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.     

Prevalence of antibodies of Leptospira serovars in beef cattle in central Queensland.

OBJECTIVE: To obtain up-to-date data on the prevalence of antibodies to Leptospira serovars in central Queensland beef herds preliminary to assessing their role in bovine subfertility and the role of cattle as a zoonotic reservoir. DESIGN: Sera from 2857 female cattle in 68 central Queensland beef herds were tested for antibodies to 14 Leptospira serovars using the microscopic agglutination test. Vaccination use and age of cattle were collected to enable the calculation of crude and age-stratified seroprevalences. RESULTS: The most commonly detected antibodies were to serovars hardjo (15.8% crude seroprevalence), tarassovi (13.9%), pomona (4.0%) and szwajizak (2.2%). Vaccinates were omitted from the hardjo and pomona seroprevalence data. The seroprevalence for hardjo and pomona tended to increase with age of the animals. CONCLUSION: These results are broadly similar to those of previous serological surveys. The data suggest that serovars other than hardjo, pomona and tarassovi, are unlikely to have a significant role in bovine subfertility and that cattle are unlikely to be a source of human infection with them in central Queensland.     

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

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

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)     

SOME ASPECTS OF THE EPIZOOTIOLOGY OF BOVINE EPHEMERAL FEVER IN AUSTRALIA

Serum samples collected from cattle in Western Australia, Northern Territory, Queensland and New South Wales in 1966 had neutralising antibodies to ephemeral fever virus, although the last major epizootic of ephemeral fever was in 1955-56. The incidence of antibodies ranged from 1.5% in Western Australia to 29.0% in Queens- land, and 57.6% of serums assayed were of low titre (2 to 5). Antibodies were not found in serums collected from cattle in Victoria, South Australia, southern Western Australia or Norfolk Island. After the 1967-68 epizootic the pro-porton of cattle with antibody ranged from 3.1% to 47.6% in herds with antibody in Victoria to 81.8% to 91.7% in herds in Queensland, and 58.2% of serums assayed had antibody titres greater than 45. Cattle with low levels of antibody in 1966 had high levels after the 1967-68 epizootic, although it is not known what pro-portion showed clinical signs of ephemeral fever during the epizootic. Serum samples collected in 1966, and which contained low levels of antibody, were fractionated by gel filtration and the neutralising activity was confined to the 7S globulin fraction. In one cow experimentally infected with ephemeral fever virus, the neutralising activity at 15 days after inoculation was confined to the 19S globulin fraction, in both the 19S and 7S fractions at 22 days but was almost totally confined to the 7S fraction by day 36. The significance of the results is discussed, and it is suggested that ephemeral fever virus remains enzootic in areas of Australia between major epizootics, but the infecting virus may be of low pathogenicity and immunogenicity for cattle, resulting mainly in subclinical infections.     

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.     

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%).     

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.     

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.     

Serological evidence of bovine leptospirosis in Malawi

Two hundred and seventy-five serum samples from cattle in Malawi were tested as a pilot survey for Leptospira antibody titres. Fifty-nine (21.4%) of the animals were positive for leptospirosis, while 35 (12.7%) animals reacted inconclusively. Titres to L. hardjo and L. pomona serovars were the most prevalent. Results are also discussed with reference to the areas where samples were collected.     

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)     

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.     

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 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%).     

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.     

A SEROLOGICAL SURVEY FOR BLUETONGUE VIRUS ANTIBODY IN WESTERN AUSTRALIA

A serological survey was carried out to detect specific (serotype 20) and a group bluetongue virus antibody in cattle and sheep serums collected in Western Australia during the period January 1 1978 to June 30 1979. Of 18,849 cattle serums examined by the gel diffusion precipitin test (GDPT), 9.7% were positive and 6.1% gave doubtful results. All 1949 sheep serums tested were negative. Precipitin antibody was demonstrated in 22.5% of serums from Kimberley cattle and 3.6% of cattle serums from the Northwest. Serums collected from cattle in the South were consistently negative in GDPT. When 915 serums that reacted in the GDPT were further tested by the complement fixation test (CFT), 164 were positive. The percentage of CFT positive serums increased as the GDPT reaction became stronger. 2467 serums collected from cattle in Kimberley and Northwest areas and tested by the CFT, 175 (7.1%) were positive. These 175 positive serums were also examined by GDPT and 164 doubtful or positive reactions were obtained. The virus neutralisation (VNT) using serotype 20 virus was carried out on 3804 serums, including all serums that reacted in the GDPT, and 57 were positive. When the VNT positive serums were examined in the other 2 tests, 47 serums were either positive or doubtful in the GDPT and 8 were positive in the CFT. The presence of bluetongue virus group antibody in cattle serums closely followed the suggested distribution pattern of Culicoides brevitarsis but specific serotype 20 neutralising antibody was limited to cattle serums from stations situated north of latitude 17 degrees S in an area of mean annual rainfall higher than 700 mm.     

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).     

Seroepidemiological studies of the detection of leptospires of the sejroe group in cattle in middle Thuringia]

From 1983 to 1989 14,361 head of cattle from the middle Thuringia region were investigated by means of microagglutination test for the presence of Leptospira serovars hardjo, saxkoebing and sejroe. 4,484 samples from cattle with abortion (1983-1985) and a random sample of 5,284 cattle sera (1985) were investigated giving priority to L. hardjo. Furthermore 3,293 samples from cattle with abortion (1986/87) and a random samples test of 1,300 cattle sera (1989) were tested giving priority to L. saxkoebing. 2.5 percent of the cows having aborted and 10.3 percent of cattle tested out of the whole livestock showed antibodies against L. hardjo. The test for presence of L. saxkoebing demonstrated in cattle with abortion a seroprevalence of 14 percent and in the random samples tests a portion of reagents of 11.3 percent. The degree of infection differed regionally, the highest degree was found in livestocks of the northern part of the Thuringia forest. The prevalence of L. saxkoebing in cattle with abortion from large dairy farms was 18.6 percent, which is higher than the average in the region examined. Antibodies against hardjo and sejroe were interpreted as caused by cross reactivity.     

FURTHER OBSERVATIONS ON THE INCIDENCE OF BRUCELLOSIS IN DAIRY CATTLE IN VICTORIA

Random blood sampling of 8,448 dairy cattle in two Shires of Victoria revealed that 194 (4.5%) serums, from south-eastern Victoria and 147 (3.6%) serums from northern Victoria were positive to the International tube agglutination test for Br. Abortus . Of these reactions at least 82 (1.9%) and 56 (1.4%) of the serums indicated that these animals were or had been infected with Br. Abortus .
The results show that 65% and 73% of the herds in the two areas did not contain Br. Abortus infected cattle. Two factors influenced the incidence of infected animals in herds. First the incidence of infected animals decreased with increasing length of time during which herd re-placement calves had been vaccinated with Br. Abortus strain 19. Second, the introduction of mature previously calved cows increased the incidence of infected animals.
Some discussion is given to the usefulness of the revaccination of animals and the control of brucellosis in Victoria with is possible effect on cattle meat export markets.     

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

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