Detection of antibodies to leptospirosis in experimentally infected dogs using the microcapsule agglutination test

Six puppies were infected with a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae and another five animals with a virulent strain of Leptospira interrogans serovar canicola, respectively. Antibodies were examined at 3, 5, 7, 11 and 14 days after infection, using the microcapsule agglutination test (MCAT) and the conventional microscopic agglutination test (MAT). Compared with the MAT, the MCAT detected early specific IgM antibody with high sensitivity. The MCAT titres reached a peak at the 7th day after infection and declined gradually after the 11th day, while the MAT titres increased up to the 14th day.     

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.     

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.     

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.     

Leptospiral vaccines: immunogenicity of protein-free medium cultivated whole cell bacterins in swine

Swine serologically negative for anti-Leptospira antibodies were given 2 doses of a pentavalent vaccine (3 weeks between doses) prepared from Leptospira serovars canicola, icterohaemorrhagiae, hardjo, pomona, and grip-potyphosa (0.2 mg/serovar/dose). Leptospires used for vaccinal production were cultivated in a protein-free medium or in a bovine albumin-containing medium. All vaccinated swine had demonstrable antibody titers within 1 week of the initial vaccination. Peak microscopic agglutination titers were between 256 and 1,024 after the 2nd vaccinal dose was given. After challenge exposure with serovar canicola, control swine had titers of at least 13,653 and the vaccinated swine had titers of 3,403 to 8,192, depending on the vaccine. Leptospiremia and kidney infections were not detected in any canicola Moulton immunized swine, but did appear in control swine. The Al(OH)3 adjuvant had no obvious influence of any of the vaccinal titers.     

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.     

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.     

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.     

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.     

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

     

Leptospirosis in dogs: a serologic survey and case series 1996 to 2001

All leptospirosis microscopic agglutination test titers for the Leptospira serovars icterohaemorrhagiae, canicola, grippotyphosa, bratislava, hardjo, and pomona conducted on 1,260 blood samples from dogs at the University of Illinois Veterinary Diagnostic Laboratory between March 1996 and March 2001 were evaluated. Low titers (1:100 to 1:400) were predominantly L. icterohaemorrhagiae and L. canicola, which represented the predominant serovars (65.4%) among all positive samples with low titers. L. grippotyphosa was the predominant serovar (72.1%) among samples with clinically significant titers (greater than 1:800). The medical records of 87 dogs with a titer greater than 1:800 that were patients at the Veterinary Teaching Hospital of the University of Illinois were reviewed. A clinical diagnosis of leptospirosis was made in 15 cases (17.2%) based on the elevated titer, appropriate clinical signs, lack of recent vaccination, and lack of concurrent disease that could explain the clinical signs present. Renal disease was present in 10 of the cases, concurrent renal and hepatic disease in two, and hepatic disease in three. In 12 cases, the predominant serovar was L. grippotyphosa; titers to L. grippotyphosa and L. bratislava were equal in magnitude in three cases.     

Leptospirosis in dogs: a serologic survey and case series 1996 to 2001

All leptospirosis microscopic agglutination test titers for the Leptospira serovars icterohaemorrhagiae, canicola, grippotyphosa, bratislava, hardjo, and pomona conducted on 1,260 blood samples from dogs at the University of Illinois Veterinary Diagnostic Laboratory between March 1996 and March 2001 were evaluated. Low titers (1:100 to 1:400) were predominantly L. icterohaemorrhagiae and L. canicola, which represented the predominant serovars (65.4%) among all positive samples with low titers. L. grippotyphosa was the predominant serovar (72.1%) among samples with clinically significant titers (greater than 1:800). The medical records of 87 dogs with a titer greater than 1:800 that were patients at the Veterinary Teaching Hospital of the University of Illinois were reviewed. A clinical diagnosis of leptospirosis was made in 15 cases (17.2%) based on the elevated titer, appropriate clinical signs, lack of recent vaccination, and lack of concurrent disease that could explain the clinical signs present. Renal disease was present in 10 of the cases, concurrent renal and hepatic disease in two, and hepatic disease in three. In 12 cases, the predominant serovar was L. grippotyphosa; titers to L. grippotyphosa and L. bratislava were equal in magnitude in three cases.     

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.     

Determination of specific anti-leptospiral immunoglobulins M and G in sera of experimentally infected dogs by solid-phase enzyme-linked immunosorbent assay

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) to detect specific anti-leptospiral IgM and IgG in sera of dogs experimentally infected with Leptospira interrogans serotype canicola are reported. In all dogs specific anti-leptospiral IgM was detected from the second half of the first week after infection, the maximum being attained during the second week. Subsequently the IgM titre gradually decreased. Specific anti-leptospiral IgG was detected later and increased gradually to reach almost the same level as the IgM titre after two to three months. During the initial stage of the infection, when the microscopic agglutination titre was still negative or very low, a high IgM titre was accompanied by a negative or very low IgG titre in every case. After the initial stage a substantial IgG titre was also detectable. It is suggested that the test is suitable for serodiagnostic purposes, particularly for the diagnosis of a current infection in an individual.     

Serologic survey for leptospirosis in Arizona beef cattle in 1981

Sera from 1,215 beef cattle in Arizona were evaluated by leptospiral microscopic agglutination test in 1981. Over 25% had agglutinins to greater than or equal to 1 of 5 serovars of Leptospira interrogans used as antigens (canicola, grippotyphosa, hardjo, icterohaemorrhagiae, and pomona) at a titer of greater than or equal to 1:100, and 8.2% had titers of greater than or equal to 1:400 to greater than or equal to 1 serovars. The most common serovar to which reactions were detected was hardjo; agglutinins were detected at titers of greater than or equal to 1:100 in 14.3% and of greater than or equal to 1:400 in 5.5%. Cross reactions were rare at serum dilutions greater than or equal to 1:100 (2%) and extremely rare at greater than or equal to 1:400 (0.7%). Because vaccination with leptospiral bacterins is seldom practiced in Arizona beef cattle, a titer of greater than or equal to 1:100 may be useful in estimating incidence and prevalence of the disease and as an aid to diagnosis of leptospirosis.     

A Microtitration Agglutination Test for Detecting Group E Streptococcus Infection in Swine

A microtitration agglutination test was developed and evaluated for detecting infection of swine with group E streptococci type IV, the most common causative agent of streptococcic lymphadenitis of swine.

Whole cell agglutinogens representing group and type antigens of group E streptococci were tested in the microtitration agglutination test against reference antisera to Streptococcus groups A, B, C, D, E, F, G. H, K, L, M, N, O, P, Q, R, S and U, as well as specific antisera to types II, IV and V of group E. Group E specific agglutinogens were unsatisfactory in the microtitration agglutination test because of cross reactions with group P and U antisera and because of poor reproducibility of the test. Type specific agglutinogens of group E streptococci reacted only with their respective homologous antisera and not with any heterologous group antisera. None of the group E streptococci agglutinogens reacted with 52 normal swine sera.

Agglutinogen made from group E streptococci type IV was selected for further evaluation in the microtitration agglutination test because group E streptococci types II and V are considered to be of minor importance in the etiology of streptococcic lymphadenitis of swine. Swine experimentally infected with a type IV strain developed significant titers in the microtitration agglutination test. All swine tested negative before exposure and seroconverted (titer ≥4) two to six weeks postexposure.

The microtitration agglutination test was used by two different laboratories to test 187 duplicate samples of serum from infected swine. A total of 94.1% of the tests were read at either the same titer (48.1%) or a difference of not more than one dilution (46.0%) at the two laboratories. There was disagreement between the two laboratories in the test-positive test-negative status of 19 of the sera (10.2%). Titers of two of the sera differed by two dilutions (<4 at one laboratory and 8 at the other). The remaining 17 sera differed in titer by only one dilution (<4 at one laboratory and 4 at the other).

     

Serological Survey of Leptospiral Antibodies in Swine in Quebec

The prevalence of reactors to different serovars of Leptospira interrogans was determined in 117 swine premises in Quebec. A total of 926 sera were tested, using the microscopic agglutination method, against six leptospiral serovars: pomona, icterohaemorrhagiae, grippotyphosa, hardjo, canicola and ballum. Of the sera tested, 30 (3.2%) reacted to one of the three following serovars: pomona (2%), icterohaemorrhagiae (1.1%) and ballum (0.1%).     

Prevalence of leptospira species among farmed and domestic animals in Greece

A total of 1527 serum samples from pigs, goats, sheep, cattle and dogs in Greece were examined by the microscopic agglutination test and 11-8 per cent of them had antibodies against one or more Leptospira serovars at titres of 1/100 or more. The predominant serovar affecting farm animal species was Bratislava, and Copenhageni was common among dogs and the second most important serovar when all animals were considered together. Another prevalent serovar was Australis, but antibodies to Pomona were detected only in goats and cattle.     

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%.     

Survey of leptospiral agglutinins in the sera of swine of southeastern Alabama

The occurrence of leptospirosis in swine of southeastern Alabama was determined. A total of 627 sera were tested, using the microscopic agglutination method, with live antigens of 12 serovars. Of the sera tested, 121 (19.3%) had a titer of 1:100 or greater to the serovars employed. The percentage distribution of sera with titers of greater than or equal to 1:100 among serovars most commonly reported was as follows: Leptospira interrogans serovars pomona, 3.8%; icterohaemorrhagiae, 3.3%; canicola, 1.6%; hardjo, 0.7%; and grippotyphosa, 0.16%. Of the less commonly recognized leptospiral serovars, the percentages reacting were as follows: ballum, 4.9%; autumnalis, 3.2%; pyrogenes, 1.1%; and bataviae, 0.4%. None of the sera reacted with antigen of serovars australis, tarassovi, or wolffi.