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)     

Leptospira antibody detection in dog serum in the years 1985 to 1988

In 1985-1988, 993 serum samples of dogs from Southern Bavaria and 408 samples from Northern Bavaria and from several Lands of the Federal Republic of Germany were tested for antibodies against the serovars canicola, icterohaemorrhagiae, grippotyphosa, bratislava, pomona, saxkoebing, sejroe and hardjo by using the microscopic agglutination test (MAT). 683 seras (48.75%) out of altogether 1401 samples showed a reaction against one up to seven serovars. The mostly low canicola- and icterohaemorrhagiae titers, having been proved in over 30% of the samples, can be put down to the fact, that usually the dogs had been vaccinated. Most frequently titers were found with the serovars grippotyphosa and bratislava--in Southern Bavaria 28.3%, in Northern Bavaria and other Lands of the Federal Republic of Germany 18.6%. The prevalence of titers against serovar saxkoebing, with or without a reaction against other serovars out of homologous and heterologous serogroups, reach up to 3.2% in sendings coming from Southern Bavaria and in other sendings up to 6.1%.     

Some leptospira agglutinins detected in domestic animals in British Columbia.

During a period of six years 7,555 bovine sera, 421 canine sera, 251 porcine sera and 135 equine sera were tested for agglutinins to Leptospira interrogans serotypes canicola, grippotyphosa, hardjo, icterohemorrhagiae, pomona and sejroe. The bovine sera reacted predominantly with hardjo and/or sejroe at a rate of 15% compared to 3.5% with pomona. Breeding or abortion problems were associated with pomona but not with sejroe/hardjo agglutinins. The canine sera reacted to canicola (9.9%y and icterohemorrhagiae (5.4%), tcted predominantly with canicola (8.9%) and icterohemorrhagiae (8.1%).     

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.     

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.     

Serological studies on leptospirosis in domestic animals in Quebec.

During a period of 30 months, from January 1977 to June 1979, Leptospira agglutinins were detected in 355 (6%) of 5841 bovine sera, 52 (10.1%) of 511 porcine sera, one (5%) of 20 equine sera and one (12.5%) of eight canine sera. Bovine, porcine and equine sera reacted predominantly with L. pomona. Reactors to L. hardjo/sejroe, L. icterohaemorrhagiae and L. grippotyphosa were also detected in cattle. One porcine serum reacted with L. grippotyphosa and one canine serum with L. icterohaemorrhagiae. Al the sera originated from suspected cases of leptospirosis.     

Leptospira Reactions in Sheep After Heat Inactivation

Sir:- In exnniininf 766 sera from imported sheep we had some difficulty interpreting certain results which showed a prozone tuctinn apinst L. sejroe and hardjo antigens in the leptospira microscopic afflutination test.     

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.     

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)     

The significance of leptospiral titres associated with bovine abortion

To investigate relationships between serological titres to 2 serovars, pomona (L. pomona) and hardjo (L. hardjo), of Leptospira interrogans and abortions, log linear and logit models were fitted to herd and individual cow data from cattle serologically negative for brucellosis. Serological titres to both serovars were significantly related to abortions in individual cows, with L. pomona having a stronger relationship than L. hardjo. L. hardjo was not significant when herd data were analysed. Differences between dairy and beef cattle in the serological titres found to both L. pomona and L. hardjo were detected when data sets of all cattle or cattle with no history of abortion were analysed. The beef/dairy differences may be due to different management practices and/or to different geographical distributions of both serovars and populations of beef and dairy cattle. If there are no cattle in a herd with a reciprocal titre of 3000 or greater for L. pomona, it is unlikely that L. pomona is associated with the abortion problem. There was no specific L. hardjo titre which separated high and low probabilities that the serum came from a cow or herd with an abortion history.     

Evaluation of an ELISA for the diagnosis of experimentally induced and naturally occurring Leptospira hardjo infections in cattle

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Leptospira interrogans serovar hardjo (hardjo) infection in cattle was compared with the microscopic agglutination test (MAT). Glutardialdehyde was used in the ELISA to couple sonicated hardjo antigen to the microtiter plate. Mouse monoclonal anti-bovine IgG1 coupled to peroxidase was used as conjugate. Sera from calves experimentally inoculated with hardjo reacted positively in the MAT as early as 10 days after inoculation; these sera did not react positively in the ELISA until 25 days after the first inoculation. Positive and negative field sera from 704 adult cattle on 90 farms were examined by the MAT and the ELISA; a 90% correlation between the two tests was demonstrated. Eighty-six sera from calves inoculated with four Leptospira serogroups other than hardjo and 227 field sera from adult cattle with naturally occurring leptospirosis other than hardjo were examined by the ELISA. Fewer than 1% of these heterologous sera reacted with hardjo antigen in the ELISA. We concluded that the ELISA described in this report is an advantageous alternative to the MAT for diagnosing 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.     

Leptospira interrogans serovar Hardjo: an abortifacient in New Zealand? A review of the literature

From experimental and clinical evidence, Leptospira interrogans serovar hardjo has been suspected as an abortifacient in cattle overseas. Early reports from North America and Australia described clinical disease with abortions occurring up to 12 weeks later associated with hardjo infections. Recent studies in the United Kingdom have found hardjo infections in nearly 69 percent of aborted foetuses from problem farms, and a prospective epidemiological study determined a relationship between hardjo infection and abortion on one property. In New Zealand, although the epidemiology of hardjo infections has been studied, little attention has been paid to the potential abortifacient role of this organism. In part this is due to the impracticality of using serology to diagnose infections that may have occurred months prior to the abortion. Future research to resolve this question should therefore revolve around hardjo isolation, cohort studies, and the examination of pathogenic mechanisms by which hardjo may induce abortion in cattle.     

Preliminary study on differentiation of Leptospira grippotyphosa and Leptospira sejroe from other common pathogenic leptospiral serovars in canine urine by polymerase chain reaction assay.

A multiplex polymerase chain reaction (PCR) method using primer sets of G1/G2 and B64-I/B64-II was validated to detect pathogenic leptospira serovars from canine urine samples. The PCR method was found to be specific and sensitive with a detection limit of 100 cells of Leptospira icterohaemorrhagiae per milliliter of urine. The primer set previously designated and erroneously transcribed B64-I/B64-II amplified a DNA fragment of 352 base pairs from Leptospira grippotyphosa and Leptospira sejroe but not from Leptospira autumnalis, Leptospira bratislava, Leptospira canicola, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. From 100 diagnostic canine urine samples, 5 were found positive for Leptospira grippotyphosalsejroe with a PCR product of 352 base pairs and 6 were positive for other pathogenic leptospira serovars with a PCR product of 285 base pairs. One 285-base pair product was sequenced and found to be 99.3% homologous to the G1/G2 PCR fragment sequence reported previously. All 352-base pair PCR products of clinical samples and pure cultures of L. grippotyphosa and L. sejroe were sequenced. The 352-base pair fragment sequences of L. grippotyphosa and L sejroe were identical. Only 2 base pairs were found different between the sequences from pure cultures and those from clinical samples. Serum samples from 3 positive cases that generated a PCR product of 352 base pairs were tested by the microscopic agglutination test, and 2 were found to be positive for L. grippotyphosa (1:10,240 and 1:5,120), 1 was positive for L. grippotyphosa (1:320) or L. icterohaemorrhagiae (1:320). The results of this study suggest that the multiplex PCR with the primer set G1/G2 and the erroneously transcribed B64-I/B64-II may be able to differentiate L. grippotyphosa or L. sejroe from other pathogenic leptospira serovars commonly tested for in Canadian diagnostic laboratories.     

Herd-level risk factors associated with Leptospira spp. seroprevalence in dairy and beef cattle in Spain.

Our aim in this cross-sectional study was to investigate the seroprevalence of Leptospira spp. infection in herds and cattle and the relationships between seroprevalence and beef versus dairy, size, replacement policy and grazing management in a representative area of beef- and dairy-cattle production in Spain. Herds were the initial sampling unit. Blood samples were collected from 762 dairy cattle belonging to 81 herds and 1238 beef cattle from 134 herds; sera were tested for antibodies against 11 serovars of Leptospira (autumnalis, ballum, bratislava, canicola, castellonis, copenhagheni, grippotyphosa, hardjo, louisiana, pomona and tarassovi) using the microagglutination test. Forty-three percent (36.2-49.5%) of the herds and 8% (6.4-8.8%) of the individuals were seropositive against one or more of the serovars studied. Bratislava was the most-prevalent serovar (24% of the herds and 4% of the individuals) followed by hardjo (11 and 1%, respectively). Grippotyphosa, copenhagheni and tarassovi were more prevalent in dairy than in beef herds (P<0.001, P<0.05, P<0.05, respectively) -- but no significant association was found between herd-size and Leptospira seroprevalence for any of the serovars considered.     

The role of the common vole (Microtus arvalis) in the epidemiology of bovine infection with Leptospira interrogans serovar hardjo

Control of leptospirosis in cattle depends on the presence of other possible maintenance hosts, with which cattle may have contact. Twenty-seven common voles (Microtus arvalis) were trapped on a dairy farm where the cattle were infected with Leptospira interrogans serovar hardjo (hardjo). In the sera of 11 voles, titres greater than or equal to 100 against serogroup Grippotyphosa were measured with the microscopical agglutination test (MAT). From 8 of these 11 voles, which also showed interstitial lymphoplasmacellular nephritis, Leptospira interrogans serovar grippotyphosa was isolated. We found no evidence that the common vole is a maintenance host for hardjo in this biotope.     

Isolation of Leptospira hardjo from kidneys of Ontario cattle at slaughter.

Kidneys from 117 cattle from 110 Ontario farms were examined at slaughter for leptospires. Leptospira hardjo (hardjo-bovis A) was isolated from 11 kidneys and L. kennewicki from one. The isolations were all made (12/89, 13.5%) from beef cattle from feedlots, no isolates being obtained from dairy or beef cattle from extensive farms (0/28). Isolations were only made from cattle with antibody titers (greater than or equal to 20) against the serovars recovered. Isolation was more sensitive than immunofluorescence in identifying leptospira, particularly in animals with low antibody titers against L. hardjo. Leptospira were isolated from two kidneys with multiple gross lesions of focal nephritis, but there was no correlation between the presence of scanty kidney lesions and isolations of leptospira. Leptospira hardjo infection appears to be common in Ontario feedlot cattle.     

Comparative serological study of Leptospira serovar hardjo genotypes for use in the microscopic agglutination test.

A comparative serological study was conducted using the Leptospira microscopic agglutination test (MAT). Genotypes hardjoprajitno (HP), hardjo-bovis A (HA), and hardjo-bovis B (HB) were compared to determine which best detects hardjo antibody in cattle serum. A total of 2,431 cattle sera were tested. Sera were collected from 4 geographic regions of the United States. Samples were obtained without knowledge of breed, age, vaccination history, or herd health status. Of the sera collected, 60.7% (1,475) were negative at the 1 : 100 dilution for all three genotypes. Serological reactivity at the 1 : 100 dilution was identified in 956 (39.3%) of the sera tested. Considering the 956 positive sera, 941 (98.4%) reacted to HP, whereas the remaining 15 sera (1.6%) reacted to only HA and/or HB. A total of 394/941 (41.9%) HP positive sera failed to react to HA or HB. The results of this study support the conclusion that HP antigen was most sensitive in detection of hardjo antibody.     

LEPTOSPIRAL AGGLUTININS IN DOGS IN SYDNEY

From June 1971 to June 1972, sera from 600 dogs in Sydney were tested for leptospiral agglutinins by a rapid slide agglutination method. The end-point titre was taken at 50 percent agglutination of the live organisms. Forty-one samples (6.8 percent) had a significant leptospiral titres (100 or greater) and 5 of these reacted to 2 serotypes. Thirty serums (5 percent) contained agglutinins against L. copenhageni, and 6 (1 percent) against L. pomona, while a few samples reacted against hardjo, tarassovi, australis, grippotyphosa or pyrogenes serotypes. No significant titres were found to L. canicola, L. hebdomadis, L. autumnalis or L. bataviae.     

The prevalence and distribution of leptospiral titres in cattle and pigs in Queensland

Serological test results for leptospiral species on serums from cattle and pigs performed by the diagnostic laboratories of the Queensland Department of Primary Industries from July 1973 to June 1976 were used to determine the prevalence and geographical distribution of 3 leptospiral serotypes in Queensland. There was a higher prevalence of antibodies to L. hardjo than to L. pomona in cattle, whereas in pigs the prevalence of antibodies to L. pomona was much higher than that for L. tarassovi or L. hardjo. Feral pigs had a particularly high prevalence of L. pomona antibodies. There is a different geographical distribution of antibodies to L. pomona and L. hardjo. L. hardjo antibodies appear to be fairly uniformly distributed but there is a higher prevalence of L. pomona antibodies in low rainfall areas. This relationship was shown to be significantly correlated.