Development of an enzyme-linked immunosorbent assay for detecting antibodies in sera of Brucella suis-infected swine.

An enzyme-linked immunosorbent assay was developed using a heat-killed Brucella suis antigen for detecting antibodies in the sera of swine from which B. suis was isolated. Optimal enzyme-linked immunosorbent assay reactions were obtained using heat-killed B. suis antigen at a concentration comparable to McFarland Standard No. 1. Statistically significant differences were observed in the enzyme-linked immunosorbent assay results of 40 animals from which B. suis was isolated and the results for 48 noninfected swine at serum dilutions of 1:25 and 1:50 (P < 0.0001). The enzyme-linked immunosorbent assay is a rapid reproducible test which can be readily automated that appears to have practical value for screening large numbers of breeding and slaughter swine for brucellosis.     

Mercury in soils and crops from fields receiving high cumulative sewage sludge applications: Validation of U. S. EPA's risk assessment for human ingestion

The Metropolitan Water Reclamation District of Greater Chicago (MWRDGC) has owned and operated a 6320 ha Dedicated Beneficial Sludge Utilization Site in Fulton County, Illinois since 1971. The site consists of calcareous strip mine spoil intermingled with placed land. Sewage sludge from Chicago is barged to the site, located approximately 185 miles southwest of the city, and utilized to reclaim the strip mined soils and to fertilize the corn and wheat crops grown on them. Fields have received as much as 1317 dry Mg ha^?1 of sewage sludge since 1971. Sludge Hg concentrations have ranged from 1.1 to 8.5 mg Hg kg^?1 with mean concentration of 3.31 mg Hg kg^?1, and maximum cumulative Hg loading rates are approximately 4 kg ha^?1. Sludge applications have significantly increased extractable soil Hg concentrations, and regression analysis indicates that from 80 to 100% of the Hg applied to soils in sewage sludge since 1971 still resides in the top 15 cm of soil. Since 1985 the MWRDGC has been monitoring Hg concentration in corn leaf and grain, wheat grain and soils at the Fulton County site. Monitoring data indicate that 98.8% of the corn grain samples, 93.0% of the wheat samples and 50.7% of the corn leaf samples collected from 1985 through 1992 had Hg concentrations below detectable limits (<25μg kg^?1). Cumulative Hg loading rates are utilized along with crop tissue concentrations to compute crop uptake response slopes (UC) for Hg into plant tissues at the Fulton County site. The UC for corn and wheat grain was zero and for corn leaf was ?0.0014 (mg Hg/kg tissue)/(kg Hg/ha soil), which indicate that sewage sludge additions did not increase plant tissue Hg concentrations at the Fulton County site. The negative UC obtained for corn leaf may actually indicate that sewage sludge applications decreased Hg uptake from mined soils possibly due to organic carbon and sulfides in the anaerobically digested sludge binding native Hg. The United States Environmental Protection Agency (U. S. EPA) has recently promulgated their 40 CFR Part 503 regulation for sewage sludge use and disposal. The rule sets risk based limits on ten metals, including Hg, in sludges that are land applied. Exposure pathways involving plant uptake of Hg are briefly discussed and it is shown that the UC used in U. S. EPA's risk assessment models for these pathways overpredict uptake of Hg by crops when compared with the UC derived from the MWRDGC's monitoring data at Fulton County.     

Specific in-vitro lymphocyte immunostimulation activities of Brucella abortus fractions obtained by column chromatography

A Brucella abortus soluble antigen (BASA) preparation and fractions obtained thereof, by column chromatography, were compared in terms of their ability to induce specific lymphocyte stimulation responses (LSR) in lymphocytes from cattle infected with B. abortus. Endotoxin and protein contents and the fractions were determined. The LSR induced by BASA and the fractions were compared in terms of correctly identifying samples from infected and non-infected cattle. The sensitivity and specificity for each preparation were determined and these two attributes were then correlated with endotoxin and protein content. The results suggest that lymphocyte stimulation had greater association with relative protein content than endotoxin content of the antigen preparations.     

Detection of brucella antibodies of different immunoglobulin classes in cow milk by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assays were conducted on milk of cows from which Brucella abortus was isolated and that of noninfected controls. Horseradish peroxidase-labeled rabbit antibovine immunoglobulins IgG, IgG1, and IgA were used as conjugates. A heat-killed whole-cell suspension of B abortus strain 19 was used as the antigen. Differences in antibody profiles were observed in milk of cows from which B abortus was isolated and in milk of noninfected cows. Antibody profiles were similar in milk of cows infected with B abortus and that of cows from which B abortus strain 19 was isolated.     

Temporal cell-mediated immune responses of cattle following experimental and natural exposure to living Brucella abortus.

A study on cell-mediated immune responses in cattle with different exposure experiences to Brucella abortus was conducted by an in vitro lymphocyte stimulation assay. The purpose of this study was to determine how soon the cell-mediated immune responses would be detected following experimental exposure to B. abortus and to study the cell-mediated immune trend following experimental and natural exposure of cattle to B. abortus. The first positive cell-mediated immune responses occurred one to two weeks after experimental inoculation with living B. abortus strain 2308. The cell-mediated immune responses in these animals appeared at least one week before the appearance of of B. abortus serum agglutinating antibodies. Animals which were naturally infected with B. abortus biotypes 1 and 2 demonstrated positive cell-mediated immune responses throughout the study.     

Prediction of phytotoxicity and uptake of metals by models in proposed USEPA 40 CFR part 503 sludge regulations: Comparison with field data for corn and wheat

The United States Environmental Protection Agency (USEPA) has recently (February 6, 1989) proposed the 40 CFR Part 503 regulations which govern sludge use and disposal. The numerical limits on cumulative soil metal loading rates in these regulations were generated through risk assessment analysis based on modeling metal flux through several terrestrial pathways. Field data from a 6300 ha dedicated land application site operated by the Metropolitan Water Reclamation District of Greater Chicago (District) since 1972 indicate that these pathway models do a poor job of predicting bioaccumulation of Cd, Cu, and Zn in corn leaf and grain and wheat grain tissue. The USEPA pathway model does not take soil pH into account and predicts that plant tissue concentration will be linearly related to metal loading rates in soil. District field data indicate that crop tissue metal concentrations are dependent on soil pH and increase at low metal loadings but then become independent of total metal loading rate. No phytotoxicity was observed in corn or wheat despite soil loading rates for Cr, Cu, Ni, and Zn that were 13, 28, 3, and 16 times higher than their respective phytotoxic thresholds predicted by the USEPA pathway model.

     

Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19.

Cell-mediated immune responses in cattle naturally infected with strains of Brucella abortus and in cattle vaccinated with B abortus strain 19 during calfhood were studied by an in vitro lymphocyte-stimulation procedure. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B abortus-soluble antigen or phytohemagglutinin, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine (3HdT) and, after harvesting, assayed for 3HdT incorporation into DNA by liquid scintillation spectrometry. Lymphocytes from cattle with bacteriologically confirmed isolation of B abortus underwent a significantly higher lymphocyte stimulation with B abortus-soluble antigen than did cattle vaccinated with B abortus strain 19 during calfhood (P less than 0.005). Standard seroagglutination tests were conducted simultaneously with lymphocyte-stimulation tests, but there was no apparent correlation between levels of humoral antibodies and the cell-mediated immune responses as measured by in vitro specific lymphocyte stimulation.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.