Comparison of surface proteins of Anaplasma marginale grown in tick cell culture, tick salivary glands, and cattle

Abstract

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate of A. marginale was propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis. Development of A. marginale in cell culture was morphologically similar to that described previously in ticks. In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derived A. marginale and the bovine erythrocytic stage, currently the source of A. marginale antigen, was determined. Structural conservation on the tick salivary-gland stage was also examined. Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted with A. marginale from cell culture and tick salivary glands. MSP1a among geographic A. marginale isolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide. The cell culture-derived A. marginale maintained the same-size MSP1a as that found on the Virginia isolate of A. marginale in bovine erythrocytes and tick salivary glands. Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture. These data show that MSPs of erythrocyte-stage A. marginale are present on culture stages and may be structurally conserved during continuous culture. The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.

FIG. 1

Antibodies to A. marginale erythrocyte-stage surface proteins react with A. marginale grown in vitro in tick cell culture. Proteins of A. marginale-infected (A) or uninfected (B) tick cells were separated by SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose membranes, and reacted with specific antibodies, and reactions were visualized by chemiluminescence. Antibodies are as follows: anti-MSP1a, R874 (1:4,000) and MAb 22B1 (10 μg/ml); anti-MSP2, R883 (1:10,000) and R884 (1:10,000); anti-MSP5, MAb F16C1 (10 μg/ml); anti-purified erythrocyte-stage A. marginale (A.m.), R821 (1:10,000); negative antibody (Ab) controls, Tryp2B1 (10 μg/ml), Tryp1E1 (10 μg/ml), and normal rabbit serum (1:4,000). Numbers to the right of each panel indicate molecular mass in kilodaltons.

FIG. 2

Antibody to MSP1a detects a 70,000-molecular-weight band in the Virginia isolate of A. marginale from erythrocytes, tick salivary glands, and in vitro culture but a 105,000-molecular-weight band in the Florida isolate of A. marginale. A. marginale organisms purified from infected bovine erythrocytes (er.) or total proteins of infected tick cell cultures (t.c.) or infected (inf.) or uninfected (uninf.) salivary glands of D. andersoni (s.g.) were separated and blotted with anti-MSP1a (MAb 22B1) or negative control (Tryp1E1) antibody. The figure shows a comparison of MSP1a in Florida and Virginia erythrocyte stages (A), in Virginia erythrocyte and cultured stages (B), and in Virginia cultured and salivary-gland stages (C). Numbers at right indicate molecular mass in kilodaltons.

FIG. 3

Antibody to MSP2 detects a 36,000-molecular-weight band in Florida and Virginia-isolate, erythrocyte-stage (er.) A. marginale and in A. marginale from tick salivary glands (s.g.) or grown in vitro (t.c.). Antibodies are as follows: anti-MSP2, R883 (1:10,000); control, normal rabbit serum (1:10,000). Numbers at right indicate molecular mass in kilodaltons.

FIG. 4

MSP2 and MSP3 are the major polypeptides recognized by bovine infection serum in A. marginale from bovine erythrocytes, tick salivary glands, and in vitro culture. (A) Virginia isolate, erythrocyte-stage A. marginale proteins were separated and reacted with a bovine infection serum (PA188, 1:300), specific antibodies to MSPs, or control antibodies. (B) PA188 (1:300) was reacted with separated proteins derived from Florida or Virginia isolate, erythrocyte-stage A. marginale (er.) or with infected or uninfected (unif.) tick salivary glands (s.g.) and in vitro cultures (t.c.). (C) As for panel B, except that the dilution of PA188 is 1:1,000. Antibodies are as follows: bovine infection serum PA188, anti-MSP2 R883 (1:10,000), anti-MSP3 R955 (1:10,000), control normal rabbit serum (1:10,000), anti-MSP1a MAb 22B1 (2 μg/ml), anti-MSP3 MAb 75C2 (2 μg/ml), and control MAb Tryp1E1 (2 μg/ml). Numbers at left indicate molecular mass in kilodaltons.

FIG. 5

Conservation of MSP2 and MSP3 polypeptides in A. marginale grown in culture for varying times. Two-dimensional electrophoresis and immunoblotting with bovine infection serum PA188 (1:300) of A. marginale analyzed after passage 9 (30 January 1995) (A), passage 13 (6 June 1996) (B), passage 17 (13 November 1996) (C), and passage 21 (24 January 1997) (D). The migration positions of molecular weight standards (lysozyme, 14,300; trypsin inhibitor, 21,500; carbonic anhydrase, 30,000; ovalbumin, 46,000; bovine serum albumin, 66,000; phosphorylase b, 97,400; and myosin, 220,000) are shown on the left of each panel, and positions of isoelectric point standards (conalbumin, 6.3; carbonic anhydrase, 5.9; bovine serum albumin, 5.5; actin, 5.1; and trypsin inhibitor, 4.5) are shown at the bottom of each panel.

FIG. 6

Diversity in MSP2 and MSP3 polypeptides in A. marginale (A.m.) from salivary glands of D. andersoni (D.a.). Two-dimensional electrophoresis and immunoblotting of infected tick salivary glands with bovine infection serum PA188 (1:300) are shown. The migration positions of standards are illustrated as described for Fig. 5.

FIG. 7

Antibody to MSP4 detects a 31,000-molecular-weight band in A. marginale (A.m.)-infected but not in uninfected tick cells grown in vitro. Proteins were separated, blotted as described for Fig. 1, and reacted with anti-MSP4 antibody (rabbit C; 1:1,000) or preimmune serum (1:1,000), and reactions were visualized by chemiluminescence. Numbers to the right of each panel indicate molecular mass in kilodaltons.

FIG. 8

Antibody to MSP5 detects a 19,000-molecular-weight band in Florida and Virginia-isolate, erythrocyte-stage (er.) A. marginale and in A. marginale from tick salivary glands (s.g.) or grown in vitro (t.c.). Antibodies are anti-MSP5 MAb F16C1 (10 μg/ml) (A) and control Tryp1E1 (10 μg/ml) (B). Numbers at right indicate molecular mass in kilodaltons.