Restriction endonuclease analysis of Leptospira interrogans serovar hardjo isolates from cattle

The genomes of 253 strains of Leptospira interrogans serovar hardjo were examined by restriction endonuclease analysis of their DNA. The strains had been isolated from cattle at an abattoir (190), milk of agalactic cows (seven) and from aborted bovine fetuses (56). Two distinct genome types, Hardjoprajitno and Hardjobovis, were detected. The majority (91 per cent) of isolates from abattoir cattle were of the Hardjobovis type while most (76 per cent) of the isolates from clinical/pathological material were similar to Hardjoprajitno.     

Development and initial evaluation of an indirect enzyme-linked immunosorbent assay for the detection of Leptospira interrogans serovar hardjo antibodies in bovine sera.

Outer sheath antigen from Leptospira interrogans serovar hardjo type hardjoprajitno and acetic acid extracted antigens from serovar hardjo types hardjoprajitno and hardjobovis were evaluated in an immunoassay for ability to detect hyperimmune rabbit serum to serovar hardjo. The degree of cross-reactivity with hyperimmune rabbit sera to L. interrogans serovars pomona, copenhageni, grippotyphosa, canicola and sejroe, and Leptospira biflexa serovar patoc was also measured for each antigen. All of the antigens reacted with the antiserum to L. interrogans serovar hardjo. The outer sheath antigen however, also showed wide cross-reactivity with the antisera to all of the serovars of L. interrogans tested and with the antiserum to L. biflexa serovar patoc. The acetic acid extracted antigen from either type hardjoprajitno, or type hardjobovis, showed a high degree of specificity for serovar hardjo antiserum. The hardjobovis acetic acid extracted antigen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting, and was incorporated into an indirect ELISA for detection of anti-serovar hardjo antibodies in bovine serum. This ELISA showed a relative specificity of 100% with 156 bovine sera which were negative at a dilution of 1:100 in the microscopic agglutination test (MAT) for L. interrogans serovars hardjo, pomona, sejroe, icterohaemorrhagiae, copenhageni, canicola, and grippotyphosa. The relative sensitivity of this assay with 192 bovine sera which had serovar hardjo MAT titres of > or = 100 was 95.3% (95% confidence limit = 2.99%). The degree of cross-reactivity with 289 bovine sera which had serovar pomona MAT titres of > or = 100 (with no detectable serovar hardjo MAT titres) was approximately 1.0%. This assay was: easily standardized, scored objectively, repeatable, semi-automated and used a non-hazardous antigen that can be routinely prepared in gram amounts.     

Monoclonal antibodies to Leptospira interrogans serovar pomona.

Three monoclonal antibodies produced against Leptospira interrogans serovar pomona have been studied for their diagnostic usefulness. All three monoclonals reacted strongly in the enzyme-linked immunosorbent assay and indirect fluorescent antibody test with serovar pomona and did not react with serovars grippotyphosa, canicola, icterohaemorrhagiae and hardjo.     

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.     

The efficacy of a Leptospira interrogans serovars pomona and copenhageni and L. borgpetersenii serovar hardjo vaccine in cattle

An experimental, trivalent, bovine, leptospiral vaccine, containing inactivated Leptospira interrogans serovars pomona and copenhageni and L. borgpetersenii serovar hardjo Hardjobovis, was developed. The experimental vaccine was shown to protect hamsters against virulent challenge with each of the component serovars. In a serological efficacy test in cattle, the experimental vaccine was compared for bioequivalence with a similar product, registered in New Zealand for veterinary use. The experimental vaccine induced higher titres in cattle than the latter mentioned product.     

Production and characterization of monoclonal antibodies specific for Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno.

Murine monoclonal antibodies were produced by immunizing BALB/c mice with a killed whole-cell antigen prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis. Six of these antibodies recognized epitopes on the homologous antigen and on whole-cell antigen prepared from Leptospira interrogans serovar hardjo type hardjoprajitno. These antibodies did not cross-react with whole-cell antigens prepared from L. borgpetersenii serovar sejroe, 10 other pathogenic Leptospira serovars, or the saprophytic Leptospira biflexa serovar patoc. Three other monoclonal antibodies reacted with antigens prepared from the 2 hardjo serovars and serovar sejroe but not with antigens from the 10 other pathogenic serovars, or serovar patoc. The epitopes recognized by all of the hardjo-specific antibodies and 2 of the 3 hardjo/sejroe-specific antibodies were susceptible to sodium meta-periodate oxidation. All of the antibodies were characterized by Western blots with the hardjobovis whole-cell antigen. Each of the 9 monoclonal antibodies was inhibited from binding to the hardjobovis antigen by bovine sera which were obtained from cattle experimentally infected with hardjobovis and from field cattle, with anti-serovar hardjo microscopic agglutination test antibody titres ranging from 100 to 12800. Some of these antibodies may be suitable for incorporation into competitive enzyme immunoassays for the specific detection of antibodies to either of the hardjo serovars.     

Detection and characterization of leptospiral antigens using a biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot.

A biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of Leptospira interrogans serovars in experimentally inoculated bovine urine samples was evaluated. Immunoglobulin G (IgG) from rabbits immunized with L. interrogans serovar hardjo type hardjobovis sonicated, whole cell, and formalinized-heated antigen preparations were purified by a protein A-superose column coupled to fast protein liquid chromatography, and evaluated for species specificity in the ELISA. The ELISA using each specific IgG detected as few as 10(4) leptospires of the homologous serovar hardjo diluted in phosphate-buffered saline solution with Tween 20 (PBSS-Tween 20). On immunoblot analysis of proteinase-K-digested whole cell leptospiral preparations, each IgG revealed the presence of bands specific to serovar hardjo, suggesting the presence of serovar-specific epitopes on the lipopolysaccharide molecules. The minimum number of cells of heterologous serovars pomona, grippotyphosa, bratislava, icterohaemorrhagiae and copenhageni detected by each ELISA was greater, ranging from 10(6) to 10(7). The common antigenic determinants observed on immunoblot analysis were different for each specific IgG, except for a major cross-reacting, possibly flagellar, protein doublet at approximately 36-36.5 kDa. Leptospires were equally well detected by the ELISA in both bovine urine and PBSS-Tween 20.     

Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT.     

A serosurvey for antibodies to Leptospira in dogs in the lower North Island of New Zealand

AIMS: To investigate the prevalence of antibodies to endemic and exotic Leptospira serovars in samples from a serum bank, collected from dogs in the lower North Island of New Zealand. METHODS: Sera (n=466), which had been collected from apparently healthy dogs, were screened using the microscopic agglutination test (MAT) for antibodies to serovars L. borgpeterseni serovar hardjo, L. interrogans serovars pomona, copenhageni and canicola, and L. kirschneri serovar grippotyphosa. RESULTS: Antibody to Leptospiral antigen was found in 14.2% of dogs tested. The highest level of reactivity was with serovar copenhageni, to which 9.5% (41/433) of sera were positive. Antibodies to serovars grippotyphosa and canicola were not detected in this population of dogs. CONCLUSIONS: Leptospira infection is relatively common in dogs in the lower North Island . CLINICAL RELEVANCE: Vaccination of dogs against leptospirosis should be considered using vaccine containing antigen to serovars hardjo, pomona and copenhageni. The presence of moderate levels of copenhageni antibody in dogs in the lower North Island raises the possibility that this serovar has become established in rodent populations in this region.     

Experimental infection of lactating goats with Leptospira interrogans serovars pomona and hardjo

Pathogenesis of Leptospira interrogans serovars pomona and hardjo was evaluated in 14 lactating goats. Although mild clinical signs of leptospiral infection characterized by pyrexia and reduction in milk yield appeared in some animals, a consistent clinical pattern was not observed in the inoculated animals. The pomona serovar was isolated from the kidney of 1 of the 4 goats inoculated with serovar pomona. The hardjo serovar (strain UI 750) was isolated in the rabbit serum-supplemented bovine albumin polysorbate-80 liquid medium only from the mammary gland of 1 of 4 goats at 13 days after inoculation with serovar hardjo. The positive culture was detected after an 8-month incubation period.     

Seroprevalence and association with abortion of leptospirosis in cattle in Ontario.

Sera were collected using a systematic random sampling from 348 cattle herds in Ontario, in proportion to the cattle population in different areas. One cow in five from 296 dairy herds and one in three from 52 beef herds were sampled. The sera were analyzed for prevalence of antibodies to Leptospira interrogans serovar grippotyphosa, hardjo, icterohaemorhagiae and pomona using the microscopic agglutination test. Herd seroprevalence (one or more animals with titer greater than or equal to 80) in beef and dairy herds combined was grippotyphosa 2%, hardjo 13.8%, icterohaemorrhagiae 10.1% and pomona 25.8%; 39% of all herds showed evidence of leptospiral infection with one or more serovars; 44.2% of 52 beef herds had serological evidence of infection with serovar hardjo compared to 8.4% of 296 dairy herds (P less than 0.0001). Seroprevalence of other serovars was not significantly different between beef and dairy herds. The proportion of beef animals seropositive for hardjo and for pomona increased with age, particularly for hardjo; 26.5% of beef animals aged nine years or over were seropositive for hardjo. Dairy animals showed a significant rise of hardjo but not pomona titers with age. The seroprevalence of pomona infection was significantly higher in dairy cattle in eastern Ontario than in other regions. Thirty-four (6.1%) of 553 aborted bovine fetuses had leptospires detected by immunofluorescence techniques. Sixty-five percent of these fetuses were from submissions made between November and January. Leptospires were identified as serovar hardjo by specific immunofluorescence. There appeared, however, to be a paradoxical serological response in that eight aborting cows had antibody titers to pomona rather than hardjo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of a pathogenic Leptospira serovar in response to combined in vitro pH and temperature stresses

Leptospira interrogans is a zoonotic pathogen hosted commensally by many mammalian species. On the Western Pacific island of American Samoa a seroprevalence survey conducted in 2004 indicated that 17% of subjects tested had been exposed to several Leptospira serovars, including L. interogans icterohaemorrhagiae. Resource management agencies promoted improved animal waste management practices, including composting, to reduce the risk of water contamination and human exposure from small pig husbandry facilities. This study simulated temperature and pH conditions expected in composting. We subjected L. interrogans serogroup icterohaemorrhagiae serovar Copenhageni strain M-20 to temperatures (25-50°C) and pHs (5.2-7.9) for varying durations of exposure (4-96h) to determine survival, indicated by culturing. Temperatures ≥45°C were lethal at the shortest duration of exposure (4h). The effects of temperature were enhanced by pH for temperatures <45°C. The results are summarized in a logistic model of the proportions (p(est)) of leptospires surviving the combined stresses of pH and temperature (p(est)=0.071e((1.091 pH-0.183 T(°C)))/1+0.071e((1.091 pH-0.183 T(°C)))). A graph of this function, with results of other studies carried out with serovar icterohaemorrhagiae, indicates that composting has the potential to kill leptospires, provided that all wastes are fully exposed to temperatures ≥45°C for at least 4h.     

The influence of maternal antibody and age of calves on effective vaccination against Leptospira interrogans serovar hardjo

Twelve seronegative cows were vaccinated with an experimental bivalent Leptospira interrogans serovars hardjo and pomona vaccine late in their first pregnancy. Calves born of these dams were divided into 4 equal groups that received this vaccine at 4, 6, 10 and 18 weeks of age, respectively. Before vaccination the group geometric mean titres of maternally-derived circulating antibodies ranged from 2 to 25 for the microscopic agglutination (MA) test and 3 to 35 for enzyme-linked immunosorbent assay (ELISA) using a serovar hardjo outer envelope antigen. Post-vaccination peak titres were 645 to 1612 for MA and 562 to 1037 for ELISA, respectively. Calves vaccinated at the youngest age, had the highest pre-vaccination circulating maternal antibody titres, but showed the smallest rise in post-vaccination antibody titres. Circulating maternal antibody was detected in calves up to 13 weeks of age. All immunised calves were protected against a virulent challenge with serovar hardjo type Hardjobovis, regardless of their age or maternally-derived antibody titres. These findings indicate that calves as young as 4 weeks old, vaccinated in the presence of maternally-derived antibody, can be fully protected against homologous virulent challenge.     

Seroprevalence of leptospirosis in domesticated Asian elephants (Elephas maximus) in north and west Thailand in 2004

Serum samples from Asian elephants (Elephas maximus) in the Kanchanaburi, Chiang Mai and Lampang provinces of Thailand were tested using the microscopic agglutination test against 22 serovars of Leptospira interrogans. A titre of more than 1:100 was used as evidence of infection. In northern Thailand, the seroprevalence was 58 per cent and the prevalent serovars were Leptospira interrogans serovar Sejroe, Leptospira interrogans serovar Tarassovi, Leptospira interrogans serovar Ranarum and Leptospira interrogans serovar Shermani. In western Thailand, the seroprevalence was 57 per cent and the prevalent serovars were L Tarassovi, L Sejroe, L Ranarum, Leptospira interrogans serovar Bataviae and L Shermani. These results were similar to studies in domestic livestock and stray dogs in the Bangkok district. Among the elephants from Kanchanaburi there were significant associations between seropositivity and between the camp and between the prevalent serovars and the camp.     

Development of a monoclonal antibody-based competitive enzyme-linked immunosorbent assay for the detection of Leptospira borgpetersenii serovar hardjo type hardjobovis antibodies in bovine sera.

A murine monoclonal antibody (designated M553) that binds to an epitope on whole cell antigens prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno, was produced and incorporated into a competitive enzyme-linked immunosorbent assay for the detection of bovine antibodies to serovar hardjo. The epitope recognized by M553 was susceptible to periodate oxidation. The M553 antibody was characterized by western blot with hardjobovis whole cell antigen. This antibody does not cross-react with whole cell antigens prepared from 11 other pathogenic Leptospira serovars, or, Leptospira biflexa serovar patoc. The sensitivity estimate of the competitive ELISA was 100% with field sera (n = 165) with serovar hardjo microscopic agglutination test (MAT) titres of > or = 100. The specificity estimate was 100% with sera (n = 128) obtained from a specific pathogen free herd of cattle that were negative in the MAT at a dilution of 1:100 for serovars hardjo, pomona, sejroe, copenhageni, canicola, and grippotyphosa. The specificity estimate with field sera (n = 301) with serovar hardjo MAT titres of < 100, was 98% (95% confidence interval = +/- 1.58%). There was no cross-reactivity with field sera (n = 306) with serovar pomona titres > or = 100 and serovar hardjo titres < 100. The specificity estimate with the combined populations of sera with serovar hardjo MAT titres of < 100 (n = 735) was 99.18% (95% confidence interval = +/- 0.65%). There was a high level of agreement (kappa = 0.977) between the results of the competitive ELISA and those of the MAT.     

Serological survey of leptospirosis in sows with premature birth and stillbirth in Chiba and Gunma prefectures of Japan

In 1996 and 1997, the seroprevalence against Leptospira in parturient sows with premature birth or stillbirth from two herds was investigated. In three out of four sow serum samples obtained in Gunma Prefecture, the antibody titers to Leptospira interrogans serovar copenhageni (M20) were higher than 10,000 (the reciprocal of the serum dilution). Furthermore, the antibody titers to L. interrogans serovar canicola (Hond Utrecht IV) were significantly high in the three sows and the titers ranged from 1,000 to 3,000. In sows obtained in Chiba Prefecture, significantly high antibody titers to serovar copenhageni (M20) were confirmed in eight out of 40 sows, and antibody titers greater than 10,000 in six of them. Significantly high antibody titers to L. interrogans serovar icterohaemorrhagiae (RGA) and L. canicola (Hond Utrecht IV) were confirmed in four and 18 out of the 40 sows, respectively, compared with the titers to the other serovars. These findings may indicate the prevalence of leptospirosis in pig herds in both Gunma and Chiba Prefectures.     

Seroprevalence of bovine leptospirosis in Garanhuns Municipal District,Pernambuco State,Brazil

The prevalence of Leptospira interrogans serovars in dairy cattle was determined by analyzing 464 serum samples from cows on 15 properties in Garanhuns municipal district, Pernambuco State, Brazil. A microscopic seroagglutination test including 12 serovars of Leptospira interrogans as antigens was used. Samples with titres 100 were considered positive. Two hundred and twenty-one (47.63%) of the samples were positive to one or more serovars. The prevalence of the serovars was hardjo (21.98%), bratislava (15.73%), castellonis (11.64%), tarassovi (10.56%), pyrogenes (1.72%), icterohaemorrhagiae (1.08%), pomona (0.86%), wolffi (0.86%), grippotyphosa (0.86%), djasiman (0.43%), canicola (0.21 %), and copenhageni (0.21%).     

Neospora caninum and Leptospira serovar serostatus in dairy cattle in Ontario

No significant association existed between Neospora caninum titer and serostatus to Leptospira serovar hardjo, icterohaemorrhagiae, or pomona in cattle on 78 dairy herds in Ontario. Leptospira titer increased with parity. Amongst herds not vaccinated against Leptospira, the proportions of herds with > or = 1 animal seropositive to serovar hardjo, icterohaemorrhagiae, or pomona were 45%, 42%, and 58%, respectively.     

Diagnostic specificity, sensitivity and cross-reactivity of an enzyme-linked immunosorbent assay for the detection of antibody against Leptospira interrogans serovars pomona, sejroe and hardjo in cattle.

Outer sheath antigen was prepared from Leptospira interrogans serovars pomona, sejroe and hardjo by treating the organisms with 1.0M NaC1 followed by 0.04% sodium dodecyl sulfate (SDS). Sodium dodecyl sulfate was removed from the SDS-protein complexes by the extraction of dodecyl sulfate anions as ion pairs with triethylammonium cations into an organic solvent. The outer sheath antigen was recovered from the organic solvent as a precipitate and used as the source of leptospiral enzyme-linked immunosorbent assay (ELISA) antigen. Utilizing this antigen, ELISA was adapted to detect bovine serum antibody to L. interrogans serovars pomona, sejroe and hardjo. The specificity of this assay in 344 bovine sera, which were negative in the microscopic agglutination test (MAT) for seven serovars, was 99.4%. In sera from 37 and 87 cattle which revealed MAT titers greater than or equal to 1:50 for L. interrogans serovars pomona and sejroe, the relative sensitivity of the test was 100%. The ELISA also showed a considerable degree of low level cross-reactivity with other serovars. Sixty-six (75.9%) out of 87 bovine sera which were MAT-positive (MAT titer of greater than or equal to 1:50) with serovars sejroe and hardjo only were ELISA positive with heterologous pomona antigen; 16 (43.2%) and six 16.2%) out of 37 bovine sera which were MAT positive MAT titer of greater than or equal to 1:50) with serovar pomona only were ELISA positive with heterologous sejroe and hardjo ELISA antigen respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leptospirosis in horses in Ontario.

Sera from Thoroughbred and Standardbred horses in southwest Ontario were tested for antibody to seven Leptospira interrogans serovars (autumnalis, bratislava, canicola, grippotyphosa, hardjo, icterohaemorrhagiae, pomona), using the microscopic agglutination test. There was significantly higher seroprevalence of bratislava than of other serovars, in which prevalence was low. Seroprevalence of bratislava increased significantly with age; only 5% of two to three year old horses had titers greater than or equal to 1:80 compared to 52% of horses older than seven years. Eight of 16 foals from two farms seroconverted at low titers to bratislava between four and eight months of age. Leptospires were not detected by immunofluorescence and isolation techniques in 50 kidneys collected from horses at slaughter. Fetal tissues from 52 aborted horse fetuses were also examined by these methods and serovar kennewicki was identified by immunofluorescence and by isolation in one fetus. Serovar bratislava appears to be widespread in horses in Ontario but unimportant in abortion. The clinical significance of this infection in horses in Ontario is unclear.