In Vivo Biofilm Formation of Pathogenic Leptospira spp. in the Vitreous Humor of Horses with Recurrent Uveitis

Equine recurrent uveitis (ERU) causes painful inflammatory attacks and oftentimes blindness in the affected eyes. The disease is considered a late sequela of systemic leptospirosis. The most effective therapy is the surgical removal of the vitreous (vitrectomy), which is not only therapeutic, but provides vitreous material that can be assessed diagnostically. For example, the lipL32 gene, culturable Leptospira spp., and anti-Leptospira antibodies have all been detected in vitreous samples obtained from eyes with chronic ERU. Despite this clear evidence of leptospiral involvement, the systemic administration of antibiotics in infected horses is ineffective at resolving ERU. This syndrome of chronic recurrent inflammation, which is unresponsive to antibiotic therapy, combined with apparent bacteria evading the immune response, is consistent with a biofilm-associated infection. The purpose of this study, therefore, was to detect the in vivo biofilm formation of Leptospira spp. in vitreous samples collected during vitrectomy and examined using a Warthin-Starry silver stain and immunohistochemistry. All known steps of biofilm formation were visualized in these samples, including individual Leptospira spp., leptospiral microcolonies and dense roundish accumulations of Leptospira spp. In many instances spirochetes were surrounded by an extracellular substance. Taken together, data from the present study show that ERU is a biofilm-associated intraocular leptospiral infection, which best explains the typical clinical course

Role of Intraocular Leptospira Infections in the Pathogenesis of Equine Recurrent Uveitis in the Southern United States

To investigate the role of intraocular leptospiral infections in horses with Equine Recurrent Uveitis (ERU), ocular fluid samples were collected from donated and client-owned horses with a history and ocular findings consistent with chronic ERU. Additionally, eyes were harvested from horses with normal ophthalmic examinations as a control group. Blood samples were obtained for Leptospira serology using microscopic agglutination test (MAT). Aqueous and vitreous humor samples were aseptically obtained and submitted for aerobic culture and Leptospira culture, PCR and MAT. Twenty-one control horses (40 eyes) and 31 ERU horses (46 eyes) were available for study. Serology results were available for 48/52 horses: 16/21 control and 23/27 affected horses were positive for at least one serovar; Bratislava was the most common serovar for both groups. Bacillus sp. and Micrococcus sp. were cultured from one control eye; Streptococcus sp. (n=1) and Leptospira (n=6) from eyes with ERU. Leptospira isolated belonged to serogroup pomona (n=4) and grippotyphosa (n=2). PCR results were positive in 14/31 (45%) horses diagnosed with ERU; no control horses were positive by PCR (p=0.0001). MAT was positive for 17/24 of ERU horses (71%) and 1/21 (4.7%) of normal horses (p\u3c0.0001). Horses with ERU had a high prevalence of Leptospira infection based on PCR and MAT results from intraocular fluids compared to controls. Leptospira infection should be considered as a cause of ERU in the southern United States. The diagnosis of these intraocular infections was not aided by serology and required specific, invasive sampling of ocular fluid

Intravitreal injection of low-dose Gentamicin: an alternative method of management for equine recurrent uveitis

Die Technik der intravitrealen Gentamicin Injektion darzulegen, die Auswirkungen dieser auf die klinischen Symptome von Uveitiden, sowie die möglichen unmittelbaren Komplikationen (innerhalb von 24 Stunden) und längerfristigen Komplikationen (30 bis 780 Tage) die mit dieser Technik verbunden sein können, zu beschreiben. Zusätzlich wurde der okuläre und systemische Leptospiren- Status ermittelt und der Einfluss dieser auf das Behandlungsergebnis untersucht.:Table of Contents 1 INTRODUCTION 1 2 LITERATURE OVERVIEW 2 2.1 Etiology and pathogenesis 2 2.1.1 Proposed etiologies 2 2.1.2 ERU: an immune mediated disease 3 2.2 Leptospirosis and ERU 4 2.2.1 Genetic predisposition for ERU 6 2.3 Definition of ERU 7 2.3.1 Classification and syndromes 7 2.3.2 Clinical symptoms 8 2.4 Diagnostic testing for ERU (Leptospira) 8 2.4.1 Sample collection (aqueous humor, vitreous humor, serum) 8 2.4.2 Methodology 9 2.4.2.1 Microagglutination test (MAT) 9 2.4.2.2 Polymerase chain reaction (PCR) 10 2.4.2.3 Cultures 10 2.5 Treatment of ERU 10 2.5.1 Medical management 10 2.5.2 Intravitreal and suprachoroidal injections 11 2.5.2.1 Intravitreal rapamycin injections 11 2.5.2.2 Intravitreal triamcinolone injections 11 2.5.2.3 Suprachoroidal triamcinolone injections 12 2.5.2.4 Low-dose intravitreal gentamicin injections 12 2.5.3 Surgical procedures 13 2.5.3.1 Suprachoroidal cyclosporine implants 13 2.5.3.2 Pars plana vitrectomy 14 3 PUBLICATIONS 16 3.1 Intravitreal injection of low-dose gentamicin for the treatment of recurrent or persistent uveitis in horses: Preliminary results 16 3.2 Medical and Surgical Management of Equine Recurrent Uveitis 29 4 DISCUSSION 47 5 ZUSAMMENFASSUNG 51 6 SUMMARY 52 7 REFERENCES 53To describe the intravitreal gentamicin injection technique, report the effects of the injection on the clinical signs of uveitis and to describe the associated peri-injection (within 24 hours) and post-injection complications (30 to 780 days). Additionally, evaluation of the systemic and ocular Leptospira status and its effects on the treatment outcome was performed.:Table of Contents 1 INTRODUCTION 1 2 LITERATURE OVERVIEW 2 2.1 Etiology and pathogenesis 2 2.1.1 Proposed etiologies 2 2.1.2 ERU: an immune mediated disease 3 2.2 Leptospirosis and ERU 4 2.2.1 Genetic predisposition for ERU 6 2.3 Definition of ERU 7 2.3.1 Classification and syndromes 7 2.3.2 Clinical symptoms 8 2.4 Diagnostic testing for ERU (Leptospira) 8 2.4.1 Sample collection (aqueous humor, vitreous humor, serum) 8 2.4.2 Methodology 9 2.4.2.1 Microagglutination test (MAT) 9 2.4.2.2 Polymerase chain reaction (PCR) 10 2.4.2.3 Cultures 10 2.5 Treatment of ERU 10 2.5.1 Medical management 10 2.5.2 Intravitreal and suprachoroidal injections 11 2.5.2.1 Intravitreal rapamycin injections 11 2.5.2.2 Intravitreal triamcinolone injections 11 2.5.2.3 Suprachoroidal triamcinolone injections 12 2.5.2.4 Low-dose intravitreal gentamicin injections 12 2.5.3 Surgical procedures 13 2.5.3.1 Suprachoroidal cyclosporine implants 13 2.5.3.2 Pars plana vitrectomy 14 3 PUBLICATIONS 16 3.1 Intravitreal injection of low-dose gentamicin for the treatment of recurrent or persistent uveitis in horses: Preliminary results 16 3.2 Medical and Surgical Management of Equine Recurrent Uveitis 29 4 DISCUSSION 47 5 ZUSAMMENFASSUNG 51 6 SUMMARY 52 7 REFERENCES 5

Cross-Reactivity of Antibodies against Leptospiral Recurrent Uveitis-Associated Proteins A and B (LruA and LruB) with Eye Proteins

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be α-crystallin B and vimentin. Similarly, mass spectrometric analyses identified β-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human α-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant β-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized α-crystallin B, β-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis

Development of septic polysynovitis and uveitis in foals experimentally infected with \u3ci\u3eRhodococcus equi\u3c/i\u3e

Rhodococcus equi is one of the most important causes of disease in foals. Infection is typically characterized by pyogranulomatous pneumonia although extrapulmonary infections occur occasionally. Uveitis and polysynovitis have been reported in foals naturally infected with R. equi and are thought to be the result of an immune-mediated process. However, the pathogenesis of these conditions is poorly understood. The objectives of this study were to document the occurrence of uveitis and polysynovitis after experimental infection with R. equi and to determine if these disorders are the direct result of infection at these sites. Foals between 3 and 4 weeks of age were infected intratracheally with virulent R. equi using inocula of 1×108 CFU (high inoculum; n = 16) or 1×107 CFU (low inoculum; n = 12). Foals were monitored twice daily and necropsy was performed 14 days post-infection. Aqueous humor and synovial fluid were collected aseptically and the percentage of affected lung was calculated. The mean (± SD) percentage of affected lung was significantly higher with the high inoculum (31.8 ± 14.6%) than with the low inoculum (14.4 ± 11.4%). Fourteen of 25 foals developed uveitis and 20 of 28 foals developed polysynovitis. R. equi was cultured from the aqueous humor of 11 foals and from the synovial fluid of 14 foals. The risk of development of polysynovitis and protein concentration in the aqueous humor were significantly higher in foals that received the high inoculum. These results indicate that polysynovitis and uveitis are septic complications associated with the severity of lung disease

AN ASSESSMENT OF THE CARRIER STATE AND A NOVEL MARKER OF \u3ci\u3eLEPTOSPIRA\u3c/i\u3e AND ABORTION IN CENTRAL KENTUCKY HORSES

Leptospirosis is a reemerging zoonotic infection of worldwide importance and affects all mammals. The bacterium is transmitted to animals and humans by urine, fetal membranes and body fluids. Leptospira shedding in the urine contaminates both soil and water, exposing both humans and animals to the bacterium. Leptospirosis in horses can cause abortion and is one of the etiologies of equine recurrent uveitis which can lead to blindness. Equine leptospiral abortion in Central Kentucky is primarily caused by serovar Pomona, with occasional cases attributed to serovar Grippotyphosa. There are a few reports in the literature attributing abortion to serovar Bratislava in the United States. Interestingly, Bratislava has the highest seropositivity in the horse in the United States. Two studies were conducted that are included in this dissertation. The first was to determine the prevalence of leptospirosis in horses located in Central Kentucky submitted for necropsy to the University of Kentucky Veterinary Diagnostic Laboratory. Heart, vitreous humor, kidney and urine samples were collected for microagglutination testing (MAT) and real-time PCR (qPCR). Heart blood and vitreous were tested using MAT for serovars Grippotyphosa, Pomona and Bratislava. Kidney, vitreous and urine samples were tested for pathogenic Leptospira by qPCR. MAT test results for heart blood indicated an increased seroprevalence for Bratislava as compared to Grippotyphosa and Pomona. Three horses had positive titers for serovar Bratislava in vitreous and heart blood samples. All urine samples tested negative by qPCR, and only one kidney sample had a weak positive result. Four vitreous samples tested positive for leptospirosis by qPCR, but all samples were negative upon MAT testing. No samples with positive MAT titers were positive by qPCR for any of the samples tested. Females were more likely to have positive MAT titers and were considerably older than males. MAT titers in females were also significantly higher as compared males. Finally, there was widespread seroprevalence in horses, regardless of the reason for necropsy submission. This suggests that exposure to Leptospira on Central Kentucky horse farms is common and the risk of exposure to humans and other animals is possible. The second study evaluated the enzyme heme oxygenase-1 (HO-1) and its potential as a marker for abortion in pregnant mares with elevated MAT titers. HO-1 is the rate-limiting step in the breakdown and degradation of heme to carbon monoxide, free iron and biliverdin, which is converted to bilirubin. Increased levels of HO-1 and its’ by-products are upregulated during inflammation and sepsis. HO-1 together with its by-products are essential in protecting the body from both increased inflammation and in reducing the risk of sepsis in humans, mice and rats. HO-1 together with its by-products are also essential in helping maintain pregnancy in humans, mice and rats. HO-1 in the serum of horses has not been previously investigated. The presence of HO-1 in the serum of healthy non-pregnant mares (NPM), pregnant mares throughout pregnancy (PMOT), pregnant mares 7 months pregnant with and without placentitis (PM) and pregnant mares with high MAT titers (MATS) was investigated. HO-1 levels in both NPM and PMOT were lower than PM, and significantly lower than MATS. Pregnancy alone increased HO-1, the same has been shown in other mammals and humans. Additionally, mares pregnant with elevated leptospiral titers had significantly higher HO-1 levels compared to other mares. This indicates that both pregnancy and high MAT titers increase the animals’ HO-1 response. Further investigation of HO-1 in horses is needed to ascertain its’ importance during infection and/or pregnancy and potential therapeutics

Leptospira seroprevalence and associations between seropositivity, clinical disease and host factors in horses

<p>Abstract</p> <p>Background</p> <p>A cross-sectional study was carried out to determine the seroprevalence of different serovars of <it>Leptospira </it>spp. and their association with clinical disease and host factors in Swedish horses.</p> <p>Methods</p> <p>Sera from 2017 horses brought to equine clinics during 1997–98 were investigated. The sera were examined by microscopic agglutination test for the presence of antibodies against the following <it>L. interrogans </it>serovars: Bratislava strain Jez, Icterohaemorrhagiae strain Kantorowicz and Pomona strain Pomona and also <it>L. kirschneri </it>sv Grippotyphosa strain Duyster and <it>L. borgpetersenii </it>sv Sejroe strain M 84. Host factors, disease factors, season, pasture access and outdoor confinement variables were analysed with respect to seropositivity to sv Bratislava and Icterohaemorrhagiae. Multivariable logistic regression was used to model seropositivity to sv Bratislava and Icterohaemorrhagiae (seroprevalence > 8%).</p> <p>Results</p> <p>The seroprevalence, at a cut-off 1:100, were for sv Bratislava (16.6%), Icterohaemorrhagiae (8.3%), Sejroe (1.2%), Pomona (0.5%) and Grippotyphosa (0.4%). In the multivariable analysis, it was demonstrated that seroprevalence increased with age for sv Bratislava and Icterohaemorrhagiae. For sv Bratislava the seasons April – June and October – December and for sv Icterohaemorrhagiae October – December had higher seroprevalences than other seasons. Horses not used for racing had higher levels of seropositivity to sv Bratislava. Furthermore, horses with respiratory problems as well as horses with fatigue had higher levels of seropositivity to sv Bratislava. Ponies and coldbloods, and horses with access to pasture, had lower seroprevalence for sv Icterohaemorrhagiae. Healthy horses had lower seroprevalence for sv Icterohaemorrhagiae, than non-healthy horses.</p> <p>Conclusion</p> <p>There was no significant association between clinical signs and disease and positive titres to sv Bratislava (except for the association between respiratory problems and fatigue and seropositivity to sv Bratislava). The results suggest that horses with increasing age and exposed to factors associated with outdoor life had an increased seroprevalence for sv Bratislava, indicating that horses get infected from outdoor and/or are exposed to shedding from other horses (management dependent). For sv Icterohaemorrhagiae, management possibly plays a role as ponies and coldbloods as well as healthy horses had lower seroprevalence. Overall, the age of the horse should be taken into consideration when evaluating the titre as the average healthy horse has a higher titre than a young horse.</p

Uveitis and blindness in a closed herd of Equidae following leptospiral infection

ObjectiveTo describe the ocular findings, chronology of disease, and serum leptospiral titers in a group of horses, mules, and donkeys following an outbreak of leptospirosis.MethodsFifty Equidae in central North Carolina had ophthalmic examinations and serum leptospiral microscopic agglutination test (MAT) titers performed every 3–6 months for 24 months followed by a final examination at 34 months.ResultsThroughout the nearly three-year study period, 17 horses (34%; 17/49 horses) developed signs of uveitis; 20 eyes (20/34; 58.8%) of these 17 horses were visual at the initial examination, but only four eyes (11.8%) remained visual at the final examination. Serum titers (serogroups Pomona and Bratislava) in horses with uveitis were significantly elevated compared to Equidae without uveitis (p &lt; 0.02). In the 32 horses, donkeys, and mules that did not develop uveitis, a subgroup of 11 horses and one donkey had negative or low serum leptospiral titers (titers ≤1:800) while a second subgroup of 16 horses, three mules, and one donkey had high leptospiral titers (&gt;1:800) but never developed uveitis. Water sources in the pasture were found to have high levels of leptospira.ConclusionApproximately 1/3 of horses on a farm exposed to Leptospira developed uveitis and blindness. Serum titers to L. Pomona and L. Bratislava were significantly elevated in horses with uveitis. However, despite exposure, some horses, even with very high serum titers, never developed ocular disease. These data indicates that further research is warranted to investigate the genetic and immunological aspects of the pathogenesis and susceptibility of leptospiral-associated uveitis

Leptospiroza konja u Hrvatskoj - supkliničke infekcije i pobačaji.

Clinical leptospirosis in horses has been primarily associated with abortion, stillbirth and equine recurrent uveitis. Severe septicaemia that includes fever, jaundice and anorexia, haematuria, renal and hepatic dysfunction, respiratory distress and pulmonary haemorrhage is rare. In order to obtain a better insight into the occurrence of equine leptospirosis in Croatia a sero-survey was performed using the microscopic agglutination test (MAT) among healthy and non-healthy horses. Non-healthy horses were represented by the mares that aborted. The aim of the study was to determine the prevalence of Leptospira infection and related serovars, as well as to point out the present clinical disease status of equine leptospirosis in Croatia. A total of 8059 horse sera samples of different breeds, age and sex were taken in 2011 from apparently healthy animals. In the same period, 56 samples were taken from mares that had aborted, representing non-healthy animals. Out of 8059 sera samples from healthy horses, 2999 (37.2) were considered seropositive. The highest seroprevalence for healthy horses was found for sv Bratislava in 1342 (44.8%) samples, sv Pomona 546 (18.2%) and sv Icterohaemorrhagiae 448 (14.9%). Out of 56 sera samples from the mares that aborted, representing non-healthy horses, 37 (66.1%) were considered seropositive. The highest seroprevalence for mares that aborted was found for sv Bratislava in 16 (43.2%) sera samples, sv Pomona in 11 (29.7%) and sv Icterohaemorrhagiae in 4 (10.8%). The results showed that horses in Croatia are commonly exposed to Leptospira infections. Regarding clinical disease status, mainly subclinical infections and fewer abortions, as the only clinically apparent form of leptospirosis, are currently present in Croatia, irrespective of the level of agglutinating antibody titres.Leptospiroza konja klinički se očituje prvenstveno pobačajima, ždrebljenjem uginule ždrebadi i mjesečnom sljepoćom. Septikemijski oblik koji se očituje groznicom, žuticom, gubitkom apetita, hematurijom, bubrežnom i jetrenom disfunkcijom, respiratornim poremećajima i krvarenjima u plućima javlja se rijetko. Kako bi dobili bolji uvid u pojavnost leptospiroze konja u Hrvatskoj provedeno je serološko istraživanje uporabom mikroskopske aglutinacije (MA) u populaciji zdravih konja, te u kobila nakon pobačaja. Cilj istraživanja bio je ustanoviti prevalenciju infekcije leptospirama i zastupljenost pojedinih serovara leptospira te ukazati na kliničku pojavnost leptospiroze konja u Hrvatskoj. Tijekom 2011. godine pretraženo je 8059 uzoraka seruma zdravih konja različitih pasmina, dobi i spola. Usporedno je pretraženo i 56 uzoraka seruma kobila zaprimljenih nakon pobačaja. Od 8059 uzoraka seruma zdravih konja protutijela za leptospire ustanovili smo u 2999 (37,2) uzoraka. Najveća je bila zastupljenost sv Bratislava u 1342 (44,8%) uzoraka, sv Pomona u 546 (18,2%) i sv Icterohaemorrhagiae u 448 (14,9%) uzoraka. Od 56 uzoraka seruma kobila koje su pobacile ustanovili smo 37 (66,1%) seropozitivnih uzoraka. Najveća je bila zastupljenost sv Bratislava u 16 (43,2%) uzoraka, sv Pomona u 11 (29,7%) i sv Icterohaemorrhagiae u 4 (10,8%). Rezultati ukazuju da su konji u Hrvatskoj vrlo izloženi infekciji leptospirama. S obzirom na kliničku pojavnost možemo zaključiti da trenutno u Hrvatskoj većina infekcija leptospirama u konja prolazi supklinički, te uz pojavu pobačaja u gravidnih kobila. Budući da je leptospiroza zoonoza, izuzetnu opasnost predstavljaju konji rekonvalescentni kliconoše koji mogu mokraćom izlučivati velike količine leptospira u okoliš te predstavljati izvor zaraze za druge životnje i ljude