biofilm in the vitreous samples from an ERU eye, scanning
electron microscopy (source: printed image: courtesy of H. Schoel; photograph: B.W.). (6) When an
unknown signal appears, several biofilm structures probably release planktonic bacteria at the same
time. The time-gap until the release is unknown and the signals are unknown. The coordination
that several planktonic bacteria emerge from the biofilm at the same time is probably due to quorum
sensing (cell-to-cell communication) via channels within the biofilm [466]. The released planktonic
Leptospira spp. may split and progress in different ways: Immune reactions during the “quiet” stage
after infection seem to be sufficiently suppressed by the intraocular mechanisms whose aim is to
protect the eye from any damage resulting from inflammations (ocular immune privilege [548]);
however, contact of a certain amount of Leptospira spp. with the uvea (a) might cause a uveitis
bout. Meanwhile, there are agglutinating antibodies and antigen-presenting cells within the vitreous
body. Consequently, some—but not all—Leptospira spp. might be eliminated (c). The agglutinating
antibodies inside the vitreous cavity might enhance the contact of Leptospira spp. with each other
and, thus, enhance new biofilm formation (b). Leptospira spp. show a fascinating motility in viscous
media [555] and might spread within the vitreous cavity to form biofilms in new places. Endoscopic
picture, pars plicata of the ciliary body (photograph: B.W.). (7) Acute uveitis attack: blepharospasm,
lacrimation, and swollen eyelids (unspecific symptoms). Ophthalmologic examination would reveal
signs of acute uveitis (Section 4.1a) and Figures S1–S5). Under anti-inflammatory therapy, there
will be a regression of uveitis. Planktonic bacteria might have been eliminated, the ocular immune
privilege is restored, and some bacteria start new biofilm formation.
To date, however, there is limited or no knowledge of the morphology and matrix

components in biofilms of leptospires formed in vivo. The in vivo composition of biofilms
is unquestionably different from that of environmental or in vitro biofilms. In the vitreous,
collagen fibrils play a role, as do host immune responses over time. It is likely that the
composition of the leptospiral biofilm matrix is critically influenced by the host immune
system. Among others, inflammatory products, macrophages, lymphocytes, PMNs, plasma
cells, fibrin, amyloids, and NETs have been detected in the vitreous of horses suffering
from ERU (Sections 7.7–7.10), all of which are in contact with the biofilm in vivo and some
of which may also be integrated into it. How quickly biofilm forms in the eye and how old
biofilm formations in the vitreous can become is still unknown.
An important point in intraocular leptospiral infection is also the presence of aggluti-
nating antibodies in the aqueous and vitreous humor (Sections 7.2 and 7.3). In addition to
the opsonization of motile leptospires, the aggregation or “agglutination” of leptospires
will occur in vivo, just as in MAT. In cases of insufficient phagocytosis, the agglutinating
antibodies lead to a progressive attachment of the leptospires to each other (the first step of
biofilm formation). The antibodies in the vitreous may thus promote, and perhaps even
accelerate, biofilm formation. More rarely, MAT results are negative in intraocular samples;
however, instead, either anti-Leptospira antibodies can be detected by ELISA and/or PCR
can be used to detect the LipL32 gene. The immune response of the host, which exhibits
individual differences, could therefore be of crucial importance for the course of the disease.
Here, a genetic predisposition could have an influence (Sections 1 and 3).
The most common infectious uveitis in humans is caused by Toxoplasma spp. [399],
but Leptospiral uveitis in humans has more in common with ERU. In humans, leptospiral
uveitis is also a non-granulomatous uveitis, often occurring months or years after systemic
leptospirosis [220], and is relatively easy to diagnose on the basis of the clinical examination,
including the characteristic vitreous opacities [327]. In cases of doubt, an aqueous humor
analysis is advised. In warm, humid areas and after flooding, leptospiral etiology is thought
to account for about 10% of human uveitis cases. Unlike in horses, leptospiral uveitis in
humans often has a milder course, and after a few episodes, is self-limiting. In addition, the
systemic administration of antibiotics is reported to be therapeutically effective. This may
be because the human vitreous volume is only about one-seventh the size of the equine
vitreous in volume. To the best of the authors’ knowledge, in humans, there is no hint of
autoimmune uveitis following leptospiral uveitis.
In exploring biofilm infections in the vitreous, some of the therapeutic methods used in
ERU have to be questioned (Section 5.2). Injections of triamcinolone into the vitreous cavity
and the placement of cyclosporine implants should be reserved for “non-ERU uveitis”. It
remains to be seen whether intravitreal injections of gentamicin will prove effective in the
long term, and whether and how frequently toxic retinal damage and other complications
will occur. For example, it could be that only transiently planktonic leptospires are killed
and the bacteria in the biofilm remain largely unaffected—especially because clearance in
the vitreous cavity does not allow the prolonged presence of gentamicin. Other prolonged
anti-inflammatory therapy options are only temporarily helpful. Elimination of the infec-
tion could succeed—if at all—only by the gentamicin injection. However, none of these
therapies result in the removal of vitreous opacities; thus, the prognosis for preserving or
even improving vision is worse than with a carefully performed vitrectomy.
11. Conclusions and Perspectives

Chronic leptospiral infection in the vitreous cavity is only possible through the forma-
tion of biofilm. Biofilm formation explains many points in the pathogenesis of ERU that
have previously been mysterious. Future ex vivo studies of vitrectomy specimens from
ERU eyes could provide insights regarding the composition of the biofilm, the interactions
of the different fibrils and fibers (physiological vitreous fibrils, fibrin, NETs, amyloid) with
the intraocular biofilm and the immune system, and an improvement in the therapy of
biofilm-associated infections. It is possible that aspects of the equine in vivo leptospiral
biofilm may be transferable to infections with other spirochaetes and could help to better
treat chronic Lyme disease, for example, in humans. ERU occurs spontaneously, is a conse-
quence of a natural leptospiral infection, and vitreous samples are a waste product of the
best possible therapy (vitrectomy); therefore, further ex vivo investigations are possible
without the need for animal experiments.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/microorganisms10020387/s1, Supplementary Material S1, Figure S1: Acute uveitis: Corneal
haziness in the periphery, beginning corneal vascularization (visible in the dorsal and temporal
aspects), miotic pupil. The accompanying conjunctivitis (hyperemia) seems to be less severe after
anesthetic eye drops which cause vasoconstriction. Figure S2: Acute uveitis and similar findings
similar to Figure 1, but the circular corneal vascularization is more severe. Figure S3: Left: Small
amount of fibrin in the anterior chamber, early stage and mild ERU bout. Right: Severe ERU, hy-
popyon and corneal vascularization. Figure S4: Severe ERU, the same eye is in both pictures. Left:
Corneal haziness in the periphery, corneal vascularization, sero-hemorrhagic inflammation, miotic
pupil. Right: After one week with rigorous conservative therapy. The corneal opacity and the
inflammatory products in the anterior chamber are decreasing, the pupil is dilated about 2/3, and the
fundus reflex indicates substantial vitreous haziness. Figure S5: Left: Very small amount of fibrin
(arrow) in the anterior chamber which might be missed if the anterior chamber is not examined
carefully. It is not possible to know if this was a mild ERU bout or a blunt trauma. Right: Acute
ERU bout after a few days of conservative therapy. The amount of fibrin (arrow) decreases, the
pupil is more dilated after the frequent administration of atropine ointments, and the fundus reflex
indicates a diffuse vitreous haziness. Figure S6: Increasing vitreous haziness in ERU. a: Normal
fundus reflex, b–f: Increasing vitreous haziness, f: The orange-red color indicates a high risk for
retinal detachment. Figure S7: Normal view of the optic nerve disc with centrifugal vessels of the
equine paurangiotic fundus, b–f: Increasing vitreous haziness, it becomes more and more difficult
to see details or even the outline the optic nerve disc. Figure S8: Chronic ERU. Left: Subacute
uveitis, rubeosis iridis and neovascularization, posterior synechia, cataract formation. Right: Quiet
interval, posterior synechia and cataract formation. Figure S9: Left: Atrophy of the globe, posterior
synechiae, cataract, and a “third corner” of the eyelids (arrow) as a sequela of the atrophy. Right:
Phthtisis after ERU (“end-stage”) with chronic ocular discharge. Figure S10: Chronic ERU. Left:
Subacute uveitis, posterior synechia, severe vitreous haziness. Right: Quiet interval, extensive pos-
terior synechia, beginning cataract formation. Figure S11: Chronic ERU. Left: Posterior synechia
and cataract formation. Right: posterior and anterior synechia and cataract formation. Anterior
synechia lead to the corneal opacities. Figure S12: Chronic ERU and vesicular cataracts subcapsular
of the posterior lens capsule. Left: Vesicular cataract in the nasal aspect. Temporally small posterior
synechia. Middle: Vesicular cataract (very small “bubbles”), especially in the ventral aspect of the
lens. At the “7 o’clock” position and at the “11 o’clock” position are small posterior synechia. In the
dorsal aspect are inflammatory products on the posterior lens capsule. Right: Vesicular cataract in
the periphery (relatively large or possibly confluent “bubbles”). At the “1 o’clock” position small
vitreous floaters. Figure S13: Chronic ERU, quiet interval. Arrowhead: Iris residue on the anterior
lens capsule. Arrow: Dense inflammatory products (“floaters”) in the vitreous cavity, very close to
the posterior lens capsule. These inflammatory products move (“float”) in the vitreous after blinking
and can best be seen and assessed using a hand-held ophthalmoscope. Figure S14: Chronic ERU.
Star-shaped retinal folds (arrows) around the optic nerve disc. This degree of retinal detachment
means an increased risk for retinal detachment. If vitrectomy is successfully performed and there
is no retinal detachment perioperatively, vision might be preserved. Figure S15: Chronic ERU. Left:
Large-scale detachment of the retina. This kind of detachment will progress, leading to blindness.
Right: Complete retinal detachment. The retina is still fixed around the optic nerve disc, but no
longer at the dorsal and lateral aspects of the Ora serrata. Figure S16: Leopard coat pattern uveitis:
cataract and posterior lens luxation in the left eye. Figure S17: Uveitis: blood and fibrin in the anterior
chamber. These findings may be due to a blunt trauma. Figure S18: Phacogenic uveitis: the protrusion
of lens material (arrow) through a circular defect in the anterior lens capsule. In the ventral aspect of
the lesion iris, pigment is left after posterior synechia. Figure S19: Chronic iritis, similar to “Fuchs’
heterochromic iritis” in humans: depigmentation in the iris and corneal edema. Figure S20: Uveitis
(both eyes affected) accompanying septicemia (Rhodococcus equi). In foals younger than 6 months,
ERU is extremely unlikely. Figure S21: Medulloepithelioma causing mild and insidious uveitis.
Figure S22: Ongoing painful uveitis despite rigorous conservative therapy in a horse with systemic
Micronema deletrix (syn: Halicephalobus deletrix) infection. The nematodes were later histologically
detected in the uveal tissue. Figure S23: Uveitis accompanying severe keratitis. The focus must be on
the corneal infection. Once the infection is removed, the uveitis will not continue. Figure S24: Septic
endophthalmitis: corneal vascularization is much denser than vascularization accompanying ERU.
Furthermore, the purulent infection of the inner eye leads to more intense corneal edema as well as
another type of cloudiness of the normally transparent media. Furthermore, the horses exhibit fever
and a significant disturbance of their general condition. Supplementary Material S2: Notes on the
side effects of topically administered atropine in horses. Supplementary Material S3: Notes on the
intravitreal injection of gentamicin. Supplementary Material S4, Table S1: Follow-up examination
of aqueous samples after vitrectomy: course of anti-Leptospira antibody titers (MAT) (unpublished
data). Figure S25: MAT titers over time after vitrectomy. In individual horses, an aqueous humor
sample could be taken at different times after surgery (e.g., when a fibrinolytic was injected after
surgery or after euthanasia due to other underlying diseases). Each arrow represents one eye. The
arrows start at the time of surgery and the arrowhead indicates follow-up aqueous humor testing.
Supplementary Material S5, Table S2: Literature references for testing intraocular samples from ERU
eyes or human uveitis eyes for leptospires (culture, PCR, histology/immunohistochemistry, and
electron microscopy). Supplementary Material S6: Results of examinations of intraocular samples
from horses suffering from ERU (excerpts from [18]). Table S3: Protein fractions determined by
electrophoresis (total protein “TP” and albumin “Alb.”) and calculated (globulins, “Ig”) from serum
(S) and vitreous (V) samples and calculation of the Goldmann–Witmer coefficient (GWC) in 46 paired
vitreous and serum samples. Results sorted by GWC (decreasing). Table S4: MAT titers in serum
(S) and undiluted vitreous samples (V) from horses with ERU and from horses with healthy eyes.
Intraocular MAT titers exceed the serum titers by several times. A clear difference between horses
suffering from ERU and horses with healthy eyes is only seen when looking at the intraocular samples.
Table S5: MAT results with intraocular and serum samples from horses with ERU (ophthalmological
findings differentiated) and from horses with healthy eyes. Figure S26: Typical SNAP Lepto results in
an ERU horse. Upper picture: SNAP Lepto with undiluted vitreous from an ERU eye. Picture below:
SNAP Lepto with serum of the same horse. The sample point was at the “12 o’clock position” and the
control point was at the “9 o’clock position”. The point of the vitreous sample is even darker than the
control point, indicating a high level of intraocular antibodies. The point of the serum sample shows
only a very weak coloring, which is barely visible. (SNAP Lepto is a commercially available rapid
ELISA test for the detection of anti-LipL32 antibodies from IDEXX company, Ludwigsburg, Germany)
(photograph: B.W.). Figure S27: Percentages of positive vitreous and serum samples from ERU eyes
and ERU horses, respectively, using MAT (titer ≥ 1:100). Figure S28: Positive leptospiral culture
results (n = 189) with vitreous samples compared with MAT results in serum samples. Percentage of
assignment to serogroups. Sometimes MAT was positive (titer ≥ 1:100) for more than one serovar.
Figure S29: Positive leptospiral culture results (n = 189) with vitreous samples compared with MAT
results in the same samples. Percentage of assignment to serogroups. Sometimes MAT was positive
(titer ≥ 1:100) for more than one serovar. Supplementary Material S7: Literature references cited
in publications on the examination of intraocular specimens from eyes affected with ERU for the
calculation of GWC or “C-values”. Figure S30: References for the calculation and interpretation of
the Goldmann–Witmer coefficient (GWC) in the literature. Arrows mean “cites”. Red references:
calculation of the “C-value” without any additional testing, e.g., for the IgG, albumin, or total protein
contents in corresponding intraocular and serum samples. Black references: no calculation of the
GWC at all—although cited for the GWC. Blue references: the exact calculation path for correct
GWC calculations is not described, but measurements of the IgG-levels in intraocular fluids and
serum samples are included in the methods and correct citations for the calculation path are given.
Green references: correct calculation paths of GWC are given. Black arrows: citation of references
which did not use or describe the GWC at for their work at all. Red arrows: citation of references
for calculations of the GWC or “C-value”, respectively, in which the calculation is not described.
Dashed arrows: citation of references in which the correct calculation of the GWC is described,
but the citing reference ignores the measurement of IgG or another protein fraction anyway. Blue
arrows: citation of references in which the methods describe measurements of IgG levels and cite
references for the correct calculation paths of the GWC. Green arrows: correct citations for calculation
of the GWC. Supplementary Material S8: Ultrastructural and histological examinations of vitreous
specimens and the ciliary body. Figure S31: Transmission electron microscopy pictures. A and B:
Leptospira grippotyphosa (WHO standard strain) were experimentally injected into the vitreous body
of a healthy eye of a euthanized horse. Afterwards, the vitreous sample was taken by vitrectomy.
A: Leptospira spp. seem to arrange themselves along with the vitreous fibers, which would be an
excellent starting position for biofilm formation. C and D: Vitreous samples taken during therapeutic
vitrectomy from a horse with naturally acquired ERU. Leptospira spp. are surrounded by a thick
layer of an extracellular osmiophilic matrix which lacks the experimentally injected Leptospira spp.
(Source: [424], reprint courtesy of Schluetersche Specialized Media GmbH, Hanover, Germany).
Figure S32: Vitreous material from an ERU eye prepared for transmission electron microscopy
showing phagocytosis of dense round structures. Two smaller structures are inside the phagocyte
(numbers 1 and 2); the large one (number 3) probably leads to a “frustrated phagocytosis”. These
dense round structures are now suspected to be the leptospiral biofilm. Inside of the phagocyte are
also structures which may be parts of Leptospira spp. (arrowheads). Bar = 2 µm (photograph courtesy
of Kristin Brandes). Figure S33: Ciliary body region of an ERU eye. Congo Red staining and polarized
light showing amyloid on the ciliary body. Subsequent immunohistology revealed amyloid A [224].
(Photographs courtesy of Maj-Britt Cielewicz). Figure S34: Dense vitreous opacities (“floaters”).
a: In the dorsal aspect of the vitreous chamber, b: Further distributed in the vitreous chamber, c:
Membrane-like vitreous opacity mimicking retinal detachment. Bottom: Syringe containing vitreous
material (and several “floaters”) obtained in the beginning of vitrectomy. (Photographs: B.W. and
H.G.). Figure S35: Amyloid: Vitreous opacities (Supplementary Material S1, Figure S13 and Supple-
mentary Material S8, Figure S34) after Congo Red staining and the presence of green birefringence
in polarized light. (Photographs courtesy of Ellen Giving). References [556–576] are cited in the
Supplementary materials.
Author Contributions: Writing—original draft preparation, B.W.; writing—review and editing, H.G.
and K.A. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Acknowledgments: We sincerely thank Jenni Kneuertz for careful language revision.
Conflicts of Interest: The authors declare no conflict of interest.
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microorganisms

Equine Clinic, Ludwig-Maximilians-University, 80539 Munich, Germany; [email protected] (H.G.);

Academic Editor: Gianfranco Donelli

Received: 31 December 2021 1. Introduction

Microorganisms 2022, 10, 387. https://doi.org/10.3390/microorganisms10020387 https://www.mdpi.com/journal/microorganisms

exclusively for leptospiral-induced uveitis, which is the main representative of infectious

3. Historical and Preliminary Remarks on the Etiology of ERU

3.1. Leptospirosis and Autoimmune Disease

4. Types of Equine Uveitis and Differential Diagnosis

4.1. Equine Recurrent Uveitis (ERU)

(a) Acute uveitis

4.2. “Leopard Coat Pattern”—Uveitis

4.3. Traumatic Uveitis

4.4. Phacogenic Uveitis

4.5. Chronic Iritis, Similar to “Fuchs’ Heterochromic Iritis” in Humans

4.6. Uveitis during Septicemia

4.7. Uveitis Accompanying Intraocular Tumors

4.8. Uveitis during Parasitic Infections

4.9. Uveitis Accompanying Severe Keratitis

4.11. Autoimmune Uveitis and Uveitis of Unknown Etiology

5.2. Chronic Course of Uveitis

6. Diagnostic Value of Testing Serum Samples for Anti-Leptospira Antibodies in

7. Examination of Intraocular Specimens in Horses Suffering from ERU

or after euthanasia of the horses due to bilateral blindness [174,307,316,368,370]. By this

7.1. Breakthrough with Vitrectomy

7.2. Antibody Detection in Aqueous Humor and Vitreous Samples

Blood–Aqueous Barrier and Intraocular Protein Content

7.3. Intraocular Antibody Production

7.3.1. Calculation of the Goldmann–Witmer Coefficient (GWC)

GWC = ((reciprocal titer intraocular sample)/(IgG amount intraocular sample))

7.3.2. Interpretation of the GWC

7.3.3. Misleading Interpretation of the GWC in Recent Publications on Equine Ophthalmology

7.3.4. Application of the GWC

7.4. Cultural Detection of Pathogenic Leptospira spp.

7.6. Comparison of Laboratory Results of Vitreous and Aqueous Humor Samples

7.7. Ultrastructural and Histological Examinations of Vitreous Specimens

vitrectomies [57] (Section 8.3). However, immunohistochemistry is unsuitable for routine

7.9. Neutrophil Extracellular Traps (NETs)

7.11. Samples from Healthy Eyes

8.1. Steps of Biofilm Formation and Dissemination of Infection

8.2. Characteristics of Biofilm-Associated Diseases

8.3. Biofilm Formation of Leptospira spp.

Leptospirosis as a biofilm infection seems to be of great contemporary interest and has

8.4. Vitreous Structure, ERU, and Biofilm

Finally, vaccination is another option [553]. No vaccine is currently available for

To date, however, there is limited or no knowledge of the morphology and matrix

11. Conclusions and Perspectives

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

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