Single-step technique for staining Anaplasma marginale in bovine blood smears.

Three available differential stains, Camco-Quik, Diff-Quik, and Wright-Giesma were compared for detection of intraerythrocytic Anaplasma marginale in bovine blood smears. In samples where < 1% to more than 51% of the RBC were infected, statistical analysis of the data indicated no significant difference in the detection of A marginale with Camco-Quik or Diff-Quik stains. However, a significantly lower percentage of infected RBC were detected when blood smears were stained with the Wright-Giemsa stain, compared with the other 2 methods.

High-yield preparation of purified Anaplasma marginale from infected bovine red blood cells.

Purification of Anaplasma marginale from infected bovine RBC was achieved through enzyme treatment and density-gradient centrifugation. A relative yield of 41.6% was obtained by dividing the number of organisms in the final purified preparation by the number of A marginale-infected RBC. Purified parasites were verified as A marginale by light microscopy, electron microscopy, and immunologic tests. The purified parasites reacted positively with calf and rabbit anti-A marginale sera in interfacial and slide agglutination tests. Anti-bovine RBC serum did not agglutinate purified A marginale, indicating absence of any contaminating RBC stroma. Anaplasma marginale was antigenic, but did not cause infection when the preparation was inoculated into a susceptible calf. The density of A marginale was determined to be 1.19 g/ml and cell diameters ranged from 0.25 to 0.63 micron. This method provided procedures for obtaining A marginale free of bovine RBC antigens for accurate biochemical assays and vaccine production.

A rapid staining procedure for Anaplasma marginale in bovine erythrocytes.

A commercial product that stains cells in less than 1 minute was compared with the Giemsa and the Wright-Giemsa methods for detection of the intraerythrocytic parasite, Anaplasma marginale, in bovine blood smears. The total number of A marginale-infected erythrocytes detected in 5 microscope fields of 18 different samples was determined for each staining procedure. In samples where less than 2% to greater than 25% of the RBC were infected, statistical analysis of the data indicated that the rapid staining method was superior to the Giemsa and Wright-Giemsa methods with regard to determination of the percentage of bovine erythrocytes infected with A marginale.

Costa as a glycogen store and probable site of energy generation in Tritrichomonas foetus.

Costae were isolated from the parasitic protozoan Tritrichomonas foetus and were found to contain 87% of the total cellular glycogen. Radiotracer studies, using [U-14C]glucose, were performed on starved and unstarved whole cells and on costae isolated from starved and unstarved cells. During cell starvation, catabolism of labeled costal glycogen was demonstrated. Preferential utilization by the cell of costal glycogen over other cellular glycogen stores was indicated.

Characterization of a flagellar sheath protein of Vibrio cholerae.

A flagellar sheath protein of Vibrio cholerae CA401 (Inaba) was characterized. Purity of the preparation was indicated by a single band on polyacrylamide gel electrophoresis gels and on Ouchterlony plates prepared with antibody against crude sheath material. The sheath protein was composed of three polypeptides with minimal molecular weights of 61,500, 60,000, and 56,500. The presence of sheath protein on the flagellum as well as on the outer membrane of the cell was demonstrated by ferritin labeling experiments with antiserum. Sheath protein antibody reacted similarly in labeling experiments and agglutination tests with a classical Ogawa strain and two nonagglutinating V. cholerae isolates, indicating that the sheath protein may represent the common Vibrio H antigen. Antibody specific for lipopolysaccharide labeled the cell but not the sheathed flagellum, which demonstrated that the sheath is not a simple extension of the outer membrane of the cell.

An immunodiffusion test for detection of bovine viral diarrhea virus antibodies in bovine serum.

An immunodiffusion test (IDT) was developed for detecting bovine viral diarrhea virus antibodies in bovine serum. The antigen utilized in the IDT was prepared from bovine viral diarrhea virus-infected monolayer cultures. Results of the IDT were obtained within 48 hours and correlated with the virus-neutralization test.

Isolation of Plasmodium berghei by hemolysin lysis of infected erythrocytes and evidence for a parasite hexokinase.

A rapid and simple procedure has been developed for the isolation of Plasmodium berghei parasites from infected-mouse erythrocytes employing the heat stable hemolysin produced by Pseudomonas aeruginosa. Using parasites isolated by this method, the presence of a parasite specific hexokinase has been demonstrated, providing an explanation for the increased glucose consumption observed with infected cells. Enzyme assays and serology were employed in determining the purity and yield of purified parasites. The enzyme assays showed that about 25% of the parasites in infected RBCs were recovered in the purified state. The purified parasites were not agglutinated by rabbit-anti-mouse RBC serum which indicated the purified parasites were not contaminated by RBC components.

Purification of the protein employed in an immunodiffusion test to diagnose infectious bovine rhinotracheitis.

The antigen used in an immunodiffusion test to diagnose infectious bovine rhinotracheitis has been purified by affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A So20,w of 0.749 was determined and a molecular weight of 8900 was calculated from sedimentation equilibrium analysis. The purified antigen formed precipitin lines of identity with crude diagnostic antigen. Purified antigen remained serologically active in the immunodiffusion test after lyophilization and subsequent reconstitution.

Costae of Tritrichomonas foetus: purification and chemical composition.

The costa is an intracellular organelle common to all trichomonads. Costae from Tritrichomonas foetus have been purified by a method which involves lysis of T. foetus with the heat-stable hemolysin produced by Pseudomonas aeruginosa, followed by differential centrifugation. Analysis of the purified costae demonstrated that the organelle is composed of 95 percent carbohydrate and 5 percent protein. The carbohydrate moiety, probably a polysaccharide, consisted of glucose (95 percent), mannose (0.4 percent), glucosamine (1.4 percent), ribose (0.6 percent), and an unidentified sugar (2.6 percent). The kinetosomal complex was attached to the costa after initial lysis of cells but was separated from the costa during purification.

Microimmunodiffusion test for detection of antibodies to infectious bovine rhinotracheitis virus in bovine serum.

A microimmunodiffusion test (MIDT) specific for detection of antibodies to infectious bovine rhinotracheitis (IBR) in bovine serum has been developed. The antigen used in the MIDT was prepared from IBR virus-infected Madin-Darby bovine kidney cells grown in tissue culture. The antigen was stable, and relatively high yields were obtained readily. Results of the MIDT were obtained within 48 hours and agreed with those of the serum-neutralization test.

Purification, characterization, and quantitation of the antigen employed in the immunodiffusion test for diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.

In vitro incorporation of 1-14C acetate by blood cells of normal ducks and ducks infected with Plasmodium lophurae.

Several investigations have been made, on a qualitative basis, of in vitro incorporation of 1-14C acetate into the lipids of whole blood from normal ducks and ducks infected with Plasmodium lophurae (1,3,4). It was found generally that the percent labeling in blood cells and plasma was higher for infected ducks than for normal ducks. The present study was concerned with quantitative determination of the incorporation of 1-14 C acetate into the lipids of purified erythrocytes (RBC) and leukocytes (WBC) of normal and infected ducks.

Extraction of equine infectious anemia immunodiffusion antigen with the aid of the chaotropic agent, thiocyanate.

Immunodiffusion antigen from spleens of horses infected with equine infectious anemia virus was prepared by methods employing freeze-thaw cycles and thiocyanate treatment. Thiocyanate (0.5 M) permitted the recovery of the greatest amount of antigen. Furthermore, it was most effective for recovery of immunodiffusion antigen from spleens which yielded unsatisfactory concentrations of antigen by the conventional freeze-thaw or water-extraction methods. The reactivity of the antigen did not appear to be affected by this chemical treatment.