Expression of Anaplasma marginale major surface protein 2 variants in persistently infected ticks

Abstract

Anaplasma marginale, an intraerythrocytic ehrlichial pathogen of cattle, establishes persistent infections in both vertebrate (cattle) and invertebrate (tick) hosts. The ability of A. marginale to persist in cattle has been shown to be due, in part, to major surface protein 2 (MSP2) variants which are hypothesized to emerge in response to the bovine immune response. MSP2 antigenic variation has not been studied in persistently infected ticks. In this study we analyzed MSP2 in A. marginale populations from the salivary glands of male Dermacentor variabilis persistently infected with A. marginale after feeding successively on one susceptible bovine and three sheep. New MSP2 variants appeared in each A. marginale population, and sequence alignment of the MSP2 variants revealed multiple amino acid substitutions, insertions, and deletions. These results suggest that selection pressure on MSP2 occurred in tick salivary glands independent of the bovine immune response.

FIG. 1

Experimental design. Calf PA432 was inoculated with 106 ml of infected blood (Virginia isolate of A. marginale) from PA431 (parasitemia = 0.9%) and served as the donor for infection of D. variabilis males. Calf PA432 was infested with 781 male D. variabilis ticks that were placed in orthopedic stockinettes attached to the calf when the ascending parasitemia was 4.4%. The ticks were allowed to feed for 7 days, after which they were removed and held in a humidity chamber for 6 days. The ticks were then allowed to feed on calf PA433 for 7 days, and then they were transferred directly and successively to feed for 7 days on sheep 1, 2, and 3. Forty ticks were removed from each host (PA433 and sheep 1, 2, and 3) on days 3 and 7 of tick feeding. The ticks were dissected, and salivary glands from the groups of 20 ticks were pooled and used for msp2 expression site cloning and sequence analysis.

FIG. 2

Alignment of MSP2 variant sequences identified in the tick salivary gland-derived A. marginale populations. Multiple amino acid substitutions, insertions, and deletions were detected. Alignment of protein sequences was performed using the program AlignX (Vector NTI Suite V 5.5; InforMax, Inc.) with an engine based on the CLUSTAL W algorithm (27). Conserved amino acids are shown in red, amino acids conserved in 11 to 20 or 21 sequences are shown in blue, and amino acid positions present in 1 to 10 or 21 sequences are shown in black.

FIG. 3

Dendogram constructed from the analysis of MSP2 variant sequences found in tick salivary gland-derived A. marginale populations based on a sequence distance method utilizing the neighbor-joining algorithm of Saitou and Nei (24) employing Vector NTI Suite 5.5. Sequence variants were assigned to the first A. marginale population in which they appeared. The clustering of similar sequences in different populations is shown.