Bovine IgM and IgM response to Leptospira interrogans serovar hardjo as measured by enzyme immunoassay

The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgG antibodies in the sera of cattle infected or immunized with Leptospira interrogans serovar hardjo. IgM appeared first but was quickly followed by IgG which persisted longer than IgM. The levels of antibody detectable by ELISA and by the microscopic agglutination test (MAT) did not correlate, suggesting that the two techniques measured different antigen-antibody systems. The transient nature of the IgM response as measured by ELISA indicates potential usefulness as a serodiagnostic test for detecting current leptospiral infections.     

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.     

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)     

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.     

Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle

Ayrshire cattle, which were infected with a stock of Trypanosoma vivax from Galana, Kenya, which produced haemorrhagic disease, were examined for the presence of antibodies to erythrocytes and platelets. Antibodies to normal erythrocytes and platelets were detected in the plasma of infected animals using the enzyme-linked immunosorbent assay (ELISA). The antibodies were detectable following the first peak of parasitaemia (10-15 days after infection) and antibody activity was maximal 30-35 days after infection. Plasma from cattle, taken 32 days after infection, precipitated radiolabelled proteins from autologous platelets and, less efficiently, from autologous erythrocytes. Fluorescence-activated cell sorter (FACS) assays demonstrated that erythrocytes and platelets from infected cattle bound IgM and IgG in vivo, and that both normal blood cell types could adsorb these antibodies following incubation in plasma from infected animals. Complement (C3) was similarly adsorbed to erythrocytes during infection. Antibodies adsorbed to infected erythrocytes could be eluted and the eluted antibodies bound to normal erythrocytes, as detected by immunofluorescence, but they did not react with the infecting trypanosome. It is hypothesised that although anti-blood cell antibodies may not be the primary cause of the severe anaemia and thrombocytopaenia which accompany the haemorrhagic syndrome, they could play an important role in the maintenance of these signs of disease, adversely affecting the outcome of T. vivax-associated haemorrhagic disease in the field.     

Development and validation of an ELISA for the detection of leptospire-specific antibodies in rodents

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies in rodents was developed and validated with the microscopic agglutination test (MAT) and leptospiral cultures. Sonicated antigen from cultures of serovars tarassovi and pyrogenes was used. As conjugate, a combination of anti-rat and anti-hamster IgG labeled with peroxidase was used. The optimal cut-off point was determined by plotting the sensitivity and specificity for various cut-off point values by means of receiver operating characteristic (ROC) curve. Concordance between ELISA and each of the MAT titers was measured by kappa (kappa). Proportions of positive results were compared by means of McNemar's test. Total 214 rodents were trapped, but only 117 could be processed by the three techniques (culture, ELISA, MAT; 1:20, 1:40, 1:50) and used for statistical analysis. Although, MAT titers in rodents infected with the serogroup Ballum tended to be lower than those infected with the serogroup Icterohaemorrhagiae, all (20/20) were ELISA-positive and almost all (19/20) were MAT-positive.The percentage of positive results obtained by ELISA, 47.0% exceeded significantly the 40.2% obtained by MAT (1:50). Difference between ELISA and MAT (1:40) was not significant and no differences were observed between ELISA and MAT (1:20). Agreement, specificity, sensitivity and the consequent area under the ROC curve between ELISA and MAT were higher as MAT cut-off points were lowered, being optimal at 1:20. The fact that differences between ELISA and MAT were significant at 1:50, but not at 1:40 or 1:20, supports the suggestion that lower MAT titers should be considered positive in rodents. The ELISA developed to detect leptospire-specific antibodies had optimal sensitivity and specificity in relation to MAT and it is concluded that it may constitute a very useful indicator for epidemiological purposes of past or present leptospiral infection in rodents.     

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.     

Use of enzyme immunoassay in a serological survey of leptospirosis in sheep

A total of 731 serums, all from Merino rams from 20 farms, were tested for antibodies against Leptospira interrogans serovars hardjo, pomona and tarassovi using the microscopic agglutination test (MAT). The enzyme immunoassay (EIA) technique was used to test all serums for IgM and IgG antibodies to serovar hardjo. In the MAT, reactions to serovar hardjo were most common with 236 rams (32.3%) reacting at 1/100 or greater. Only 1.9% of serums reacted against serovar tarassovi and 1.1% against serovar pomona. The percentage of sheep with positive MAT reactions to serovar hardjo ranged from 0 0 to 94.9 between farms. When using EIA, 46 (6.2%) of the serums were positive for IgM antibody and 246 (33.6%) were positive for IgG antibody. Correlation of the EIA for detection of IgG antibody with the MAT was good. The EIA detection of IgG antibody was considered to be a good alternative test to the MAT for epidemiological studies in sheep.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Bovine leptospirosis: the effects of vaccination on serological responses as determined by complement fixation and microscopic agglutination tests

Fifty-one calves were divided into six trial groups of seven to eleven animals and vaccinated with a commercial leptospiral vaccine containing serovars pomona and hardjo. Vaccinations were given at 6,7,14 or 21 months of age and animals in various groups were vaccinated on one to four occasions. Antibody responses were determined by the microscopic agglutination test (MAT) and the complement fixation test (CFT) at one to four weeks intervals until 66 weeks after the start of the trial. Fifty percent or greater agglutination in serum diluted 1:100 or more and 50% or greater fixation of complement in serum diluted 1:20 or more were considered positive titres in the MAT or CFI respectively. Positive titres were still present in some animals six weeks after vaccination at 21 months of age. In other cases MAT titres (range 1:100-1:3000) persisted for 7-23 weeks and CFI titres (range 1:20-1540) persisted for 1-14 weeks. Marked individual variation in serological findings occurred using either test. In general the number of animals producing a positive titre, and the magnitude and persistence of titres was related to the number of doses of vaccine given. It was concluded that for diagnostic purposes neither the CFT nor the MAT could reliably differentiate titres due to vaccination from those following natural infections.     

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log(10) titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature.The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2-3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.     

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.     

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

The serological response of calves to Leptospira interrogans serovar hardjo vaccines and infection as measured by the microscopic agglutination test and anti-IgM and anti-IgG enzyme-linked immunosorbent assay

The microscopic agglutination test (MAT) and the anti-IgM and anti-IgG enzyme-linked immunosorbent assays (ELISA) were used to examine sera taken over the course of 16 weeks from 35 calves vaccinated and/or infected with Leptospira interrogans serovar hardjo. The relationship between the IgM and IgG responses to vaccination and infection were determined. The rapid and high rise in IgM levels following challenge made the anti-IgM ELISA a potentially good indicator of recently established infection although some transitory high levels were seen where infection did not become established. The slow IgG response to infection made the anti-IgG ELISA of limited diagnostic use.     

Seroprevalence of leptospirosis in Korean municipal zoo animals

From 2002 to 2005, we collected 118 serum samples from 34 species belonging to 13 families of zoo animals in Korea and determined the prevalence of antibodies for 18 serovars of Leptospira spp. using the microscopic agglutination test (MAT). Twenty-nine (25%) of the serum samples tested were positive for one or more of the serovars. There were no significant differences in relation to genders: 23% and 26% of positives occurring in male and female animals, respectively (P>0.05). However, the seroprevalence for the Leptospira spp. was significantly higher (P<0.05) in herbivores (45%) than in either carnivores (17%) or omnivores (17%). Among the 5 serovars detected in this study, the most common was sejroe (n=27; 87% of all positive reactions). All positive reactions showed low titers (< or = 1:200) and the positives were most frequently detected in 1:25 (58%) and 1:50 (23%) serum dilutions. The highest antibody titer (1:200) was observed for the serovars sejroe (n=1) and bratislava (n=1). We conclude that the exposure of zoo animals to Leptospira spp. is relatively common in Korea and produces low MAT titers, with sejroe being the most commonly encountered serovar.     

Antibody response of cattle to rinderpest vaccine

Antibody production was studied in cattle infected with rinderpest vaccine virus. Vaccinated cattle produced both IgM and IgG serum antibodies. The IgG antibodies were mainly those of IgG2 subclass. No IgA antibody response was detected in vaccinated animals.     

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

     

A microplate enzyme-linked immunorsorbent assay for measuring antibody to Anaplasma marginale in cattle serum

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.