35 leptospira isolated from the vitreous body of 32 horses with recurrent uveitis (ERU)

130 vitreous samples, systematically collected in 1998 from 117 horses during vitrectomy, were cultured for the presence of leptospires. All horses suffered from equine recurrent uveitis (ERU), also known as periodic ophthalmia or moon blindness, and were treated surgically to combat painful attacks, and to preserve vision. In 35 out of 130 vitreous samples (35/130 = 26.9%), leptospires could be isolated. These isolates belong to the grippotyphosa serogroup (n = 31) and to the australis serogroup (n = 4). So, for the first time, leptospires were recovered from eyes in vivo in a large number of horses with ERU. Vitreous samples and one serum sample from each horse were also tested for leptospiral antibodies using the microscopic agglutination test (MAT). In 92 vitreous samples (92/130 = 70.7%) and 96 serum samples (96/117 = 82.0%) leptospiral antibodies were detected at a dilution of > 1:100. The presence of intact leptospires and specific antibodies in eyes affected with ERU demonstrates a local antibody production to leptospiral antigen. These results indicate an important etiological role of leptospires in equine recurrent uveitis.     

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

Equine recurrent uveitis (ERU) has been linked to leptospirosis in Europe; however, regional differences exist in reports from the United States. The objective of this study was to investigate the role of intraocular leptospiral infections in horses with ERU in the Southern United States. Blood and ocular fluid samples were collected from horses with ERU and normal controls. Leptospira serology was performed using microscopic agglutination test (MAT). Aqueous and vitreous humor samples were obtained and submitted for aerobic and Leptospira culture, polymerase chain reaction (PCR), and MAT. Twenty-one control horses (40 eyes) and 31 ERU horses (46 eyes) were available. Serology was available for 48 of 52 horses: 16 of 21 control and 23 of 27 affected horses were positive for at least one serovar; bratislava was the most common serovar identified. Bacillus sp. and Micrococcus sp. were cultured from one control horse's eye; Streptococcus sp. (n = 1) and Leptospira (n = 6) were cultured from the eyes of six ERU horses. Leptospira isolates belonged to serogroup pomona (n = 4) and grippotyphosa (n = 2). Polymerase chain reaction results were positive in 14 of 31 (45%) horses with ERU; no control horses were positive by PCR (P = .0001). Microscopic agglutination test was positive for 17 of 24 ERU horses (71%) and one 21 (4.7%) normal horses (P < .0001). Horses with ERU had a high prevalence of Leptospira infection based on PCR and MAT results from intraocular fluids compared with control horses. The diagnosis of intraocular infections was not aided by serology and required specific invasive sampling of ocular fluid. Leptospira infection should be considered as a cause of ERU in the Southern United States.     

Detection of leptospira in the vitreous body of horses without ocular diseases and of horses with equine recurrent uveitis (ERU) using transmission-electron microscopy

Equine recurrent uveitis (ERU) is caused by persistent intraocular leptospira, which appear to use the vitreous body as a refuge. The detection of leptospira in the vitreous body of horses with spontaneous ERU by histological methods has not yet been described. Thirty eight vitreous body samples from 36 horses with ERU (collected during vitrectomy), and 10 vitreous body samples obtained from 5 horses without ocular disease (control group) were examined by transmission electron microscopy. Prior to sample collection, 2 ml of a leptospira culture suspension were injected into the vitreous body of 2 eyes enucleated from horses of the control group. The detection of leptospira in samples, experimentally inoculated with these bacteria was uncomplicated; in vitreous body samples from horses with spontaneous ERU the detection was successful in only a few cases (3/38). The morphologically varying envelope of leptospira in vitreous body samples of horses which developed ERU spontaneously suggests the existence of a bacterial masquerade in vivo.     

Equine recurrent uveitis: A clinical manifestation of leptospirosis

Leptospirosis is a zoonosis of worldwide distribution affecting domestic animals, wildlife and man. The bacterial disease is caused by pathogenic Leptospira spp., which are transmitted from reservoir hosts to accidental hosts. Horses are accidental hosts and can become susceptible to leptospiral infections. Widespread exposure to leptospires exists and is significantly more common than clinical disease. Leptospirosis can have different clinical manifestations including abortion, still birth, systemic disease with hepatic or renal dysfunction, and equine recurrent uveitis (ERU). ERU is the most frequently encountered clinical manifestation and this article will focus on the review of leptospira‐associated ERU. Equine recurrent uveitis is the most common cause of vision impairment and blindness in horses. The pathogenesis of leptospira‐associated ERU involves direct bacterial effects and immune‐mediated responses. Clinical signs vary between the acute and chronic phases of the disease and progress over time. The diagnosis of leptospira‐associated ERU can be difficult and usually requires a combination of diagnostic tests. Medical and surgical treatments have been described with varying outcomes. The prognosis for sight is usually poor, although core vitrectomy may improve the outcome. Avoidance of leptospiral exposure of horses is the only reliable prevention of leptospira‐associated disease.     

A Comparison Between Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay Methods for Detecting Leptospira in Equine Recurrent Uveitis

Leptospirosis is a widespread zoonotic disease that affects humans and many species of animals. Leptospiral organisms have long been presumed to be associated with the presence of equine recurrent uveitis (ERU). The connection between ERU and leptospirosis can be established using various techniques. In the current study, we used a polymerase chain reaction (PCR) assay to help establish a diagnosis of ERU caused by Leptospira infection and compared the results with those of enzyme-linked immune assay (ELISA). A total of 31 and 30 serum samples were taken from horses with ERU (based on clinical diagnosis) and horses that were healthy, respectively, between June 2007 and December 2008. The results showed that from 31 affected horses, a total of seven and five samples were positive for leptospiral infection using PCR and ELISA, respectively, whereas there was no evidence of infection with Leptospira spp. in 30 serum samples of healthy horses. All of the positive samples in ELISA were also positive by PCR, whereas PCR detected two positive cases that were negative by ELISA. Although there was no significant difference between these two methods, it is apparent that PCR may be a more reliable tool for detecting the presence of leptospires in ERU.     

Comparison of diagnostic procedures for porcine leptospirosis.

Kidneys and matched serum samples were obtained from 368 pigs slaughtered at three Victorian abattoirs, and originating from 42 farms. Macroscopic lesions (white spots) were observed on 102 of the kidneys. Serum samples were tested by the microscopic agglutination test (MAT) and by an IgM enzyme immunoassay (EIA). Kidneys were cultured for leptospires, examined histologically after Warthin-Starry silver staining and after immunogold silver staining (IGSS), and tested for leptospiral DNA by DNA hybridization. Forty-four infected pigs were identified by culture or immunogold silver staining of kidneys or by high MAT titres (greater than or equal to 1024). Infection was demonstrated in 7.5% of visibly normal kidneys, in 23.5% of kidneys with white spots, and in 48% of kidneys with large white spots, of 1 cm diameter or greater. The apparent (maximum) sensitivities of diagnostic procedures for detecting infection were as follows: MAT (at a titre of either 64 or 1024) 95%; IgM EIA 82%; culture 61%; presence of white spots 55%; IGSS 52%; presence of large white spots 30%; Warthin-Starry silver staining 20%. IGSS, Warthin-Starry staining and DNA hybridization all appeared to be highly specific. Of 22 kidney sections identified as positive by IGSS, 13 showed intact leptospires, and these kidneys were all culture-positive. Nine others showed leptospiral antigen in the kidney tubules but no intact leptospires. Only five of these kidneys were culture-positive.     

Depiction of the structure of the vitreous body in horses without ocular diseases and in horses with equine recurrent uveitis (ERU) using transmission electron microscopy

Neither the ultrastructure of the vitreous body from horses without ocular diseases, nor the pathomorphological changes in the vitreous body associated with equine recurrent uveitis (ERU) have been described. However, the vitreous body plays an important role in the pathogenesis of ERU. Ten vitreous body samples obtained from 5 horses without ocular disease, and 38 vitreous body samples from horses with ERU (collected during vitrectomy) were examined by transmission electron microscopy. The vitreous body samples of horses without ocular diseases were characterized by a loose network of unbranched fibrils 10-12 nm in width. In the vitreous body samples of horses with ERU numerous dense bundles of fibrils, mononuclear inflammatory cells and necrotic cells represent the destruction of the vitreous fibrillar network. In this study, equine vitreous body ultrastructure was described for the first time. Thus, demonstrating ultramorphologically, the clinically apparent changes of the vitreous body associated with ERU.     

Serodiagnosis of leptospirosis in pigs using an axial filament enzyme-linked immunosorbent assay.

The axial filament (AF) from Leptospira interrogans serovar canicola was isolated by cesium chloride density gradient centrifugation of 2% sarcosyl treated whole cells. Isolation of AF was confirmed by electron microscopic examination, by protein-A immunogold labelling, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. Analysis by SDS-PAGE of the purified preparation showed relatively weak bands of molecular size 41 kDa and 21 kDa, and strong bands of 35 kDa and 34.5 kDa. Immunoblot analysis using antiserum to the AF against sonicated leptospires of a variety of serovars showed prominent reaction against the 41, 35, and 34.5 kDa protein bands, as well as against minor bands of molecular weight 43, 39, and 37 kDa. Antisera prepared against leptospiral serovars also identified minor bands at 33 and 32 kDa. Immunoblots with antiserum to whole cells of serovar bratislava detected the 35 and 34.5 kDa AF bands of Borrelia burgdorferi moderately and of Treponema hyodysenteriae only slightly in comparison to leptospiral AF. Antibody to B. burgdorferi did not detect the leptospiral AF antigen. Immunoblots with antiserum to T. hyodysenteriae showed a marked reaction with a 41 kDa band of B. burgdorferi but only a very minor reaction with leptospiral AF. The AF was tested in an AF-ELISA against sera from 260 pigs, many of which reacted in the microscopic agglutination test (MAT) against one or more leptospiral serovars. A sensitivity of 97.1% and a specificity of 93.1% was determined in comparison to the MAT. Only moderate correlation was observed between titres detected in the AF-ELISA and the MAT (r = 0.4). When sonicated whole cells (WC) of serovar canicola were used in an ELISA (WC-ELISA), high correlation was observed between AF-ELISA and WC-ELISA (r = 0.97). These findings show that the AF-ELISA can be used effectively as a species-specific antigen for the serological diagnosis of leptospirosis in swine and that sonicated whole cells can substitute excellently for purified AF as the antigen source. These findings may be extrapolated to the use of AF in immunodiagnosis of leptospirosis in other species.     

Comparison between specific immunoperoxidase staining and bacteriological culture in the diagnosis of renal leptospirosis of pigs

Seventy-two pigs were examined for the presence of leptospires in the kidney by both bacteriological culture and an immunoperoxidase procedure performed on formalin-fixed, paraffin-embedded sections of tissue with a primary antibody raised in rabbits against serovar pomona. The methods were in accordance in 62 of 70 (89 per cent) of the specimens. Compared with culture the sensitivity of the immunoperoxidase procedure was 30 of 38 (78 per cent) and its specificity 100 per cent; the predictive value of a positive result was 100 per cent, of a negative result, 80 per cent. The major advantages of the immunoperoxidase procedure are specificity, speed of execution and the possibility of simultaneous visualisation of leptospiral antigen and microscopic lesion.     

Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States

OBJECTIVE: To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. SAMPLE POPULATION: Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). PROCEDURES: Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreous humor from clinically normal horses (n = 12) and horses with chronic (> 3-month) ERU (28). Aqueous humor and serum were also evaluated for anti-Leptospira antibody titers from clinically normal horses (n = 12), horses with non-ERU inflammation (17), and horses with confirmed chronic ERU (24). RESULTS: Bacterial DNA was not detected in aqueous humor or vitreous humor of horses with ERU or clinically normal horses. No significant difference was found in titers of anti-Leptospira antibodies in serum or aqueous humor among these 3 groups. Only 2 horses, 1 horse with ERU and 1 horse with non-ERU inflammation, had definitive intraocular production of antibodies against Leptospira organisms. CONCLUSIONS AND CLINICAL RELEVANCE: In horses from the southeastern United States, Leptospira organisms may have helped initiate ERU in some, but the continued presence of the organisms did not play a direct role in the pathogenesis of this recurrent disease.     

Identification of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain reaction analysis.

Studies were carried out to determine the cause of death in a prematurely born Thoroughbred foal that died 24 hours after birth. Necropsy revealed gross lesions suggestive of septicemia. A commercial Leptospira polymerase chain reaction (PCR) assay designed to specifically amplify the hemolysis-associated protein 1 (hap1) gene present only in pathogenic Leptospira strains detected the presence of Leptospira DNA in various tissues of the foal. Histologic examination of lung, liver, kidney, and myocardium revealed numerous spirochetes in Warthin-Starry-stained tissue sections. Results of PCR analysis and histologic examination suggested a leptospiral infection in the newborn foal. At the moment of death, the infection coexisted with a streptococcal-associated aspiration bronchopneumonia and postpartum septicemia. These findings indicate that the PCR assay based on the amplification of the hap1 gene represents a useful tool for specific detection of pathogenic leptospira in field samples taken from horses.     

Occurrence of various immunoglobulin isotopes in horses with equine recurrent uveitis (ERU)

We investigated 30 healthy eyes and 41 eyes with ERU from 57 horses. The total immunoglobulin titers and titers of IgGa, IgGb, IgM were measured in aqueous humour, vitreous and serum using different ELISA techniques. Every sample investigated contained detectable amounts of immunoglobulins. Compared to control eyes significantly increased titers were found in the aqueous humour and vitreous of the ERU eyes for all immunoglobulin isotypes studied (p < or = 0.01). While IgM was detected in only 2 out of thirty aqueous humour and in none of the thirty vitreous samples of healthy eyes, 79.6% of samples of ERU eyes revealed considerable IgM titers. Changes of the IgGa/IgGb ratio in the eye as compared to that in the autologous serum was more frequent in affected than in healthy eyes. In contrast to the intraocular immunoglobulins there were no significant differences in immunoglobulin serum titers in healthy horses and those affected with ERU (p > 0.05). In conclusion, the results argue for a physiological appearance of immunoglobulins in the healthy eye. The increased titers of immunoglobulins in eyes stricken with ERU might be signs either of a local ocular production of antibodies and/or an increased permeability of intraocular barriers.     

Serodiagnosis of bovine leptospirosis by IgG-Enzyme-Linked Immunosorbent Assay and Latex Agglutination Test

The efficacy of a recombinant leptospiral outer membrane protein LipL41 as an antigen for conducting IgG-Enzyme linked immunosorbent assay (ELISA) and latex agglutination test (LAT) for serodiagnosis of bovine leptospirosis was evaluated. The recombinant LipL41 antigen developed and used for detecting the antibodies was specific in detection of the pathogenic serovars of Leptospira, as the expression of the LipL41 antigen is restricted only to pathogenic leptospires. A total of 430 bovine serum samples were subjected to IgG-ELISA and LAT, and the sensitivity and specificity were assessed in comparison with microscopic agglutination test (MAT). The sensitivity and specificity of IgG-ELISA and LAT were 86.84% and 93.16%, and 95.42% and 98.33% respectively. Both the tests are found to be sensitive, specific and concurred with the standard MAT. The study concluded that the rLipL41 protein could be used as a potential diagnostic antigen in different assay formats for bovine leptospirosis.     

Detection of leptospires in bovine semen by polymerase chain reaction

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.     

Use of furosemide to obtain bovine urine samples for leptospiral isolation

The use of the diuretic furosemide made it possible to obtain samples of urine from cattle for leptospiral isolations. The drug was injected IV at a dose level of 0.8 mg/kg for heifers and 0.5 mg/kg for calves. The average time to first voiding in heifers was 19 minutes. The average time from the first to the second voiding was 17 minutes. The average time to the first voiding in four calves was 12 minutes; the average time from the first to the second voiding was 10 minutes. A decrease in urinary osmolarity provoked by furosemide created a more favorable condition for the survival of leptospires. Leptospires were isolated in 24 (72.7%) of 33 weekly cultural attempts with the aid of furosemide in three experimentally infected adult cattle. Serovar hardjo was isolated in 16 (57.1%) of 28 weekly cultural attempts with aid of the diuretic in four experimentally infected calves. The recovery frequency was 28.5% from the first voiding and 50% from the second. Leptospires were not isolated from urine obtained from the calves by manual stimulation. Untoward side effects that might have been attributable to furosemide were not observed. Furosemide appears to be well suited to obtain urine samples from cattle for leptospiral isolation.     

The epidemiological interpretation of serological responses to leptospiral serovars in sheep

Serum samples were examined for evidence of leptospiral agglutinins from 928 sheep from 45 lines and kidneys from 12 of these lines for evidence of leptospiral infection. All sheep had been submitted for slaughter at meat works in the Manawatu. Serological results were analysed using the results at a minimum serum dilution in the microscopic agglutination test (MAT) of 1:24 and at a minimum dilution of 1:48. It was shown that a minimum dilution of 1:24 resulted in many non-specific or cross-reactions. A minimum dilution of 1:48 was more accurate for detecting the serological prevalence of specific agglutinins to leptospires in ovine sera. Twenty percent of the sheep had titres of 1:48 or greater to hardjo, 3.8% to pomona, 2.6% to tarassovi, 2.3% to copenhageni and 2.7% to ballum. No titres of 1:48 or greater to australis were detected. Serovar hardjo was isolated from the kidneys of three animals in one line. Eighteen months later 291 serum samples and 95 urine samples were collected from live animals on the property from which the three hardjo infected animals originated. No titres to hardjo were detected in the sera of lambs, but a serological prevalence of 44% and 84% to this serovar was demonstrated in the hoggets and ewes respectively. No leptospires were demonstrated in any of the urine samples. These results show that sporadic infection of sheep with hardjo can occur but they also indicate that infection with this serovar is not endemic and that sheep are unlikely to act as maintenance hosts for hardjo in New Zealand.     

Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis.

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.     

Surgical management of equine recurrent uveitis with single port pars plana vitrectomy

Current information suggests that equine recurrent uveitis (ERU) is an immune-mediated reaction to infectious agents or to autologous ophthalmic tissue. Recurrences are associated with progression of irreversible ocular damage. This report describes the intraoperative technique, complications, and long-term results of 38 eyes in 35 horses with ERU that underwent pars plana vitrectomy. The majority of the horses were warm-blooded. Recurrence of ERU was prevented in 35 of the 38 eyes. Some horses, especially in patients with incipient cataracts, developed vision loss in postoperative, quiescent eyes which was usually associated with cataract formation. Vision was stable in 85% of all eyes that underwent vitrectomy. Pars plana vitrectomy in horses appears successful in interrupting the cycle of repeated episodes of ERU, and the subsequent globe destruction in the majority of eyes. Removal of uveitis-induced 'immunologic memory' in the vitreous by vitrectomy may reduce adverse interaction between the vitreous and the uveal tract, and therefore reduce the recurrence of ERU.     

Detection of Leptospira spp. in wildlife reservoir hosts in Ontario through comparison of immunohistochemical and polymerase chain reaction genotyping methods

A total of 460 kidney samples from wildlife (beavers, coyotes, deer, foxes, opossums, otters, raccoons, skunks) were obtained from road-kill and hunter/trapper donations in Ontario between January 2010 and November 2012. The objectives of the study were to detect Leptospira spp. by immunohistochemistry and polymerase chain reaction (PCR), to map presence of leptospires in wildlife relative to livestock and human populations, and to characterize positive samples by sequencing and comparison to leptospires known to affect domestic animals and humans. The proportion of samples that tested positive ranged from 0% to 42%, with the highest rates in skunks and raccoons. Leptospira spp. were present in kidneys of wildlife across Ontario, particularly in areas of high human density, and areas in which livestock populations are abundant. The PCR was too weak in most samples to permit genotyping and examination of the relationship between the leptospires found in this study and those affecting domestic animals and humans.     

Detection of leptospires in biological fluids using DNA hybridisation

DNA extracted from Leptospira interrogans serovar pomona was labelled with phosphorus-32 by nick translation and used as a genomic probe to detect leptospiral DNA. The sensitivity of detection in a 10-microliter spot on nylon membranes was 160 pg of leptospiral DNA or 1.1 X 10(3) leptospires and assays with nylon membranes were somewhat more sensitive than assays with nitrocellulose membranes. The probe reacted with the pathogenic hardjo and tarassovi leptospiral serovars, but not with other genera of bacteria. To detect leptospires in body fluids, these were treated to free leptospiral DNA and then concentrated on membranes using a Bio-Dot apparatus. Neither serum nor urine interfered with the assay system. The DNA of leptospires added to pig urine was stable for at least 2 h at room temperature and for at least 20 h at -20 degrees C.