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.     

Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomona

Monoclonal antibodies (mAb) were produced by fusing Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c and ND4 mice that were immunized with killed Leptospira interrogans serovar pomona whole cells. Thirty hybridomas which produced antibodies (of the IgG1, IgG2a, IgG2b, or IgG3 isotype) that bound to epitopes on the serovar pomona whole cell antigen were identified by an indirect enzyme-linked immunosorbent assay (ELISA). Twenty-eight of these 30 mAbs cross-reacted in the indirect ELISA with at least one whole cell antigen prepared from 12 other pathogenic Leptospira serovars, and/or with whole cell antigen from the non-pathogenic Leptospira biflexa serovar patoc. The two serovar pomona-specific mAbs, which were designated M897 and M898, were obtained from the ND4 mouse and were both of the IgG1 isotype. In competitive ELISAs, M897 and M898 were inhibited from binding to the pomona antigen by bovine sera with anti-serovar pomona microscopic agglutination test (MAT) titres ranging from 100 to 6400. No significant inhibition was observed with pomona MAT-negative sera or with sera from animals experimentally infected with serovars canicola, copenhageni, grippotyphosa, hardjo type hardjobovis or sejroe. The epitopes recognized by M897 and M898 were both highly susceptible to sodium meta-periodate oxidation, indicating a carbohydrate composition. Neither of these mAbs reacted in immunoblots with the separated components of the serovar pomona whole cell antigen.     

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.     

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.     

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)     

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.     

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.     

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%).     

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.     

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%).     

Serological reactions to Leptospira species in game animals of northern Natal

Fifty sera collected from 12 different species of free-living game animals in game parks in the Northern Natal were tested against 8 Leptospira interrogans antigens using the microscopic agglutination test (MAT). Six out of 50 animals had titres, all less than 100. Three of these animals had titres to serovar mini, 1 animal to tarrasovi, and 3 animals had multi-serovar reactions, 1 to mini and hardjo, and 1 to tarrasovi, copenhageni and pomona.     

An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G₁ that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay.

At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation

     

Antibody response to genus- and serovar-specific leptospiral antigens in Leptospira-infected cows

In cows inoculated with Leptospira interrogans serovar pomona or hardjo, the 2-mercaptoethanol-sensitive microscopic agglutination test (MAT) antibody to the serovar appeared 3 to 8 days after inoculation and peaked at 10 to 20 days, whereas the 2-mercaptoethanol-resistant MAT antibody was predominant at 35 to 80 days. A persistent antibody response, probably associated with serovar-specific leptospiral antigens, was detected in the cows inoculated with serovar pomona, using a sonicated or an alkaline-extracted antigen derived from serovar pomona in the enzyme-linked immunosorbent assay (ELISA). In contrast, a short-lived antibody response to the same antigens was demonstrated in cows inoculated with serovar hardjo, probably more typical of the response to genus-specific leptospiral antigens. Antigens derived from L biflexa serovar patoc only detected the latter type of antibody response in cows inoculated with serovar pomona or hardjo. Correlative studies revealed that the antigens derived from serovar patoc seem to be genus specific and serologically closely related, but not identical. The antigens derived from serovar pomona were genus specific on the basis of the early antibody response to leptospiral inoculation in the cows, but serovar specific based on the subsequent more persistent response to leptospiral inoculation. These antigens were also serologically closely related, but not identical. Examination of sera from cows that aborted and were MAT-positive for serovar pomona or hardjo revealed a more serovar-specific antibody response, indicating that there may have been a less recent leptospiral antigenic stimulus, thus emphasizing the caution with which results of the ELISA and other serologic assays for the detection of bovine leptospirosis must be interpreted.     

Serologic responses of dogs given a commercial vaccine against Leptospira interrogans serovar pomona and Leptospira kirschneri serovar grippotyphosa

OBJECTIVE: To evaluate serum titers obtained by use of the microscopic agglutination test (ie, MAT titers) to Leptospira interrogans serovar pomona and autumnalis and Leptospira kirschneri serovar grippotyphosa in dogs given a commercial vaccine against serovars pomona and grippotyphosa. ANIMALS: Forty 12-week-old puppies and 20 mature Beagles. PROCEDURE: Puppies received a commercial vaccine against serovars pomona and grippotyphosa at 12 weeks of age, then received a booster vaccine and 3 weeks later; mature dogs received the vaccine once. Serum MAT titers to serovars pomona, autumnalis, and grippotyphosa were measured before vaccination and at 2, 4, 6, 10, and 16 weeks after the first or only vaccination. RESULTS: Of the 40 puppies vaccinated, 40, 0, and 40 developed MAT titers of > 100 after vaccination to serovars pomona, grippotyphosa, and autumnalis, respectively. Microscopic agglutination test titers to serovar autumnalis were higher than MAT titers to serovars pomona and grippotyphosa and persisted in some dogs for 16 weeks (6 weeks longer than for titers to serovar pomona). Of the 20 mature dogs, 13, 5, and 20 developed MAT titers of > 100 at 2 weeks to serovars pomona, grippotyphosa, and autumnalis, respectively. Titers to serovar pomona were higher and persisted in some dogs beyond 16 weeks after vaccination, compared with titers to serovars pomona and grippotyphosa, which persisted for 10 and 6 weeks, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Subunit vaccines against serovars pomona and grippotyphosa induce MAT titers not only to homologous antigens but also to serovar autumnalis, which could lead to a misdiagnosis of leptospirosis caused by serovar autumnalis.     

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.     

An old disease with a new face: canine leptospirosis does not lose its relevance

The clinical features of the disease are presented based on retrospective analysis of the records of eleven dogs diagnosed with leptospirosis using clinical signs and results of the microagglutination test (MAT) between 1991 and 1996. Additionally, Leptospira titres were determined in 30 healthy dogs and 20 hospitalised dogs without clinical or laboratory evidence of leptospirosis. A positive titre for L. grippotyphosa, L. pomona, L. bratislava, L. australis, L. icterohaemorrhagiae and/or L. canicola was found in 16 normal dogs and only one hospitalised patient. Eight of these dogs had titres of > or = 1:800. Only one of them had been vaccinated shortly before sampling. These results suggest that many dogs from the surroundings of Bern, Switzerland have contact with various Leptospira interrogans serovars. In ten healthy dogs, the Leptospira titre was determined before and four weeks after vaccination with leptospiral antigen. Only two of the dogs showed a serologically measurable response to the antigen contained in the vaccine. In dogs MAT titers presumably do not reliably reflect the immune status against leptospiral infections.     

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.     

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%.     

Serodiagnosis of canine leptospirosis by solid-phase enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Leptospira interrogans serotype canicola in dogs was developed and evaluated. Comparison of the ELISA with the microscopic agglutination test (MAT) showed that, during the first two weeks after an experimental infection with serotype canicola, the ELISA detected antibody at higher dilutions than the MAT. After the second week post-infection both tests detected antibody at almost equal titres (r = 0.89). The outer envelope (OE) antigen of serotypes icterohaemorrhagiae, copenhageni and canicola was fairly serotype-specific, whereas the pellet (P) antigen showed more cross-reactivity. Both OE and P antigen of Leptospira biflexa strain Patoc I could be used as cross-reacting antigen in the ELISA. Compared to the MAT, the ELISA has some technical advantages. It is suggested that the ELISA would be useful as a screening test.     

Levels of some reproductive diseases in the dairy cattle of Colombia

Of 4,144 serum samples collected from cows on 113 farms from eight areas of Colombia 3.3% had positive and 8.8% inconclusive titres to Brucella abortus, 21.7, 6.3, 1.6, 0.6 and 0.7% of cows had positive titres to Leptospira serovars hardjo, pomona, canicola, icterohaemorrhagiae and grippotyphosa respectively. Questionnaires completed on 110 farms revealed that 6, 2.5 and 4.6% of cows had had metritis, aborted or retained their placentas respectively in the previous 12 months. Trichomonas foetus and Campylobacter fetus were isolated from 13.7% and 15% of the bulls sampled on 103 farms. Six and two bulls had inconclusive and positive titres to Brucella abortus. Eight and 23 bulls had positive titres to pomona and hardjo. The results were discussed and remedies for control suggested.