This study documents the examination of 17 horses (both sexes, 3-18 years old) suffering from spontaneous equine recurrent uveitis (ERU). Vitreal samples obtained by pars plana vitrectomy were examined macroscopically and ultrastructurally, and in most cases also by cultural examination, by microscopic agglutination test (MAT) and by polymerase chain reaction. In 24% (4/17) of the animals, ultrastructural examination by electron microscopy revealed intact leptospiral bacteria in the vitreous. The leptospires were detected freely in the vitreous and also incorporated by a phagocyte. They were surrounded by a rim of proteinaceous material which was reduced around a phagocytosed leptospira. Ninety-four per cent (16/17) of the vitreal samples presented significant antibody levels in the MAT, mostly against leptospiral serovar Grippotyphosa. Seventy-five per cent (9/12) of bacterial culture examinations were positive for leptospira. Polymerase chain reaction was positive in all (16/16) examinations performed. Our findings support previous reports suggesting that leptospires play an important role in the pathogenesis of ERU. Interestingly, this study found leptospires after secondary and later acute episodes. A persistent leptospiral infection is therefore suggested as the cause of ERU.
ZusammenfassungDie Literatur zur Ätiologie der equinen rezidivierenden Uveitis (ERU) erscheint seit Jahrzehnten verwirrend, da einerseits wegen nachweisbaren Autoimmunreaktionen eine "Autoimmunkrankheit" beschrieben wird, andererseits jedoch wiederholt Nachweise einer intraokularen Leptospireninfektion erfolgt sind. Die Etablierung der Vitrektomie bei an ERU erkrankten Pferden ermöglicht erstmals die routinemäßige Entnahme von Glaskörperproben aus vielen der betroffenen Augen. Diese im Rahmen der Vitrektomie gewonnen Glaskörperproben können sowohl zur Untersuchung auf zelluläre und humorale Autoimmunphänomene als auch zu mikrobiologischen Studien verwendet werden und zur Klärung der Ätiologie und Pathogenese der ERU dienen. Neben Serumproben von 724 an ERU erkrankten und von 132 augengesunden Pferden konnten von 1998 bis 2001 bei Vitrektomien von 426 eindeutig an ERU erkrankten Augen unverdünnte Glaskörperproben unter sterilen Kautelen entnommen werden. Als Kontrolle dienten 54 Glaskörperproben aus gesunden Pferdeaugen. Alle Proben (Serum und Glaskörper) wurden mittels Mikroagglutinationsreaktion (MAR) auf Antikörper gegen Leptospiren getestet. Für die Berechnung des Goldmann-Witmer-Koeffizienten (C) wurden bei 36 Glaskörper-und Serumprobenpaaren Elektrophoresen durchgeführt. Zur Anzüchtung von Leptospiren wurden 41 Glaskörperproben aus gesunden und 358 Glaskörperproben aus an ERU erkrankten Augen kultiviert. Von den Glaskörperproben aus an ERU erkrankten Augen wurden zudem 55 mittels PCR auf Leptospiren-DNA untersucht. Aus 6 vitrektomierten Augen konnten in unterschiedlichen Abständen nach der Operation erneut Proben entnommen und mittels MAR untersucht werden. Der Krankheitsverlauf von 150 der operierten Pferdeaugen wurde im Mittel bis 2,7 Jahre nach der Vitrektomie weiter verfolgt. Zwischen Höhe und Vorkommen von Antikörpertitern gegen Leptospiren im Serum von augengesunden und an ERU erkrankten Pferden bestand ein geringer, jedoch nicht signifikanter Unterschied. Die MAR konnte in 3/54 (6 %) der Glaskörperproben aus gesunden Pferdeaugen sowie in 382/426 (90 %) der Glaskörperproben aus an ERU erkrankten Augen Antikörper gegen Leptospiren nachweisen. Die Berechnung des Goldmann-Witmer-Koeffizienten konnte für 34/36 (94 %) der Probenpaare eine intraokulare Antikörperproduktion nachweisen (C > 8). In 189/358 (53 %) Glaskörperproben aus an ERU erkrankten Pferdeaugen und in keiner der 41 Glaskörperproben aus gesunden Augen konnten kulturell Leptospiren nachgewiesen werden. Die positiven Kulturergebnisse konnten sowohl aus Augen erzielt werden, die erst wenige Wochen erkrankt waren, als auch aus Augen, die schon über viele Jahre rezidivierende Entzündungen gehabt hatten. Bei 18/189 (9,5 %) der Serum-und Glaskörperprobenpaare mit positivem Kulturbefund in den Glaskörperproben konnten in den zugehörigen Serumproben keine Antikörper gegen Leptospiren mehr nachgewiesen werden. Die Polymerase-Ketten-Reaktion (PCR) wies in 39/55 (71 %) Glaskörperproben aus an ERU erkrankten Augen Leptospiren-DNA nach. In den 6 Glaskörper...
Most of the dogs with leptospirosis in southern Germany had sera reacting to serogroups other than icterohaemorrhagiae and canicola, which are contained in the vaccine. Thus, currently available vaccines in Europe do not protect against the most common Leptospira organisms associated with clinical disease.
Summary The pathogenesis of equine recurrent uveitis (ERU) is not completely understood. Persistent infection of pathogenic leptospires in the eyes is the probable primary cause. Consistently, we isolated and characterized in this study leptospires from 32.2% of the intraocular samples collected from 501 horses from the following Western European countries: Germany, Switzerland, Austria, The Netherlands, Luxembourg, Great Britain, Italy and Poland. The vast majority of the leptospiral isolates belonged to serogroup Grippotyphosa (78.2%). The other isolates belonged to the Australis group (14.2%), the Sejroe group (3.6%), the Pomona group (2.5%) and the Javanica group (1.5%). Apart from three isolates that likely belong to Leptospira kirschneri, serovar Dadas, all Grippotyphosa group isolates belonged to L. kirschneri, serovar Grippotyphosa. Analysis with monoclonal antibodies revealed that the isolates differed from Moskva V, the reference strain of serovar Grippotyphosa. However, the Grippotyphosa isolates closely resembled serovar Grippotyphosa, strain Duyster, which has been isolated from a Dutch patient. Because of apparent phenotypic and genotypic differences between the strains Moskva V and Duyster, we propose two different types within serovar Grippotyphosa, namely type Moskva and type Duyster. Distribution of type Duyster is restricted to Western Europe. Strikingly, only a few serovars were isolated from the eyes of horses with ERU. A number of other commonly occurring serovars, such as Copenhageni, were not found, whereas the presence of antibodies in blood and eye fluids against such serovars suggests that infection occurs. Therefore, we hypothesize that, while many pathogenic serovars might be able to penetrate the eyes, only few are capable to persist.
Equine recurrent uveitis (ERU) is a frequently occurring disease, at least in horses in Germany. A vitrectomy is predominantly the course of action in cases where a chronic intraocular leptospiral infection is present. In some cases, the clinical and ophthalmological diagnosis is not conclusive and in these cases a preoperative laboratory test using anterior chamber fluid is indicated. However, false negative results occur when only MAT is used. The aim of this study was to investigate intraocular samples from horses with recurrent uveitis by using ELISA to detect anti-leptospiral antibodies and to compare the results to those of more established laboratory tests, i. e. MAT and PCR. Preliminary investigations indicated a high sensitivity of ELISA in the detection of leptospiral uveitis in horses. 80 eyes of 72 diseased horses were included in the study. The patients' history was taken and a thorough ophthalmologic examination was performed. Intraocular fluids were obtained by diagnostic anterior chamber paracentesis (n = 29) or therapeutic vitrectomy (n = 80) and submitted for MAT, ELISA and PCR to detect leptospiral antibodies and/or DNA. The ELISA allowed the separate determination of the immunoglobulin classes M, G and A. For statistical evaluation Cohen's kappa and the chi-square tests were applied and a p-value of 0.05 or less was considered to be significant. 22 healthy horses (n = 42 eyes) served as controls. 78 % of vitreous samples from horses with ERU and 10 % of healthy controls reacted positively in the MAT, whereas in the ELISA it was 85 % and 0 %, and in the PCR it was 61% and 0 %, respectively. Specific IgA was detected in 84 % of the vitreous samples from horses with ERU, and in 11% of cases, IgA was solely detectable. Differences were highly significant (p<0.001). None of the aqueous samples in the control group contained leptospiral antibodies. Up to 64 % of aqueous humour specimens that were obtained for diagnostic purposes yielded a positive result in the MAT. For the ELISA, it was even 89 %. Anti-leptospiral IgA was the most frequently detected immunoglobulin class. There was a high level of agreement in the MAT and ELISA results. However, ELISA provided a greater number of positive results. The detection of IgA against leptospires proved to be especially sensitive for an intraocular leptospiral infection and, thus, important for the laboratory diagnosis of leptospiral uveitis in horses. Looking at the cost involved in laboratory tests, it would be reasonable to start with an IgA ELISA. Only if there are negative results, additional tests could be performed. In single IgA negative cases there might be positive reactions using MAT, PCR or ELISA for IgG or IgM.
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