Bovine leptospirosis: Microbiological and histological findings in cattle slaughter

Kidneys from cattle at slaughter were examined for the presence of leptospires. Of 218 (8.3%) kidneys leptospires were isolated from 18; all were identified as Leptospira interrogans serovar hardjo. None of the leptospire-infected kidneys had histopathological lesions indicative of leptospirosis and leptospires were demonstrated in only 2 by immunogold silver staining. Leptospires infected kidneys remained viable for at least 21 days when stored at 4 degrees but became non-viable within 14 days when stored frozen at -15 degrees.     

Prevalence of pathogenic Leptospira spp. in sheep in a sheep-only abattoir in New Zealand

AIM: To determine the prevalence of the two most commonly diagnosed pathogenic Leptospira spp. serovars, Hardjobovis and Pomona, in sheep in a sheep-only abattoir in New Zealand, and to determine the prevalence of kidneys which were leptospire culture-positive collected from sheep seropositive or seronegative to the microscopic agglutination test (MAT). METHODS: A repeated cross-sectional observational study was conducted of serological and kidney culture prevalences of Leptospira borgpetersenii serovar Hardjobovis and Leptospira interrogans serovar Pomona. Lines of sheep and individual sheep were systematically randomly selected at a sheep-only abattoir during 18 May 2004 to November 2004 and 06 December 2004 to 14 June 2005. Additionally, a cross-sectional study examined prevalences in a purposively selected line of sheep from a flock with clinical evidence of an outbreak of leptospirosis. RESULTS: In the study population of 15,855 sheep of which 2,758 were sampled, 5.7 (95% CI=4.9-6.7)% were seropositive to one or both serovars; 44.2 (95% CI=34.6-54.2)% of 95 lines of sheep and 44.9 (95% CI=35.0-55.3)% of 89 farms showed serological evidence of infection. The serological prevalence of serovar Hardjobovis was significantly higher than that of serovar Pomona both at line (33% and 4%, respectively) and individual (5% and 1%, respectively) levels. A low but persistent seroprevalence of Hardjobovis throughout both years suggested low-level endemicity to this serovar, whereas Pomona infections appeared to be sporadic. Leptospires were isolated from kidneys of 8/37 (22%) Hardjobovis- and 1/6 (17%) Pomona-seropositive, and 5/499 (1%) seronegative animals. Of the animals purposively sampled from a farm with a clinical outbreak of leptospirosis, all kidneys from the 13 seropositive animals were culture-positive, indicating a high risk of exposure of meat workers in outbreak situations. Kidneys of MAT-seropositive sheep were 21.7 (95% CI=7.6-61.9) times more likely to test culture-positive than kidneys from animals with negative MAT titres. In general, the results indicated that 13/1,000 sheep slaughtered were potentially shedding leptospires. CONCLUSIONS: The study demonstrated the presence of a definite risk of occupational exposure of meat workers in a sheep-only slaughterhouse to the two most commonly diagnosed pathogenic Leptospira spp. serovars in New Zealand.     

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.     

Leptospira in slaughtered fattening pigs in southern Vietnam: presence of the bacteria in the kidneys and association with morphological findings

One kidney was collected from each of 32 fattening pigs at an abattoir in southern Vietnam in 2001 in order to demonstrate infecting Leptospira serovar and to associate renal macro- and microscopic findings with the presence of renal leptospires. Leptospires were demonstrated in 22 (69%) of the investigated kidneys by immunofluorescence. Multifocal interstitial nephritis (MFIN) and gross renal lesions (white spots) were each demonstrated in 24 (75%) kidneys. Leptospira interrogans serovar bratislava was isolated from one kidney. There was no association between presence of leptospires and MFIN (P=0.19), respectively and white spots (P=0.98), respectively. These data suggest that Leptospira infection is common among fattening pigs in the study area and that these animals may be considered as an occupational human health hazard. It is also suggested that the presence of white spots is an unreliable indicator of the presence of renal leptospires.     

An improved immunohistochemical diagnostic technique for canine leptospirosis using antileptospiral antibodies on renal tissue.

The purpose of this study was to compare the immunoreactivity in canine renal tissues stained with antisera specific for 3 leptospiral antigens and those processed with traditional staining methods. In addition, immunoglobulin staining was done on tissues with immunoreactivity to leptospiral antigens. Formalin-fixed renal sections from 12 dogs with chronic interstitial nephritis suspected or proven to have leptospirosis (6 dogs with silver-stained leptospires and 6 dogs in which silver-stained leptospires were not detected) were used. Antibodies consisted of a monoclonal antibody to Leptospira kirschneri serovar grippotyphosa lipopolysaccharide (LPS) and 2 polyclonal antibodies to outer membrane proteins, including OmpL1, a leptospiral porin, and LipL41, an outer membrane lipoprotein. The murine monoclonal antisera against LPS (F71C2-1) had the most abundant and consistent immunoreactivity. Immunoreactive areas were present in 6 of 6 sections positive by silver staining and included extracellular granular debris in intertubular areas, debris in macrophages, organisms in tubular lumina, and cytoplasmic granules in tubular epithelia. Antisera with specificity for the outer membrane proteins OmpL1 and LipL41 detected only intact organisms in tubular lumina. Immunoreactivity to OmpL1 (polyclonal 338) occurred in 4 of 5 sections positive by silver staining, but immunoreactivity to LipL41 (polyclonal 813) occurred in only 1 of 6 silver-positive sections. Each of the kidney sections in which leptospiral antigens were detected by immunohistochemistry also was positive by silver staining. Sections negative by silver staining were also negative by immunostaining. Although immunohistochemistry did not enhance sensitivity, amplification of signal by secondary antibody and hematoxylin counterstaining improved the ease of diagnosis and allowed better evaluation of tissue morphology than did silver staining methods. IgG was the most abundant immunoglobulin. IgG immunoreactivity occurred predominantly in plasma cells within interstitial infiltrates. Interstitial infiltrates contained abundant immunoreactivity to LPS, but immunoreactivity to OmpL1 and LipL41 was not noted.     

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.     

Immunohistochemical detection of porcine rotavirus using immunogold silver staining (IGSS).

Immunogold silver staining (IGSS) was applied for the detection of porcine group A rotavirus in formalin-fixed paraffin-embedded tissue sections of small intestine. Prior to the application of IGSS, the reactivity of protein A-gold as a marker was tested with group-specific antiserum in immunogold electron microscopy. Immune aggregates were intensely and specifically labeled with the gold complex. Application of IGSS to tissue sections resulted in specific dark staining of villous enterocytes infected by group A rotavirus. This method also proved effective for the detection of rotaviral antigen in infected cultured cells. The IGSS method may be suitable for routine diagnostic detection of rotaviral infections and may have application for detection of other viral pathogens of veterinary importance.     

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.     

Leptospira interrogans serovar hardjo is not a major cause of bovine abortion in Victoria

The aim of this study was to determine whether evidence could be obtained of foetal infection with Leptospira interrogans serovar hardjo in aborted foetuses collected from dairy farms. Material from 197 abortions occurring over a wide area of Victoria was collected over 3 years. None of 195 foetal kidney cultures or 7 cultures from membranes was positive for leptospiral organisms. Immunogold silver staining for leptospires was performed on sections of kidneys, lungs or heart from 156 foetuses, with negative results. Evidence of transient leptospiral infection in 11 of 123 foetuses was obtained by foetal heart blood serology. Two isolates of L. interrogans serovar hardjo were obtained from the urine of milking cows. These strains were examined by restriction endonuclease analysis and both were shown to be of the genotype Hardjobovis, as have been all Australian isolates studied so far. It appears that foetal infection with serovar hardjo is not associated with any substantial proportion of bovine abortions in Victoria, in contrast to the situation in Northern Ireland. The apparent absence from Victoria of the pathogenic genotype Hardjoprajitno is a possible explanation.     

Recurrent uveitis in horses: vitreal examinations with ultrastructural detection of leptospires

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.     

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.     

Detection of leptospires in tissue using an immunoperoxidase staining procedure

An immunoperoxidase technique for the localisation of leptospires in sections of formalin fixed paraffin embedded kidney tissue is described. The procedure utilises a two-layered antibody sandwich with rabbit anti-leptospiral immunoglobin. Using antiserum to specific leptospiral serovars the presence and distribution of specific serovar in the tissue could be determined. The technique was also used to detect leptospires of given serovars in smears made from infected tissues and fluids. There was good correlation between culture results and results of the immunoperoxidase staining method on kidneys infected with leptospires. The diagnostic possibilities of the technique on formalin fixed tissue specimens are discussed.     

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.     

柞蚕微孢子虫单克隆抗体的研制及诊断

研究报告了柞蚕微孢子虫Nosemaantheraeae(简称N .a)单克隆抗体制备及免疫胶体金银染色法 (IGSS)诊断的结果。用抗N .a的单抗结合间接免疫胶体金银染色法 (IGSS)对 6种微孢子虫进行检测 ,在光学显微镜下柞蚕微孢子呈现特异的褐色 ,能与包括家蚕微孢子虫在内的其它 5种微孢子虫加以区别。     

Impacts of intensification of pastoral agriculture on soils: Current and emerging challenges and implications for future land uses

Abstract

AIM: To find evidence for localisation in the uterus, and fetal infection, of Leptospira spp. in farmed deer in the lower North Island of New Zealand during and shortly after the breeding season.

METHODS: Between February and July 2008, 116 blood samples, 120 kidneys, 120 uteri and 27 fetuses were collected from 120 mixed-age hinds from lines from nine farms, at a deer slaughter premises. Serum samples were tested for antibodies against Leptospira borgpetersenii serovar Hardjo-bovis and Leptospira interrogans serovar Pomona, using the microscopic agglutination test (MAT). For both serovars, a titre of >1:48 was considered positive. Samples from kidneys, uteri and fetal tissue were subjected to bacterial culture, using Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and real-time PCR, using DNA gyrase subunit B gene primers.

RESULTS: Thirty-four of 116 (29.3%) serum samples were positive for serovar Hardjo-bovis, and 13 (11.2%) for serovar Pomona. Seven of 120 kidneys were positive for serovar Hardjo-bovis by culture, and five of these, but no others, were positive by real-time PCR. Of 120 uteri, none was culture- or PCR-positive. None of 27 fetal samples was culture-positive but one was positive by real-time PCR. The dam of the PCR-positive fetus was culture-negative from the kidney, but had an MAT titre of 1:192 for Hardjo-bovis.

CONCLUSIONS: Attempts to isolate Leptospira spp. from the genital tracts and early fetuses of farmed deer were unsuccessful. However, molecular evidence suggested fetal infection in one case. This finding justifies further study of the role of leptospires in the genital tract and fetus and its association with reproductive loss in farmed deer.     

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.     

Bovine abortion associated with Leptospira interrogans serovar hardjo infection

During 1981, 265 bovine abortions were investigated by serological and histological methods for evidence of leptospiral infection. Leptospires were demonstrated in the tissues of 10 foetuses by a Levaditi silver impregnation technique. Serological testing of maternal sera indicated that Leptospira interrogans serovar hardjo was associated with 5 of the abortions while the remaining 5 were due to L. interrogans serovar pomona infection. In cases of abortion associated with L. interrogans serovar hardjo leptospires were readily demonstrated in foetal liver, kidney, intestine and heart. They were demonstrated less often in lung and placenta and could not be found in foetal brain. Autolysis did not appear to interfere with the demonstration of leptospires by silver impregnation. No lesions attributable to leptospiral infection were seen in placentas but mild interstitial nephritis was found in some of the foetuses. Fourteen other cows had serological evidence of recent leptospiral infection but leptospires were not detected in foetal tissues. Histological examination of silver impregnated foetal tissues in combination with the microscopic agglutination test was shown to be an effective method for diagnosing abortion associated with L. interrogans serovar hardjo in cattle.     

Leptospirosis in sheep in Western Canada

A survey of 930 ovine sera and kidneys from 33 sheep was conducted to assess the rate of leptospiral infection in sheep slaughtered in Alberta. Sera were tested for the presence of agglutinins to indigenous serovars of Leptospira interrogans. Kidneys with gross lesions were examined for the presence of leptospires by means of an indirect fluorescent antibody test (FAT) and by culture. Antibodies to serovars pomona and hardjo were present at rates of 1.0% and 0.4%, respectively, in sheep from Saskatchewan, Alberta and British Columbia. Sera from 120 feedlot lambs shipped from Oregon reacted to serovars pomona, hardjo and grippotyphosa at rates of 1.7%, 61.7% and 59.1%, respectively. Fluorescent antibody test detected serovars (presumptively) hardjo in 52% of Oregon feedlot lambs and grippotyphosa in 32% of the same group, a finding supported by the isolation of both these serovars from a pool of two fluorescent antibody test-positive kidneys. The grippotyphosa strain was highly virulent for hamsters, producing intense icterus and death. Leptospires, presumptively serovar grippotyphosa were demonstrated by fluorescent antibody test in one Alberta lamb kidney. The possibility of spreading leptospirosis by movement of breeding stock through public facilities and by assembling lambs in feedlots is discussed.     

The prevalence of leptospirosis and its association with multifocal interstitial nephritis in swine at slaughter.

An abattoir survey was undertaken to determine the prevalence of leptospirosis and its association with lesions of multifocal interstitial nephritis (so-called "white spotted kidneys") in swine at slaughter. Both cross-sectional and case-control study designs were used. Of 197 kidneys from hogs randomly selected at slaughter, 11 (5.6%) had generalized grey-white foci typical of multifocal interstitial nephritis (MFIN). Antibody titers greater than or equal to 1:80 against Leptospira pomona were detected in nine (4.6%) hogs and against L. bratislava in 63 (32%) of these hogs. Leptospira pomona (kennewicki) was detected by immunofluorescence in 5/197 (2.5%) of randomly selected hogs. Leptospires identified as genotype kennewicki were isolated from six (9.8%) of 61 kidneys cultured. Leptospira bratislava was not detected by immunofluorescence or culture. There was a highly significant (p = 0.00) and strong association (odds ratio (OR) = 195) between high L. pomona titer (greater than or equal to 1:80) and the presence of leptospires in the kidneys, as detected by culture. There was also a significant (p = 0.046) and strong (OR = 8.10) association between multifocal interstitial nephritis and the presence of renal leptospires as detected by culture. The association between leptospiral titer and MFIN lesions in the prevalence survey group of animals was statistically significant (p = 0.031), but this association was not significant in the case-control study group (p = 0.071) The failure to identify L. bratislava despite serological evidence of infection suggests that some of these seropositive animals may have been transiently infected at an early age, that serological findings were falsely positive, or that immunofluorescence and isolation attempts failed to detect L. bratislava if they were indeed present in the kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical identification and pathologic findings in natural cases of equine abortion caused by leptospiral infection

The aim of this study was to examine the utility of immunohistochemistry (IHC) in the diagnosis of leptospiral equine abortion and to compare IHC to silver staining and serology of the aborted mares. Ninety-six fetuses from 57 farms were examined using all 3 diagnostic techniques, revealing evidence of leptospiral infection in 3 fetuses (3.1%) from 3 (5.3%) different farms. A new finding in 1 of these confirmed cases of leptospiral abortion was the presence of macroscopic pinpoint grayish-white nodules that had a histologic correlate of hepatic necrosis; other histologic findings were consistent with those previously reported. IHC performed using 2 different leptospiral antisera (multivalent whole-cell rabbit antiserum and rabbit antiserum against the major outer membrane protein LipL32) yielded similar results. IHC was more sensitive (19/21 [90.5%] tissue samples) than silver staining (8/21 [38.1%] tissue samples), and more specific than serology performed using the microscopic agglutination test. The primary advantage of IHC over silver staining was the ability of IHC to identify leptospiral antigen not only as morphologically intact spiral forms.