Analysis of protein compositions and surface protein epitopes of Anaplasma centrale and Anaplasma marginale.

Protein composition was compared and epitopes were analyzed among the isolates of Anaplasma centrale and A. marginale by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using bovine antisera and monoclonal antibodies, and enzyme-linked immunosorbent assay. Common and unique proteins were found among the isolates. All isolates tested had a major surface protein with an apparent molecular weight of 38 to 40 kilodalton which had slight molecular size variations between species. This protein was also a dominant immunogen to the host. At least two species-common epitopes, one of which might contain carbohydrate(s), were present on the major surface protein. One species-specific epitope was identified on the major surface protein of A. marginale isolates.

Nucleic acid levels in Anaplasma marginale during experimentally induced bovine anaplasmosis.

The DNA and RNA concentrations in uninfected and Anaplasma marginale-infected bovine erythrocytes were determined. Bovine anaplasmosis was experimentally induced and the nucleic acid levels were followed during the development of the A. marginale infection. Two distinct growth stages were found, the "multiplication" stage and the "transfer" stage. Based on this hypothetycal developmental cycle, the amount of DNA and RNA per Anaplasma initial body was estimated as 73.1 x 10(-3) pg and 45.7 x 10(-3) pg respectively.

Detection of an Anaplasma marginale common surface protein present in all stages of infection.

The establishment and maintenance of anaplasmosis-free cattle herds is impaired due to the lack of a rapid, sensitive, and specific serologic test to detect persistently infected cattle which serve as carriers for the organism. To develop an improved diagnostic test for anaplasmosis we screened Anaplasma marginale initial body proteins to identify a protein common to antigenically different isolates that is recognized by the host immune system at all stages of infection. Seronegative cattle were infected with either the Florida, Virginia, or North Texas isolate of A. marginale and monitored for infection by daily examination of Wright-stained blood smears for parasitized erythrocytes. Sera from cattle at different stages of infection, from acute through persistent, were used to immunoprecipitate A. marginale proteins that were metabolically radiolabeled with [35S]methionine or surface radiolabeled with 125I. Multiple A. marginale proteins were recognized by using sera either undiluted or at 1:10; however, only four or five proteins were sufficiently antigenic to elicit antibody reactive with a 1:1,000 serum dilution. A single protein with an apparent molecular mass of 86 kilodaltons was consistently recognized at all stages of infection regardless of the isolate used to infect the cattle. This protein was demonstrated to be on the surface of the A. marginale initial body and to be water soluble. We propose use of this 86-kilodalton protein to develop an improved serologic test for diagnosis of bovine anaplasmosis.

Comparison of proteins synthesized by two different isolates of Anaplasma marginale.

We present results on the initial definition of proteins synthesized by two isolates of Anaplasma marginale. Bovine erythrocytes infected with A. marginale were radioactively labeled with [35S]methionine or a 3H-amino acid mixture during short-term in vitro culture. The labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed protein bands of various apparent molecular weights from less than 14,000 to greater than 200,000. The bands observed represented A. marginale proteins because (i) uninfected erythrocytes from the same animal did not incorporate radioisotope during identical culture conditions, and (ii) the incorporation of radioisotope into proteins during culture of infected erythrocytes was inhibited by tetracycline but not by cycloheximide. The radioactive protein profiles of two different isolates of A. marginale, from Washington and Florida, were compared by two-dimensional gel electrophoresis. About 200 proteins were resolved in each case. Several proteins differed in position when the two-dimensional gel maps were compared, indicating variations in protein structure between the two A. marginale isolates.

Deoxyribonucleic acid of Anaplasma marginale.

Deoxyribonucleic acid from isolated marginal bodies and calf erythrocytes infected with Anaplasma marginale is found to be double stranded and to contain 51 moles per cent guanine plus cytosine.