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.     

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.     

Serological studies on leptospirosis in cattle in east central Alabama.

Serological surveys of leptospiral antibodies in cattle were carried out in Macon and the surrounding counties of East Central Alabama. A total of 286 bovine serum samples were screened for the presence of antibodies against live antigens from twelve pathogenic leptospiral serotypes using a microscopic agglutination test. The most frequently encountered serotypes were Leptospira hardjo (47%), Leptospira wolffi (34%), Leptospira canicola (12%), Leptospira pomona (10%) and Leptospira ballum (10%). Leptospira autumnalis, Leptospira grippotyphosa, Leptospira icterohemorrhagiae, Leptospira pyrogenes and Leptospira tarassovi were observed in less than 5% of the samples.     

THE PREVALENCE OF ANTIBODIES TO MEMBERS OF LEPTOSPIRA INTERROGANS IN CATTLE

A total of 1,355 random samples taken from bovine serums submitted for brucellosis testing in Victoria were submitted to the microscopic agglutination test for the presence of antibody to 12 serovars of Leptospira interrogans . The most common reaction obtained was to serovar hardjo , although the percentage of reactors varied from 24.8% in the metropolitan area to 56.3% in north-eastern Victoria (mean 40.6%). A total of 86.3% of farms from which 3 or more samples were taken had at least one reactor to serovar hardjo . The prevalence of antibody to other serovars was tarassovi (7.8% of reactors), ballum (3.7%), pomona (2.4%), autumnalis (1.8%) and bataviae (1.2%). Reactions to other serovars were observed in serums of less than 1% of cattle tested; serums from 50.8% of cattle did not react to any antigen.     

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.     

Observations of leptospirosis in farmed deer

AIMS: Slaughterhouse and on-farm surveys were undertaken to investigate some aspects of leptospirosis (Leptospira interrogans) in farmed deer in the lower North Island of New Zealand. METHODS: Blood samples and kidneys were collected at slaughter from 601 l-year and older red and red X Wapiti stags and 21 adult hinds from 53 farms (10 or 12 deer per farm). Serum samples were analysed for up to seven Leptospiral serovars. Gross and histological examinations of kidneys were undertaken. Kidneys from 202 deer were cultured for leptospires. A follow-up postal questionnaire (68% response) indicated one herd had been vaccinated prior to the survey. Serological analyses were also carried out on serum bank samples from a previous on-farm survey involving male and female weaner, yearling and adult red deer from 16 commercial deer farms in March and November. RESULTS: Serological reactions at titres > or = 96 to serovar hardjo were present in 73.6%, pomona in 41.5%, copenhageni in 11.3% and tarassovi in 15.1% of farms from the slaughterhouse survey. Antibodies to serovars australis, ballam and balanica were present in three, one and four of six herds studied, respectively. Titre prevalence to hardjo was higher than that of pomona and other serovars within farms. Cultures for Leptospira were positive in 10 stags from six lines with similar prevalence across age groups. Histological examination showed many gross lesions were associated with mild interstitial cellular infiltration characteristic of subclinical Leptospiral infections. Some sections from culture-positive kidneys contained spirochetes in renal tubules. The on-farm survey showed a 10-30% within-herd prevalence of pomona and hardjo titres in 56% of 3-month-old deer herds, but by 11 months of age, 100% of herds were titre-positive with high prevalences to one or both serovars. Concurrently, herds of 1-year-old and adult deer on the same farms were all seropositive. CONCLUSION: This study has shown that Leptospiral infections are common in farmed deer in the survey area.     

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.     

Leptospirosis: II. Investigation of clinical disease in dairy cattle in the Waikato district of New Zealand

Investigations were carried out in 1975, 1976 and 1977 in 16 dairy herds where leptospiral abortions were suspected and in five other herds where clinical disease was not present. Both Leptospira interrogans serovars pomona and hardjo were isolated from cattle in herds with leptospirosis, but only pomona was recovered from those that had aborted. There was no evidence that hardjo caused clinical disease in dairy cattle in the Waikato district. It was found that 73% of the cows that aborted and 19% of other animals in the same herds had microscopic agglutination test titres to pomona of 1:2,000 or greater. By contrast, only 2% of cattle in herds without clinical evidence of leptospirosis had such titres. One cow retained a titre of 1:2,000 or greater to pomona for 7 months; titres of this order had a shorter duration in other cows. Leptospiruria occurred in 50% of cows that had aborted and in 9% of in-contact cows in the same herds. Only 0.7% of cows had leptospiruria in the herds with no clinical disease. Ten of 35 cows shedding pomona still had leptospiruria one month later. It was concluded that clinical leptospirosis should be diagnosed by testing a sample of the herd, rather than just individual cows, because of the variability and persistence of leptospiruria and serological titres in cows with and without clinical signs. Although hardjo is common in cattle in the Waikato district, it was not found to cause abortion in cattle.     

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

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

Neospora caninum and Leptospira serovar serostatus in dairy cattle in Ontario

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

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

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

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.     

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.     

The use of the enzyme-linked immunosorbent assay (ELISA) to detect the IgM and IgG antibody response to Leptospira interrogans serovars hardjo, pomona and tarassovi in cattle

Leptospira interrogans serovars pomona, hardjo and tarassovi were each used to inoculate 6 cattle. Three-hundred and ninety-nine sera collected from the inoculated animals and from a control group over a 3-month period were tested using the microscopic agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA). Leptospiruria was monitored by microscopic examination and culture. The ELISA detected specific IgM antibody against the serovars in all infected cattle 1 week after inoculation. This IgM antibody persisted in most of the animals for 3-5 weeks. Specific IgG antibody appeared at the same time or just after IgM, but persisted for much longer. Levels of antibody detected by the ELISA and the MAT did not correlate with each other, nor with the periods of leptospiruria found in the infected cattle.     

Experimental demonstration of an antigenic relationship between Leptospira and equine cornea

Horses inoculated with either equine cornea or killed Leptospira interrogans serovars pomona, tarassovi, icterohaemorrhagiae, wolffi and hardjo, developed corneal opacity and produced antibodies which made it possible to demonstrate partial antigenic identity between equine cornea and four of those serovars employed. These antibodies were isolated by means of immunoadsorptions, purified by ion-exchange chromatography (DEAE-Sephadex A-50) and run by immuno-electrophoresis in agar gel. Both antibodies, anti-equine cornea and anti-leptospira, showed that they corresponded to the IgGb subclass. They bound themselves to equine cornea in vivo and in vitro as was proved by immunofluorescence. This antigenic relationship may be in part responsible for pathogenesis of corneal opacity in leptospirosis of horses.     

Survey to estimate prevalence of Leptospira interrogans infection in mature cattle in the United States.

A total of 5,142 kidney tissue samples and 5,111 serum samples from mature cattle in 49 states and Puerto Rico were collected at slaughter. Age of cattle ranged from 1 to 16 years (mean, 6.6 years). Leptospires were isolated from 88 (1.7%) kidney tissues, and 2,493 (49%) sera contained antibodies against 1 or more of 12 Leptospira interrogans serovars. Leptospires were observed by immunofluorescence in 41 (0.8%) kidney tissues. Using agglutinin-absorption tests, 73 (83%) isolates were identified as serovar hardjo, 11 (12.5%) as serovar pomona, and 4 (4.5%) as serovar grippotyphosa. By use of restriction endonuclease analysis studies of chromosomal DNA, all isolates differed from reference serovars but were identical to strains previously isolated from cattle or swine in the United States. Of the serovar hardjo isolates, 85% were identical to restriction endonuclease analysis type (genotype) hardjo-bovis A and 11 (15%) were identical to genotype hardjo-bovis B. Serovar pomona isolates were identical to genotypes kennewicki A (64%) or kennewicki B (36%), and serovar grippotyphosa isolates were identical to the RM 52 strain. Isolation rates were significantly (P less than 0.001) higher for beef cattle than for dairy cattle and were higher (P less than 0.001) for bulls than for cows. Combined culture and immunofluorescence results indicated that 2% of mature cattle were renal carriers of leptospires.     

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.     

Serological prevalence of bovine leptospirosis in Plateau State, Nigeria

Serum samples obtained from 1.537 cattle in the 14 local government areas (LGAs) of Plateau State of Nigeria were screened for the presence of leptospiral antibodies using 13 serovars in a modified microscopic agglutination test (MAT). Two hundred and twenty-two (14.4 p.100) of the cattle tested had leptospiral antibody titres of 1:100 or higher to one or more of the test antigens. The prevalence rates of antibodies to individual serovars were: hardjo (35.6 p.100), pomona (11.7 p.100), pyrogenes (11.7 p.100), canicola (9.5 p.100), grippotyphosa (7.7 p.100), bratislava (5.9 p.100), icterohaemorrhagiae (5.9 p.100), ballum (4.5 p.100), autumnalis (3.6 p.100), bataviae (2.3 p.100) and tarassovi (1.8 p.100). The serological prevalence of bovine leptospirosis in the various local government areas of Plateau State of Nigeria differed significantly (P less than 0.05; X2).     

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.